Proteomic profile of culture filtrate from the Brazilian vaccine strain <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG Moreau compared to <Emphasis Type="Italic">M. bovis</Emphasis> BCG Pasteur

Background  

Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection.     

Rapid kinetics of regulator of G-protein signaling (RGS)-mediated Galphai and Galphao deactivation. Galpha specificity of RGS4 AND RGS7

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits speeding deactivation. Galpha deactivation kinetics mediated by RGS are too fast to be directly studied using conventional radiochemical methods. We describe a stopped-flow spectroscopic approach to visualize these rapid kinetics by measuring the intrinsic tryptophan fluorescence decrease of Galpha accompanying GTP hydrolysis and Galpha deactivation on the millisecond time scale. Basal k(cat) values for Galpha(o), Galpha(i1), and Galpha(i2) at 20 degrees C were similar (0.025-0.033 s(-1)). Glutathione S-transferase fusion proteins containing RGS4 and an RGS7 box domain (amino acids 305-453) enhanced the rate of Galpha deactivation in a manner linear with RGS concentration. RGS4-stimulated rates could be measured up to 5 s(-1) at 3 microm, giving a catalytic efficiency of 1.7-2.8 x 10(6) m(-1) s(-1) for all three Galpha subunits. In contrast, RGS7 showed catalytic efficiencies of 0.44, 0.10, and 0.02 x 10(6) m(-1) s(-1) toward Galpha(o), Galpha(i2), and Galpha(i1), respectively. Thus RGS7 is a weaker GTPase activating protein than RGS4 toward all Galpha subunits tested, but it is specific for Galpha(o) over Galpha(i1) or Galpha(i2). Furthermore, the specificity of RGS7 for Galpha(o) does not depend on N- or C-terminal extensions or a Gbeta(5) subunit but resides in the RGS domain itself.     

Guanine nucleotide effects on catecholamine secretion from digitonin-permeabilized adrenal chromaffin cells

The nonhydrolyzable GTP analogue guanosine 5'-(beta, gamma-imido)triphosphate (GMP-PNP) produced an ATP-dependent but Ca2+-independent stimulation of [3H]norepinephrine release from permeabilized chromaffin cells. This stimulation of secretion was 25-35% of the secretion induced by 10 microM Ca2+. A similar Ca2+-independent stimulation was produced by other non-hydrolyzable GTP analogues. No effect was seen with a variety of other nucleotides, including GTP. The GMP-PNP effect was specifically inhibited by low concentrations of guanine nucleotides. Addition of cAMP did not mimic the Ca2+-independent GMP-PNP effect, but did slightly enhance Ca2+-dependent secretion. Pretreatment with pertussis toxin had no effect on Ca2+-dependent secretion or on the GMP-PNP effect. There was no detectable diglyceride or inositol phosphate produced during GMP-PNP treatment, and addition of diglyceride and inositol trisphosphate did not induce secretion. Guanosine 5'-(beta-thio)diphosphate (GDP-beta-S), in addition to its ability to inhibit the GMP-PNP effect, partially inhibited Ca2+-dependent secretion. At 10 microM free Ca2+, the effects of GMP-PNP and Ca2+ were nonadditive. In fact, secretion in the presence of both GMP-PNP and 10 microM Ca2+ was slightly less than secretion due to Ca2+ alone. These data suggest that a guanine nucleotide-dependent process interacts in some way with one or more components of the normal Ca2+-dependent secretory pathway. However, it may not be an intrinsic part of the mechanism underlying Ca2+-dependent secretion.     

Molecular models of NS3 protease variants of the Hepatitis C virus

Background  

Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed.     

The mitogenome of Onchocerca volvulus from the Brazilian Amazonia focus

We report here the first complete mitochondria genome of Onchocerca volvulus from a focus outside of Africa. An O. volvulus mitogenome from the Brazilian Amazonia focus was obtained using a combination of high-throughput and Sanger sequencing technologies. Comparisons made between this mitochondrial genome and publicly available mitochondrial sequences identified 46 variant nucleotide positions and suggested that our Brazilian mitogenome is more closely related to Cameroon-origin mitochondria than West African-origin mitochondria. As well as providing insights into the origins of Latin American onchocerciasis, the Brazilian Amazonia focus mitogenome may also have value as an epidemiological resource.     

Regulation of G protein signaling by the 70 kDa heat shock protein

G protein-coupled receptors (GPCRs) transduce extracellular signals to the interior of the cell by activating membrane-bound guanine nucleotide-binding regulatory proteins (G proteins). An increasing number of proteins have been reported to bind to and regulate GPCRs. We report a novel regulation of the alpha_2A adrenergic receptor (α_2A-R) by the ubiquitous stress-inducible 70 kDa heat shock protein, hsp70. Hsp70, but not hsp90, attenuated G protein-dependent high affinity agonist binding to the α_2A-R in Sf9 membranes. Antagonist binding was unchanged, suggesting that hsp70 uncouples G proteins from the receptor. As hsp70 did not bind G proteins but complexed with the α_2A-R in intact cells, a direct interaction with the receptor seems likely. In the presence of hsp70, α_2A-R-catalyzed [³⁵S]GTPγS binding was reduced by approximately 70%. In contrast, approximately 50-fold higher concentrations of hsp70 were required to reduce agonist binding to the stress-inducible 5-hydroxytryptamine_1A receptor (5-HT_1A-R). In heat-stressed CHO cells, the α_2A-R was significantly uncoupled from G proteins, coincident with an increased localization of hsp70 at the membrane. The contrasting effect of hsp70 on the α_2A-R compared to the 5-HT_1A-R suggests that during stress, upregulation of hsp70 may attenuate signaling from specific GPCRs as part of the stress response to foster survival.     

In vitro protein kinase activity measurement by flow cytometry

Protein kinases are important drug targets, and a wide variety of methods have been developed for assessing their activity. A key element in developing selective kinase inhibitors is the ability to rapidly compare the effects of an inhibitor on several related or unrelated kinases. We describe a simple, nonradioactive, bead-based method for detecting kinase activity in vitro. Biotinylated peptide substrates are immobilized on beads and phosphorylation is detected with anti-phosphopeptide antibodies with no separation steps required. Phosphorylation is dependent on the amount of kinase in the assay and can be inhibited by known kinase inhibitors in a concentration-dependent manner. Using Luminex technology, we measured the activity of three kinases (PKA, PKC-μ, and Akt) on multiple substrates simultaneously. We also discuss conditions necessary to optimize measurement of the activity of several kinases in a single sample.