Extracellular Hsp72 concentration relates to a minimum endogenous criteria during acute exercise-heat exposure

Extracellular heat-shock protein 72 (eHsp72) concentration increases during exercise-heat stress when conditions elicit physiological strain. Differences in severity of environmental and exercise stimuli have elicited varied response to stress. The present study aimed to quantify the extent of increased eHsp72 with increased exogenous heat stress, and determine related endogenous markers of strain in an exercise-heat model. Ten males cycled for 90 min at 50% O2peak in three conditions (TEMP, 20°C/63% RH; HOT, 30.2°C/51%RH; VHOT, 40.0°C/37%RH). Plasma was analysed for eHsp72 pre, immediately post and 24-h post each trial utilising a commercially available ELISA. Increased eHsp72 concentration was observed post VHOT trial (+172.4%) (P<0.05), but not TEMP (-1.9%) or HOT (+25.7%) conditions. eHsp72 returned to baseline values within 24hrs in all conditions. Changes were observed in rectal temperature (Trec), rate of Trec increase, area under the curve for Trec of 38.5°C and 39.0°C, duration Trec ≥ 38.5°C and ≥ 39.0°C, and change in muscle temperature, between VHOT, and TEMP and HOT, but not between TEMP and HOT. Each condition also elicited significantly increasing physiological strain, described by sweat rate, heart rate, physiological strain index, rating of perceived exertion and thermal sensation. Stepwise multiple regression reported rate of Trec increase and change in Trec to be predictors of increased eHsp72 concentration. Data suggests eHsp72 concentration increases once systemic temperature and sympathetic activity exceeds a minimum endogenous criteria elicited during VHOT conditions and is likely to be modulated by large, rapid changes in core temperature

Quantification of Rapid Myosin Regulatory Light Chain Phosphorylation Using High-Throughput In-Cell Western Assays: Comparison to Western Immunoblots

Quantification of phospho-proteins (PPs) is crucial when studying cellular signaling pathways. Western immunoblotting (WB) is commonly used for the measurement of relative levels of signaling intermediates in experimental samples. However, WB is in general a labour-intensive and low-throughput technique. Because of variability in protein yield and phospho-signal preservation during protein harvesting, and potential loss of antigen during protein transfer, WB provides only semi-quantitative data. By comparison, the "in-cell western" (ICW) technique has high-throughput capacity and requires less extensive sample preparation. Thus, we compared the ICW technique to WB for measuring phosphorylated myosin regulatory light chain (PMLC(20)) in primary cultures of uterine myocytes to assess their relative specificity, sensitivity, precision, and quantification of biologically relevant responses.ICWs are cell-based microplate assays for quantification of protein targets in their cellular context. ICWs utilize a two-channel infrared (IR) scanner (Odyssey(R)) to quantify signals arising from near-infrared (NIR) fluorophores conjugated to secondary antibodies. One channel is dedicated to measuring the protein of interest and the second is used for data normalization of the signal in each well of the microplate. Using uterine myocytes, we assessed oxytocin (OT)-stimulated MLC(20) phosphorylation measured by ICW and WB, both using NIR fluorescence. ICW and WB data were comparable regarding signal linearity, signal specificity, and time course of phosphorylation response to OT.ICW and WB yield comparable biological data. The advantages of ICW over WB are its high-throughput capacity, improved precision, and reduced sample preparation requirements. ICW might provide better sensitivity and precision with low-quantity samples or for protocols requiring large numbers of samples. These features make the ICW technique an excellent tool for the study of phosphorylation endpoints. However, the drawbacks of ICW include the need for a cell culture format and the lack of utility where protein purification, concentration or stoichiometric analyses are required

SDF1-Induced Antagonism of Axonal Repulsion Requires Multiple G-Protein Coupled Signaling Components That Work in Parallel

SDF1 reduces the responsiveness of axonal growth cones to repellent guidance cues in a pertussis-toxin-sensitive, cAMP-dependent manner. Here, we show that SDF1's antirepellent effect can be blocked in embryonic chick dorsal root ganglia (DRGs) by expression of peptides or proteins inhibiting either Gαi, Gαq, or Gβγ. SDF1 antirepellent activity is also blocked by pharmacological inhibition of PLC, a common effector protein for Gαq. We also show that SDF1 antirepellent activity can be mimicked by overexpression of constitutively active Gαi, Gαq, or Gαs. These results suggest a model in which multiple G protein components cooperate to produce the cAMP levels required for SDF1 antirepellent activity

COPI Is Required for Enterovirus 71 Replication

Enterovirus 71 (EV71), a member of the Picornaviridae family, is found in Asian countries where it causes a wide range of human diseases. No effective therapy is available for the treatment of these infections. Picornaviruses undergo RNA replication in association with membranes of infected cells. COPI and COPII have been shown to be involved in the formation of picornavirus-induced vesicles. Replication of several picornaviruses, including poliovirus and Echovirus 11 (EV11), is dependent on COPI or COPII. Here, we report that COPI, but not COPII, is required for EV71 replication. Replication of EV71 was inhibited by brefeldin A and golgicide A, inhibitors of COPI activity. Furthermore, we found EV71 2C protein interacted with COPI subunits by co-immunoprecipitation and GST pull-down assay, indicating that COPI coatomer might be directed to the viral replication complex through viral 2C protein. Additionally, because the pathway is conserved among different species of enteroviruses, it may represent a novel target for antiviral therapies

Astrocyte pathology in the prefrontal cortex impairs the cognitive function of rats

Interest in astroglial cells is rising due to recent findings supporting dynamic neuron-astrocyte interactions. There is increasing evidence of astrocytic dysfunction in several brain disorders such as depression, schizophrenia or bipolar disorder; importantly these pathologies are characterized by the involvement of the prefrontal cortex and by significant cognitive impairments. Here, to model astrocyte pathology, we injected animals with the astrocyte specific toxin L-a-aminoadipate (L-AA) in the medial prefrontal cortex (mPFC); a behavioral and structural characterization two and six days after the injection was performed. Behavioral data shows that the astrocyte pathology in the mPFC affects the attentional set-shifting, the working memory and the reversal learning functions. Histological analysis of brain sections of the L-AA-injected animals revealed a pronounced loss of astrocytes in the targeted region. Interestingly, analysis of neurons in the lesion sites showed a progressive neuronal loss that was accompanied with dendritic atrophy in the surviving neurons. These results suggest that the L-AA-induced astrocytic loss in the mPFC triggers subsequent neuronal damage leading to cognitive impairment in tasks depending on the integrity of this brain region. These findings are of relevance to better understand the pathophysiological mechanisms underlying disorders that involve astrocytic loss/dysfunction in the PFC.This work was supported by the Marie Curie Fellowship FP7-PEOPLE-2010-IEF 273936, BIAL Foundation Grants 138/2008 and 61/2010, FEDER funds through Operational program for competitiveness factors-COMPETE -, ON2 Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN/FEDER, and by national funds through FCT-Foundation for Science and Technology-project (PTDC/SAU-NSC/118194/2010) and fellowships (SFRH/BPD/66151/2009 and SFRH/BD/89714/2012)

Influence of aerobic fitness on gastrointestinal barrier integrity and microbial translocation following a fixed-intensity military exertional heat stress test

Purpose: Exertional-heat stress adversely disrupts gastrointestinal (GI) barrier integrity, whereby subsequent microbial translocation (MT) can result in potentially serious health consequences. To date, the influence of aerobic fitness on GI barrier integrity and MT following exertional-heat stress is poorly characterised. Method: Ten untrained (UT; VO2max = 45 ± 3 ml·kg−1·min−1) and ten highly trained (HT; VO2max = 64 ± 4 ml·kg−1·min−1) males completed an ecologically valid (military) 80-min fixed-intensity exertional-heat stress test (EHST). Venous blood was drawn immediately pre- and post-EHST. GI barrier integrity was assessed using the serum dual-sugar absorption test (DSAT) and plasma Intestinal Fatty-Acid Binding Protein (I-FABP). MT was assessed using plasma Bacteroides/total 16S DNA. Results: UT experienced greater thermoregulatory, cardiovascular and perceptual strain (p < 0.05) than HT during the EHST. Serum DSAT responses were similar between the two groups (p = 0.59), although Δ I-FABP was greater (p = 0.04) in the UT (1.14 ± 1.36 ng·ml−1) versus HT (0.20 ± 0.29 ng·ml−1) group. Bacteroides/Total 16S DNA ratio was unchanged (Δ; -0.04 ± 0.18) following the EHST in the HT group, but increased (Δ; 0.19 ± 0.25) in the UT group (p = 0.05). Weekly aerobic training hours had a weak, negative correlation with Δ I-FABP and Bacteroides/total 16S DNA responses. Conclusion: When exercising at the same absolute workload, UT individuals are more susceptible to small intestinal epithelial injury and MT than HT individuals. These responses appear partially attributable to greater thermoregulatory, cardiovascular, and perceptual strain

Complex involvement of pertussis toxin-sensitive G proteins in the regulation of type 1 alpha metabotropic glutamate receptor signaling in baby hamster kidney cells

Previously, we demonstrated that the coupling of the metabotropic glutamate receptor mGlu1 alpha to phosphoinositide hydrolysis is enhanced by pertussis toxin (PTX) in stably transfected baby hamster kidney cells (BHK). Here, we show that the PTX effect on agonist-stimulated [H-3]inositol phosphate accumulation can be resolved into two components: an immediate increase in agonist potency, and a more slowly developing increase in the magnitude of the response observed at maximally effective agonist concentrations. Using G(q)/(11)alpha- and G(i/o)alpha-selective antibodies to immunoprecipitate [S-35]guanosine-5'-O-(3-thio)triphosphate-bound G alpha proteins, we also show that agonist stimulation of mGlu1a in BHK membranes increases specific [S-35]guanosine-5'-O-(3-thio)triphosphate binding to both G(q/11) and G(i/o) proteins. Preincubation of BHK-mGlu1 alpha with L-glutamate (300 mu M) results in a progressive loss (60% in 30 min) of L-quisqualate-induced [H-3]inositol phosphate accumulation (without a change in potency), providing evidence for agonist-induced receptor desensitization. Although such desensitization of mGlu receptor signaling was mimicked by a phorbol ester, agonist-induced phosphorylation of the receptor was not observed and protein kinase C inhibition by Ro 31-8220 did not prevent L-glutamate-mediated desensitization. In contrast, PTX treatment of the cells almost completely prevented L-glutamate-mediated desensitization. Together, these data provide evidence for a multifunctional coupling of mGlu1a to different types of G proteins, including PTX-sensitive G(i)-type G proteins. The latter are involved in the negative control of phospholipase C activity while also influencing the rate of desensitization of the mGlu1 alpha receptor

5-HT receptors mediating contraction in the rat-tail artery

We studied the effects of 5-HT in resistance vessels. Compounds acting on 5-HT2A and 5-HT1B receptors were tested on rat-tail arterial rings in varying experimental conditions. We tested 5-HT from 50 nM to 100 microM: the pD2 was 6.4 +/- 0.1. We evaluated vasoconstriction by 5-HT in tissues slightly depolarized with 30 mM KCl. In this condition, the 5-HT concentration-related contraction started at lower concentration in comparison to control tissues. Preincubation with 50 nM ketanserin, a 5-HT2A-antagonist, and 1 microM prazosin, an alpha1-antagonist, strongly inhibited concentration-related contraction by 5-HT: the pD2 was 3.2 +/- 0.2. Moreover, we experimented alpha-methyl-5-HT (alpha-me-5-HT) and 5-carboxamidotryptamine (5-CT), selective agonists at 5-HT2A and 5-HT1B receptors, respectively. Both agonists induced concentration-related contraction; the potency order observed was 5-HT > alpha-me-5-HT > 5-CT. Finally, we studied SB 224289, a selective 5-HT1B-antagonist, on contraction by 5-HT in control and in depolarized conditions. 0.2 microM SB 224289 significantly inhibited vasoconstriction induced by 5-HT in depolarized vascular tissues. The data indicate that vasoconstriction induced by 5-HT is mainly dependent on 5-HT2A receptors; however, 5-HT1B receptors are also present in rat-tail artery

5-HT receptors mediating contraction in the rat-tail artery

none45-HT receptors mediating contraction in the rat-tail artery. Studies on the regulation of vasoconstriction by 5-hydroxytryptamine.noneNICOLETTI P.; RAGAZZI E.; CAPARROTTA L.; FROLDI G.Nicoletti, P.; Ragazzi, Eugenio; Caparrotta, Laura; Froldi, Guglielmin