Mechanisms of Arg-Pro-Pro-Gly-Phe inhibition of thrombin

Investigations determined the mechanism(s) by which Arg-Pro-Pro-Gly-Phe (RPPGF) inhibits thrombin-induced platelet activation. High concentrations of RPPGF inhibit thrombin-induced coagulant activity. RPPGF binds to the active site of thrombin by forming a parallel beta-strand with Ser214-Gly216 and interacts with His57, Asp189, and Ser195 of the catalytic triad. RPPGF competitively inhibits alpha-thrombin from hydrolyzing Sar-Pro-Arg-paranitroanilide with a Ki = 1.75 +/- 0.03 mM. Other mechanisms were sought to explain why RPPGF inhibits thrombin activation of platelets at concentrations below that which inhibits its active site. Soluble RPPGF blocks biotinylated NATLDPRSFLLR of the thrombin cleavage site on protease-activated receptor (PAR)1 from binding to the peptide RPPGC (IC50 = 20 microM). The soluble recombinant extracellular domain of PAR1 (rPAR1EC) blocks biotinylated RPPGF binding to rPAR1EC (IC50 = 50 microM) bound to microtiter plates, but rPAR1EC deletion mutants missing the sequence LDPR or PRSF do not. RPPGF and related forms prevent the thrombin-like enzyme thrombocytin from proteolyzing rPAR1EC at concentrations that do not block thrombocytin's active site. These studies indicate that RPPGF is a bifunctional inhibitor of thrombin: it binds to PAR1 to prevent thrombin cleavage at Arg41 and interacts with the active site of alpha-thrombin.     

A revised mechanism for the inactivation of bovine liver enoyl-CoA hydratase by (methylenecyclopropyl)formyl-CoA based on unexpected results with the C114A mutant

The compound (methylenecyclopropyl)formyl-CoA (MCPF-CoA) has been reported earlier as a potent active site-directed inactivator of bovine liver enoyl-CoA hydratase (ECH). It is believed that the mechanism of inactivation involves the attack of Cys114 at C-2' of MCPF-CoA, resulting in ring cleavage and permanent covalent modification of the enzyme. Here, we describe studies with the C114A mutant of bovine liver ECH, which was constructed and purified to determine the role of this residue in the catalytic mechanism of the enzyme. The C114A mutant, which is catalytically competent, shows an unexpected susceptibility to inactivation by MCPF-CoA, indicating that Cys114 is not the primary nucleophile responsible for the inactivation of the enzyme. To determine if catalytic residues Glu115 and Glu135 play a role in the inactivation of the enzyme, the E115Q and E135Q mutants were also constructed and purified. It was determined that these mutants did not react with MCPF-CoA, indicating a possible role for both residues in the inactivation of the wild-type enzyme. Pepsin digestion and subsequent LC-MS/MS analysis of the inactivated wild-type enzyme and C114A mutant revealed that Glu115 was modified in each case, supporting the hypothesis that this residue is the true nucleophile that traps MCPF-CoA and indicating that the covalent modification of Cys114 reported earlier may be a postinactivation artifact. We propose a modified mechanism of inactivation involving Glu115 and Glu135, and suggest that MCPF-CoA may be a mechanism-based inhibitor for bovine liver ECH.     

Stimulatory effect of cooling tower biocides on amoebae.

Two species of amoebae were isolated from the cooling tower of an air-conditioning system and examined for effects of exposure to four cooling tower biocides, a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds, another quaternary ammonium compound alone, and an isothiazolin derivative. The amoebae isolated were Acanthamoeba hatchetti and a Cochliopodium species. Two other amoeba cultures, an A. hatchetti culture and Cochliopodium bilimbosum, were obtained from the American Type Culture Collection (ATCC) and were also tested. The cooling tower isolates were more resistant to most of the biocides than the ATCC isolates were. The isothiazolin derivative was the least inhibitory to all four amoeba isolates, and tributyltin neodecanoate mixed with quaternary ammonium compounds was the most inhibitory to three of the four isolates. After exposure to lower concentrations of the biocides, including for one strain the manufacturer's recommended concentration of one biocide, the cooling tower amoeba populations increased significantly compared with unexposed controls, whereas the ATCC isolates were not stimulated at any of the concentrations tested. In some cases, concentrations which stimulated cooling tower amoebae inhibited the growth of the ATCC isolates. These results suggest that cooling tower amoebae may adapt to biocides, underscoring the need to use freshly isolated cooling tower organisms rather than organisms from culture collections for testing the efficacy of such biocides. The stimulatory effect of biocides on amoeba populations is an alarming observation, since these organisms may be reservoirs for legionellae. Biocides used to control microbial growth may actually enhance populations of host organisms for pathogenic bacteria.     

Pyk2 regulates cell-edge protrusion dynamics by interacting with Crk

Focal adhesion kinase (FAK) is well established as a regulator of cell migration, but whether and how the closely related proline-rich tyrosine kinase 2 (Pyk2) regulates fibroblast motility is still under debate. Using mouse embryonic fibroblasts (MEFs) from Pyk2^–/– mice, we show here, for the first time, that lack of Pyk2 significantly impairs both random and directed fibroblast motility. Pyk2^–/– MEFs show reduced cell-edge protrusion dynamics, which is dependent on both the kinase and protein–protein binding activities of Pyk2. Using bioinformatics analysis of in vitro high- throughput screens followed by text mining, we identified CrkI/II as novel substrates and interactors of Pyk2. Knockdown of CrkI/II shows altered dynamics of cell-edge protrusions, which is similar to the phenotype observed in Pyk2^–/– MEFs. Moreover, epistasis experiments suggest that Pyk2 regulates the dynamics of cell-edge protrusions via direct and indirect interactions with Crk that enable both activation and down-regulation of Crk-mediated cytoskeletal signaling. This complex mechanism may enable fine-tuning of cell-edge protrusion dynamics and consequent cell migration on the one hand together with tight regulation of cell motility, a process that should be strictly limited to specific time and context in normal cells, on the other hand.     

5-N-Substituted-2-(substituted benzenesulphonyl) glutamines as antitumor agents. Part II: synthesis, biological activity and QSAR study

Cancer is a major killer disease throughout human history. Thus, cancer becomes a major point of interest in life science. It was proved that cancer is a nitrogen trap and tumor cells are avid glutamine consumers. The non-essential amino acid glutamine, which is a glutamic acid derivative, supplies its amide nitrogen to tumor cells in the biosynthesis of purine and pyrimidine bases of nucleic acids as well as takes part in protein synthesis. Based on these and in continuation of our composite programme of development of new potential anticancer agents through rational drug design, 17 new 5-N-Substituted-2-(substituted benzenesulphonyl) glutamines were selected for synthesis. These compounds as well as 36 earlier synthesized glutamine analogues were screened for antitumor activity using percentage inhibition of tumor cell count as the activity parameter. QSAR study was performed with 53 compounds in order to design leads with increased effectiveness for antitumor activity using both physicochemical and topological parameters. QSAR study showed that steric effect on the aromatic ring is conducive to the activity. n-butyl substitution on aliphatic side chain and atom no 12 is important for antitumor activity of glutamine analogues.     

A novel family of transporters mediating the transport of glutathione derivatives in plants

Uptake and compartmentation of reduced glutathione (GSH), oxidized glutathione (GSSG), and glutathione conjugates are important for many functions including sulfur transport, resistance against biotic and abiotic stresses, and developmental processes. Complementation of a yeast (Saccharomyces cerevisiae) mutant (hgt1) deficient in glutathione transport was used to characterize a glutathione transporter cDNA (OsGT1) from rice (Oryza sativa). The 2.58-kb full-length cDNA (AF393848, gi 27497095), which was obtained by screening of a cDNA library and 5'-rapid amplification of cDNA ends-polymerase chain reaction, contains an open reading frame encoding a 766-amino acid protein. Complementation of the hgt1 yeast mutant strain with the OsGT1 cDNA restored growth on a medium containing GSH as the sole sulfur source. The strain expressing OsGT1 mediated [3H]GSH uptake, and this uptake was significantly competed not only by unlabeled GSSG and GS conjugates but also by some amino acids and peptides, suggesting a wide substrate specificity. OsGT1 may be involved in the retrieval of GSSG, GS conjugates, and nitrogen-containing peptides from the cell wall.     

Changes in Fatty Acid Composition of Chromohalobacter israelensis with Varying Salt Concentrations

The adaptation of fatty acid composition of Chromohalobacter israelensis, a euryhalophilic bacterium, grown at different salt concentrations was studied. C. israelensis tolerated NaCl up to concentrations of 20% (w/v) and showed optimal growth at 7% (w/v). Major fatty acids of this bacterium were palmitic acid (16:0), stearic acid (18:0), palmetoleic acid (16:1cis9), and cis-vaccenic acid (18:111). The salt concentration strongly influenced the fatty acid composition. In the presence of sub-optimal salt concentrations, the degree of saturation decreased, suggesting the importance of salt in maintaining the osmotic balance of the cell with its environment.     

Quantitative structure activity relationship (QSAR) studies of some substituted benzenesulphonyl glutamines as tumour suppressors

As a part of a composite programme of rational drug design (RDD), we had synthesized some substituted benzenesulphonyl glutamines and evaluated their inhibitory activities against Ehrlich Ascites Carcinoma (EAC) cell line in Swiss albino mice. Quantitative structure activity relationship (QSAR) studies of these inhibitory activities using Fujita-Ban model as well as Modified Hansch-Fujita model gave excellent correlations (correlation coefficient r = 0.89 and 0.82 respectively). These results could be useful in designing 'lead' compound with potent inhibitory activity on DNA and RNA synthesis and tumour development.     

IL-10 Accelerates Re-Endothelialization and Inhibits Post-Injury Intimal Hyperplasia following Carotid Artery Denudation

The role of inflammation on atherosclerosis and restenosis is well established. Restenosis is thought to be a complex response to injury, which includes early thrombus formation, acute inflammation and neo-intimal growth. Inflammatory cells are likely contributors in the host response to vascular injury, via cytokines and chemokines secretion, including TNF-alpha (TNF). We have previously shown that IL-10 inhibits TNF and other inflammatory mediators produced in response to cardiovascular injuries. The specific effect of IL-10 on endothelial cell (ECs) biology is not well elucidated. Here we report that in a mouse model of carotid denudation, IL-10 knock-out mice (IL-10KO) displayed significantly delayed Re-endothelialization and enhanced neo-intimal growth compared to their WT counterparts. Exogenous recombinant IL-10 treatment dramatically blunted the neo-intimal thickening while significantly accelerating the recovery of the injured endothelium in WT mice. In vitro, IL-10 inhibited negative effects of TNF on ECs proliferation, ECs cell cycle, ECs-monocyte adhesion and ECs apoptosis. Furthermore, IL-10 treatment attenuated TNF-induced smooth muscle cells proliferation. Our data suggest that IL-10 differentially regulate endothelial and vascular smooth cells proliferation and function and thus inhibits neo-intimal hyperplasia. Thus, these results may provide insights necessary to develop new therapeutic strategies to limit vascular restenosis during percutaneous coronary intervention (PCI) in the clinics.