Advanced glycation end-products induce calpain-mediated degradation of ezrin

Advanced glycation end-products (AGEs) are important mediators of diabetic complications via incompletely understood pathways. AGEs bind to intracellular ERM proteins (ezrin, radixin and moesin) that modulate cell shape, motility, adhesion and signal transduction. AGEs bind to the N-terminal domain of ezrin but not full-length ezrin. The AGE binding site may be made accessible either by proteolysis releasing an N-terminal fragment or ezrin activation by phosphorylation. Increased intracellular calcium is a primary event in cell activation by high glucose or AGEs. Calpain activity is increased concomitantly, and ezrin is a calpain substrate. The present study assessed whether glycated proteins affect ezrin cleavage and activation in renal tubule epithelial cells. After 7?days, AGE-BSA decreased ezrin levels in MDCK renal tubular cells to 66?±?4% of control. AGE-RNAse, ribosylated fetal bovine serum and methylglyoxal-BSA all had similar effects. The AGE-BSA-induced decrease in ezrin was abolished by calpastatin peptide, a specific calpain inhibitor, and 1,2-bis-aminophenoxyethane-tetraacetic acid acetoxymethyl ester (BAPTA-AM), a calcium chelator. Ezrin breakdown products were increased in AGE-BSA-treated cells, with a main fragment of ～?43?kDa. In?vitro, calpain?1 cleaved recombinant human ezrin, generating breakdown fragments including an N-terminal fragment of ～?43?kDa. Studies with ezrin mutants showed that non-phosphorylated ezrin was more susceptible to calpain cleavage. AGE-BSA decreased phosphorylated ERM levels to 31?±?12% in MDCK cells. Thus, AGE-BSA promotes calpain-mediated proteolysis of ezrin in MDCK cells by both increasing calpain activity and reducing phosphorylation. Therapies targeting both glycated proteins and calpain may provide protection against diabetic complications. Structured digital abstract ? Calpain-1?cleaves?Ezrin?by?protease assay?(View Interaction:?1,?2).     

An opioid basis for early-phase isoflurane-induced hypotension in rats

This study was conducted to more clearly delineate the possible role of endogenous opioid receptors and opioid peptides in general anesthesia-associated hypotension in rats. Exposure to 2% isoflurane in oxygen produced a triphasic change in mean arterial pressure (MAP), including an early phase in which MAP fell by -28.4 +/- 2.2%. The magnitude of this early-phase hypotension was attenuated in rats pretreated with intravenous (i.v.) mu-subtype-selective doses of either naloxone or methylnaloxone but not central doses of the selective mu-opioid antagonist beta-funaltrexamine. This early hypotensive phase was also reduced following i.v. pretreatment with antiserum against methionine-enkephalin but not beta-endorphin. These findings suggest that early-phase isoflurane-induced hypotension may be due to activation of peripheral mu-opioid receptors by an endogenous opioid peptide, possibly related to methionine-enkephalin.     

Short-term temporal dynamics of yeast abundance on the tall fescue phylloplane

Six replicate trials were conducted to determine the short-term temporal dynamics and the effects of foliar applications of nutrients on the phylloplane yeast community of tall fescue (Festuca arundinacea Schreb.). In each trial, 2% sucrose + 0.5% yeast extract solution or sterile deionized water (control) was applied to the experiment plots. Twelve hours post-treatment (at 0600 hours), leaf samples were collected and yeast colony-forming units (cfu) were enumerated by dilution plating. This process was repeated at 1200, 1800, and 2400 hours in each trial. Significant differences were observed between the number of yeast cfu and the time at which the samples were collected. On average, the number of yeast cfu recovered was significantly less at 1800 hours and significantly greatest at 2400 hours when compared with all other sampling times. Averaged over all time intervals, we observed a trend of increased yeast abundance in turf treated with the nutrient solution compared with control treatments. In a separate investigation, atmospheric yeast abundance above the canopy of tall fescue was assessed in the morning (0900) and in the afternoon (1500) using a Thermo Andersen single stage viable particle sampler. In 5 of the 6 trials of this experiment, atmospheric yeast abundance was significantly greater in the morning than in the afternoon. Results suggest the following colonization model: phylloplane yeasts on tall fescue reproduce during the late evening and early morning, stabilize during the late morning and early afternoon through exchange of immigrants and emigrants, and decline during the late afternoon and (or) early evening.     

The mouth of a dense-core vesicle opens and closes in a concerted action regulated by calcium and amphiphysin

Secretion of hormones and peptides by neuroendocrine cells occurs through fast and slow modes of vesicle fusion but the mechanics of these processes are not understood. We used interference reflection microscopy to monitor deformations of the membrane surface and found that both modes of fusion involve the tightly coupled dilation and constriction of the vesicle. The rate of opening is calcium dependent and occurs rapidly at concentrations <5 microM. The fast mode of fusion is blocked selectively by a truncation mutant of amphiphysin. Vesicles do not collapse when fusion is triggered by strontium, rather they remain locked open and membrane scission is blocked. In contrast, constriction of the vesicle opening continues when endocytosis is blocked by inhibiting the function of dynamin. Thus, fast and slow modes of fusion involve similar membrane deformations and vesicle closure can be uncoupled from membrane scission. Regulation of these processes by calcium and amphiphysin may provide a mechanism for controlling the release of vesicle contents.     

AutoDecon, a deconvolution algorithm for identification and characterization of luteinizing hormone secretory bursts: description and validation using synthetic data

Hormone signaling is often pulsatile, and multiparameter deconvolution procedures have long been used to identify and characterize secretory events. However, the existing programs have serious limitations, including the subjective nature of initial peak selection, lack of statistical verification of presumed bursts, and user-unfriendliness of the application. Here we describe a novel deconvolution program, AutoDecon, which addresses these concerns. We validate AutoDecon for application to serum luteinizing hormone (LH) concentration time series using synthetic data mimicking real data from normal women and then comparing the performance of AutoDecon with the performance of the widely employed hormone pulsatility analysis program Cluster. The sensitivity of AutoDecon is higher than that of Cluster (∼ 96% vs. 80%, P = 0.001). However, Cluster had a lower false-positive detection rate than did AutoDecon (6% vs. 1%, P = 0.001). Further analysis demonstrated that the pulsatility parameters recovered by AutoDecon were indistinguishable from those characterizing the synthetic data and that sampling at 5- or 10-min intervals was optimal for maximizing the sensitivity rates for LH. Accordingly, AutoDecon presents a viable nonsubjective alternative to previous pulse detection algorithms for the analysis of LH data. It is applicable to other pulsatile hormone concentration time series and many other pulsatile phenomena. The software is free and downloadable at http://mljohnson.pharm.virginia.edu/home.html.     

Eimeria tenella: utilization of a nasal vaccine with sporozoite antigens incorporated into Iscom as protection for broiler breeders against a homologous challenge

The aim of this study was to evaluate a nasal vaccine using antigens derived from sporozoites of Eimeria tenella incorporated into Iscom to protect broiler chicks. Forty-five one-day-old chickens (Cobb), unvaccinated against coccidiosis, were used in this experiment. The birds were maintained in separated battery cages and divided into three groups: G1 (n = 15), G2 (n = 15), and G3 (n = 15). G1 received 50 μg of sporozoites + Iscom vaccine, G2 received Iscom without antigens, and G3 received only PBS. The treatments were administered by nasal route on days 0, 7, and 21 of the experiment. On the 28th day, all birds were challenged with 105 sporulated oocysts of E. tenella. On the challenge day, three birds from each group were euthanized to evaluate lymphocyte proliferation. Lesion scores were obtained from five birds from each group, 7 days after challenge. The remaining animals were euthanized on the 50th day. The mean lymphocyte proliferation responses were significantly different (P = 0.03); G1 was 2.3-2.6 times more elevated than G2, and G3 (P < 0.001). 83% of the birds from G1 showed an IgY antibody reaction by ELISA at challenge. The means for oocysts shedding were 16,890 ± 20,511, 48,080 ± 50,047, and 65,020 ± 74,461, for G1, G2 and G3 birds, respectively. There was no significant difference (P > 0.17) in oocysts shedding between groups. However, the G1 and G2 chicks demonstrated reduction in percentage of oocyst shedding when compared to control birds (G3) by 74.02% and 26.05%, respectively. The average lesion scores were G1 = 0.4, G2 = 1, and G3 = 2. This study demonstrated that the lowest lesion score and oocyst shedding were observed in the birds from the group that received antigens derived from sporozoite with an Iscom adjuvant (G1). These results suggest that this vaccine can induce protection against avian coccidiosis.     

Detection of infectious prions in urine

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). The mechanism of prion transmission is unknown. In this study, we attempted to detect prions in urine of experimentally infected animals. PrP(Sc) was detected in approximately 80% of the animals studied, whereas no false positives were observed among the control animals. Semi-quantitative calculations suggest that PrP(Sc) concentration in urine is around 10-fold lower than in blood. Interestingly, PrP(Sc) present in urine maintains its infectious properties. Our data indicate that low quantities of infectious prions are excreted in the urine. These findings suggest that urine is a possible source of prion transmission.