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The radical allylation of a series of -halo or -phenylseleno--alkoxy esters in the presence of MgBr2·OEt2is reported and compared with analogous reactions under non-chelating conditions. The addition of MgBr2·OEt2gives excellent selectivity favoring antiproducts; in some cases ratios >100:1 are obtained. Varying the substrate substituents reveals that these reactions are quite tolerant of alkyl functionalities at the -position. Changes to the alkoxy function indicate that a chelate is involved in the reaction. The reactions are successful with secondary iodides, bromides, and phenylselenides, as well as tertiary iodides, which all give very high ratios under chelation control. Performing less well under the same conditions are substrates with a radical exocyclic to a tetrahydrofuran ring. EDTA titration is used to determine the amount of Mg2+dissolved in the allylation reaction mixture, and 13C NMR is employed to better define the nature of the complex formed (chelate or monodentate) prior to the reaction. Competition experiments suggest that the chelate and monodentate pathways are in competition for the radical allylation with allyltributyltin.Key words: allylation, radicals, Lewis acid, stereoselectivity, 1,2-induction.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.

The numbers of aerobic and aero-anaerobic germs per milliliter of stallion semen were converted into a decimal logarithm (log C). This parameter was used to determine factors associated with different levels of microbial contamination and to test efficacy of hygienic practices.Raw fresh semen. The log C was different among stud farms and among stallions. Microbial contamination was decreased when ejaculates were collected at the first intromission (5.7 ± 0.1, n = 47) compared to ejaculates collected after ≥ 2 intromissions (6.1 ± 0.1, n = 30). Semen was less contaminated when collected with an open vagina (5.0 ± 0.2, n = 10) compared to a closed-type artificial vagina (6.2 ± 0.1, n = 7). Washing the penis before collection resulted in no decrease in the log C value.Artificial insemination (AI) samples of extended fresh semen. Raw semen was more contaminated (5.5 ± 0.2, n = 20) than extended semen (4.4 ± 0.1, n = 20). The log C of extended semen was decreased after 6 h at 4°C (3.6 ± 0.2; antibiotic effect), but not thereafter. The combination of penicillin (50 UI/ml) and gentamicin (50 μg/ml) was the most effective tested antibiotic to reduce bacterial contamination (3.8 ± 0.1, n = 14 compared to 5.1 ± 0.1, n = 14 without antibiotics).AI samples of frozen semen. Raw semen was more contaminated (5.7 ± 0.1, n = 24) than frozen semen (4.1 ± 0.1, n = 24). Microbial contamination rates were different among ejaculates but repeatable among the straws of an ejaculate.

Analyses of the endogenous cytokinin contents of established tissue strains of Mercurialis annua are reported. The strains were derived from three individuals (strong male, weak male, female), differing by one of the three genes determining sex. The data are compared with the endogenous cytokinins of male and female shoot apices. Tissue strains are characterized by the disappearance of natural cytokinin metabolites in the female; in both males, ?2-isopentenyl-adenosine and only trans-ribosylzeatin exist but in different quantities. Benzyladenine and ribosylbenzyladenine were identified in the three strains but the quantities also differed as a function of the genotype. The marked differences in cytokinin metabolism of tissue strains indicate that sex genes continue to function in the dedifferentiated state. Each strain also exhibited persistent morphological and histological characteristics, and a different sensitivity to the withdrawal of 2-4-dichlorophenoxyacetic acid or benzyladenine from the medium. Each had a specific and characteristic effect on the organogenesis of nodes cultivated in close proximity to callus pieces. These data complement the above results and show that sex genes act at the callus-tissue level. The possibility that these genes act at the early stages of embryogenesis of male and female individuals is also discussed.

All five subunits of yeast mitochondrial F1-ATPase have been isolated by reverse-phase high performance liquid chromatography. This procedure allows micro-preparative purification of all the subunits with 60% recoveries. The complete amino acid sequence of the epsilon-subunit has been established. This has been achieved by the sequence analysis of subnanomole amounts of the intact molecule and that of peptides derived by enzymatic digestion with endoproteinase Arg-C and by chemical cleavage with hydroxylamine. Yeast ATP synthase epsilon-subunit is composed of 61 residues with a calculated molecular mass of 6612 Da. This polypeptide is rather basic since it contains 7 basic residues and 3 acidic residues. This study shows a slight similarity with the bovine epsilon-subunit ATP synthase since there are 16 identical residues.

ATP induced swelling of isolated yeast mitochondria suspended in an isoosmotic solution of potassium gluconate. Valinomycin stimulated the swelling rate, indicating that K+ influx in the presence of ATP is rate-controlling. This swelling was inhibited by ADP, phosphate (probably acting on the external face of the inner membrane), and Mg2+, which forms a complex with ATP. ATP-induced swelling did not require working F0-F1-ATPase since it was not inhibited by oligomycin and uncoupler. CTP and GTP also induced a swelling. ATP also induced mitochondrial swelling in potassium glutamate, chloride, and acetate but not in phosphate solutions. Sodium, but not ammonium, can replace potassium ion. It is probable that the ATP-channel opening also necessitates an electrogenic cation influx. Respiration also induced swelling of mitochondria suspended in isoosmotic potassium gluconate solution. ATP- or respiration-induced swelling were inhibited equally by N,N'-dicyclohexylcarbodiimide, propranolol, and Zn2+ but not by quinine; all these drugs inhibit the H+/K+ exchange. It was concluded that this unspecific channel is not open under conditions used to measure oxidative phosphorylation. Its physiological role remains unknown.

Knowledge of vocal tract acoustic transfer functions is important for the understanding of phenomena occurring during speech production. We present here a new measurement method based on the external excitation of the vocal tract with a known pseudo-random sequence, where the transfer function is obtained as the FFT of the crosscorrelation between the sequence and the signal measured at the lips. The advantages of this method over methods based on sweep-tones or white noise excitation are (1) a much shorter measurement time (about 100 ms), (2) the possibility of speech sound production during the measurement, and (3) a more accurate definition of bandwidths. This method has been checked against classical methods through systematic comparisons on a small corpus of vowels and fricative consonants. Moreover, it has been verified that simultaneous speech sound production does not perturb significantly the measurements. This method should thus be a very helpful tool for the investigation of the acoustic properties of the vocal tract in various cases such as vowels, fricatives and nasals.

The energetic metabolism of rat C6 glioma cells has been investigated as a function of the proliferative and differentiation states under three-dimensional (3-D) growing conditions on microcarrier beads. First, the transient deprivation of glutamine from the culture medium induced a marked decrease in the growth rate and a differentiation of C6 cells through the oligodendrocytic phenotype. Second, the respiratory capacity of the C6 cells during short-term subcultures with or without glutamine continuously declined as a function of the cell density, in part due to the mitochondrial content decrease. During the transition from the early exponential to the plateau growth phase in glutamine-containing medium, the oxygen consumption rate per single cell decreased concomitantly with a decrease in the glucose consumption and lactate production rates. This phenomenon led to a sixfold decrease in the total ATP production flux, without significantly affecting the cellular ATP/ADP ratio, thus indicating that some ATP-consuming processes were simultaneously suppressed during C6 proliferation. In glutamine-free medium, the cellular ATP/ADP ratio transiently increased due to growth arrest and to a reduced ATP turnover. Moreover, the results indicated that glutamine is not an essential respiratory substrate for rat C6 glioma under short-term glutamine deprivation. Worth noting was the high contribution of the mitochondrial oxidative phosphorylation toward the total ATP synthesis (about 80%), regardless of the proliferation or the differentiation status of the C6 cells.

The mitochondrial phosphate transport protein (PTP) has been purified in a reconstitutively active form from Saccharomyces cerevisiae and Candida parapsilosis. ADP/ATP carriers that copurify have been identified. The PTP from S. cerevisiae migrates as a single band (35 kDa) in sodium dodecyl sulfate gels with the same mobility as the N-ethylmaleimide-alkylated beef heart PTP. It does not cross-react with anti-sera against beef heart PTP. The CNBr peptide maps of the yeast and beef proteins are very different. The rate of unidirectional phosphate uptake into reconstituted proteoliposomes is stimulated about 2.5-fold to a Vmax of 170 mumol of phosphate min-1 (mg PTP)-1 (22 degrees C) by increasing the pHi of the proteoliposomes from 6.8 (same as pHe) to 8.0. The Km for Pi of this reconstituted activity is 2.2 mM. The transport is sensitive to mersalyl (50% inhibition at 60 microM) and insensitive to N-ethylmaleimide. We have purified peptides matching the highly conserved motif Pro-X-(Asp/glu)-X-X-(Lys/Arg)-X-(Arg/lys) (X is an unspecified amino acid) of the triplicate gene structure sequence of the beef heart PTP. The N-ethylmaleimide-reactive Cys42 of the beef heart protein, located between the two basic amino acids of this motif (Lys41-Cys42-Arg43), is replaced with a Thr in the yeast protein. This substitution most likely is responsible for the lack of N-ethylmaleimide sensitivity of the yeast protein and mersalyl thus reacts with another cysteine to inhibit the transport. Finally it is concluded that Cys42 has no essential role in the catalysis of inorganic phosphate transport by the mitochondrial phosphate transport protein.

This paper reports studies of bioenergetic modifications in a TTR1 single-nuclear mutant, isolated as resistant to triethyltin, an inhibitor of mitochondrial ATPase, and effective in cAMP-dependent protein phosphorylation. This mutant appears to have lost the wildtype cell ability to respond to a decrease of oxygen concentration in the growth medium by a decrease of cytochrome concentration in the cell. ATP synthesis rate in mutant cells in both the prestationary and stationary phase of growth appeared increased in comparison to wild-type cells, as too was respiration rate. A comparative study of mitochondria extracted from wild-type and from TTR1 mutant cells showed an increase in respiration rate, an increase in ATP synthesis rate, and an increase in TPP+ uptake in mutant mitochondria. The specific ATPase activity, as well as its sensitivity to TET, appears to be similar for mitochondria extracted from both strains. It was proposed that the modification of mitochondrial biogenesis in the TTR1 mutant may be due to a response of the cell to an increase in ATP hydrolysis caused by the mutation. It is also possible that the modification in cAMP-dependent protein kinase regulation which appeared to occur in this mutant affects protein(s) involved in mitochondrial biogenesis.

Three mutants of Saccharomyces cerevisiae resistant to triethyltin (an inhibitor of mitochondrial ATPase) on non-fermentative media, and non-resistant to this drug on fermentative media, were isolated and named TTR1, TTR2 and TTR3. Apart from triethyltin resistance, these mutants show the following common characteristics: (1) Increased intracellular cytochrome c concentration. (2) Increased respiration rate. (3) Decreased growth yield. (4) Increased growth sensitivity to several drugs inhibiting oxidative phosphorylation: namely, CCCP (permeabilizing inner mitochondrial membrane to protons), valinomycin (permeabilizing inner mitochondrial membrane to potassium) and oligomycin (inhibitor of mitochondrial ATPase). (5) Increased sensitivity to carbon source starvation. For each mutant, these characteristics appeared to be due to a single pleiotropic nuclear mutation. Mutation TTR1 causes additional phenotypic characteristics which do not appear in mutants TTR2 and TTR3: (1) Pinkish coloration of colonies which is more pronounced after a long growth period. (2) Inability of the cells to store glycogen. (3) Growth defect of the cells on a galactose-containing medium. (4) Inability of a diploid homozygote mutant strain to sporulate. All these phenotypic characteristics have already been described in yeast mutants deregulated in cAMP-dependant protein phosphorylation. Crossing of a strain bearing the TTR1 mutation with a strain mutated in the adenylate cyclase structural gene suggested that the TTR1 phenotype is due to a modification in regulation of cAPK by cAMP, making cell multiplication possible without intracellular cAMP.

With the aid of a two-mass model of the vocal source loaded by the input impedance of the vocal tract, we first study theoretically, then by simulation, the effects of source-vocal tract coupling on the intrinsic frequencies F0 of the French oral vowels. We explain the correlation which exists between F0 and the frequency of the first formant as well as the results from the experiment described by Ishizaka and Flanagan [1972]. In conclusion, we show that the acoustic coupling cannot explain the different values of F0, intrinsic in the vowels, but on the contrary, a physiological coupling has to compensate for the effects of acoustic coupling to explain the characteristics revealed in natural speech.

La barrière hémato-encéphalique (BHE) de par sa présence limite l’acheminement d’agents thérapeutiques au système nerveux central (SNC). La présence de pompes à efflux au niveau de la BHE constitue un important mécanisme d’exclusion d’agents lipophiles tentant t’atteindre le SNC. La glycoprotéine-P (p-gp), l’une de ses pompes à efflux, est exprimée à la BHE et par les cellules tumorales et a pour substrats plusieurs agents de chimiothérapie. Il a été démontré que l’ouverture osmotique de la BHE (OBHE) est efficace pour acheminer des agents n’étant pas des substrats de la PGP. Cependant, il n’a pas été démontré que l’OBHE permet d’augmenter également l’acheminement d’agents antinéoplasiques substrats de la PGP. Cette étude a été mise sur pied afin de déterminer si l’OBHE permet d’augmenter l’acheminement de substrats de la PGP.

An Agrobacterium tumefaciens suspension induces a strong agglutination of aldehyde-fixed pig erythrocytes at pH 5.0. The agglutination is inhibited by some polysaccharides, such as fucoidin, and also when the pH is raised to 7.0. Lectins (sugar-binding proteins) associated with the bacterial cell wall of A.tumefaciens strain 84.5 were directly evidenced by spectrofluorimetry using fluoresceinylated neoglycoproteins. The specific binding of the fluorescein-labelled neoglycoprotein bearing α-L-fucoside residues was also optimal at pH 5.0. A lectin was purified by affinity chromatography on agarose substituted with α-L-fucopyranoside. Furthermore, the haemagglutination activity of this lectin was inhibited by polysaccharides isolated from poplar leaves.