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This manuscript is a summary of my activities from my PhD, until the fist years of my research in the French technological research institute, namely the CEA.Particularly, the following items are mentioned: the NMR characterization of biomolecules such as sugars, proteins, either dia or paramagnetic, water-protein interactions, cellular metabolism, and electron transfer rates in proteins belonging several redox centers.Then, I mention my recent work in a lab specialized in the hyperpolarized xenon applied to liquid phase, particularly the characterization of hydrophobic cavities of proteins and the high complexation of xenon into a family of cage-molecules named cryptophanes.; Ce manuscrit résume mes activités de recherche en thèse, dans mes différents séjours post-doctoraux et mes premières années de recherche au Commissariat à l'Energie Atomique. Elles concernent la caractérisation de biomolécules par RMN, comme des sucres, des protéines, dia ou paramagnétiques. Le manuscrit aborde aussi la caractérisation des interactions protéines-eau, du métabolisme cellulaire, et la mesure de vitesses électroniques impliquant des protéines à plusieurs centres d'oxydoréduction. J'aborde ensuite mon travail récent au sein d'une équipe spécialisée dans le xénon hyperpolarisé appliqué à la phase liquide, en particulier la caractérisation de cavités hydrophobes de protéines et la forte complexation du xénon par une famille de molécules-cage nommée cryptophanes.

Nitric oxide (NO) is produced from L-Arginine by the NO synthase family. Dysregulation of the synthesis of this signal molecule is involved in the development of numerous pathologies. The cellular effects of NO are partly due to the inhibition of the complexes of the mitochondrial respiratory chain. We studied the NO regulation of the oxidative phosphorylation and the permeability transition, two mitochondrial processes controlling cellular survival. We have demonstrated that the inhibition of cytochrome c oxidase (COX) by NO was associated with an inhibition of the respiration and the ATP synthesis. However, NO increased the yield of oxidative phosphorylation (ATP/O) due to a reduction in the « slipping ». Our work highlights an effect of NO which depends on the substrate used. Indeed, in the presence of NADH-linked substrates, NO does not have the beneficial effect on the yield of the oxidative phosphorylation, that obtained in the presence of FADH2-linked substrates with or without NADH-linked subtrates. The study of the redox status of the cytochromes of the COX revealed that in mitochondria energized with FADH2-linked substrates with or without NADH-linked subtrates, the cytochromes aa3 are more reduced than in the presence of NADHlinked subtrates alone.We can suggest that the « slipping » depends on the redox status of the cytochrome aa3. NO could thus physiologically increase oxidative phosphorylation efficiency. We also sought to support the hypothesis that the inhibition of complex I would have an antiapoptotic effect by inhibiting the PTP opening and the cytochrome c release. Because NO acts on the whole respiratory chain, we could not certify that this activating effect of the pore opening is due to an inhibition of a complex in particular. It seems that the permeability transition is sensitive to the complex II inhibition. Inhibition of the complex II by inhibitors like TTFA or malonate would have a pro-apoptotic effect despite the presence of powerful inhibitor of PTP such as cyclosporine. This results suggests that complex II belongs to the regulating proteic complexes of the PTP and that its inhibition prevails over the effect induced by the inhibition of complex I.; Le monoxyde d'azote (NO) est formé à partir de L-arginine par la famille des NO synthases. La dysrégulation de la synthèse de cette molécule de signalisation cellulaire est impliquée dans le développement d'un grand nombre de pathologies. Les effets cellulaires du NO sont en partie dus à une inhibition des complexes de la chaîne respiratoire mitochondriale. Nous nous sommes intéressés aux effets du NO sur la régulation de l'oxydation phosphorylante et de le transition de perméabilité, deux processus mitochondriaux gouvernant la survie cellulaire. Nous avons ainsi démontré que l'inhibition de la cytochrome c oxydase (COX) par le NO était associée à une inhibition de la respiration et de la synthèse d'ATP. Cependant le NO augmente l'efficacité de l'oxydation phosphorylante (ATP/O) en diminuant le processus de « slipping ». Toutefois, notre travail a mis en exergue un effet du NO dépendant du substrat utilisé. En présence de NADH seul, le NO n'a pas l'effet bénéfique sur le rendement de l'oxydation phosphorylante, obtenu avec le FADH2 seul ou associé au NADH. L'étude de l'état d'oxydoréduction des cytochromes de la COX a révélé que dans les mitochondries énergisées avec du FADH2 seul ou en association avec le NADH, les cytochromes aa3 sont plus réduits qu'en présence de NADH seul. Nous pouvons suggérer que le « slipping » au niveau de la COX soit dépendant de l'état d'oxydoréduction de ses cytochromes. Le NO pourrait donc être un régulateur physiologique du processus d'oxydation phosphorylante. Nous avons également cherché à étayer l'hypothèse selon laquelle l'inhibition du complexe I aurait un effet anti-apoptotique en inhibant l'ouverture du PTP et la libération de cytochrome c. Le NO ayant un effet sur la chaîne respiratoire dans sa globalité, nous n'avons pu certifier que son effet activateur sur l'ouverture du pore soit dû à l'inhibition d'un complexe en particulier. Il semble que le phénomène de transition de perméabilité soit sensible à l'inhibition du complexe II. L'inhibition du complexe II par un inhibiteur comme le TTFA ou le malonate aurait un effet pro-apoptotique et ce malgré la présence d'inhibiteurs puissants du PTP comme la ciclosporine. Ces résultats suggèrent que le complexe II ferait partie des complexes protéiques régulateurs du PTP et que son inhibition soit dominante sur l'effet induit par l'inhibition du complexe I.

Non disponible / Not available, Le stress oxydant est un processus essentiel dans l'initiation et le développement des nombreuses pathologies liées à l'âge, en particulier l'athérosclérose, les pathologies neuro-dégénératives, le vieillissement accéléré, le cancer. Des données épidémiologiques suggèrent que les antioxydants pourraient avoir une influence bénéfique dans la prévention de ces pathologies, ainsi que dans la longévité. Dans une première partie, notre travail a consisté à obtenir 5 extraits et deux principes actifs présents dans trois plantes de la médecine traditionnelle chinoise et ouïgour (Glycyrrhiza glabra, Cistanche deserticola et Lycium Barbarum) et dans le thé médicinal ouïgour (UMT) ; nous avons également synthétisé 6 substances chimiquement définies de structure oleane (acide glycyrrhétinique, GA) pour évaluer leur potentialité antioxydante. Nous avons réalisé aussi, une modélisation du pharmacophore hypolipémiant de ce squelette par "mapping" avec 3 séries desmolécules normolipémiantes introduites en thérapeutique (statines, fibrates, inhibiteurs de l' ACAT), et proposé un mécanisme d'action de type fibrates. Notre double objectif a consisté, d'une part, à corréler l'utilisation de telles substances, avec la prévention du vieillissement et des pathologies liées à l'âge, et d'autre part, à développer de nouveaux archétypes structuraux antioxydants associés avec un effet hypolipémiant. Dans une deuxième partie, nous avons étudié les effets antioxydants de ces substances sur deux modèles in vitro : la lignée cellulaire HL-60 humaine (système d'oxydation myéloperoxydase/H2O2/HOCI) et les microsomes hépatiques de rats (système d'oxydation cytochrome P450/NADPH réductase). Nous avons observé que, parmi les dérivés synthétisés, deux triols (II et IV) et deux diènes (III et V) présentent des propriétés antioxydantes plus puissantes que celles de leur chef de file GA et de la vitamine E. Dans les mêmes conditions, la glabridine (Glab) et l'extrait de l'UMT revèlent aussi une activité antioxydante dans ces deux systemes, de niveau comparable à celle de l'extrait EGB 761 de Ginkgo biloba. L'ensemble des extraits de plantes étudiées (dont les cistanosides- CD, les licoflavonoïde -LF et le Lycium barbarum polysaccharide- LBP), présentent une activité antioxydante s'inscrivant dans la séquence : EGB?UMT?Glab>CD>LF>LBP. La troisième partie de ce travail présente une enquête préliminaire sur la faisabilité d'une étude épidemlologique dans la population de centenaires : 30 sujets âgés dont 10 d'origine ouïgour avec une étude comparative des paramètres lipidiques, antioxydants et nutritionnels. Nos résultats suggèrent le rôle possible de la consommation habituelle d'antioxydants dans la prévention de pathologies liées à l'âge. Ce travail s'inscrit dans un grand projet d'étude épidémiologique concernant un nombre éleve de sujets chinois (dont ouïgour) et français.

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Eleven strains of coryneform bacteria were isolated from soil samples by enrichment culture in a mineral medium containing beta-caryophyllene as the sole energy and carbon source. Ten of them could also metabolize longifolene. Numerical taxonomy, based on the use of 147 characteristics, revealed a large diversity. The DNA G + C content was found to be in the range of 62.5-68.3 mol%. Three strains were placed in "The National Collection of Industrial and Marine Bacteria".

Studies of absorbance related to the cytochrome b-559 photooxidation induced by FCCP, with and without addition of 3-p-chlorophenyl-1, 1-dimethylurea (CMU), DBMIB or p-benzoquinone, in whole cells and in chloroplast fragments of Chlamydomonas reinhardti, were carried out. In addition to the wild type, three strains of non-photosynthetic mutants were used: Fl 5, which lacks P 700; Fl 9 and Fl 15, which are deficient in bound cytochrome c-553 and in cytochrome b-563. In the presence of FCCP, whole cells and chloroplast fragments of the four strains showed a System II-dependent photooxidation of cytochrome b-559. This photooxidation was inhibited by CMU but it occurred again in presence of FCCP, CMU and DBMIB. In chloroplast fragments, cytochrome b-559 photooxidation was also inhibited by an excess of FCCP; it was recovered, likewise, by addition of DBMIB. In whole cells, the highest measured redox changes were: 1 mu mol oxidized cytochrome b-559 per 1 mmol chlorophyll, corresponding approximately to about one seventh (wild type, Fl5) or one fifth (Fl 9, Fl 15) of the total amount of this cytochrome. Another kind of cytochrome b-559 photooxidation, CMU-insensitive, also occurred in the mutants Fl 9 and Fl 15 and in the wild type, but not in the mutant Fl 5. This latter kind of photooxidation was observed with chloroplast fragments in the presence of FCCP and CMU and also with whole cells in the presence of FCCP, CMU and p-benzoquinone. These reactions can be attributed to the Photosystem I; they do not require the intervention of the cytochrome c-553. A high-potential form of cytochrome b-559, hydroquinone-reducible, was involved in these two kinds of photooxidation. In addition, a lower potential form, reducible only by ascorbate, appeared to be able to interfere also. An interpretation is attempted, taking into consideration the various effects of FCCP and DBMIB, at different concentrations, on photosynthetic electron transport.

Ellipticine is able to induce cytochrome P448 synthesis in Rat liver microsomes which converts the drug into 9-hydroxy derivative, with a higher rate than microsomal cytochrome P450 of non-treated or phenobarbital induced Rats. In vivo, ellipticine is also transformed into a second hydroxylated product on the indole aromatic ring.

The molecular evolution is considered in several protein families. It can be studied with the data of an entirely known structure, like in hemoglobin or cytochrome, or of only partial structural data, as for proteases or immunoglobulins. In the case of isozymes the study of physico-chemical and kinetic properties is more indirect. Finally, it is shown that the molecular mechanisms are always the same, happening probably at the same frequency, the differences being due to a variable selection by molecular constraints and adaptation to the environment.

Sychronous divisions of Euglena gracilis strain Z can be obtained by various methods. When the cells are cultivated in a medium containing lactate as the sole carbon source, synchronous divisions are observed, independent of the conditions of illumination. Nevertheless, there exists a relationship between the phase of cell division and ther periods of light and darkness applied to the culture. During the cell cycle, the synthesis of macromolecules is discontinuous--this is true of nuclear and mitochondrial DNA, ribosomal and nonribosomal RNA, and certain proteins (cytochrome c 558). Cyclic variations in the structure of mitochondria and chloroplasts are observed. In the course of the cell cycle, sequential metabolic processes accompany structural modifications of the organelles. Also, at the beginning of the cycle, at the start of phase G1, the cytoplasmic ribosomes are synthesized, and then, in green euglenids, nonribosomal RNAs are formed. These syntheses of RNA precede enlargement of the chondriome and plastids. In mid-G1 phase, a new synthesis of RNA begins, which precedes synthesis of nuclear and mitochondrial DNA. At the end of G1 phase, division of organelles starts, beginning with the chondriome and plastids, arranged in a network.

Liver mitochondria were isolated from normal and thyroidectomized rats and their protein components analyzed by polyacrylamide gel electrophoresis. In whole mitochondria 35 protein fractions with MW ranging from 10,000 to 135,000 were characterized. In the absence of thyroid hormone secretion, the amount of a MW 54,000 fraction was always decreased. Injection of small doses of 3,5,3'-triiodo-L-thyronine to the thyroidectomized animal restored the quantity of that protein fraction to normal. Isolated outer mitochondrial membranes showed the presence of 20 protein fractions. These fractions revealed no change after thyroidectomy. The mitoplast, which contained 35 fractions, exhibited a decrease of the MW 54,000 component in thyroidectomized rats. The mitoplast was separated into several fractions. Water soluble matrix proteins presented molecular weights ranging between 40,000 and 55,000. Proteins, which were slightly bound to the inner mitochondrial membrane and could be extracted by KCl, presented molecular weights between 25,000 and 45,000. Structural proteins showed a principal specific component of MW equals 23,000. Electrophoretic patterns obtained with these submitochondrial fractions were similar in normal and thyroidectomized animals. The mitoplast fraction which contained the insoluble cytochromes (a, a3, b, c1) was isolated ; its principal constituent, of MW 54,000 was significantly decreased after thyroidectomy. Thus, the lack of thyroid hormone secretion lowered the level of a protein constituent bound to the inner membrane of liver mitochondria. The synthesis of this constituent could be controlled by mitochondrial nucleic acids.

Kinetic studies of cytochrome reduction show that only two beta-type cytochromes reducible in the presence of succinate are present in A. campestris mitochondria. At 25 degrees C, the difference spectrum reduced in the presence of antimycin minus oxidized shows the two beta-type cytochrome have a common, asymetric alpha-band at 560 nm which splits into two peaks at 559 and 562 nm at liquid nitrogen temperature (-196 degrees C).

Specific antibody has been obtained against cytochrome b (pig heart mitochondria). It inhibits the electron transport of the respiratory chain in the intact mitochondria at the cytochrome b site of the inner mitochondrial membrane. It has no effect on the isolated submitochondrial particles which are inside-out inner membrane vescicles free of any outer membrane or outside-out inner membrane. These findings indicate a probably not transmembranous topologic localization of cytochrome b; this component of the respiratory chain seems located near the outer side of the inner mitochondrial membrane.

Twenty encapsulated strains of Flavobacterium multivorum were isolated from soil by elective culture in minimal medium containing inulin as the sole source of carbon and energy. These strains were compared with the type strain and with 5 other strains (including 4 clinical strains) of F. multivorum. Of 168 substrates tested, 18 carbohydrates were used as the sole carbon and energy source by all 26 strains. A few amino acids were used by some strains. The yellow pigment produced was found to be a carotene and its production was photo-inducible. The presence of a cytochrome c oxidase of the aa3 type was suggested by absorption spectra. The major cell lipids were phosphatidylethanolamine and sphingolipids; the major fatty acids were 13-methyltetradecanoic and 2-hydroxy-13-methyltetradecanoic acids. Soil and clinical strains of F. multivorum showed roughly similar patterns of antibiotic multiresistance. The average G + C content of the DNA of 11 strains was 40.8 +/- 1.5 mol%.

During chronic hypoxia at sea level (FiO2 : 0,08) the liver concentrations of cytochrome P 450 and b5 decreased. Phenytoin administration induces an increase in cytochrome P 450 and b5 liver content. The hydroxylation of phenytoin that was decreased at low level of cytochrome P 450 and oxygen, reached again a normal level after induction of cytochrome P 450 in spite of the persistency of a low level of available oxygen. Liver cytochrome P 450 concentration is the main limiting factor of phenytoin hydroxylation.

Five chromosomal genes, CYPI to CYP5 involved in the regulation of the synthesis of iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2 are described. The function of these genes was studied either by varying the proportion of the mutated and wild type alleles in the cell vy varing the growth conditions, or else by transforming the mutants into sigma-cytoplasmic petites. We have shown a network of genetic interactions which regulate the synthesis of three structurally different proteins : iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2, by two unlinked genes : CYC1 and CYP1, one of which (CYC1) is the structural gene by iso-1-cytochrome c. Within this network the interactions are proportional to the gene dosage and are either antagonistic or synergistic depending on the allele combination and the protein studied. The mutated alleles cyp1 stimulate the synthesis of iso-2-cytochrome c, inhibit the synthesis of iso-1-cytochrome c, while the cytochrome b2 synthesis is also inhibited but by a combination of cyp1 mutated alleles CYC1 wild type allele. Other loci, CYP2, CYP3, CYP4 and CYP5 were also studied in various allelic combinations. They show some interactions between them or with CYC1 locus but these interactions are different and less pronounced than those involving loci CYP1 and CYC1.

Mitochondria used in the present study were isolated from skeletal muscle of normal and thyroidectomized rats. The preparations were controlled by electron microscopy. It was not possible to find any morphological change induced by thyroidectomy, nevertheless, some difference appeared in the cytochrome contents which were slightly decreased. Oxygen consumption rates of thyroidectomized rat mitochondria were decreased when the particles were maintained in states 3 and 4 in the presence of various substrates, but the P/O ratios were not modified. The activities of mitochondrial enzymes were in general slightly affected by thyroidectomy except for glycerol-1-phosphate cytochrome c reductase and NADH rotenone sensitive cytochrome c reductase which were decreased and for glutamate dehydrogenase activity which was increased. The tRNA nucleotidyltransferase activity found in the mitochondrial matrix was not influenced by the absence of thyroid secretion. Normal rat muscle mitochondria incorporate 14C-leucine with an artificial ATP-generating system or with a respiratory substrate. The amino acid incorporation was decreased by thyroidectomy. Muscle mitochondria analyzed by polyacrylamide gel electrophoresis contained more than 30 protein components with MW ranging from 10.000 to 135.000. Thyroidectomy lowered the amount of a fraction of about 54.000 MW. It is not impossible that all the data observed in the absence of thyroid secretion are in relation with changes induced in the mitochondrial genome as previously shown in mitochondria isolated from liver or thyroidectomized rats.

DNA sequence studies of mutated and wild type alleles of an intron in the mosaic mitochondrial gene for cytochrome b have revealed the possible existence of a protein coded in the intron and involved in RNA splicing. This protein would be endowed with properties of intrinsic autotomy of its own messenger RNA.

Nabam (fungicide dithiocarbamate) has been incorporated with the diet of rats during six months (the doses were: 0, 10, 50, 100, 500, 1000 and 2000 ppm). It decreases significantly the hepatic microsomal enzymes activity (aniline hydroxylase and aminopyrine N-demethylase and the liver P 450 content, but the cytochrome b 5 concentration doesn't seem to be modified. The microsomal lipidic and proteic content is only modified with the highest Nabam dose. Several hypotheses may be proposed to explain the Nabam effects: one is the inhibition of the monooxygenases and their biosynthesis repression.

During the "respiratory adaptation" of Bacillus coagulans, it was possible to dissociate the kinetics of cytochrome a and a3 synthesis with carbon monoxide. The synthesis of cytochrome a3 is preferentially repressed when the pH of the incubation medium is pH 6.5 instead of pH 5.5. However, though the total synthesis of tetrapyrrole compounds is the same at both pH values, the excretion of coproporphyrin III is much increased at pH 6.5. Bacillus coagulans, sensitive to the "glucose effect", shows the "pH effect" only in the presence of high glucose concentrations. The repression of the oxidase complex synthesis by a slight increase of the extracellular pH appears directly related to the increase of the extracellular coproporphyrin III.

During a eight days period of chronic hypoxia (FiO2 : 0,06) the cytochrome P 450 content of mice'liver decreases the 1st day, increases the 2nd and the 3rd day and decreases again and remains at a low levels. During a 13 days period of chronic hypoxia (FiO2 = 0,08) the P 450 level remains inchanged during 5 days. It decreases after until the 13teen day. G6Pase, an endoplasmic reticulum enzyme marker is quite inchanged in these conditions.

In a first part, an elementary theoretical introduction to ESR is given. In a second part, selected examples illustrate the possibilities of this method. Studies of hemoproteins, flavoproteins, model complexes and phospholipid systems are described. This review is made for biochemists who have no experience in ESR.

Once the pair nitrate-nitrite--in quantity respectively set by the ingestion level or transformation level by intestinal bacteria--has entered through the intestinal mucosa, it may react with active biochemical groups. Nitrite, owing to its high oxido-reduction potential, may induce the oxidation of a large number of compounds, as for example the Fe++ heme-Fe+++ hemine system, reduced cytochromes-oxidized cytochromes system, etc. If the presence of nitrite in blood is not clearly established, this is due to the nitrite high chemical reactivity. Moreover, a transformation by the tissues of nitrates into nitrites after a nitro-reduction is quite possible.