Apoptotic extinction of germ cells in testes of Cyp26b1 knockout mice

Cyp26b1 encodes a retinoic acid (RA) metabolizing cytochrome P450 enzyme that is expressed in embryonic tissues undergoing morphogenesis, including the testes. We have generated transgenic mice lacking Cyp26b1 and have observed increased RA levels in embryonic testes. Cyp26b1(-/-) germ cells prematurely enter meiosis at embryonic d 13.5 and appear to arrest at pachytene stage. Furthermore, after embryonic d 13.5, a rapid increase in apoptosis is observed in male germ cells derived from Cyp26b1(-/-) embryos; germ cells are essentially absent in mutant male neonates. In contrast, testicular somatic cells appear to develop normally in the absence of Cyp26b1. Moreover, ovarian germ and somatic cells appear unaffected by the lack of CYP26B1. We also show that the synthetic retinoid Am580, which is resistant to CYP26 metabolism, induces meiosis of male germ cells in cultured gonads, suggesting that abnormal development of germ cells in the Cyp26b1(-/-) testes results from excess RA rather than the absence of CYP26B1-generated metabolites of RA. These results provide evidence that CYP26B1 maintains low levels of RA in the developing testes that blocks entry into meiosis and acts as a survival factor to prevent apoptosis of male germ cells.     

Functional desensitization of the extracellular calcium-sensing receptor is regulated via distinct mechanisms: role of G protein-coupled receptor kinases, protein kinase C and beta-arrestins

The extracellular calcium-sensing receptor (CaR) senses small fluctuations of the extracellular calcium (Ca(2+)(e)) concentration and translates them into potent changes in parathyroid hormone secretion. Dissecting the regulatory mechanisms of CaR-mediated signal transduction may provide insights into the physiology of the receptor and identify new molecules as potential drug targets for the treatment of osteoporosis and/or hyperparathyroidism. CaR can be phosphorylated by protein kinase C (PKC) and G protein-coupled receptor kinases (GRKs), and has been shown to bind to beta-arrestins, potentially contributing to desensitization of CaR, although the mechanisms by which CaR-mediated signal transduction is terminated are not known. We used a PKC phosphorylation site-deficient CaR, GRK and beta-arrestin overexpression or down-regulation to delineate CaR-mediated desensitization. Fluorescence-activated cell sorting was used to determine whether receptor internalization contributed to desensitization. Overexpression of GRK 2 or 3 reduced Ca(2+)(e)-dependent inositol phosphate accumulation by more than 70%, whereas a GRK 2 mutant deficient in G alpha(q) binding (D110A) was without major effect. Overexpression of GRK 4-6 did not reduce Ca(2+)(e)-dependent inositol phosphate accumulation. Overexpression of beta-arrestin 1 or 2 revealed a modest inhibitory effect on Ca(2+)(e)-dependent inositol phosphate production (20-30%), which was not observed for the PKC phosphorylation site-deficient CaR. Agonist-dependent receptor internalization (10-15%) did not account for the described effects. Thus, we conclude that PKC phosphorylation of CaR contributes to beta-arrestin-dependent desensitization of CaR coupling to G proteins. In contrast, GRK 2 predominantly interferes with G protein-mediated inositol-1,4,5-trisphosphate formation by binding to G alpha(q).     

Electromagnetic navigation diagnostic bronchoscopy in peripheral lung lesions

BACKGROUND: Electromagnetic navigation bronchoscopy (ENB) with biopsy under fluoroscopic guidance has enhanced the yield of flexible bronchoscopy in the diagnosis of peripheral lung lesions. However, the accuracy of ENB navigation suggests that the addition of fluoroscopy is redundant. OBJECTIVES: Data were prospectively collected to determine the yield of ENB without fluoroscopy in the diagnosis of peripheral lung lesions. METHOD: ENB was performed via flexible bronchoscopy (superDimension/Bronchus system; superDimension Inc; Plymouth, MN). Biopsy specimens were obtained through the extended working channel after navigation. Fluoroscopy was not utilized, but post-transbronchial biopsy chest radiographs were obtained to exclude pneumothorax. The primary end point was diagnostic yield, and the secondary end points were navigation accuracy, procedure duration, and safety. Analysis by lobar distribution was also performed to assess performance in different lobes of the lung. RESULTS: Ninety-two peripheral lung lesions were biopsied in the 89 subjects. The diagnostic yield of ENB was 67%, which was independent of lesion size. Total procedure time ranged from 16.3 to 45.0 min (mean [+/- SD] procedure time, 26.9 +/- 6.5 min). The mean navigation error was 9 +/- 6 mm (range, 1 to 31 mm). There were two incidences of pneumothorax for which no intervention was required. When analyzed by lobar distribution, there was a trend toward a higher ENB yield in diagnosing lesions in the right middle lobe (88%). CONCLUSIONS: ENB can be used as a stand-alone bronchoscopic technique without compromising diagnostic yield or increasing the risk of pneumothorax. This may result in sizable timesaving and avoids radiation exposure.     

Diagnostic performance of the platelet function analyzer (PFA-100) for the detection of disorders of primary haemostasis in patients with a bleeding history-a systematic review and meta-analysis

The Platelet Function Analyzer (PFA-100) is increasingly being used in the workup of patients with a bleeding diathesis. A profound knowledge of the possible diagnostic performance of this test is essential in order to make sound clinical decisions based on its results. It was the aim of this study to systematically review the published literature and provide valid estimates of the diagnostic performance of the PFA-100 for detecting disorders of primary haemostasis in newly presenting patients with a bleeding diathesis. A comprehensive literature search was performed for studies published between January 1994 and February 2006. Studies were eligible for the systematic review if they provided data supposed to be applicable to the determination of the diagnostic performance of the PFA-100. Furthermore, they were included in a meta-analysis if study reporting allowed calculation of sensitivity and specificity and if study quality ensured minimized biases of these estimates for the described clinical setting. Pooled weighted sensitivity, specificity and diagnostic odds ratio were calculated applying random effects modelling and constructing summary operator characteristic curves. This was done separately for the available test modifications using either collagen/epinephrine (PFA-EPI) or collagen/adenosine-diphosphate (PFA-ADP) for platelet activation. Thirty-six articles were included in the systematic review. Six studies met our eligibility criteria for a meta-analysis. The major reason for exclusion from the meta-analysis was a case-control design. A total of 1486 and 1259 patients were included in the meta-analysis of the diagnostic performance of the PFA-EPI and PFA-ADP, respectively. Pooled weighted sensitivity and specificity of the PFA-EPI/PFA-ADP in detecting a disorder of primary haemostasis were: 82.5/66.9% (95%-confidence interval (95%-CI): 76.0-88.9%/57.9-75.9%), and 88.7/85.5% (95%-CI: 84.3-93.1%/82.0-89.1%). 83/75% of patients with a positive PFA-EPI/PFA-ADP result do have a disorder of primary haemostasis whereas 88/79% with a negative PFA-EPI/PFA-ADP result do not. The PFA-EPI appeared to have a higher sensitivity and better predictive values than the PFA-ADP in detecting disorders of primary haemostasis, although a rigorous gold standard definition for a disorder of primary haemostasis, particularly for platelet disorders, was not applied in most studies. The majority of the studies lacked important requirements for quality and reporting, precluding a more precise and definitive characterization of the clinical utility of the PFA-100. This emphasizes the need for an evidence-based critical appraisal of diagnostic studies in haemostasis research in order to promote the conducting of studies that produce clinically relevant results.     

Effect of an invasive strategy on in-hospital outcome in elderly patients with non-ST-elevation myocardial infarction

Aims: We sought to investigate the impact of an invasive treatmentin elderly patients presenting with non-ST elevation myocardialinfarction (NSTEMI) in clinical practice. Methods and results: We analysed data of consecutive elderly patients (75 years)with NSTEMI who were prospectively enrolled in the German AcuteCoronary Syndromes registry between July 2000 and November 2002.Overall 1936 patients were divided into two groups: 1005 (51.9%)underwent coronary angiography and/or revascularization, 931(48.1%) received conservative treatment. In the invasive group,percutaneous coronary intervention was performed in 37.5% within48 h and in 17.6% after 48 h, whereas 9.8% underwent coronaryartery bypass grafting within the hospital stay. In-hospitaldeath (12.5 vs. 6.0%, P < 0.0001) and death/myocardial infarction(17.3 vs. 9.6%, P < 0.0001) occurred significantly less oftenin patients with invasive strategy. After adjustment of theconfounding factors in the propensity score analysis the invasivestrategy remained superior for mortality (OR 0.55, 95% CI 0.35–0.86)and death and non-fatal myocardial infarction (OR 0.51, 95%CI 0.35–0.75) and 1 year mortality (OR 0.56, 95% CI 0.38–0.81).Major bleeding complications tended to be more frequent in theinvasive group (8.8 vs. 5.8%, P = 0.07). Conclusion: In clinical practice, in elderly patients with NSTEMI, an invasivestrategy is associated with an improved in-hospital and 1 yearoutcome but a trend towards more bleeding complications.     

Directed selection of a conformational antibody domain that prevents mature amyloid fibril formation by stabilizing Abeta protofibrils

The formation of amyloid fibrils is a common biochemical characteristic that occurs in Alzheimer's disease and several other amyloidoses. The unifying structural feature of amyloid fibrils is their specific type of beta-sheet conformation that differentiates these fibrils from the products of normal protein folding reactions. Here we describe the generation of an antibody domain, termed B10, that recognizes an amyloid-specific and conformationally defined epitope. This antibody domain was selected by phage-display from a recombinant library of camelid antibody domains. Surface plasmon resonance, immunoblots, and immunohistochemistry show that this antibody domain distinguishes Abeta amyloid fibrils from disaggregated Abeta peptide as well as from specific Abeta oligomers. The antibody domain possesses functional activity in preventing the formation of mature amyloid fibrils by stabilizing Abeta protofibrils. These data suggest possible applications of B10 in the detection of amyloid fibrils or in the modulation of their formation.