Hypohydration effects on skeletal muscle performance and metabolism: a 31P-MRS study

The purpose of this study was to determinewhether hypohydration reduces skeletal muscle endurance and whetherincreased H⁺ andP_i might contribute to performancedegradation. Ten physically active volunteers (age 21-40 yr)performed supine single-leg, knee-extension exercise to exhaustion in a1.5-T whole body magnetic resonance spectroscopy (MRS) system wheneuhydrated and when hypohydrated (4% body wt).³¹P spectra were collected at arate of one per second at rest, exercise, and recovery, and weregrouped and averaged to represent 10-s intervals. The desired hydrationlevel was achieved by having the subjects perform 2-3 h ofexercise in a warm room (40°C dry bulb, 20% relative humidity)with or without fluid replacement 3-8 h before the experiment.Time to fatigue was reduced (P < 0.05) by 15% when the subjects were hypohydrated [213 ± 12 vs. 251 ± 15 (SE) s]. Muscle strength was generally notaffected by hypohydration. Muscle pH andP_i/-ATP ratio were similarduring exercise and at exhaustion, regardless of hydration state. The time constants for phosphocreatine recovery were also similar betweentrials. In summary, moderate hypohydration reduces muscle endurance,and neither H⁺ norP_i concentration appears to berelated to these reductions.

     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

Human embryonic stem cells passaged using enzymatic methods retain a normal karyotype and express CD30

Human embryonic stem cells (hESCs) are thought to be susceptible to chromosomal rearrangements as a consequence of single cell dissociation. Compared in this study are two methods of dissociation that do not generate single cell suspensions (collagenase and EDTA) with an enzymatic procedure using trypsin combined with the calcium-specific chelator EGTA (TEG), that does generate a single cell suspension, over 10 passages. Cells passaged by single cell dissociation using TEG retained a normal karyotype. However, cells passaged using EDTA, without trypsin, acquired an isochromosome p7 in three replicates of one experiment. In all of the TEG, collagenase and EDTA-treated cultures, cells retained consistent telomere length and potentiality, demonstrating that single cell dissociation can be used to maintain karyotypically and phenotypically normal hESCs. However, competitive genomic hybridization revealed that subkaryotypic deletions and amplifications could accumulate over time, reinforcing that present culture regimes remain suboptimal. In all cultures the cell surface marker CD30, reportedly expressed on embryonal carcinoma but not karyoptically normal ESCs, was expressed on hESCs with both normal and abnormal karyotype, but was upregulated on the latter.     

Effect of neutrophil elastase and its inhibitor EPI-hNE4 on transepithelial sodium transport across normal and cystic fibrosis human nasal epithelial cells

Background

Hyperactivity of the epithelial sodium (Na⁺) channel (ENaC) and increased Na⁺ absorption by airway epithelial cells leading to airway surface liquid dehydration and impaired mucociliary clearance are thought to play an important role in the pathogenesis of cystic fibrosis (CF) pulmonary disease. In airway epithelial cells, ENaC is constitutively activated by endogenous trypsin-like serine proteases such as Channel-Activating Proteases (CAPs). It was recently reported that ENaC activity could also be stimulated by apical treatment with human neutrophil elastase (hNE) in a human airway epithelial cell line, suggesting that hNE inhibition could represent a novel therapeutic approach for CF lung disease. However, whether hNE can also activate Na⁺ reabsorption in primary human nasal epithelial cells (HNEC) from control or CF patients is currently unknown.

Methods

We evaluated by short-circuit current (I_sc) measurements the effects of hNE and EPI-hNE4, a specific hNE inhibitor, on ENaC activity in primary cultures of HNEC obtained from control (9) and CF (4) patients.

Results

Neither hNE nor EPI-hNE4 treatments did modify I_sc in control and CF HNEC. Incubation with aprotinin, a Kunitz-type serine protease inhibitor that blocks the activity of endogenous CAPs, decreased I_sc by 27.6% and 54% in control and CF HNEC, respectively. In control and CF HNEC pretreated with aprotinin, hNE did significantly stimulate I_sc, an effect which was blocked by EPI-hNE4.

Conclusions

These results indicate that hNE does activate ENaC and transepithelial Na⁺ transport in both normal and CF HNEC, on condition that the activity of endogenous CAPs is first inhibited. The potent inhibitory effect of EPI-hNE4 on hNE-mediated ENaC activation observed in our experiments highlights that the use of EPI-hNE4 could be of interest to reduce ENaC hyperactivity in CF airways.     

Genetic snapshots of the Rhizobium species NGR234 genome

Background  

In nitrate-poor soils, many leguminous plants form nitrogen-fixing symbioses with members of the bacterial family Rhizobiaceae. We selected Rhizobium sp. NGR234 for its exceptionally broad host range, which includes more than 112 genera of legumes. Unlike the genome of Bradyrhizobium japonicum, which is composed of a single 8.7 Mb chromosome, that of NGR234 is partitioned into three replicons: a chromosome of about 3.5 Mb, a megaplasmid of more than 2 Mb (pNGR234b) and pNGR234a, a 536,165 bp plasmid that carries most of the genes required for symbioses with legumes. Symbiotic loci represent only a small portion of all the genes coded by rhizobial genomes, however. To rapidly characterize the two largest replicons of NGR234, the genome of strain ANU265 (a derivative strain cured of pNGR234a) was analyzed by shotgun sequencing.     

VKORC1L1, an Enzyme Rescuing the Vitamin K 2,3-Epoxide Reductase Activity in Some Extrahepatic Tissues during Anticoagulation Therapy

Vitamin K is involved in the γ-carboxylation of the vitamin K-dependent proteins, and vitamin K epoxide is a by-product of this reaction. Due to the limited intake of vitamin K, its regeneration is necessary and involves vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is known to be supported by VKORC1 protein, but recently a second gene, VKORC1L1, appears to be able to support this activity when the encoded protein is expressed in HEK293T cells. Nevertheless, this protein was described as being responsible for driving the vitamin K-mediated antioxidation pathways. In this paper we precisely analyzed the catalytic properties of VKORC1L1 when expressed in Pichia pastoris and more particularly its susceptibility to vitamin K antagonists. Vitamin K antagonists are also inhibitors of VKORC1L1, but this enzyme appears to be 50-fold more resistant to vitamin K antagonists than VKORC1. The expression of Vkorc1l1 mRNA was observed in all tissues assayed, i.e. in C57BL/6 wild type and VKORC1-deficient mouse liver, lung, and testis and rat liver, lung, brain, kidney, testis, and osteoblastic cells. The characterization of VKOR activity in extrahepatic tissues demonstrated that a part of the VKOR activity, more or less important according to the tissue, may be supported by VKORC1L1 enzyme especially in testis, lung, and osteoblasts. Therefore, the involvement of VKORC1L1 in VKOR activity partly explains the low susceptibility of some extrahepatic tissues to vitamin K antagonists and the lack of effects of vitamin K antagonists on the functionality of the vitamin K-dependent protein produced by extrahepatic tissues such as matrix Gla protein or osteocalcin.     

Brief exposure to sensory cues elicits stimulus-nonspecific general sensitization in an insect

The effect of repeated exposure to sensory stimuli, with or without reward is well known to induce stimulus-specific modifications of behaviour, described as different forms of learning. In recent studies we showed that a brief single pre-exposure to the female-produced sex pheromone or even a predator sound can increase the behavioural and central nervous responses to this pheromone in males of the noctuid moth Spodoptera littoralis. To investigate if this increase in sensitivity might be restricted to the pheromone system or is a form of general sensitization, we studied here if a brief pre-exposure to stimuli of different modalities can reciprocally change behavioural and physiological responses to olfactory and gustatory stimuli. Olfactory and gustatory pre-exposure and subsequent behavioural tests were carried out to reveal possible intra- and cross-modal effects. Attraction to pheromone, monitored with a locomotion compensator, increased after exposure to olfactory and gustatory stimuli. Behavioural responses to sucrose, investigated using the proboscis extension reflex, increased equally after pre-exposure to olfactory and gustatory cues. Pheromone-specific neurons in the brain and antennal gustatory neurons did, however, not change their sensitivity after sucrose exposure. The observed intra- and reciprocal cross-modal effects of pre-exposure may represent a new form of stimulus-nonspecific general sensitization originating from modifications at higher sensory processing levels.     

Influence of lipolysis and fatty acid availability on fuel selection during exercise

The aim of the present study was to investigate the influence of substrate availability on fuel selection during exercise. Eight endurance-trained male cyclists performed 90-min exercise at 70 % of their maximal oxygen uptake in a cross-over design, either in rested condition (CON) or the day after 2-h exercise practised at 70 % of maximal oxygen uptake (EX). Subjects were given a sucrose load (0.75 g kg^?1 body weight) 45 min after the beginning of the 90-min exercise test. Lipolysis was measured in subcutaneous abdominal adipose tissue (SCAT) by microdialysis and substrate oxidation by indirect calorimetry. Lipid oxidation increased during exercise and tended to decrease during sucrose ingestion in both conditions. Lipid oxidation was higher during the whole experimental period in the EX group (p?=?0.004). Interestingly, fuel selection, assessed by the change in respiratory exchange ratio (RER), was increased in the EX session (p?=?0.002). This was paralleled by a higher rate of SCAT lipolysis reflected by dialysate glycerol, plasma glycerol, and fatty acids (FA) levels (p?<?0.001). Of note, we observed a significant relationship between whole-body fat oxidation and dialysate glycerol in both sessions (r ²?=?0.33, p?=?0.02). In conclusion, this study highlights the limiting role of lipolysis and plasma FA availability to whole-body fat oxidation during exercise in endurance-trained subjects. This study shows that adipose tissue lipolysis is a determinant of fuel selection during exercise in healthy subjects.     

The chick embryo: hatching a model for contemporary biomedical research

Animal models play a crucial role in fundamental and medical research. Progress in the fields of drug discovery, regenerative medicine and cancer research among others are heavily dependent on in vivo models to validate in vitro observations, and develop new therapeutic approaches. However, conventional rodent and large animal experiments face ethical, practical and technical issues that limit their usage. The chick embryo represents an accessible and economical in vivo model, which has long been used in developmental biology, gene expression analysis and loss/gain of function experiments. It is also an established model for tissue/cell transplantation, and because of its lack of immune system in early development, the chick embryo is increasingly recognised as a model of choice for mammalian biology with new applications for stem cell and cancer research. Here, we review novel applications of the chick embryo model, and discuss future developments of this in vivo model for biomedical research.