Isolation and characterization of the yeast aspartyl-tRNA synthetase gene

A yeast genomic library in Escherichia coli, constructed by insertion of Sau3A restriction fragments into the hybrid Saccharomyces cerevisiae-E. coli plasmid pFL1, was screened by a radioimmunoassay (RIA) for colonies expressing yeast aspartyl-tRNA synthetase (AspRS). Four clones were isolated by this technique. Data obtained by Southern and restriction analysis of the inserts showed a common 3.8-kb BamHI restriction fragment which, when inserted into the plasmid pFLl, gave a positive RIA. Several controls showed that this 3.8-kb insert codes for the entire AspRS : (i) S. cerevisiae transformed by the PFL1 plasmid carrying the 3.8-kb fragment overproduces AspRS activity by a factor of ten compared to the wild-type yeast strain; and (ii) a new protein with electrophoretic behaviour similar to AspRS and immuno-reactive toward anti-AspRS appears in crude extracts of transformed yeast and E. coli.     

Nucleotide sequence of the gene coding for yeast cytoplasmic aspartyl-tRNA synthetase (APS); mapping of the 5'' and 3'' termini of AspRS mRNA.

A 3.8 Kb DNA fragment, which contains the structural gene of aspartyl-tRNA synthetase (AspRS) and its flanking regions, has been fully sequenced by the combined M13/dideoxy chain terminator method. From the single open reading frame of correct length (1671 bp) we deduced an amino acid sequence consistent with that of several peptides of AspRS. No significant internal sequence repeats were observed in the primary structure of the protein. The AspRS gene (APS) has a codon usage pattern typical of non abundant proteins. S1 nuclease analysis of APS mRNA showed a major start 17 bases downstream from a "TATA box" and stops near an RNA polymerase terminator sequence.     

Enrichment and characterization of the mRNAs of four aminoacyl-tRNA synthetases from yeast.

We have partially purified the messenger RNAs for yeast arginyl-, aspartyl-, valyl-, alpha and beta subunits of phenylalanyl-tRNA synthetases in order to study their biosynthesis and ultimately, to isolate their genes. Sucrose gradient fractionation of poly U-Sepharose selected mRNAs resulted in a ten fold enrichment of the in vitro translation activity of these mRNAs. The translation products of messenger RNAs for arginyl- and valyl-tRNA synthetases have the same molecular weight as the purified enzymes; translation of aspartyl-tRNA synthetase messenger RNA yielded a 68 kD molecular weight polypeptide (while the purified cristallisable enzyme appears as a 64-66 kD doublet, which, as we showed is a proteolysis product). The translation of the mRNAs for alpha and beta phenylalanyl-tRNA synthetase gave polypeptides having the same molecular weight as those obtained from the purified enzyme, but the major translation products are slightly heavier, indicating that they may be translated as precursors. As estimated from centrifugation experiments mRNAs of arginyl-, aspartyl-, alpha and beta subunits of phenylalanyl-tRNA synthetase were 1700-2000 nucleotides long, indicating that alpha and beta are translated from two different mRNAs.     

Dissection of the TssB-TssC Interface during Type VI Secretion Sheath Complex Formation

The Type VI secretion system (T6SS) is a versatile machine that delivers toxins into either eukaryotic or bacterial cells. At a molecular level, the T6SS is composed of a membrane complex that anchors a long cytoplasmic tubular structure to the cell envelope. This structure is thought to resemble the tail of contractile bacteriophages. It is composed of the Hcp protein that assembles into hexameric rings stacked onto each other to form a tube similar to the phage tail tube. This tube is proposed to be wrapped by a structure called the sheath, composed of two proteins, TssB and TssC. It has been shown using fluorescence microscopy that the TssB and TssC proteins assemble into a tubular structure that cycles between long and short conformations suggesting that, similarly to the bacteriophage sheath, the T6SS sheath undergoes elongation and contraction events. The TssB and TssC proteins have been shown to interact and a specific α-helix of TssB is required for this interaction. Here, we confirm that the TssB and TssC proteins interact in enteroaggregative E. coli. We further show that this interaction requires the N-terminal region of TssC and the conserved α-helix of TssB. Using site-directed mutagenesis coupled to phenotypic analyses, we demonstrate that an hydrophobic motif located in the N-terminal region of this helix is required for interaction with TssC, sheath assembly and T6SS function.     

Effet du climat et de l’exposition sur la dynamique des populations de la cochenille violette,Parlatoria oleae Colvée (Hemiptera : Diaspididae), en conditions arides

Résumé

Ce travail a pour objectif l’étude de quelques aspects bio-écologiques de la cochenille violette, Parlatoria oleae Colvée 1880, bio-agresseur des cultures de l’Olivier en régions arides. Le suivi du cycle biologique ainsi que la démo-écologie de ce ravageur ont été réalisés grâce à des dénombrements périodiques des populations sur les différents organes de l’arbre (méthodes de Vasseur &; Schvester) de décembre 2010 à décembre 2011 dans la région d’Ain Touta (nord-est algérien). L’espèce y a montré deux générations par an : une génération printanière se développant entre avril et juillet et une génération automnale évoluant entre août et octobre. La ponte débuta en avril et s’échelonna jusqu‘à la fin septembre. L’exposition nord est la plus favorable à cette diaspine qui y trouve des conditions microclimatiques optimales pour son développement. La ponte moyenne est de 8 à 9 ?ufs par femelle. L’analyse statistique de l’effet des conditions climatiques étudiées (températures minimale, maximale et moyenne ; précipitations, gelée et indice d’aridité De Martone) sur les effectifs des différents stades, montre une grande variabilité d’un stade à un autre. L’analyse statistique établie révèle également que les effectifs de l’espèce présentent des variations très hautement significatives selon l’orientation dans l’arbre d’olive colonisé.     

The Pluripotency Factor-Bound Intron 1 of Xist Is Dispensable for X Chromosome Inactivation and Reactivation In Vitro and In Vivo

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A dialysis system with phytoplankton for monitoring chemical pollution in freshwater ecosystems by alkaline phosphatase assay

The effects of three groups of chemicals, heavy metals, pesticides and phenolics, on alkaline phosphatase activity of intact Chlorella vulgaris cells were investigated. There was a marked inhibitory effect of heavy metals and a slight one due to phenolics, but the pesticides tested showed no effect. In order to detect heavy metals in freshwater ecosystems, we propose a dialysis system, which can be placed in the field and then provide early warning signals of toxicity. This phosphatase inhibition test is recommended the first stage of an enzymatic screening system for chemical pollution in water.     

High androgen receptor immunoexpression in human "Sertoli cell only" testis

Our purpose was to evaluate cellular androgen receptor (AR) distribution and intensity of immunostaining in the human azoospermic testis. Thirty six biopsy specimens from azoospermic men were immunostained, using a monoclonal antibody of human AR. The localization and the intensity of AR immunostaining was evaluated in Sertoli Cell Only (SCO) testis (G1, n = 21), in spermatogenesis arrest testis (G2, n = 11) and in histologically normal testis (G3, n = 4). We found an AR immunostaining in Sertoli, peritubular myoid and Leydig cells, but not in germ cells. The intensity of the immunostaining varied substantially between biopsy specimens of different patients. Sertoli and Leydig cells AR immunostaining (score and intensity) in SCO group was higher than in the other groups. For Sertoli cells, the score means of AR immunoreactivity were 20 +/- 2.36, 10.18 +/- 1.0 and 1 +/- 1, for G1, G2 and G3 groups, respectively. For Leydig cells, the score means were 10.24 +/- 1.37, 6 +/- 0.71 and 0, for G1, G2 and G3 groups, respectively. We found significant differences between G1 and G2 (p = 0.0008), between G1 and G3 (p = 1.54 10-7) and G2 and G3 (p = 0.00032). These results suggest that in the testis AR is located exclusively in somatic cells and its expression is higher in SCO syndrome than in normal and in arrest spermatogenesis testes.     

Microtubule-associated protein MAP1A, MAP1B, and MAP2 proteolysis during soluble amyloid beta-peptide-induced neuronal apoptosis. Synergistic involvement of calpain and caspase-3

A growing body of evidence supports the notion that soluble oligomeric forms of the amyloid beta-peptide (Abeta) may be the proximate effectors of neuronal injuries and death in the early stages of Alzheimer disease. However, the molecular mechanisms associated with neuronal apoptosis induced by soluble Abeta remain to be elucidated. We recently demonstrated the involvement of an early reactive oxygen species-dependent perturbation of the microtubule network (Sponne, I., Fifre, A., Drouet, B., Klein, C., Koziel, V., Pincon-Raymond, M., Olivier, J.-L., Chambaz, J., and Pillot, T. (2003) J. Biol. Chem. 278, 3437-3445). Because microtubule-associated proteins (MAPs) are responsible for the polymerization, stabilization, and dynamics of the microtubule network, we investigated whether MAPs might represent the intracellular targets that would enable us to explain the microtubule perturbation involved in soluble Abeta-mediated neuronal apoptosis. The data presented here show that soluble Abeta oligomers induce a time-dependent degradation of MAP1A, MAP1B, and MAP2 involving a perturbation of Ca2+ homeostasis with subsequent calpain activation that, on its own, is sufficient to induce the proteolysis of isoforms MAP2a, MAP2b, and MAP2c. In contrast, MAP1A and MAP1B sequential proteolysis results from the Abeta-mediated activation of caspase-3 and calpain. The prevention of MAP1A, MAP1B, and MAP2 proteolysis by antioxidants highlights the early reactive oxygen species generation in the perturbation of the microtubule network induced by soluble Abeta. These data clearly demonstrate the impact of cytoskeletal perturbations on soluble Abeta-mediated cell death and support the notion of microtubule-stabilizing agents as effective Alzheimer disease drugs.     

Effect of freezing–thawing process and quercetin on human sperm survival and DNA integrity

We aimed in the first part of our work to study the effect of cryopreservation on the human sperm DNA integrity and the activation of caspase 3, the main apoptosis indicator. In the second part, we were interested in testing the effect of quercetin, as an antioxidant, in preventing sperm damage during the freeze–thawing process. Seventeen semen samples were obtained from 17 men recruited for infertility investigations. Liquefied sperm was cryopreserved using spermfreeze®. Nine of the used samples were divided into two aliquots; the first one was cryopreserved with spermfreeze only (control) and the second one was cryopreserved with spermfreeze supplemented with quercetin to a final concentration of 50 μM. Sperm motility and viability were assessed according to WHO criteria. We used TUNEL assay and the Oxy DNA assay to assess sperm DNA integrity. Activated caspase 3 levels were measured in spermatozoa using fluorescein-labeled inhibitor of caspase (FLICA). Cryopreservation led to a significant increase in sperm DNA fragmentation, DNA oxidation and caspase 3 activation (p < 0.01). Supplementation of the cryopreservation medium with quercetrin induced a significant improvement in post thaw sperm parameters, compared to those of control, regarding sperm motility (p = 0.007), viability (p = 0.008) and DNA integrity (p = 0.02); however, it had no effect on caspase 3 activation (p = 0.3). We conclude that oxidative stress plays a major role in inducing sperm cryodamage but implication of apoptosis in this impairment requires further investigations. Quercetin could have protective effect during cryopreservation but further research is needed to confirm this effect.