Chronic-moderate ethanol exposure of L(tk-) cells expressing ? 4? 3? GABAA receptors reduces potency of allopregnanolone potentiation of GABA-evoked inward currents: Possible role of PKC

Aim: To investigate the effect of chronic-moderate ethanol (CME) treatment upon direct activation and allosteric modulation of GABAA receptors, and to assess the sensitivity of these parameters to PKC inhibition in control and ethanol-treated cells. Methods: L(tk-) cells were exposed to 20mM ethanol in culture media for 14 days prior to induction of stable expression of human recombinant ?4?3? receptors using dexamethasone. Concentration-response curves for GABA (1nM - 100µM), and allopregnanolone (ALLO; 1nM - 30µM) co-applied with 200nM GABA were obtained using whole-cell patch-clamp electrophysiology at a holding voltage of -60 mV. 400nM calphostin C (CphC) or vehicle (DMSO) was administered to cells via the pipette solution, which was prepared with 2mM Mg.ATP. SDS-PAGE and western blotting were used to compare levels of whole-cell expression, quantified relative to ?-actin, of ?4 and ? GABAA receptor subunits, and several isoforms of PKC (?, ?, ?, and ?), in control and CME-treated cells. Data was reported as Mean ± SEM and significance determined by either one-way ANOVA with Newman-Keuls multiple comparison tests, or two-way, unpaired t-tests. Results: Expression of ?4 subunits was reduced 35% (P0.05) and pEC50 (control: 6.18±0.04, n=13, CME: 6.17±0.04, n=4, P>0.05) of GABA were unchanged. CphC increased the GABA pEC50 relative to control (6.62±0.08, n=3, P<0.001) but had no effect upon responses at pEC20 GABA. Following CME, the potency of GABA was unaltered in the presence of CphC. The magnitude of ALLO-induced potentiation in control cells (7.34±0.6 fold, n=19) was unchanged by CME (ATP: 7.39±1.0 fold, n=9, P>0.05), and CphC had no significant effect in control or CME-treated cells. The ALLO pEC50 in control cells (6.23±0.05, n=19) was unaffected by CphC. Following CME, the potency of ALLO was reduced (5.68±0.06, n=9, P<0.001) but was enhanced in the presence of CphC, which restored potency almost back to control levels (5.94±0.09, n=5, P<0.05 relative to control). Expression of the ?, ?, ?, and ? isoforms of PKC was detected in whole-cell lysates of L(tk-) cells but only PKC? was significantly altered by CME treatment, exhibiting a nearly 4-fold increase (3.9±0.47 fold P<0.01) when compared with that in controls. Discussion: CME of un-induced L(tk-) cells was sufficient to alter sensitivity of ?4?3? receptor function to alterations of the balance of phosphorylation induced by CphC. The increased expression of PKC? after CME may have been directly related to the absence of effect of CphC upon GABA potency. As direct interaction of GABAA receptors with PKC? has not been determined, the effects observed for potency and efficacy of ALLO following CME may be indicative of changes to the phosphorylation of accessory proteins or other PKC isoforms by PKC?

Current Perspective on the Location and Function of Gamma- Aminobutyric Acid (GABA) and its Metabolic Partners in the Kidney.

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter located in the mammalian central nervous system, which binds to GABAA and GABAB receptors to mediate its neurological effects. In addition to its role in the CNS, an increasing number of publications have suggested that GABA might also play a role in the regulation of renal function. All three enzymes associated with GABA metabolism; glutamic acid decarboxylase, GABA ?-oxoglutarate transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) have been localised to the kidney providing the necessary machinery for localised GABA synthesis and metabolism. Moreover GABA receptors have been localised to both tubular and vascular structures in the kidney, and GABA is excreted in urine (~3 ?M) in humans. Despite the collective evidence describing the presence of a GABA system in the kidney, the precise function of such a system requires further clarification. Here we provide an overview of the current renal GABA literature and provide novel data that indicates GABA can act at contractile pericyte cells located along vasa recta capillaries in the renal medulla to potentially regulate medullary blood flow

Quinine blocks 5-HT and 5-HT3 receptor mediated peristalsis in both guinea pig and mouse ileum tissue

Introduction. Quinine is commonly used to treat malaria; however one of the principal side effects is gastrointestinal disturbances (White, 1992). 5-HT3 receptors modulate gut peristalsis (Chetty et al., 2006), and, as quinine has been shown to act as a 5-HT3 receptor antagonist (Thompson and Lummis, 2008) it is possible that these side effects result from actions at gut 5-HT3 receptors. To address this question, we examined the ability of quinine to antagonise 5-HT and 5-HT3 mediated peristalsis in guinea pig and mouse ileum. Methods. Ileum was excised from male guinea pigs (200-300g) and C57BL/6 mice (25-35g) following cervical dislocation. Ileum segments (3-5 cm) were mounted in 50 ml organ baths containing Tryode’s solution at 35-37 °C. Concentration-response curves were constructed for 5-HT and the selective 5-HT3 agonist 2-Me-5-HT (non-cumulative doses). Quinine was pre-applied for 10 min and inhibition measured using agonist concentrations that elicited a submaximal response. Results. Concentration-dependent contractions produced by 5-HT (pEC50 = 5.45 ± 0.17, n = 8) and the selective 5-HT3 agonist 2-Me-5-HT (5.01 ± 0.17, n = 11) were not significantly different (Student’s t-test, t = 0.619, df = 17, p = 0.544) in guinea pig ileum. Increasing concentrations of quinine were able to antagonise the activities of both 5-HT (pIC50 = 5.03 ± 0.2, n = 6) and 2-Me-5HT (pIC50 = 4.59 ± 0.26, n = 4). At mouse ileum, 5-HT (pEC50 = 7.57 ± 0.33, n = 9) was more potent (Student’s t-test, t = 3.6, df = 12, p = 0.004) than 2-Me-5-HT (pEC50 = 5.45 ± 0.58, n = 5). Quinine antagonised both the 5-HT (pIC50 = 4.87 ± 0.31, n = 7) and 2-Me-5-HT-induced (pIC50 = 6.18 ± 1.14, n = 4) contractions. Conclusions. These results support previous electrophysiological studies that identified quinine as an antagonist at recombinant 5-HT3 receptors with IC50 values comparable with those reported here (pIC50 = 4.87, Thompson et al., 2007). Further, we found that quinine completely blocked 5-HT induced contractions in mouse and guinea pig, raising the possibility that quinine targets other 5-HT receptors in the gut (e.g., 5-HT4 receptors) and may influence intestinal function

Urinary ATP and visualization of intracellular bacteria: a superior diagnostic marker for recurrent UTI in renal transplant recipients?

Renal transplant recipients (RTR) are highly susceptible to urinary tract infections (UTIs) with over 50% of patients having at least one UTI within the first year. Yet it is generally acknowledged that there is considerable insensitivity and inaccuracy in routine urinalysis when screening for UTIs. Thus a large number of transplant patients with genuine urine infections may go undiagnosed and develop chronic recalcitrant infections, which can be associated with graft loss and morbidity. Given a recent study demonstrating ATP is released by urothelial cells in response to bacteria exposure, possibly acting at metabotropic P2Y receptors mediating a proinflammatory response, we have investigated alternative, and possibly more appropriate, urinalysis techniques in a cohort of RTRs.Mid-stream urine (MSU) samples were collected from 53 outpatient RTRs. Conventional leukocyte esterase and nitrite dipstick tests, and microscopic pyuria counts (in 1 ?l), ATP concentration measurements, and identification of intracellular bacteria in shed urothelial cells, were performed on fresh unspun samples and compared to ‘gold-standard’ bacterial culture results.Of the 53 RTRs, 22% were deemed to have a UTI by ‘gold-standard’ conventional bacteria culture, whereas 87%, 8% and 4% showed evidence of UTIs according to leukocyte esterase dipstick, nitrite dipstick, and a combination of both dipsticks, respectively. Intracellular bacteria were visualized in shed urothelial cells of 44% of RTRs, however only 1 of the 23 RTRs (44%) was deemed to have a UTI by conventional bacteria culture. A significant association of the ‘gold-standard’ test with urinary ATP concentration combined with visualization of intracellular bacteria in shed urothelial cells was determined using the Fisher’s exact test.It is apparent that standard bedside tests for UTIs give variable results and that seemingly quiescent bacteria in urothelial cells are very common in RTRs and may represent a focus of subclinical infection. Furthermore, our results suggest urinary ATP concentration combined with detection of intracellular bacteria in shed urinary epithelial cells may be a sensitive means by which to detect ‘occult’ infection in RTRs

Structure and properties of [(4,6-tBu2C6H2O)2Se]2An(THF)2, An = U, Np, and their reaction with p-benzoquinone.

The synthesis and characterization of U(iv) and Np(iv) selenium bis(phenolate) complexes are reported. The reaction of two equivalents of the U(iv) complex with p-benzoquinone results in the formation of a U(v)-U(v) species with a bridging reduced quinone. This represents a rare example of high-valent uranium chemistry as well as a rare example of a neptunium aryloxide complex

The Community-Acquired Pneumonia Organization (CAPO) Cloud-Based Research Platform (the CAPO-Cloud): Facilitating Data Sharing in Clinical Research

Background: Pneumonia is a costly and deadly respiratory disease that afflicts millions every year. Advances in pneumonia care require significant research investment and collaboration among pneumonia investigators. Despite the importance of data sharing for clinical research it remains difficult to share datasets with old and new investigators. We present CAPOCloud, a web-based pneumonia research platform intended to facilitate data sharing and make data more accessible to new investigators. Methods: We establish the first two use cases for CAPOCloud to be the automatic subsetting and constraining of the CAPO database and the automatic summarization of the database in aggregate. We use the REDCap data capture software and the R programming language to facilitate these use cases. Results: CAPOCloud allows CAPO investigators to access the CAPO clinical database and explore subsets of the data including demographics, comorbidities, and geographic regions. It also allows them to summarize these subsets or the entire CAPO database in aggregate while preserving privacy restrictions. Discussion: CAPOCloud demonstrates the viability of a research platform combining data capture, data quality, hypothesis generation, data exploration and data sharing in one interface. Future use cases for the software include automated univariate hypothesis testing, automated bivariate hypothesis testing, and principal component analysis

A meta-analytical assessment of the efficacy of immediate release methylphenidate to reduce hyperactivity in children with autistic spectrum disorder (ASD)

Psychostimulant medications, such as Methylphenidate (MPH) have become main stay treatments for Attention Deficit Hyperactivity Disorder (ADHD) symptoms, such as hyperactivity, impulsivity and inattention in children. Such symptoms frequently also co-exist in children with Autistic Spectrum Disorders (ASD), thereby complicating a differential diagnosis. The presence of ADHD-like symptoms in children with ASD can have a significant detrimental impact on learning and social interaction, and can limit the outcome of behavioural interventions to address areas of core ASD difficulties. Therefore, it is of clinical relevance and potential benefit to address the issue of effectiveness and tolerability/safety of stimulants such as methylphenidate in children with co-morbid diagnoses of ASD + ADHD. This study performed a meta-analysis of three randomized controlled trials homogeneously measuring the effect of immediate release Methylphenidate (MPH) to reduce scores of hyperactivity in children with comorbid ADHD + ASD. The findings that IR-MPH produces an overall moderate significant benefit to reduce hyperactivity in ASD are discussed in relation to the risk/benefit question of medication use in this population

Raising the unification scale in supersymmetry

In the minimal supersymmetric standard model, the three gauge couplings appear to unify at a mass scale near $2 \times 10^{16}$ GeV. We investigate the possibility that intermediate scale particle thresholds modify the running couplings so as to increase the unification scale. By requiring consistency of this scenario, we derive some constraints on the particle content and locations of the intermediate thresholds. There are remarkably few acceptable solutions with a single cleanly defined intermediate scale far below the unification scale.Comment: 22 pages, macros included. One figure, available at ftp://ftp.phys.ufl.edu/incoming/rais.ep