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Mitochondria are the major source of cellular energy metabolism. In the cardiac cells, mitochondria produce byway of the oxidative phosphorylation more than 90% of the energy supply in the form of ATP, which is utilized in many ATP- dependent processes, like cycling of the contractile proteins or maintaining ion gradients. Reactive oxygen species (ROS) are by-products of cellular metabolism and their levels are controlled by intracellular antioxidant systems. Imbalance between ROS and the antioxidant defense leads to oxidative stress and oxidative changes to cellular biomolecules. Molecular hydrogen ([H.sub.2]) has been proved as beneficial in the prevention and therapy of various diseases including cardiovascular disorders. It selectively scavenges hydroxyl radical and peroxynitrite, reduces oxidative stress, and has anti-inflammatory and anti-apoptotic effects. The effect of [H.sub.2] on the myocardial mitochondrial function and coenzyme Q levels is not well known. In this paper, we demonstrated that consumption of [H.sub.2]-rich water (HRW) resulted in stimulated rat cardiac mitochondrial electron respiratory chain function and increased levels of ATP production by Complex I and Complex II substrates. Similarly, coenzyme [Q.sub.9] levels in the rat plasma, myocardial tissue, and mitochondria were increased and malondialdehyde level in plasma was reduced after HRW administration. Based on obtained data, we hypothesize a new metabolic pathway of the [H.sub.2] effect in mitochondria on the Q-cycle and in mitochondrial respiratory chain function. The Q- cycle contains three coenzyme Q forms: coenzyme Q in oxidized form (ubiquinone), radical form (semiquinone), or reduced form (ubiquinol). [H.sub.2] may be a donor of both electron and proton in the Q-cycle and thus we can suppose stimulation of coenzyme Q production. When ubiquinone is reduced to ubiquinol, lipid peroxidation is reduced. Increased [CoQ.sub.9] concentration can stimulate electron transport from Complex I and Complex II to Complex III and increase ATP production via mitochondrial oxidative phosphorylation. Our results indicate that [H.sub.2] may function to prevent/treat disease states with disrupted myocardial mitochondrial function. Key words: coenzyme [Q.sub.9], energy, mitochondrial respiratory chain, molecular hydrogen, myocardium. Les mitochondries sont la principale source d'energie pour le metabolisme de la cellule. Dans les cellules cardiaques, les mitochondries produisent, grace a la phosphorylation oxydative, plus de 90 % de l'apport energetique sous forme d'ATP utilisee dans de nombreux processus dependant de l'ATP, comme le cycle des proteines contractiles et le maintien des gradients ioniques. Les derives reactifs de l'oxygene proviennent du metabolisme cellulaire et leurs taux sont maintenus par les systemes antioxydants intracellulaires. Un desequilibre entre les derives reactifs de l'oxygene et la defense antioxydante mene a du stress oxydatif et a des modifications oxydatives des biomolecules cellulaires. On a montre que l'hydrogene moleculaire ([H.sub.2]) apporte des bienfaits dans la prevention et le traitement de diverses maladies, y compris les maladies cardiovasculaires. Il piege selectivement le radical hydroxyl et le peroxynitrite, attenue le stress oxydatif, est dote de proprietes anti-inflammatoires et anti- apoptotiques. On ne connait pas bien l'effet de l'[H.sub.2] sur la fonction mitochondriale myocardique et les taux de coenzyme Q. Dans cet article, nous avons montre que la consommation d'eau a teneur elevee en [H.sub.2] entraine la stimulation du fonctionnement de la chaine respiratoire electronique dans les mitochondries cardiaques chez le rat, avec une augmentation des degres de production d'ATP par les substrats Complexe I et Complexe II. De maniere similaire, apres l'administration d'eau a teneur elevee en [H.sub.2] chez le rat, les taux de coenzyme Q9 dans le plasma, ainsi que dans le tissu et les mitochondries myocardiques etaient en hausse, avec un abaissement des taux de malondialdehyde dans le plasma. A partir des donnees obtenues, nous presumons la presence d'une nouvelle voie metabolique de l'effet de l'[H.sub.2] dans les mitochondries sur le cycle Q, ainsi que sur le fonctionnement de la chaine respiratoire mitochondriale. Le cycle Qcomprend trois formes de coenzyme Q: le coenzyme Qsous forme oxydee (ubiquinone), sous forme radicale (semiquinone) ou sous forme reduite (ubiquinol). L'[H.sub.2] pourrait etre un donneur d'electrons comme de protons dans le cycle Q, et nous pouvons donc presumer qu'il stimule la production de coenzyme Q. Quand l'ubiquinone est reduite en ubiquinol, la peroxydation lipidique diminue. L'augmentation de la concentration de CoQ9 peut stimuler le transport des electrons du Complexe I et du Complexe II vers le Complexe III, et faire augmenter la production d'ATP par l'intermediaire de la phosphorylation oxydative mitochondriale. Nos resultats montrent que par son fonctionnement, l'[H.sub.2] pourrait permettre de prevenir ou de traiter des pathologies comprenant des perturbations du fonctionnement des mitochondries myocardiques. [Traduit par la Redaction] Mots-cles: coenzyme Q9, energie, chaine respiratoire mitochondriale, hydrogene moleculaire, myocarde., Introduction Mitochondria play an important role in the physiology of the heart function and in the pathogenesis and development of various types of cardiovascular diseases (CVD) that belong to the [...]

Generation of free radicals through incomplete reduction of oxygen during ischemia-reperfusion (I/R) is well described. On the other hand, molecular hydrogen ([H.sub.2]) reduces oxidative stress due to its ability to react with strong oxidants and easily penetrate cells by diffusion, without disturbing metabolic redox reactions. This study was designed to explore cardioprotective potential of hypoxic postconditioning (HpostC) against I/R (30 min global I-120 min R) in isolated rat hearts using oxygen-free Krebs-Henseleit buffer (KHB). Furthermore, the possibility to potentiate the effect of HpostC by [H.sub.2] using oxygen-free KHB saturated with [H.sub.2] ([H.sub.2] + HpostC) was tested. HPostC was induced by 4 cycles of 1-minute perfusion with oxygen-free KHB intercepted by 1-minute perfusion with normal KHB, at the onset of reperfusion. [H.sub.2] + HPostC was applied in a similar manner using [H.sub.2]-enriched oxygen-free KHB. Cardioprotective effects were evaluated on the basis of infarct size (IS, in % of area at risk, AR) reduction, post-I/R recovery of heart function, and occurrence of reperfusion arrhythmias. HPostC significantly reduced IS/AR compared with non-conditioned controls. [H.sub.2] present in KHB during HPostC further decreased IS/AR compared with the effect of HPostC, attenuated severe arrhythmias, and significantly restored heart function (vs. controls). Cardioprotection by HpostC can be augmented by molecular hydrogen infusion. Key words: ischemia-reperfusion injury, ischemic postconditioning, hypoxic postconditioning, molecular hydrogen, antioxidants. La production de radicaux libres par la reduction incomplete de l'oxygene pendant l'ischemie-reperfusion (I/R) est un phenomene qui est bien decrit. Par ailleurs, l'hydrogene moleculaire ([H.sub.2]) permet de reduire le stress oxydatif en raison de sa capacite de reagir avec des oxydants puissants et de penetrer facilement dans les cellules par diffusion, sans perturber les reactions d'oxydoreduction metabolique. Cette etude a ete concue en vue d'explorer le potentiel cardioprotecteur du postconditionnement hypoxique (postCH) contre l'I/R (I globale de 30 min -Rde 120 min) dans des creurs isoles de rat a l'aide de tampon Krebs-Henseleit (TKH) sans oxygene. En outre, nous avons etudie la possibilite de potentialiser l'effet du postCH par l'[H.sub.2] a l'aide de TKH sans oxygene sature d'[H.sub.2] (postCH + [H.sub.2]). Nous avons obtenu le postCH en procedant a 4 cycles de perfusion d'une minute avec du TKH sans oxygene interceptes par des perfusions d'une minute avec du TKH normal au debut de la reperfusion. Nous avons applique la postCH + [H.sub.2] d'une maniere semblable a l'aide de TKH sans oxygene enrichi d'[H.sub.2]. Nous avons evalue les effets cardioprotecteurs sur la base de la reduction de la taille de l'infarctus (TI, en % de la zone a risque, ZR), de la recuperation de la fonction cardiaque apres l'I/R et de l'apparition d'arythmies de reperfusion. La postCH a permis de reduire le rapport TI/ZR de facon plus marquee que chez les temoins non conditionnes. L'[H.sub.2] present dans le TKH pendant la postCH a permis de diminuer encore plus le rapport TI/ZR qu'avec la postCH, d'attenuer les arythmies graves et de retablir la fonction cardiaque de facon notable (vs. les temoins). En somme, il est possible d'augmenter la cardioprotection par le postconditionnement hypoxique en perfusant de l'hydrogene moleculaire. [Traduit par la Redaction] Mots-cles : lesion d'ischemie-reperfusion, postconditionnement ischemique, postconditionnement hypoxique, hydrogene moleculaire, antioxydants., Introduction The strategies that may improve prognosis of patients after infarction or cardiosurgical interventions are intensively studied. Ischemic preconditioning (IP) induced by short-term ischemic periods has been shown to enhance [...]

We describe the steady-state function of the ubiquitous mammalian Na/H exchanger (NHE) 1 isoform in voltage-clamped Chinese hamster ovary cells, as well as other cells, using oscillating pH-sensitive microelectrodes to quantify proton fluxes via extracellular pH gradients. Giant excised patches could not be used as gigaseal formation disrupts NHE activity within the patch. We first analyzed forward transport at an extracellular pH of 8.2 with no cytoplasmic Na (i.e., nearly zero-trans). The extracellular Na concentration dependence is sigmoidal at a cytoplasmic pH of 6.8 with a Hill coefficient of 1.8. In contrast, at a cytoplasmic pH of 6.0, the Hill coefficient is

The photosynthetic bacterium Rhodobacter capsulatus contains two [NiFe] hydrogenases: an energy-generating hydrogenase, HupSL, and a regulatory hydrogenase, HupUV. The synthesis of HupSL is specifically activated by [H.sub.2] through a signal transduction cascade comprising three proteins: the [H.sub.2]-sensing HupUV protein, the histidine kinase HupT, and the transcriptional regulator HupR. Whereas a phosphotransfer between HupT and HupR was previously demonstrated, interaction between HupUV and HupT was only hypothesized based on in vivo analyses of mutant phenotypes. To visualize the in vitro interaction between HupUV and HupT proteins, a six-His ([His.sub.6])-HupU fusion protein and the HupV protein were coproduced by using a homologous expression system. The two proteins copurified as a [His.sub.6]-HupUHupV complex present in dimeric and tetrameric forms, both of which had [H.sub.2] uptake activity. We demonstrated that HupT and HupUV interact and form stable complexes that could be separated on a native gel. Interaction was also monitored with surface plasmon resonance technology and was shown to be insensitive to salt concentration and pH changes, suggesting that the interactions involve hydrophobic residues. As expected, [H.sub.2] affects the interaction between HupUV and HupT, leading to a weakening of the interaction, which is independent of the phosphate status of HupT. Several forms of HupT were tested for their ability to interact with HupUV and to complement hupT mutants. Strong interaction with HupUV was obtained with the isolated PAS domain of HupT and with inactive HupT mutated in the phosphorylable histidine residue, but only the wild-type HupT protein was able to restore normal [H.sub.2] regulation.

Atom depth, originally defined as the distance between a protein atom and the nearest water molecule surrounding a protein, is a simple but valuable geometrical descriptor of the protein interior. It can be easily computed from the 3D structure of a protein, thus complementing the information provided by the calculation of the solvent accessible surface area and buried surface area. Depth has been found to be correlated with several molecular, residue and atomic properties, such as average protein domain size, protein stability, free energy of formation of protein complexes, amino acid type hydrophobicity, residue conservation and hydrogen/deuterium amide proton exchange rates.

A sulfur reductase (SR) and a hydrogenase were purified from solubilized membrane fractions of anaerobically grown cells of the sulfur-dependent archaeon Acidianus ambivalens and the corresponding genes were sequenced. The SR reduced elemental sulfur with hydrogen as electron donor [45 U [(mg protein).sup.-1]] in the presence of hydrogenase and either 2,3-dimethylnaphthoquinone (DMN) or cytochrome c in the enzyme assay. The SR could not be separated from the hydrogenase during purification without loss of activity, whereas the hydrogenase could be separated from the SR. The specific activity of the hydrogenase was 170 U [(mg protein).sup.-1] with methyl viologen and 833 U [(mg protein).sup.-1] with DMN as electron acceptors. Both holoenzymes showed molecular masses of 250 kDa. In SDS gels of active fractions, protein bands with apparent masses of 110 (SreA), 66 (HynL), 41 (HynS) and 29 kDa were present. Enriched hydrogenase fractions contained 14 [micro]mol Fe and 2 [micro]mol Ni [(g protein).sup.-1]; in addition, 2-5 [micro]mol Mo [(g protein).sup.-1] was found in the membrane fraction. Two overlapping genomic cosmid clones were sequenced, encoding a five-gene SR cluster (sre) including the 110 kDa subunit gene (sreA), and a 12-gene hydrogenase cluster (hyn) including the large and small subunit genes and genes encoding proteins required for the maturation of NiFe hydrogenases. A phylogenetic analysis of the SR amino acid sequence revealed that the protein belonged to the DMSO reductase family of molybdoenzymes and that the family showed a novel clustering. A model of sulfur respiration in Acidianus developed from the biochemical results and the data of the amino acid sequence comparisons is discussed.

The photophysical properties of complexes of recombinant human serum albumin (rHSA) with protoporphyrin IX (ZnPP) and their activities in the photosensitized reduction of water to hydrogen [(H.sub.2)] are presented. It is shown that in the presence of the colloidal PVA-Pt as a catalyst and triethanolamine (TEOA) as a sacrificial electron donor, the photosensitized reduction of water to [H.sub.2] takes place.

Research has been conducted on LOV1 domain from the green alga. The authors report the introduction of methylated thiol group into this domain, and demonstrate that the replacement of the reactive cysteine with methionine makes the transfer of a proton or hydrogen atom from the thiol group to the flavin mononucleotide impossible.

Research has been conducted on enzyme rho which is responsible for Escherichia coli growth and survival. The authors have demonstrated that metal-1,4-dithio-2,3-dihydroxybutane chelates inhibit rho via a unique pathway, and that these agents are more potent that the only other known rho inhibitor, bicyclomycin.

Research has been conducted on Podospora anserina HET-s prion protein. The authors describe the study of this protein by hydrogen/deuterium exchange carried out via the use of matrix assisted laser desorption-ionization mass spectrometry.

Research has been conducted on photoactive yellow protein, which is in pH-dependent equilibrium with the bleached form in the dark, and its mutants. The study of E46Q mutant of this protein has demonstrated that its photocycle kinetics is affected by the relaxation of the equilibrium.

The authors have investigated trans-3-chloroacrylic acid dehalogenase which converts 3-bromo- and 3-chloroacrylate trans-isomers to malonate semialdehyde. They describe the synthesis of dehalogenase's alpha- and beta-subunit genes carried out via the use of polymerase chain reaction-based strategy and proteins purified as functional heterohexamers.

Research has been conducted on cyclodextrin glycosyltransferase which catalyzes transglycosylation reactions. The authors have identified amino acid residues which are important for transglycosylation reaction specificity, and have described the use of error-prone PCR mutagenesis for screening Bacillus circulans strain 251 cyclodextrin glycosyltransferase's mutants with increase hydrolytic activity.

Research has been conducted on 4-hydroxyphenylacetate 3-monooxygenase. The authors report the the production and utilization of flavin adenine dinucleotide by 4-hydroxyphenylacetate 3-monooxygenase have been coordinately coupled.

Research has been conducted on Na (super)+/H (super)+ exchanger. The authors describe the characterization of the exchanger's cytoplasmic tail carried out via the use of histidine-tagged protein containing C-terminal amino acids.