Hypoxia transiently affects skeletal muscle hypertrophy in a functional overload model.

Hypoxia induces a loss of skeletal muscle mass, but the signaling pathways and molecular mechanisms involved remain poorly understood. We hypothesized that hypoxia could impair skeletal muscle hypertrophy induced by functional overload (Ov). To test this hypothesis, plantaris muscles were overloaded during 5, 12, and 56 days in female rats exposed to hypobaric hypoxia (5,500 m), and then, we examined the responses of specific signaling pathways involved in protein synthesis (Akt/mTOR) and breakdown (atrogenes). Hypoxia minimized the Ov-induced hypertrophy at days 5 and 12 but did not affect the hypertrophic response measured at day 56. Hypoxia early reduced the phosphorylation levels of mTOR and its downstream targets P70(S6K) and rpS6, but it did not affect the phosphorylation levels of Akt and 4E-BP1, in Ov muscles. The role played by specific inhibitors of mTOR, such as AMPK and hypoxia-induced factors (i.e., REDD1 and BNIP-3) was studied. REDD1 protein levels were reduced by overload and were not affected by hypoxia in Ov muscles, whereas AMPK was not activated by hypoxia. Although hypoxia significantly increased BNIP-3 mRNA levels at day 5, protein levels remained unaffected. The mRNA levels of the two atrogenes MURF1 and MAFbx were early increased by hypoxia in Ov muscles. In conclusion, hypoxia induced a transient alteration of muscle growth in this hypertrophic model, at least partly due to a specific impairment of the mTOR/P70(S6K) pathway, independently of Akt, by an undefined mechanism, and increased transcript levels for MURF1 and MAFbx that could contribute to stimulate the proteasomal proteolysis.

Combinations of ketamine and atropine are neuroprotective and reduce neuroinflammation after a toxic status epilepticus in mice.

Epileptic seizures and status epilepticus (SE) induced by the poisoning with organophosphorus nerve agents (OP), like soman, are accompanied by neuroinflammation whose role in seizure-related brain damage (SRBD) is not clear. Antagonists of the NMDA glutamate ionotropic receptors are currently among the few compounds able to arrest seizures and provide neuroprotection even during refractory status epilepticus (RSE). Racemic ketamine (KET), in combination with atropine sulfate (AS), was previously shown to counteract seizures and SRBD in soman-poisoned guinea-pigs. In a mouse model of severe soman-induced SE, we assessed the potentials of KET/AS combinations as a treatment for SE/RSE-induced SRBD and neuroinflammation. When starting 30min after soman challenge, a protocol involving six injections of a sub-anesthetic dose of KET (25mg/kg) was evaluated on body weight loss, brain damage, and neuroinflammation whereas during RSE, anesthetic protocols were considered (KET 100mg/kg). After confirming that during RSE, KET injection was to be repeated despite some iatrogenic deaths, we used these proof-of-concept protocols to study the changes in mRNA and related protein contents of some inflammatory cytokines, chemokines and adhesion molecules in cortex and hippocampus 48h post-challenge. In both cases, the KET/AS combinations showed important neuroprotective effects, suppressed neutrophil granulocyte infiltration and partially suppressed glial activation. KET/AS could also reduce the increase in mRNA and related pro-inflammatory proteins provoked by the poisoning. In conclusion, the present study confirms that KET/AS treatment has a strong potential for SE/RSE management following OP poisoning. The mechanisms involved in the reduction of central neuroinflammation remain to be studied.

Pitfalls of reverse transcription quantitative polymerase chain reaction standardization: Volume-related inhibitors of reverse transcription.

A large part of the reliability of reverse transcription quantitative polymerase chain reaction (RT-qPCR) data depends on technical variations. Such variations are mainly attributable to the reverse transcription step. Standardization is a key factor in decreasing the intersample variability. However, an ideal standardization is not always possible, and compromises must be found. Due to technical requirements, the current consensus is that a constant amount of total RNA should be used for the RT step (CA-RT). Because RNA isolation yields are variable, such a practice requires the use of variable volumes of nucleic acid extracts in RT reaction. We demonstrate that some RNA extracts contain both exogenous and endogenous inhibitors. These inhibitors induce a decrease in RT efficiency that significantly impairs the reliability of RT-qPCR data. Conversely, these inhibitors have a slight effect on the qPCR step. To overcome such drawbacks, we proposed to carry out the RT reaction with a constant volume of RNA extract by preserving a constant RNA amount through the supplementation of yeast transfer RNA (CV-RT). We show that CV-RT, compared with the usual CA-RT, allows us to decrease the RT-qPCR variability induced by intersample differences. Such a decrease is a prerequisite for the reliability of messenger RNA quantification.

Inflammatory changes during epileptogenesis and spontaneous seizures in a mouse model of mesiotemporal lobe epilepsy.

PURPOSE: Neuroinflammation appears as a prominent feature of the mesiotemporal lobe epilepsy syndrome (MTLE) that is observed in human patients and animal models. However, the precise temporal relationship of its development during epileptogenesis remains to be determined. The aim of the present study was to investigate (1) the time course and spatial distribution of neuronal death associated with seizure development, (2) the time course of microglia and astrocyte activation, and (3) the kinetics of induction of mRNAs from neuroinflammatory-related proteins during the emergence of recurrent seizures. METHODS: Experimental MTLE was induced by the unilateral intrahippocampal injection of kainate in C57BL/6 adult mice. Microglial and astrocytic changes in both ipsilateral and contralateral hippocampi were examined by respectively analyzing griffonia simplicifolia (GSA) lectin staining and glial fibrillary acidic protein (GFAP) immunoreactivity. Changes in mRNA levels of selected genes of cytokine and cytokine regulatory proteins (interleukin-1ß, IL-1ß; interleukin-1 receptor antagonist, IL-1Ra; suppressor of cytokine signaling 3, SOCS3) and enzymes of the eicosanoid pathway (group IVA cytosolic phospholipase A2, cPLA(2)-α; cycloxygenase-2, COX-2) were studied by reverse transcription-quantitative real time polymerase chain reaction. KEY FINDINGS: Our data show an immediate cell death occurring in the kainate-injected hippocampus during the initial status epilepticus (SE). A rapid increase of activated lectin-positive cells and GFAP-immunoreactivity was subsequently detected in the ipsilateral hippocampus. In the same structure, Il-1ß, IL-1Ra, and COX-2 mRNA were specifically increased during SE and epileptogenesis with a different time course. Conversely, the expression of SOCS3 mRNA, a surrogate marker of interleukin signaling, was mainly increased in the contralateral hippocampus after SE. SIGNIFICANCE: Our data show that specific neuroinflammatory pathways are activated in a time- and structure-dependent manner with putative distinct roles in epileptogenesis.

Selection of reference genes for real-time quantitative reverse transcription-polymerase chain reaction in hippocampal structure in a murine model of temporal lobe epilepsy with focal seizures.

Reference genes are often used to normalize expression of data from real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and only a validation of their stability during a given experimental paradigm leads to reliable interpretations. The present study was thus designed to validate potential reference genes in a mouse model of mesiotemporal lobe epilepsy (MTLE) with focal seizures after unilateral intrahippocampal injection of kainate (KA). Ipsilateral and contralateral hippocampi were removed during nonconvulsive status epilepticus (5 hr), epileptogenesis (7 days), and the chronic period of recurrent focal seizures (21 days). Naive animals were equally studied. The stability of eight potential reference genes (hypoxanthine phosphoribosyltransferase, Hprt1; peptidylprolyl isomerase A, Ppia; TATA box binding protein, Tbp; beta-actin, Actb; acidic ribosomal phosphoprotein P0, Arbp; glyceraldehyde-3-phosphate dehydrogenase, Gapdh; ribosomal RNA 18S, 18S rRNA; and glucuronidase beta, Gusb) were determined using geNorm and NormFinder software. The first five (Hprt1, Ppia, Tbp, Actb, and Arbp) were found to be stable across the different phases of the disease and appeared adequate for normalizing RT-qPCR data in this model. This was in contrast to the other three (18S rRNA, Gapdh, and Gusb), which showed unstable expressions and should be avoided. The analysis of KA-induced changes in the expression of glial fibrillary acidic protein (Gfap) gene resulted in various relative expressions or even a completely different pattern when unstable reference genes were used. These results highlight the absolute need to validate the reference genes for a correct interpretation of mRNA quantification.

Downregulation of Akt/mammalian target of rapamycin pathway in skeletal muscle is associated with increased REDD1 expression in response to chronic hypoxia.

Although it is well established that chronic hypoxia leads to an inexorable loss of skeletal muscle mass in healthy subjects, the underlying molecular mechanisms involved in this process are currently unknown. Skeletal muscle atrophy is also an important systemic consequence of chronic obstructive pulmonary disease (COPD), but the role of hypoxemia in this regulation is still debated. Our general aim was to determine the molecular mechanisms involved in the regulation of skeletal muscle mass after exposure to chronic hypoxia and to test the biological relevance of our findings into the clinical context of COPD. Expression of positive and negative regulators of skeletal muscle mass were explored 1) in the soleus muscle of rats exposed to severe hypoxia (6,300 m) for 3 wk and 2) in vastus lateralis muscle of nonhypoxemic and hypoxemic COPD patients. In rodents, we observed a marked inhibition of the mammalian target of rapamycin (mTOR) pathway together with a strong increase in regulated in development and DNA damage response 1 (REDD1) expression and in its association with 14-3-3, a mechanism known to downregulate the mTOR pathway. Importantly, REDD1 overexpression in vivo was sufficient to cause skeletal muscle fiber atrophy in normoxia. Finally, the comparative analysis of skeletal muscle in hypoxemic vs. nonhypoxemic COPD patients confirms that hypoxia causes an inhibition of the mTOR signaling pathway. We thus identify REDD1 as a negative regulator of skeletal muscle mass during chronic hypoxia. Translation of this fundamental knowledge into the clinical investigation of COPD shows the interest to develop therapeutic strategies aimed at inhibiting REDD1.

Down-regulation of Akt/mammalian target of rapamycin signaling pathway in response to myostatin overexpression in skeletal muscle.

Myostatin, a member of the TGF-beta family, has been identified as a master regulator of embryonic myogenesis and early postnatal skeletal muscle growth. However, cumulative evidence also suggests that alterations in skeletal muscle mass are associated with dysregulation in myostatin expression and that myostatin may contribute to muscle mass loss in adulthood. Two major branches of the Akt pathway are relevant for the regulation of skeletal muscle mass, the Akt/mammalian target of rapamycin (mTOR) pathway, which controls protein synthesis, and the Akt/forkhead box O (FOXO) pathway, which controls protein degradation. Here, we provide further insights into the mechanisms by which myostatin regulates skeletal muscle mass by showing that myostatin negatively regulates Akt/mTOR signaling pathway. Electrotransfer of a myostatin expression vector into the tibialis anterior muscle of Sprague Dawley male rats increased myostatin protein level and decreased skeletal muscle mass 7 d after gene electrotransfer. Using RT-PCR and immunoblot analyses, we showed that myostatin overexpression was ineffective to alter the ubiquitin-proteasome pathway. By contrast, myostatin acted as a negative regulator of Akt/mTOR pathway. This was supported by data showing that the phosphorylation of Akt on Thr308, tuberous sclerosis complex 2 on Thr1462, ribosomal protein S6 on Ser235/236, and 4E-BP1 on Thr37/46 was attenuated 7 d after myostatin gene electrotransfer. The data support the conclusion that Akt/mTOR signaling is a key target that accounts for myostatin function during muscle atrophy, uncovering a novel role for myostatin in protein metabolism and more specifically in the regulation of translation in skeletal muscle.

Immune function during and after 60 min of moderate exercise wearing protective clothing.

INTRODUCTION: As exercise while wearing protective clothing exacerbates body heat storage compared to exercise in the heat, and as exercise alters immune responses, it appeared worthwhile to examine immune and stress responses while wearing protective clothing during moderate exercise. METHODS: Eight subjects completed two bouts of exercise at 45% Vo2(max) in a thermoneutral environment: once while wearing shorts only (Control trial, CON) and again while wearing protective clothing (PRO). Venous blood samples were taken to analyze TNF-alpha mRNA by RT-PCR in LPS stimulated blood, plasma catecholamines, and cortisol. Blood cell count was analyzed by flow cytometry. Rectal temperature (T(re)) was monitored continuously. RESULTS: Exercise with PRO resulted in significantly greater increases in T(re) (39.2 +/- 0.2 degrees C in PRO vs. 38.0 +/- 0.1 degrees C in CON) and plasma epinephrine and norepinephrine (+70% and 150%, respectively). Plasma cortisol increased only at the end of PRO exercise (+33%). Leukocyte and lymphocyte cell count was 14% and 18% higher, respectively, but there were no significant changes in T cytotoxic and NK cell counts compared to the CON trial. Only T helper lymphocyte count was lower (-29%). During both exercise trials, T helper lymphocytes were significantly decreased at the end of exercise and recovery. With or without protective clothing, exercise was associated with an inhibition of TNF- alpha expression in stimulated monocytes (approximately -50% at min 20 and 40, and approximately -30% at min 60). DISCUSSION: Protective clothing wearing induces significant thermal challenge during exercise. The inhibition of TNF-alpha appears to be mediated primarily by exercise and not the added thermal load associated with protective clothing.

GFP-transduced CD34+ and Lin- CD34- hematopoietic stem cells did not adopt a cardiac phenotype in a nonhuman primate model of myocardial infarct.

OBJECTIVE: Studies in mice have reported contradictory results on the contribution of bone marrow cells to myocardial regeneration. This study aims to evaluate their ability to differentiate into cells of cardiac lineage in a nonhuman primate mode of myocardial infarct. MATERIALS AND METHODS: Lin(-)CD34(-) and CD34(+)-enriched bone marrow cells or mobilized peripheral blood cells were transduced with green fluorescent protein (GFP) and injected directly into ischemic myocardium. The fate of the transplanted cells was evaluated using quantitative reverse transcription polymerase chain reaction (QRT-PCR) and immunohistology. Animals were followed-up using echocardiography. RESULTS: QRT-PCR analysis detected from 3% to 10% of the original number of administered GFP(+) cells after 7 days. These GFP(+) cells did not express cardiac tissue-specific markers, but were immunophenotypically consistent with undifferentiated hematopoietic cells. The local production of vascular endothelial growth factor, measured by QRT-PCR, was approximately doubled as compared to the untreated infarcted control heart. Three months after hematopoietic stem cell (HSC) administration, no GFP(+) cells were detected and no evidence of regeneration of the infarcted region was found by histological examination. In contrast, a high level of matrix metalloproteinase 2 was measured in infarct and peri-infarct area. At this time, an improved ejection fraction and decreased left ventricular chamber dimension, which might be also related to a natural course after reperfusion, were observed. CONCLUSIONS: Our data show that GFP(+) CD34(+) and Lin(-)CD34(-)-enriched HSC do not differentiate into cardiomyocytes or into endothelial cells in the infarcted myocardium and that the local production of some growth factors had no positive effect on myocardial regeneration after 3 months.

Stress-induced hippocampus Npas4 mRNA expression relates to specific psychophysiological patterns of stress response.

Neuronal Per-Arnt-Sim (PAS) domain protein 4 (Npas4) is a key protein that intervenes in GABA synapse scaling and neurotrophicity enhancing. Since GABA and neurotrophicity are implicated in stress response and Npas4-deficient rodents exhibit behavioral alterations, an investigation was designed in rats to verify whether stress-induced spontaneous hippocampus Npas4 mRNA expression would be associated with specific patterns of stress response. The rats were exposed to one of three stressor levels: no stress (CTL, n = 15), exposure to a footshock apparatus (Sham, S, n = 40) and footshock (F, n = 80). After stress exposure the S and F rats were tested in an activity cage, and subsequently in an elevated plus maze (EPM), just prior to the sacrifice. Using cluster analysis, the animals already assigned to a stress level were also distributed into 2 subgroups depending on their Npas4 mRNA levels. The low (L) and high (H) Npas4 expression subgroups were identified in the S and F groups, the CTL group being independent of the Npas4 levels. The Npas4 effect was studied through the interaction between stress (S and F) and Npas4 level (L and H). The biological stress response was similar in H and L rats, except blood corticosterone that was slightly lower in the H rats. The H rats were more active in the actimetry cage and presented higher levels of exploration in the EPM. They also exhibited higher hippocampus activation, as assessed by the c-fos, Egr1 and Arc mRNA levels. Therefore high Npas4 expression favors stress management.

Ectopic expression of myostatin induces atrophy of adult skeletal muscle by decreasing muscle gene expression.

Myostatin is a master regulator of myogenesis and early postnatal skeletal muscle growth. However, myostatin has been also involved in several forms of muscle wasting in adulthood, suggesting a functional role for myostatin in the regulation of skeletal muscle mass in adult. In the present study, localized ectopic expression of myostatin was achieved by gene electrotransfer of a myostatin expression vector into the tibialis anterior muscle of adult Sprague Dawley male rats. The corresponding empty vector was electrotransfected in contralateral muscle. Ectopic myostatin mRNA was abundantly present in muscles electrotransfected with myostatin expression vector, whereas it was undetectable in contralateral muscles. Overexpression of myostatin elicited a significant decrease in muscle mass (10 and 20% reduction 7 and 14 d after gene electrotransfer, respectively), muscle fiber cross-sectional area (15 and 30% reduction 7 and 14 d after gene electrotransfer, respectively), and muscle protein content (20% reduction). No decrease in fiber number was observed. Overexpression of myostatin markedly decreased the expression of muscle structural genes (myosin heavy chain IIb, troponin I, and desmin) and the expression of myogenic transcription factors (MyoD and myogenin). Incidentally, mRNA level of caveolin-3 and peroxisome proliferator activated receptor gamma coactivator-1alpha was also significantly decreased 14 d after myostatin gene electrotransfer. To conclude, our study demonstrates that myostatin-induced muscle atrophy elicits the down-regulation of muscle-specific gene expression. Our observations support an important role for myostatin in muscle atrophy in physiological and physiopathological situations where myostatin expression is induced.

Lack of evidence of sustained hematopoietic reconstitution after transplantation of unmanipulated adult liver stem cells in monkeys.

The aim of this study was to search for hematopoietic potential in the liver of non-human primates. Lethally irradiated (2 x 5 Gy gamma) macaque monkeys were given autologous hepatic mononuclear cells (HMNC) isolated from a liver lobe by perfusion and digestion with 0.1% collagenase. Two monkeys were given intramedullary injections of HMNC (18.6 x 10(6)/kg, 20.4 x 10(6)/kg) and two others were co-transplanted with HMNC (14.35 x 10(6)/kg, 96.5 x 10(6)/kg) and bone marrow mesenchymal stem cells (0.42 x 10(6)/kg, 1.16 x 10(6)/kg). All monkeys exhibited a transient neutrophil recovery from day 22 for 10 days, but failed to produce platelets and remained transfusion-dependent. In conclusion, adult liver stem cells from a monkey model show a low level of in vivo hematopoietic potential, suggesting ex vivo manipulation will be required before clinical use of such cells.

Effect of glucocorticoid depletion on heat-induced Hsp70, IL-1beta and TNF-alpha gene expression.

When exposed to heat, conscious naive rats may develop lethal heatstroke, depending on heat load, i.e., time spent at high body core temperature. The occurrence of heatstroke was hypothesized to result from a defective glucocorticoid secretion related to altered heat-stress responses. We thus investigated the potential involvement of glucocorticoids in heat tolerance and its consequences on physiological responses, heat shock protein 70 (Hsp70), and cytokine mRNA expressions. Two hours before heat exposure, the animals were injected either with metyrapone, an inhibitor of corticosterone synthesis, or with its vehicle. Heat exposure lasted for 15, 30, 45 or 60 min. Thereafter, the rats were distributed into three groups according to their heat load: null, moderate (without any lethal risk) and intense (with lethal risk). Physiological responses were evaluated with colonic temperature, plasma lactate and hematocrit. Brain responses were assessed in frontal cortex through Hsp70, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) mRNA expressions. The animals with a severe heat load exhibited a high hematocrit, increased plasma lactate level and enhanced brain IL-1beta and Hsp70 mRNA expressions. Independent of the heat load, Metyrapone rats showed the same thermophysiological responses and IL-1beta and Hsp70 mRNA expressions when compared with vehicle rats. However, the Metyrapone rats experiencing an intense heat load exhibited an increased TNF-alpha mRNA expression. In conclusion, these data (i) confirm that heat load is important in the calibration of the risk attached to heat exposure; and (ii) suggest that corticosterone synthesis inhibition may favor TNF-alpha mRNA expression without any effect on Hsp70 mRNA expression.

Prolonged inflammatory gene response following soman-induced seizures in mice.

Following exposure to the organophosphorus nerve agent soman, the development of long-lasting seizures and build-up of irreversible seizure-related brain damage (SRBD) still represent a therapeutic challenge. A neuro-inflammatory reaction takes place in the brain after poisoning but its characteristics and potential role in SRBD and post-status epilepticus epileptogenesis is not well understood. In the present study we have analyzed by quantitative RT-PCR the time course of changes in mRNA levels of IL-1beta, TNFalpha, IL-6, ICAM-1 and SOCS3 in hippocampus, whole cortex and cerebellum in a mouse model of severe seizures and neuropathy up to 7 days after poisoning. Mice received an injection of the oxime HI-6 (50mg/kg) 5 min prior to the administration of a convulsive dose of soman (172 microg/kg). An important and highly significant increase of the five mRNA levels was recorded in cortex and hippocampus. In the cortex, the activation was generally detected as early as 1h post-intoxication with a peak response recorded between 6 and 24h. In the hippocampus, the gene up-regulation was delayed to 6h post-soman and the peak response observed between 24 and 48 h. After peaking, the response declined (except for ICAM in the hippocampus) but remained elevated, some of them significantly, at day 7. Interestingly, in the cerebellum, some changes were also observed but were several fold smaller. In conclusion, the present study indicates a quick neuro-inflammatory gene response that does not subside over 7 days suggesting a potential role in the neurological consequences of soman-induced status epilepticus.

Endogenous expression and in vitro study of CRF-related peptides and CRF receptors in the rat gastric antrum.

In vivo studies suggest that corticotrophin-releasing factor (CRF) and CRF-like peptides, urocortin 1 (UCN 1) and UCN 2, inhibit gastric emptying and stimulate colonic motility through CRF2 and CRF1 receptors, respectively. We evaluated expression and functions of CRF, UCN 1, UCN 2 and CRF1 and CRF2 receptors in the rat gastric antrum. Tissues were processed for immunohistochemistry and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). In vitro studies were performed to test the functional significance of CRF, UCN 1 and UCN 2. Some experiments were realized in the presence of specific CRF1 or CRF2 receptors antagonists. CRF1 and CRF2 receptors-like immunoreactivity (CRF1 and CRF2 receptors-LI) was localized in fibers and neurons of the myenteric ganglia. CRF1 and CRF2 receptors-LI was also found in nerve fibers distributed in the muscle layers. CRF- and UCN 1-LI was observed in neuronal cell bodies of the myenteric ganglia and in numerous nerve fibers running parallel to smooth muscle cells. Quantitative RT-PCR demonstrated UCN 2, CRF1 and CRF2 receptors expressions in both muscle layers and mucosa of the gastric antrum. Functional studies showed that CRF, UCN 1 and UCN 2 decreased antral phasic contractions. CRF(1) receptor antagonist (CP-154,526) did not block CRF-like peptides-induced inhibition of antral motility. In contrast, a CRF2 receptor antagonist (Astressin2-B) blocked the effects of CRF-like peptides on the antral muscle contractions. These results demonstrate (1) the presence of CRF, UCN and CRF1 and CRF2 receptors in the rat gastric antrum; (2) that, in vitro, CRF-like peptides inhibit phasic contractions of the antrum through CRF2 receptor. These results strongly suggest that CRF-like peptides play a major role in the regulatory mechanisms that underlie the neural control of gastric motility through CRF2 receptor.

Role of hypoxia-induced anorexia and right ventricular hypertrophy on lactate transport and MCT expression in rat muscle.

To dissect the independent effects of altitude-induced hypoxemia and anorexia on the capacity for cardiac lactate metabolism, we examined the effects of 21 days of chronic hypobaric hypoxia (CHH) and its associated decrease in food intake and right ventricle (RV) hypertrophy on the monocarboxylate transporter 1 and 4 (MCT) expression, the rate of lactate uptake into sarcolemmal vesicles, and the activity of lactate dehydrogenase isoforms in rat muscles. In comparison with control rats (C), 1 mmol/L lactate transport measured on skeletal muscle sarcolemmal vesicles increased by 33% and 58% in hypoxic (CHH, barometric pressure = 495 hPa) and rats pair-fed an equivalent quantity of food to that consumed by hypoxic animals, respectively. The increased lactate transport was higher in PF than in CHH animals ( P < .05). No associated change in the expression of MCT1 protein was observed in skeletal muscles, whereas MCT1 mRNA decreased in CHH rats, in comparison with C animals (42%, P < .05), partly related to caloric restriction (30%, P < .05). MCT4 mRNA and protein increased during acclimatization to hypoxia only in slow-oxidative muscles (68%, 72%, P < .05, respectively). The MCT4 protein content did not change in the plantaris muscle despite a decrease in transcript levels, related to hypoxia and caloric restriction. In both the left and right ventricles, the MCT1 protein content was unaffected by ambient hypoxia or restricted food consumption. These results suggest that MCT1 and MCT4 gene expression in fast-glycolytic muscles is mainly regulated by posttranscriptional mechanisms. Moreover, the results emphasize the role played by caloric restriction on the control of gene expression in response to chronic hypoxia and suggest that hypoxia-induced right ventricle hypertrophy failed to alter MCT proteins.

Ageing effects on the expression of cell defence genes after UVA irradiation in human male cutaneous fibroblasts using cDNA arrays.

Ageing is a multifactorial process in which reactive oxygen species (ROS) are thought to be implicated. ROS cause oxidative alterations on cell constituents, and damage accumulation can lead to mutations in DNA. Modulation of gene expression during ageing is now quite documented but results are often controversial and/or incomplete. As ultraviolet A is one of the exogenous factors involved in skin ageing, by the production of ROS, we further document the modifications in gene expression during ageing process and response to an oxidative stress. For this purpose, we used a cDNA macroarray containing 82 genes related to cell defence, essentially represented by antioxidant and DNA repair proteins. Ageing-associated gene expression was assessed in normal skin human fibroblasts from three age groups: children (n=4), adults (n=4) and olders (n=3), at the basal state and after a 5J/cm2 UVA irradiation. Analysis revealed that 22 genes were never detected, whereas certain were always expressed such as those related to antioxidant defence, extracellular matrix (ECM) regulator and XPC. Transcripts related to ECM, MMP1 and MMP3 were increased with age and after UVA irradiation, independently of age. It appeared that transcripts involved in the redox status control (TXN and APEX) decreased as a function of age, at the basal state and after irradiation, respectively. Most of transcripts involved in DNA repair were not detected but repression of POLD1 in the adult group and induction of XRCC5 and LIG4 were observed after UVA irradiation, as a function of age. In the basal state, the transcript of GAS1, regulator of cell cycle arrest in G1 phase was found to be decreased with age. HMOX1 increased after UVA irradiation. In conclusion, the decrease in expression of some antioxidant system, cell cycle control gene and extracellular matrix enzymes, particularly after UV exposure can explain the occurrence of photoaging.

2,3-Diarylimidazo[1,2-a]pyridines as potential inhibitors of UV-induced keratinocytes apoptosis: synthesis, pharmacological properties and interactions with model membranes and oligonucleotides by NMR.

Four 2,3-diarylimidazo[1,2-a]pyridines (I, 1a-c) were synthesized as inhibitors of UV-induced apoptosis and showed quite different properties. First, only the pyridinyl derivative I showed protection in molt cells. From the supposed intracellular target, phospholipid membrane models were studied by (1)H, (2)H and (31)P NMR spectroscopy. All these molecules can incorporate the membrane bilayer of small unilamellar vesicles of lecithin (SUV). However, I is clearly closed to the external polar head of the lipids, and is relatively mobile in the layer. Conversely, the other molecules are strongly immobilized in the deep part of the external layer. (31)P solid-state NMR spectra recorded on phospholipid dispersions (multilayers vesicles (MLV)) completely excluded any detergent effect or any modification of temperature transition. The only structural or dynamic effect observed was a homogeneous, but limited, reduction in the chemical shift anisotropy in the presence of I, in agreement with its superficial location. (2)H NMR experiment performed on the same model using perdeuterated phospholipids showed no significant fluidity reduction at the level of terminal CD(3) groups in the presence of 1a-c, according to their deep location. Finally, their interactions with synthetic oligonucleotide, d(CGATCG)(2) was studied showing non specific interactions of 1a on the external GC pair, while no interaction was observed with the other derivatives.

iNOS is a mediator of the heat stress-induced preconditioning against myocardial infarction in vivo in the rat.

OBJECTIVE: The inducible isoform of nitric oxide synthase (iNOS) is known to be a trigger of the heat stress (HS)-induced cardioprotection. Since iNOS also appears to mediate various forms of myocardial preconditioning, the goal of this study was to investigate its role as a mediator of the HS response. METHODS AND RESULTS: Male Wistar rats were divided in six groups, subjected or not to HS (42 degrees C internal temperature, for 15 min). Twenty-four hours later, they were treated or not with either L-NAME, a non-selective inhibitor of NO synthase isoforms, or 1400W, a selective iNOS inhibitor, 10 min before being subjected to a 30-min left coronary artery occlusion followed by a 120-min reperfusion, in vivo. The infarct size (tetrazolium staining) reducing effect conferred by heat stress (from 46.0+/-1.4% in sham to 26.8+/-3.8% in HS groups) was completely abolished by both L-NAME (53.9+/-3.1%) and 1400W (51.8+/-3.3%). Additional studies using Western blot analysis demonstrated a 3.8-fold increase in myocardial iNOS protein expression 24 h after HS. CONCLUSION: These results suggest an involvement of iNOS as a mediator of the protection conferred by heat stress against myocardial ischaemia.

Time course of skin features and inflammatory biomarkers after liquid sulfur mustard exposure in SKH-1 hairless mice.

Sulfur mustard (SM) is a strong bifunctional alkylating agent that produces severe tissue injuries characterized by erythema, edema, subepidermal blisters and a delayed inflammatory response after cutaneous exposure. However, despite its long history, SM remains a threat because of the lack of effective medical countermeasures as the molecular mechanisms of these events remain unclear. This limited number of therapeutic options results in part of an absence of appropriate animal models. We propose here to use SKH-1 hairless mouse as the appropriate model for the design of therapeutic strategies against SM-induced skin toxicity. In the present study particular emphasis was placed on histopathological changes associated with inflammatory responses after topical exposure of dorsal skin to three different doses of SM (0.6, 6 and 60mg/kg) corresponding to a superficial, a second-degree and a third-degree burn. Firstly, clinical evaluation of SM-induced skin lesions using non invasive bioengineering methods showed that erythema and impairment of skin barrier increased in a dose-dependent manner. Histological evaluation of skin sections exposed to SM revealed that the time to onset and the severity of symptoms including disorganization of epidermal basal cells, number of pyknotic nuclei, activation of mast cells and neutrophils dermal invasion were dose-dependent. These histopathological changes were associated with a dose- and time-dependent increase in expression of specific mRNA for inflammatory mediators such as interleukins (IL1ß and IL6), tumor necrosis factor (TNF)-α, cycloxygenase-2 (COX-2), macrophage inflammatory proteins (MIP-1α, MIP-2 and MIP-1αR) and keratinocyte chemoattractant (KC also called CXCL1) as well as adhesion molecules (L-selectin and vascular cell adhesion molecule (VCAM)) and growth factor (granulocyte colony-stimulating factor (Csf3)). A dose-dependent increase was also noted after SM exposure for mRNA of matrix metalloproteinases (MMP9) and laminin-γ2 which are associated with SM-induced blisters formation. Taken together, our results show that SM-induced skin histopathological changes related to inflammation is similar in SKH-1 hairless mice and humans. SKH-1 mouse is thus a reliable animal model for investigating the SM-induced skin toxicity and to develop efficient treatment against SM-induced inflammatory skin lesions.