PKA Mediates Constitutive Activation of CFTR in Human Sweat Duct

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels are constitutively activated in sweat ducts. Since phosphorylation-dependent and -independent mechanisms can activate CFTR, we sought to determine the actual mechanism responsible for constitutive activation of these channels in vivo. We show that the constitutively activated CFTR Cl− conductance (gCFTR) in the apical membrane is completely deactivated following α-toxin permeabilization of the basolateral membrane. We investigated whether such inhibition of gCFTR following permeabilization is due to the loss of cytoplasmic glutamate or due to dephosphorylation of CFTR by an endogenous phosphatase in the absence of kinase activity (due to the loss of kinase agonist cAMP, cGMP or GTP through α-toxin pores). In order to distinguish between these two possibilities, we examined the effect of inhibiting the endogenous phosphatase activity with okadaic acid (10−8 M) on the permeabilization-induced deactivation of gCFTR. We show that okadaic acid (1) inhibits an endogenous phosphatase responsible for dephosphorylating cAMP but not cGMP or G protein-activated CFTR and (2) prevents deactivation of CFTR following permeabilization of the basolateral membrane. These results indicate that distinctly different phosphatases may be responsible for dephosphorylating different kinase-specific sites on CFTR. We conclude that the phosphorylation by PKA alone appears to be primarily responsible for constitutive activation of gCFTR in vivo

Guideline adherence and patient satisfaction in the treatment of inflammatory bowel disorders – an evaluation study

Background: Crohn's disease (CD) and ulcerative colitis (UC) are the most frequent inflammatory bowel disorders (IBD). IBD cause a significant burden to society due to extensive health care utilization from the first clinical symptoms until diagnosis and thereafter due to direct and indirect costs. Besides the socio-economic impact of CD and UC, gastrointestinal and extraintestinal symptoms affect quality of life, but there is remarkably little data about the quality of treatment as assessed by patient satisfaction, quality of life and adherence to guidelines. Thus the aim of this study was to identify variables that influence quality of treatment and quality of life as well as patient satisfaction. Methods: The Essener Zirkel Study was a cross sectional study of 86 IBD-patients with a confirmed diagnosis of CD or UC. They were recruited at primary, secondary and tertiary care settings. Quality of treatment, quality of life and patient satisfaction were evaluated. Consulting behaviour and number of examinations, duration of disease and variables regarding adherence to guidelines were evaluated, too. Results: 59 (69%) patients had CD and 27 had UC (31%). 19% spent more than four years until the suspected diagnosis of IBD was confirmed and visited more than five physicians. All patients showed a significantly reduced quality of life compared to the 1998 German normative population. In spite of being under medical treatment, nearly half of the patients suffered from strong quality of life restricting symptoms. Over all, 35% described their treatment as moderate or bad. Patients who consulted psychotherapists and non-medical practitioners suffered significantly less from depression. Conclusion: Besides structural deficiencies due to the health care policy, we revealed the adherence to guidelines to be a problem area. Our findings support the assumption, that providing better health care and especially maintaining constant patient-physician communication improves patient satisfaction.Claudia Pieper, Sebastian Haag, Stefan Gesenhues, Gerald Holtmann, Guido Gerken and Karl-Heinz Jöcke

Biodegradation of Pig Manure by the Housefly, Musca domestica: A Viable Ecological Strategy for Pig Manure Management

The technology for biodegradation of pig manure by using houseflies in a pilot plant capable of processing 500–700 kg of pig manure per week is described. A single adult cage loaded with 25,000 pupae produced 177.7±32.0 ml of eggs in a 15-day egg-collection period. With an inoculation ratio of 0.4–1.0 ml eggs/kg of manure, the amount of eggs produced by a single cage can suffice for the biodegradation of 178–444 kg of manure. Larval development varied among four different types of pig manure (centrifuged slurry, fresh manure, manure with sawdust, manure without sawdust). Larval survival ranged from 46.9±2.1%, in manure without sawdust, to 76.8±11.9% in centrifuged slurry. Larval development took 6–11 days, depending on the manure type. Processing of 1 kg of wet manure produced 43.9–74.3 g of housefly pupae and the weight of the residue after biodegradation decreased to 0.18–0.65 kg, with marked differences among manure types. Recommendations for the operation of industrial-scale biodegradation facilities are presented and discussed

Negative Correlation between Brain Glutathione Level and Negative Symptoms in Schizophrenia: A 3T 1H-MRS Study

BACKGROUND: Glutathione (GSH), a major intracellular antioxidant, plays a role in NMDA receptor-mediated neurotransmission, which is involved in the pathophysiology of schizophrenia. In the present study, we aimed to investigate whether GSH levels are altered in the posterior medial frontal cortex of schizophrenic patients. Furthermore, we examined correlations between GSH levels and clinical variables in patients. METHODS AND FINDINGS: Twenty schizophrenia patients and 16 age- and gender-matched normal controls were enrolled to examine the levels of GSH in the posterior medial frontal cortex by using 3T SIGNA EXCITE (1)H-MRS with the spectral editing technique, MEGA-PRESS. Clinical variables of patients were assessed by the Global Assessment of Functioning (GAF), Scale for the Assessment of Negative Symptoms (SANS), Brief Psychiatric Rating Scale (BPRS), Drug-Induced Extra-Pyramidal Symptoms Scale (DIEPSS), and five cognitive performance tests (Word Fluency Test, Stroop Test, Trail Making Test, Wisconsin Card Sorting Test and Digit Span Distractibility Test). Levels of GSH in the posterior medial frontal cortex of schizophrenic patients were not different from those of normal controls. However, we found a significant negative correlation between GSH levels and the severity of negative symptoms (SANS total score and negative symptom subscore on BPRS) in patients. There were no correlations between brain GSH levels and scores on any cognitive performance test except Trail Making Test part A. CONCLUSION: These results suggest that GSH levels in the posterior medial frontal cortex may be related to negative symptoms in schizophrenic patients. Therefore, agents that increase GSH levels in the brain could be potential therapeutic drugs for negative symptoms in schizophrenia

Regulation of Plant Developmental Processes by a Novel Splicing Factor

Serine/arginine-rich (SR) proteins play important roles in constitutive and alternative splicing and other aspects of mRNA metabolism. We have previously isolated a unique plant SR protein (SR45) with atypical domain organization. However, the biological and molecular functions of this novel SR protein are not known. Here, we report biological and molecular functions of this protein. Using an in vitro splicing complementation assay, we showed that SR45 functions as an essential splicing factor. Furthermore, the alternative splicing pattern of transcripts of several other SR genes was altered in a mutant, sr45-1, suggesting that the observed phenotypic abnormalities in sr45-1 are likely due to altered levels of SR protein isoforms, which in turn modulate splicing of other pre-mRNAs. sr45-1 exhibited developmental abnormalities, including delayed flowering, narrow leaves and altered number of petals and stamens. The late flowering phenotype was observed under both long days and short days and was rescued by vernalization. FLC, a key flowering repressor, is up-regulated in sr45-1 demonstrating that SR45 influences the autonomous flowering pathway. Changes in the alternative splicing of SR genes and the phenotypic defects in the mutant were rescued by SR45 cDNA, further confirming that the observed defects in the mutant are due to the lack of SR45. These results indicate that SR45 is a novel plant-specific splicing factor that plays a crucial role in regulating developmental processes

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

Hepatocyte growth factor enhances death receptor-induced apoptosis by up-regulating DR5

<p>Abstract</p> <p>Background</p> <p>Hepatocyte growth factor (HGF) and its receptor c-MET are commonly expressed in malignant gliomas and embryonic neuroectodermal tumors including medulloblastoma and appear to play an important role in the growth and dissemination of these malignancies. Dependent on cell context and the involvement of specific downstream effectors, both pro- and anti-apoptotic effects of HGF have been reported.</p> <p>Methods</p> <p>Human medulloblastoma cells were treated with HGF for 24–72 hours followed by death receptor ligand TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) for 24 hours. Cell death was measured by MTT and Annexin-V/PI flow cytometric analysis. Changes in expression levels of targets of interest were measured by Northern blot analysis, quantitative reverse transcription-PCR, Western blot analysis as well as immunoprecipitation.</p> <p>Results</p> <p>In this study, we show that HGF promotes medulloblastoma cell death induced by TRAIL. TRAIL alone triggered apoptosis in DAOY cells and death was enhanced by pre-treating the cells with HGF for 24–72 h prior to the addition of TRAIL. HGF (100 ng/ml) enhanced TRAIL (10 ng/ml) induced cell death by 36% (<it>P </it>< 0.001). No cell death was associated with HGF alone. Treating cells with PHA-665752, a specific c-Met receptor tyrosine kinase inhibitor, significantly abrogated the enhancement of TRAIL-induced cell death by HGF, indicating that its death promoting effect requires activation of its canonical receptor tyrosine kinase. Cell death induced by TRAIL+HGF was predominately apoptotic involving both extrinsic and intrinsic pathways as evidenced by the increased activation of caspase-3, 8, 9. Promotion of apoptosis by HGF occurred via the increased expression of the death receptor DR5 and enhanced formation of death-inducing signal complexes (DISC).</p> <p>Conclusion</p> <p>Taken together, these and previous findings indicate that HGF:c-Met pathway either promotes or inhibits medulloblastoma cell death via pathway and context specific mechanisms.</p

The total synthesis of (-)-cyanthiwigin F by means of double catalytic enantioselective alkylation

Double catalytic enantioselective transformations are powerful synthetic methods that can facilitate the construction of stereochemically complex molecules in a single operation. In addition to generating two or more stereocentres in a single reaction, multiple asymmetric reactions also impart increased enantiomeric excess to the final product in comparison with the analogous single transformation. Furthermore, multiple asymmetric operations have the potential to independently construct several stereocentres at remote points within the same molecular scaffold, rather than relying on pre-existing chiral centres that are proximal to the reactive site. Despite the inherent benefits of multiple catalytic enantioselective reactions, their application to natural product total synthesis remains largely underutilized. Here we report the use of a double stereoablative enantioselective alkylation reaction in a concise synthesis of the marine diterpenoid (-)-cyanthiwigin F (ref. 8). By employing a technique for independent, selective formation of two stereocentres in a single stereoconvergent operation, we demonstrate that a complicated mixture of racemic and meso diastereomers may be smoothly converted to a synthetically useful intermediate with exceptional enantiomeric excess. The stereochemical information generated by means of this catalytic transformation facilitates the easy and rapid completion of the total synthesis of this marine natural product

The antioxidant and antiproliferative activities of methanolic extracts from Njavara rice bran

<p>Abstract</p> <p>Background</p> <p>Free radical-induced oxidative stress is the root cause for many human diseases. Naturally occurring antioxidant supplements from plants are vital to counter the oxidative damage in cells. The main objective of the present study was to characterize the antioxidant and antiproliferative potential of rice bran extracted from an important Indian rice variety, Njavara and to compare the same with two commercially available basmati rice varieties: Vasumathi, Yamini and a non medicinal variety, Jyothi.</p> <p>Methods</p> <p>Methanolic extracts of rice bran from four varieties; Vasumathi, Yamini, Jyothi and Njavara were used to study their total phenolic and flavonoid contents, <it>in vitro </it>antioxidant activities including total antioxidant activity, scavenging of nitric oxide and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical, reducing power and cytotoxic activity in C6 glioma cells. Correlation coefficient and regression analysis were done by using Sigmastat version 3.1 and Stata statistical package respectively.</p> <p>Results</p> <p>Rice bran methanolic extract from Njavara showed the highest antioxidant and cell cytotoxic properties compared to the other three rice varieties. IC₅₀values for scavenging DPPH and nitric oxide were in the range of 30.85-87.72 μg/ml and 52.25-107.18 μg/ml respectively. Total antioxidant activity and reducing power were increased with increasing amounts of the extract. Total phenolic and flavonoid contents were in the range of 3.2-12.4 mg gallic acid-equivalent (GAE)/g bran and 1.68-8.5 mg quercetin-equivalent (QEE)/g bran respectively. IC₅₀values of cytotoxic assay (MTT assay) were 17.53-57.78 μg/ml. Correlation coefficient and regression analysis of phenolic content with DPPH and NO scavenging, MTT (-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, total antioxidant assay and reducing power showed a highly significant correlation coefficient values (96-99%) and regression values (91-98%).</p> <p>Conclusion</p> <p>The results of the present study show that the crude methanolic extract from Njavara rice bran contains significantly high polyphenolic compounds with superior antioxidant activity as evidenced by scavenging of free radicals including DPPH and NO. Njavara extracts also showed highest reducing power activity, anti-proliferative property in C6 glioma cells. In conclusion, it is conceivable that the Njavara rice variety could be exploited as one of the potential sources for plant - based pharmaceutical products.</p