SmgGDS is a guanine nucleotide exchange factor that specifically activates RhoA and RhoC

SmgGDS is an atypical guanine nucleotide exchange factor (GEF) that promotes both cell proliferation and migration and is up-regulated in several types of cancer. SmgGDS has been previously shown to activate a wide variety of small GTPases, including the Ras family members Rap1a, Rap1b, and K-Ras, as well as the Rho family members Cdc42, Rac1, Rac2, RhoA, and RhoB. In contrast, here we show that SmgGDS exclusively activates RhoA and RhoC among a large panel of purified GTPases. Consistent with the well known properties of GEFs, this activation is catalytic, and SmgGDS preferentially binds to nucleotide-depleted RhoA relative to either GDP- or GTPγS-bound forms. However, mutational analyses indicate that SmgGDS utilizes a distinct exchange mechanism compared with canonical GEFs and in contrast to known GEFs requires RhoA to retain a polybasic region for activation. A homology model of SmgGDS highlights an electronegative surface patch and a highly conserved binding groove. Mutation of either area ablates the ability of SmgGDS to activate RhoA. Finally, the in vitro specificity of SmgGDS for RhoA and RhoC is retained in cells. Together, these results indicate that SmgGDS is a bona fide GEF that specifically activates RhoA and RhoC through a unique mechanism not used by other Rho family exchange factors.     

A novel method to analyze leukocyte rolling behavior <Emphasis Type="Italic">in vivo</Emphasis>

Leukocyte endothelial cell interaction is a fundamentally important process in many disease states. Current methods to analyze such interactions include the parallel-plate flow chamber and intravital microscopy. Here, we present an improvement of the traditional intravital microscopy that allows leukocyte-endothelial cell interaction to be studied from the time the leukocyte makes its initial contact with the endothelium until it adheres to or detaches from the endothelium. The leukocyte is tracked throughout the venular tree with the aid of a motorized stage and the rolling and adhesive behavior is measured off-line. Because this method can involve human error, methods to automate the tracking procedure have been developed. This novel tracking method allows for a more detailed examination of leukocyte-endothelial cell interactions. Published: August 27, 2004.     

Effects of zinc on factor I cofactor activity of C4b-binding protein and factor H

Complement inhibition is to a large extent achieved by proteolytic degradation of activated complement factors C3b and C4b by factor I (FI). This reaction requires a cofactor protein that binds C3b/C4b. We found that the cofactor activity of C4b-binding protein towards C4b/C3b and factor H towards C3b increase at micromolar concentrations of Zn(2+) and are abolished at 2 mM Zn(2+) and above. 65Zn(2+) bound to C3b and C4b molecules but not the cofactors or FI when they were immobilized in a native form on a nitrocellulose membrane. Zn(2+) binding constants for C3met (0.2 microM) and C4met (0.1 microM) were determined using fluorescent chelator. It appears that higher cofactor activity at low zinc concentrations is due to an increase of affinity between C4b/C3b and cofactor proteins as assessed by surface plasmon resonance. Inhibition of the reaction seen at higher concentrations is due to aggregation of C4b/C3b.     

Structural features associated with the binding of glutamine-containing peptides to Factor XIII

Activated Factor XIII a2 catalyzes the formation of intermolecular gamma-glutamyl- epsilon -lysyl cross-links in the fibrin network. Solution NMR studies were carried out to characterize, the structural features associated with the binding of glutamine-containing peptides to Factor XIII. A coupled uv/vis kinetic assay demonstrated that K9 peptide (1-10), alpha2-antiplasmin (1-15), and alpha2-antiplasmin (1-15 Q4N) all function as glutamine-containing substrates for activated Factor XIII a2. 2D TOCSY spectra of the peptides exhibit upfield chemical shifts for the glutamine protons in the presence of Factor XIII. These results indicate that the reactive peptide glutamines are encountering a distinctive environment within the Factor XIII active site. 1D proton line-broadening and 2D transferred-NOESY studies reveal that the glutamines and residues located C-terminally come in direct contact with the enzyme and adopt an extended conformation. Substrates with sequences similar to alpha2-antiplasmin (1-15) are proposed to bind both at the catalytic site and at a neighboring apolar region.     

Metalloproteinase with factor X activating and fibrinogenolytic activities from Vipera berus berus venom

We have previously shown that Vipera berus berus venom contains several factor X activating enzymes. In the present study we have investigated one of them. The enzyme was separated from venom by gel filtration on Sephadex G-100 superfine and chromatography on agarose HPS-7 and phenyl-agarose. The enzyme is a glycosylated metalloproteinase containing hexoses, hexosamines and neuraminic acid. The purified factor X activating enzyme consists of two equal chains (59 kDa). The specificity studies have shown that enzyme is nonspecific factor X activating proteinase hydrolysing also proteins such as azocasein, gelatin and fibrinogen. The enzyme hydrolyses oxidized insulin B-chain at the positions Ala¹⁴–Leu¹⁵ and Tyr¹⁶–Leu¹⁷ but it is inactive on fibrin, plasminogen and prothrombin. We used 8–10 amino acid residues containing peptides, which reproduce the sequence around the cleavage sites in factor X, factor IX and fibrinogen, as potential substrates for enzyme. Cleavage products of peptide hydrolysis were determined by MALDI-TOF MS. The peptide Asn–Asn–Leu–Thr–Arg–Ile–Val–Gly–Gly—factor X fragment was cleaved by enzyme at positions Leu³–Thr⁴ and Arg⁵–Ile⁶. The fibrinogen peptide fragment Glu–Tyr–His–Thr–Glu–Lys–Leu–Val–Thr–Ser was hydrolysed at position Lys⁶–Leu⁷.     

Evidence on inhibition of Listeria monocytogenes by divercin V41 action

AIMS: The aim of this study was to investigate the role of divercin V41 in inhibition and prevention of Listeria monocytogenes. METHODS AND RESULTS: Carnobacterium divergens V41 deficient in bacteriocin production was isolated and characterized by enzyme-liked immunosorbent assay, multiplex polymerase chain reaction and bacteriocin diffusion test. Carnobacterium divergens V41 (divercin+) and Carnobacterium divergens V41C9 (divercin-) were grown in the presence of L. monocytogenes in smoked salmon model medium. Carnobacterium divergens V41, but not C. divergens V41C9, was able to inhibit growth of L. monocytogenes. The results indicate that inhibition of L. monocytogenes in the presence of C. divergens V41 is because of the production of divercin V41 and not to a nutritional advantage. CONCLUSIONS: Carnobacterium divergens V41 may be a promising agent in food safety. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates a potential use of a bacteriocin producing lactic acid bacteria in the area food protection.     

Salmonella typhimurium forms adenylcobamide and 2-methyladenylcobamide, but no detectable cobalamin during strictly anaerobic growth

Under microaerophilic conditions Salmonella typhimurium LT2 synthesizes cobalamin, during which 5,6-dimethylbenzimidazole is formed from riboflavin. We report here that in an anoxic environment S. typhimurium did not form cobalamin, but rather adenylcobamide, 2-methyladenylcobamide, and cobyric acid. This indicated that S. typhimurium, like other microorganisms that synthesize 5,6-dimethylbenzimidazole from riboflavin, requires oxygen for the formation of the cobalamin base. Received: 29 June 1999 / Accepted: 29 September 1999     

Evolutionary Rate and Genetic Heterogeneity of Human T-Cell Lymphotropic Virus Type II (HTLV-II) Using Isolates from European Injecting Drug Users

Seven new Italian and two new British HTLV-II isolates were obtained from injecting drug users and the entire long terminal repeat (LTR) region was sequenced. Restriction analysis showed that all the Italian isolates are of the IIb subtype, whereas the British isolates are of the IIa subtype. To understand whether the further differentiation of each two principal HTLV-II subtypes in several subgroups could be statistically supported by phylogenetic analysis, the neighbor-joining, parsimony, and maximum likelihood methods were used. The separation between IIa and IIb is very well supported by all three methods. At least two phylogenetic subgroups exist within the HTLV-IIa and at least three within the HTLV-IIb subtype. In the present analysis, no statistical support was obtained for additional phylogroups. Two particular subgroups seem interesting because they include all European and North American injecting drug user strains within the IIa and IIb subtypes, respectively. These data confirm that European HTLV-II infection among drug users is probably derived from North America. They also suggest that though a certain differentiation by restriction analysis in different subgroups is possible, carefully interpreted phylogenetic analyses remain necessary. Using the likelihood ratio test, a molecular clock for the drug user strains was calibrated. A fixation rate between 1.08 × 10⁻⁴ and 2.7 × 10⁻⁵ nucleotide substitutions per site per year was calculated for the IIa and IIb injecting drug user strains. This is the lowest fixation rate so far reported for RNA viruses, including for HIV, which typically range between 10⁻² and 10⁻⁴.