Transcriptional analysis of the Azospirillum brasilense indole-3-pyruvate decarboxylase gene and identification of a cis-acting sequence involved in auxin responsive expression

Expression of the Azospirillum brasilense ipdC gene, encoding an indole-3-pyruvate decarboxylase, a key enzyme in the production of indole-3-acetic acid (IAA) in this bacterium, is upregulated by IAA. Here, we demonstrate that the ipdC gene is the promoter proximal gene in a bicistronic operon. Database searches revealed that the second gene of this operon, named iaaC, is well conserved evolutionarily and that the encoded protein is homologous to the Escherichia coli protein SCRP-27A, the zebrafish protein ES1, and the human protein KNP-I/GT335 (HES1), all of unknown function and belonging to the DJ-1/PfpI superfamily. In addition to this operon structure, iaaC is also transcribed monocistronically. Mutation analysis of the latter gene indicated that the encoded protein is involved in controlling IAA biosynthesis but not ipdC expression. Besides being upregulated by IAA, expression of the ipdC-iaaC operon is pH dependent and maximal at acidic pH. The ipdC promoter was studied using a combination of deletion analyses and site-directed mutagenesis. A dyadic sequence (ATTGTTTC(GAAT)GAAACAAT), centered at -48 was demonstrated to be responsible for the IAA inducibility. This bacterial auxin-responsive element does not control the pH-dependent expression of ipdC-iaaC.     

Binding kinetics,potency, and selectivity of the hepatitis C virus NS3 protease inhibitors GS-9256 and vedroprevir

Background

GS-9256 and vedroprevir are inhibitors of the hepatitis C virus NS3 protease enzyme, an important drug target. The potency, selectivity, and binding kinetics of the two compounds were determined using in vitro biochemical assays.

Methods

Potency of the compounds against NS3 protease and selectivity against a panel of mammalian proteases were determined through steady-state enzyme kinetics. Binding kinetics were determined using stopped-flow techniques. Dissociation rates were measured using dilution methods.

Results

GS-9256 and vedroprevir had measured K_i values of 89 pM and 410 pM, respectively, against genotype 1b NS3 protease; K_i values were higher against genotype 2a (2.8 nM and 39 nM) and genotype 3 proteases (104 nM and 319 nM) for GS-9256 and vedroprevir, respectively. Selectivity of GS-9256 and vedroprevir was > 10,000-fold against all tested off-target proteases. Association rate constants of 4 × 10⁵ M^− 1 s^− 1 and 1 × 10⁶ M^− 1 s^− 1, respectively, were measured, and dissociation rate constants of 4.8 × 10^− 5 s^− 1 and 2.6 × 10^− 4 s^− 1 were determined.

Conclusions

GS-9256 and vedroprevir are potent inhibitors of NS3 protease with high selectivity against off-target proteases. They have rapid association kinetics and slow dissociation kinetics.

General Significance

The NS3 protease is a key drug target for the treatment of hepatitis C. The potency, selectivity, and binding kinetics of GS-9256 and vedroprevir constitute a biochemical profile that supports the evaluation of these compounds in combination with other direct-acting antivirals in clinical trials for hepatitis C.     

Identification and characterization of PKNbeta, a novel isoform of protein kinase PKN: expression and arachidonic acid dependency are different from those of PKNalpha.

The cDNA clone encoding a novel isoform of protein kinase PKN, termed PKNbeta, was isolated from a HeLa cDNA library. PKNbeta had high sequence homology with PKNalpha, originally isolated PKN, especially in the repeats of charged amino acid-rich region with leucine-zipper like sequences (CZ region/HR1), in the carboxyl-terminal catalytic domain, and in approximately 130 amino acid stretch (D region/HR2), located between CZ region/HR1 and the catalytic domain. However, the amino acid sequence of PKNbeta differed from that of PKNalpha in the region immediately amino-terminal to the catalytic domain, which contained two distinct proline-rich sequences consistent with the class II consensus sequence, PXXPXR, for binding to SH3 domain. Distribution of PKNbeta differed from that of PKNalpha in the following two respects: (1) Northern blotting indicated that PKNbeta mRNA could not be detected in human adult tissues, but was expressed abundantly in human cancer cell lines; (2) immunochemical analysis indicated that PKNbeta localized in nucleus and perinuclear Golgi apparatus, and was almost absent in cytoplasmic region in NIH3T3 cells. Recombinant PKNbeta expressed in COS7 cells displayed autophosphorylation and peptide kinase activity, but was found to be significantly less responsive to arachidonic acid than PKNalpha. The identification of this novel isoform underscores the diversity of PKN signaling pathway.     

A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly

The activated form of Ran (Ran-GTP) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-alpha and -beta. We report here that Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. We show that TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-alpha and -beta, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. As the phosphorylation of Eg2 stimulated by the Ran-GTP-TPX2 pathway is essential for spindle assembly, we hypothesize that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics.     

Microsatellite markers for standardized genetic management of captive colonies of rhesus macaques (Macaca mulatta)

To preserve genetic variability and minimize genetic subdivision among captive Macaca mulatta at each of the U.S. National Institutes of Health (NIH)-sponsored regional research colonies, the genetic structure of each colony must be characterized. To compare population genetic and demographic parameters across colonies and generations, one standard panel of highly informative genetic markers is required. We assembled a core marker set of four multiplex polymerase chain reaction (PCR) panels comprising 15 autosomal short tandem repeat (STR) loci with high information content selected from existing panels of well-characterized markers that are currently used for parentage assessment and genetic management of rhesus macaques. We then assessed the effectiveness of these loci for providing high probabilities of individual identification and parentage resolution, and for estimating population genetic parameters that are useful for genetic management.     

Malonate and 3-nitropropionic acid neurotoxicity are reduced in transgenic mice expressing a caspase-1 dominant-negative mutant

Increasing evidence implicates caspase-1-mediated cell death as a major mechanism of neuronal death in neurodegenerative diseases. In the present study we investigated the role of caspase-1 in neurotoxic experimental animal models of Huntington's disease (HD) by examining whether transgenic mice expressing a caspase-1 dominant-negative mutant are resistant to malonate and 3-nitropropionic acid (3-NP) neurotoxicity. Intrastriatal injection of malonate resulted in significantly smaller striatal lesions in mutant caspase-1 mice than those observed in littermate control mice. Caspase-1 was significantly activated following malonate intrastriatal administration in control mice but significantly attenuated in mutant caspase-1 mice. Systemic 3-NP treatment induced selective striatal lesions that were significantly smaller within mutant caspase-1 mice than in littermate control mice. These results provide further evidence of a functional role for caspase-1 in both malonate- and 3-NP-mediated neurotoxin models of HD.     

Xgrip109: A γ Tubulin–Associated Protein with an Essential Role in γ Tubulin Ring Complex (γTuRC) Assembly and Centrosome Function

Previous studies indicate that γ tubulin ring complex (γTuRC) can nucleate microtubule assembly and may be important in centrosome formation. γTuRC contains approximately eight subunits, which we refer to as Xenopus gamma ring proteins (Xgrips), in addition to γ tubulin. We found that one γTuRC subunit, Xgrip109, is a highly conserved protein, with homologues present in yeast, rice, flies, zebrafish, mice, and humans. The yeast Xgrip109 homologue, Spc98, is a spindle–pole body component that interacts with γ tubulin. In vertebrates, Xgrip109 identifies two families of related proteins. Xgrip109 and Spc98 have more homology to one family than the other. We show that Xgrip109 is a centrosomal protein that directly interacts with γ tubulin. We have developed a complementation assay for centrosome formation using demembranated Xenopus sperm and Xenopus egg extract. Using this assay, we show that Xgrip109 is necessary for the reassembly of salt-disrupted γTuRC and for the recruitment of γ tubulin to the centrosome. Xgrip109, therefore, is essential for the formation of a functional centrosome.     

Conservation of context-dependent splicing activity in distant Muscleblind homologs

The Muscleblind (MBL) protein family is a deeply conserved family of RNA binding proteins that regulate alternative splicing, alternative polyadenylation, RNA stability and RNA localization. Their inactivation due to sequestration by expanded CUG repeats causes symptoms in the neuromuscular disease myotonic dystrophy. MBL zinc fingers are the most highly conserved portion of these proteins, and directly interact with RNA. We identified putative MBL homologs in Ciona intestinalis and Trichoplax adhaerens, and investigated their ability, as well as that of MBL homologs from human/mouse, fly and worm, to regulate alternative splicing. We found that all homologs can regulate alternative splicing in mouse cells, with some regulating over 100 events. The cis-elements through which each homolog exerts its splicing activities are likely to be highly similar to mammalian Muscleblind-like proteins (MBNLs), as suggested by motif analyses and the ability of expanded CUG repeats to inactivate homolog-mediated splicing. While regulation of specific target exons by MBL/MBNL has not been broadly conserved across these species, genes enriched for MBL/MBNL binding sites in their introns may play roles in cell adhesion, ion transport and axon guidance, among other biological pathways, suggesting a specific, conserved role for these proteins across a broad range of metazoan species.     

Essential oils of Bidens tripartita L. collected during period of 3 years composition variation analysis

The aim of this study was to evaluate the variation of essential oils composition of Bidens tripartita L. collected during three successive years (2009–2011). Essential oils were obtained by supercritical CO₂ extraction, applying gas chromatographic–mass spectrometric (GC–MS) analysis for identification of volatile compounds afterwards. The essential oils of B. tripartita showed a characteristic chemical composition from year to year, observing both quantitative and qualitative compounds differences. The yield of essential oils was 22 and 35 % higher in 2010 year material than in 2009 and 2011 year, respectively. The main compounds found in the B. tripartita essential oils were α-pinene (3.7–12.1 %), p-cymene (2.8–8.0 %), β-ocimene (40.5–45.9 %), β-elemene (9.9–15.6 %), iso and α-caryophyllenes (4.3–6.8 % and 5.2–8.2 %), and α-bergamotene (3.3–9.4 %). To determine the significance of changes in the identified compounds in the samples, representing different plant collection year, statistical hypothesis testing was applied. For classification of these samples to the groups and evaluation of similarity between them principal component analysis, multivariate analysis of variance and hierarchical cluster analysis techniques were used. The correlation analysis helped to find out the strength of linear relationship between amount of each compound and meteorological data (temperature and precipitations).