Distribution, quantitation, and origin of immunoreactive neuropeptide Y in the human gastrointestinal tract

A radioimmunoassay for measurement of immunoreactive neuropeptide Y has been developed using antiserum from a rabbit (221) immunized with porcine neuropeptide Y. Antibody 221 has been characterized for both sensitivity and specificity. To determine the distribution of neuropeptide Y in the human gastrointestinal tract, fresh tissue specimens were separated by microdissection into the muscularis externa and the mucosa-submucosa. To examine the origin of neuropeptide Y in human colon, specimens of aganglionic and ganglionic colon were obtained from patients with Hirschsprung's disease. Immunoreactive neuropeptide Y in human gut was present in highest concentrations in the muscularis externa of the stomach and in lowest concentrations in the muscularis externa of the ileum and descending colon. Neuropeptide Y in the stomach was present in higher concentrations in the muscularis externa than in the mucosa-submucosa, but in the descending colon there were lower concentrations of neuropeptide Y in the muscularis externa than in the mucosa-submucosa. In Hirschsprung's disease, concentrations of neuropeptide Y were increased in aganglionic colon in both the muscularis externa and the mucosa-submucosa, compared to corresponding layers from proximal ganglionic colon. Extracts of the gastric muscularis externa and the colonic mucosa-submucosa were separated by C18 reverse-phase high-performance liquid chromatography. One major immunoreactive species was identified by radioimmunoassay which eluted in a position similar to synthetic human neuropeptide Y. These results demonstrated both regional and layer differences in concentrations of neuropeptide Y in human gut. Increased concentrations of neuropeptide Y in aganglionic colon from Hirschsprung's disease most likely result from enlargement of neuropeptide Y-containing extrinsic nerve fibers in both the mucosa-submucosa and the muscularis externa.     

Adrenomedullary Secretion of DOPA, Catecholamines, Catechol Metabolites, and Neuropeptides

Abstract: Catecholamines and their metabolites have been proposed as markers of sympathetic nervous system stimulation. However, the adrenal medulla is a rich source of catecholamines and catecholamine metabolites and may play a significant role in plasma levels of these compounds. In addition to adrenal catecholamine metabolite efflux, the role of the catecholamine precursor 3,4-dihydroxyphenylalanine (DOPA) has not been fully evaluated. The simultaneous effluxes of catecholamines, metabolites, DOPA, and neuropeptides were measured in perfusates from isolated dog adrenals. The relative abundance of compounds detected consistently during unstimulated conditions was epinephrine ≫ norepinephrine > 3,4-dihydroxyphenylglycol > metanephrine > normetanephrine > dopamine > 3,4-dihydroxyphenylacetic acid > 3-methoxy-4-hydroxyphenylglycol ≥ DOPA ≫ [Met]enkephalin ≫ neuropeptide Y. Effluxes of analytes were not affected by cocaine and the ratios of catecholamines to metabolites increased dramatically with carbachol stimulation, consistent with negligible reuptake into adrenal cells. Thus, most of the 3,4-dihydroxyphenylglycol is expected to be derived from epinephrine and norepinephrine subsequent to translocation from chromaffin vesicles into the cytosol. The efflux of DOPA increased dramatically during stimulation with 30 µ M carbachol in a calcium-dependent manner. Efflux of DOPA during the initial stabilization period of the perfusion preparation declined exponentially, in parallel with the effluxes of the catecholamines and neuropeptides but not with metabolites. Evoked release of DOPA was Ca²⁺-dependent. These data suggest that DOPA can be stored and released exocytotically from chromaffin granules.     

Intraluminal androgen binding protein alters 3H-androgen uptake by rat epididymal tubules in vitro

Previous experiments have shown that androgen binding protein (ABP) and androgens exist in high concentrations in the tissue and the lumen of the rat caput epididymis. The present experiments were performed to determine whether or not intraluminal APB affects tubule net uptake of androgens. Caput epididymal tubules were dissected into 2-cm segments, subjected to microperfusion into the tubule lumen, and incubated for 2.5 h in 35 degrees C minimum essential medium (MEM) containing 2.0 ng tritiated testosterone (3H-T) per ml. 14C-polytheylene glycol [PEG] was included as a contamination marker. In the first series of experiments, caput tubules were perfused with a control, artificial perfusion (MKB) containing no ABP or fresh rat rete testis fluid (RTF), which is known to contain ABP. Tubules incubated while containing RTF took up 138% of the tritiated androgens taken up by control tubules. In the second series of experiments, tubules were perfused with fresh caput epididymal lumen content, MKB alone, MKB containing either 5.0 ng purified rat ABP/microliters or 50 ng ABP/microliters. Tubules incubated while containing perfused MKB took up only 47% of the tritiated androgens taken up by tubules containing perfused native lumen content. Increasing intraluminal ABP concentrations in the MKB medium increased 3H-androgen uptake in a stepwise fashion. Intraluminal ABP at a concentration of 50 ng/microliters was associated with a 71% return of 3H-androgen uptake towards that amount of 3H-androgen taken up by tubules perfused with native lumen content. Intraluminal ABP enhances net androgen uptake by caput epididymal tubules from their surrounding medium in vitro.     

Towards Clinical Molecular Diagnosis of Inherited Cardiac Conditions: A Comparison of Bench-Top Genome DNA Sequencers

Background

Molecular genetic testing is recommended for diagnosis of inherited cardiac disease, to guide prognosis and treatment, but access is often limited by cost and availability. Recently introduced high-throughput bench-top DNA sequencing platforms have the potential to overcome these limitations.

Methodology/Principal Findings

We evaluated two next-generation sequencing (NGS) platforms for molecular diagnostics. The protein-coding regions of six genes associated with inherited arrhythmia syndromes were amplified from 15 human samples using parallelised multiplex PCR (Access Array, Fluidigm), and sequenced on the MiSeq (Illumina) and Ion Torrent PGM (Life Technologies). Overall, 97.9% of the target was sequenced adequately for variant calling on the MiSeq, and 96.8% on the Ion Torrent PGM. Regions missed tended to be of high GC-content, and most were problematic for both platforms. Variant calling was assessed using 107 variants detected using Sanger sequencing: within adequately sequenced regions, variant calling on both platforms was highly accurate (Sensitivity: MiSeq 100%, PGM 99.1%. Positive predictive value: MiSeq 95.9%, PGM 95.5%). At the time of the study the Ion Torrent PGM had a lower capital cost and individual runs were cheaper and faster. The MiSeq had a higher capacity (requiring fewer runs), with reduced hands-on time and simpler laboratory workflows. Both provide significant cost and time savings over conventional methods, even allowing for adjunct Sanger sequencing to validate findings and sequence exons missed by NGS.

Conclusions/Significance

MiSeq and Ion Torrent PGM both provide accurate variant detection as part of a PCR-based molecular diagnostic workflow, and provide alternative platforms for molecular diagnosis of inherited cardiac conditions. Though there were performance differences at this throughput, platforms differed primarily in terms of cost, scalability, protocol stability and ease of use. Compared with current molecular genetic diagnostic tests for inherited cardiac arrhythmias, these NGS approaches are faster, less expensive, and yet more comprehensive.     

Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.     

In vivo effects of anacetrapib on preβ HDL: improvement in HDL remodeling without effects on cholesterol absorption

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol and lowers LDL cholesterol in dyslipidemic patients. We previously demonstrated that ANA increases macrophage-to-feces reverse cholesterol transport and fecal cholesterol excretion in hamsters, and increased preβ HDL-dependent cholesterol efflux via ABCA1 in vitro. However, the effects of ANA on in vivo preβ HDL have not been characterized. In vitro, ANA inhibited the formation of preβ, however in ANA-treated dyslipidemic hamsters, preβ HDL levels (measured by two-dimensional gel electrophoresis) were increased, in contrast to in vitro findings. Because changes in plasma preβ HDL have been proposed to potentially affect markers of cholesterol absorption with other CETP inhibitors, a dual stable isotope method was used to directly measure cholesterol absorption in hamsters. ANA treatment of hamsters (on either dyslipidemic or normal diet) had no effect on cholesterol absorption, while dalcetrapib-treated hamsters displayed an increase in cholesterol absorption. Taken together, these data support the notion that ANA promotes preβ HDL functionality in vivo, with no effects on cholesterol absorption.     

Tracking fatty acid kinetics in distinct lipoprotein fractions in vivo: a novel high-throughput approach for studying dyslipidemia in rodent models

Isotopic tracers have been used to examine lipid trafficking for many years, and data from those studies have typically yielded novel insight regarding the pathophysiology of dyslipidemia. Previous experimental designs were suitable for studies in humans because relatively large volumes of plasma could be regularly sampled. We have expanded on the earlier logic by applying high-throughput analytical methods that require reduced sample volumes. Specifically, we have examined the possibility of coupling gel-based separations of lipoproteins (e.g., lipoprint) with LC-MS/MS analyses of complex lipid mixtures as a way to routinely measure the labeling profiles of distinct lipids in discrete lipoprotein subfractions. We demonstrate the ability to measure the incorporation of [U-¹³C]oleate into triglycerides (TG), PLs (PL), and cholesterol esters (CE) in VLDL, LDL, and HDL particles in mice. Although rodent models of dyslipidemia are inherently different from humans because of alterations in enzyme activities and underlying metabolism, rodent models can be used to screen novel compounds for efficacy in altering a given biochemical pathway and therein enable studies of target engagement in vivo. We expect that it is possible to translate our approach for application in other systems, including studies in humans.     

Hydraulic conductance and the maintenance of water balance in flowers

Flowers face desiccating conditions, yet little is known about their ability to transport water. We quantified variability in floral hydraulic conductance (K_flower) for 20 species from 10 families and related it to traits hypothesized to be associated with liquid and vapour phase water transport. Basal angiosperm flowers had trait values associated with higher water and carbon costs than monocot and eudicot flowers. K_flower was coordinated with water supply (vein length per area, VLA) and loss (minimum epidermal conductance, g_min) traits among the magnoliids, but was insensitive to variation in these traits among the monocots and eudicots. Phylogenetic independent contrast (PIC) correlations revealed that few traits had undergone coordinated evolution. However, VLA and the desiccation time (T_des), the quotient of water content and g_min, had significant trait and PIC correlations. The near absence of stomata from monocot and eudicot flowers may have been critical in minimizing water loss rates among these clades. Early divergent, basal angiosperm flowers maintain higher K_flower because of traits associated with high rates water loss and water supply, while monocot and eudicot flowers employ a more conservative strategy of limiting water loss and may rely on stored water to maintain turgor and delay desiccation.     

SAVVY? (C31G) Gel for Prevention of HIV infection in Women: A Phase 3, Double-Blind,Randomized, Placebo-Controlled Trial in Ghana

Objective

The objective of this trial was to determine the effectiveness of 1.0% C31G (SAVVY) in preventing male-to-female vaginal transmission of HIV infection among women at high risk.

Methodology/Principal Findings

This was a Phase 3, double-blind, randomized, placebo-controlled trial. Participants made up to 12 monthly visits for HIV testing, adverse event reporting, and study product supply. The study was conducted between March 2004 and February 2006 in Accra and Kumasi, Ghana. We enrolled 2142 HIV-negative women at high risk of HIV infection, and randomized them to SAVVY or placebo gel. Main outcome measures were the incidence of HIV-1 and HIV-2 infection as determined by detection of HIV antibodies from oral mucosal transudate specimens and adverse events. We accrued 790 person-years of follow-up in the SAVVY group and 772 person-years in the placebo group. No clinically significant differences in the overall frequency of adverse events, abnormal pelvic examination findings, or abnormal laboratory results were seen between treatment groups. However, more participants in the SAVVY group reported reproductive tract adverse events than in the placebo group (13.0% versus 9.4%). Seventeen HIV seroconversions occurred; eight in participants randomized to SAVVY and nine in participants receiving placebo. The Kaplan-Meier estimates of the cumulative probability of HIV infection through 12 months were 0.010 in the SAVVY group and 0.011 in the placebo group (p = 0.731), with a hazard ratio (SAVVY versus placebo) of 0.88 (95% confidence interval 0.33, 2.27). Because of a lower-than-expected HIV incidence, we were unable to achieve the required number of HIV infections (66) to obtain the desired study power.

Conclusions/Significance

SAVVY was not associated with increased adverse events overall, but was associated with higher reporting of reproductive adverse events. Our data are insufficient to conclude whether SAVVY is effective at preventing HIV infection relative to placebo.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00129532","term_id":"NCT00129532"}}NCT00129532