The fibrinogen-derived peptide (RGDS) prevents proteolytic degradation of protein kinase C in platelets by inhibiting platelet aggregation

The effects of the fibrinogen-derived tetrapeptide, Arg-Gly-Asp-Ser (RGDS), on platelet activation processes was studied. At concentrations of 100-300 microM, RGDS completely prevented platelet aggregation induced by all the common platelet agonists, 'weak' and 'strong'. In agreement with earlier views on the aggregation-dependency of weak agonist-induced thromboxane synthesis and 5-hydroxytryptamine (5HT) secretion, RGDS (100-300 microM) inhibited these events induced by ADP, adrenaline and low concentrations of thrombin and collagen but not that induced by high concentrations of thrombin and collagen. 5HT secretion induced by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), was also not affected by RGDS, but proteolytic degradation of the translocated membrane-bound enzyme in PMA-treated platelets, due to the actions of the Ca2+-dependent protease (Ca-DP), was completely prevented such that in the presence of RGDS, sustained increases in membrane-bound PKC activity were observed. PMA alone caused only transient increases in membrane-bound PKC. This effect of RGDS was similar to the effect of E64-d, a recently described inhibitor of Ca-DP in platelets, or the effects seen with PMA in unstirred non-aggregating platelets. It is concluded that RGDS inhibits the actions of Ca-DP in platelets via inhibition of aggregation.     

Inhibition of agonist-induced platelet aggregation, Ca2+ mobilization and granule secretion by guanosine 5''-[beta-thio]diphosphate and GDP in intact platelets. Evidence for an inhibitory mechanism unrelated to the inhibition of G-protein-GTP interaction.

(1) We [Muir, Offord & Davies (1986) Biochem. J. 237, 631-637 and Davies, Muir & Offord (1986) Biochem. J. 240, 609-612] have previously identified a major product in the degradation of insulin by insulin proteinase (the N-terminal fragment produced by cleavage between residues LeuA13 and TyrA14, SerB9 and HisB10) together with evidence for a minor cleavage site between HisB10 and LeuB11 or between LeuB11 and ValB12. (2) We now present evidence for minor sites of cleavage between TyrA14 and GlnA15, GluB13 and AlaB14 as well as HisB10 and LeuB11.     

Phorbol ester treatment inhibits thrombin but not stable GTP analogue-induced platelet granule secretion despite inhibition of phosphatidate formation with both agonists

Previous work has demonstrated that pre-treatment of platelets with phorbol esters that activate protein kinase C eg phorbol 12-myristate 13-acetate (PMA) results in an inhibition of inositol phospholipid breakdown and granule secretion induced by physiological agonists such as thrombin and collagen. In the present study, the effect of pre-treatment with PMA on granule secretion and [32P]-phosphatidate (PA) formation induced by the stable GTP analogue, guanosine 5'-[gamma thio] triphosphate (GTP gamma S) was examined in saponin-permeabilized platelets. A low concentration of PMA ie 1.6nM, that did not induce significant 5-hydroxytryptamine (5HT) secretion on its own, but inhibited low-dose thrombin-induced 5HT secretion totally and PA formation by 30-40% in intact as well as permeabilised platelets was chosen. Our results demonstrate a lack of inhibition of GTP gamma S (40 microM)-induced 5HT secretion by PMA in permeabilised platelets, despite significant inhibition (70%) of PA formation, suggesting that apart from the diacylglycerol pathway of secretion which may be common to thrombin and GTP analogues, secretion induced by physiological agonists such as thrombin may involve another mechanism that is inhibitable by phorbol esters.     

Synergistic potentiation of 5-hydroxytryptamine secretion by platelet agonists and phorbol myristate acetate despite inhibition of agonist-induced arachidonate/thromboxane and beta-thromboglobulin release and Ca2+ mobilization by phorbol myristate acetate.

Previous studies have demonstrated an inhibition of agonist-induced inositol phospholipid breakdown and intracellular Ca2+ ([Ca2+]i) mobilization by phorbol esters in platelets. In this study, we have examined the effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced granule secretion and correlated it with agonist-induced [Ca2+]i mobilization, arachidonate and thromboxane (Tx) release in human platelets. With increasing times of incubation with PMA (10 s-5 min), the rise in [Ca2+]i induced by thrombin and the TxA2 mimetic, U46619, was increasingly inhibited (90-100% with 5 min incubation) and, correlating with this, thrombin-induced [3H]arachidonate, TxB2 and beta-thromboglobulin (beta TG) release were also inhibited. In addition, the conversion of exogenously added arachidonate to TxB2 was inhibited (50-80%) by a 10 s-5 min pretreatment with PMA. However, secretion of 5-hydroxy[14C]tryptamine (5HT) induced by thrombin or U46619 was not inhibited by 10 s-2 min incubations with PMA and, on the contrary, with low agonist concentrations, was potentiated by PMA in the absence of a significant rise in [Ca2+]i or endogenous Tx formation, to levels significantly greater than or equal to the sum of that obtained when agonist and PMA were added separately. With longer times of incubation with PMA (5 min), these synergistic effects became less pronounced as inhibitory effects of PMA on agonist-induced [14C]5HT secretion became apparent. The results indicate that, while PMA may cause an inhibition of agonist-induced [Ca2+]i mobilization resulting in an inhibition of agonist-induced arachidonate, TxB2 and beta TG release, its effects on agonist-induced 5HT secretion may be complicated by [Ca2+]i-independent synergistic effects of agonist and PMA.     

Arthrobacter pokkalii sp nov,a Novel Plant Associated Actinobacterium with Plant Beneficial Properties,Isolated from Saline Tolerant Pokkali Rice,Kerala, India

A novel yellow colony-forming bacterium, strain P3B162^T was isolated from the pokkali rice rhizosphere from Kerala, India, as part of a project study aimed at isolating plant growth beneficial rhizobacteria from saline tolerant pokkali rice and functionally evaluate their abilities to promote plant growth under saline conditions. The novel strain P3B162^T possesses plant growth beneficial traits such as positive growth on 1-aminocyclopropane-1-carboxylic acid (ACC), production of indole acetic acid (IAA) and siderophore. In addition, it also showed important phenotypic characters such as ability to form biofilm and utilization of various components of plant root exudates (sugars, amino acids and organic acids), clearly indicating its lifestyle as a plant rhizosphere associated bacterium. Taxonomically, the novel strain P3B162^T was affiliated to the genus Arthrobacter based on the collective results of phenotypic, genotypic and chemotaxonomic analyses. Moreover, molecular analysis using 16S rRNA gene showed Arthrobacter globiformis NBRC 12137^T, Arthrobacter pascens DSM 20545^T and Arthrobacter liuii DSXY973^T as the closely related phylogenetic neighbours, showing more than 98% 16S rRNA similarity values, whereas the recA gene analysis displayed Arthrobacter liuii JCM 19864^T as the nearest neighbour with 94.7% sequence similarity and only 91.7% to Arthrobacter globiformis LMG 3813^T and 88.7% to Arthrobacter pascens LMG 16255^T. However, the DNA-DNA hybridization values between strain P3B162^T, Arthrobacter globiformis LMG 3813^T, Arthrobacter pascens LMG 16255^T and Arthrobacter liuii JCM 19864^T was below 50%. In addition, the novel strain P3B162^T can be distinguished from its closely related type strains by several phenotypic characters such as colony pigment, tolerance to NaCl, motility, reduction of nitrate, hydrolysis of DNA, acid from sucrose, cell wall sugars and cell wall peptidoglycan structure. In conclusion, the combined results of this study support the classification of strain P3B162^T as a novel Arthrobacter species and we propose Arthrobacter pokkalii sp.nov.as its name. The type strain is P3B162^T (= KCTC 29498^T = MTCC 12358^T).     

Bacillus enclensis sp. nov., isolated from sediment sample

A novel bacterial strain, designated SGD-1123^T was isolated from Chorao Island, in Goa Province, India. The strain was found to be able to grow at 15–42 °C, pH 5–12 and 0–12 % (w/v) NaCl. The whole cell hydrolysates were found to contain meso-diaminopimelic acid, galactose and arabinose. The major fatty acids were identified as iso-C_15:0 and anteiso-C_15:0, MK-7 was identified as the predominant menaquinone and the predominant polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified aminolipid. The genomic DNA G+C content was determined to be 44.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences placed the isolate within the genus Bacillus and further revealed that strain SGD-1123^T had highest sequence similarity with Bacillus aquimaris, and forms a separate clade with its closest relatives i.e. B. aquimaris, Bacillus vietnamensis and Bacillus marisflavi, with which it shares 94.5, 94.1 and 94.1 % similarity respectively. The phylogenetic, chemotaxonomic and phenotypic analyses indicated that strain SGD-1123^T represents a novel species within the genus Bacillus, for which the name Bacillus enclensis is proposed. The type strain is SGD-1123^T (NCIM 5450^T=CCTCC AB 2011125^T).     

Therapeutic efficacy of Kalpaamruthaa on reactive oxygen/nitrogen species levels and antioxidative system in mammary carcinoma bearing rats

Reactive oxygen species play an important role in cancer and metastasis. Kalpaamruthaa is a modified Siddha preparation, which has been formulated in our laboratory. The preparation is an amalgamation of Semecarpus anacardium (SA), Emblica officinalis (EO) and honey, which gives an extra protectiveness to mammary carcinoma bearing animals (Sprague-Dawley stains were used for this study). The aim of our research is to determine the therapeutic efficiency of the drug with respect to lipid peroxidation and antioxidant status. The levels of lipid peroxides and antioxidant levels were measured in blood, and vital organs (liver, kidney and breast tissue) of control and experimental animals. In cancer condition, the LPO was increased and antioxidant levels were decreased. On drug (SA and KA) administration, decreased LPO and increased antioxidant levels were seen in control and experimental animals. This may be due to additive property of the drugs (SA, Emblica and honey), which possesses anticancer effect. The present study shows the good therapeutic efficacy of Kalpaamruthaa against mammary carcinoma.     

Gender differences in human immunodeficiency virus type 1-specific CD8 responses in the reproductive tract and colon following nasal peptide priming and modified vaccinia virus Ankara boosting

Induction of mucosal anti-human immunodeficiency virus type 1 (HIV-1) T-cell responses in males and females will be important for the development of a successful HIV-1 vaccine. An HIV-1 envelope peptide, DNA plasmid, and recombinant modified vaccinia virus Ankara (rMVA) expressing the H-2D(d)-restricted cytotoxic T lymphocyte P18 epitope were used as immunogens to test for their ability to prime and boost anti-HIV-1 T-cell responses at mucosal and systemic sites in BALB/c mice. We found of all prime-boost combinations tested, an HIV-1 Env peptide subunit mucosal prime followed by systemic (intradermal) boosting with rMVA yielded the maximal induction of gamma interferon (IFN-gamma) spot-forming cells in the female genital tract and colon. However, this mucosal prime-systemic rMVA boost regimen was minimally immunogenic for the induction of genital, colon, or lung anti-HIV-1 T-cell responses in male mice. We determined that a mucosal Env subunit immunization could optimally prime an rMVA boost in female but not male mice, as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tracts, colon, and lung. Defective mucosal priming in male mice could not be overcome by multiple mucosal immunizations. However, rMVA priming followed by an rMVA boost was the optimal prime-boost strategy for male mice as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tract and lung. Thus, prime-boost immunization strategies able to induce mucosal antigen-specific IFN-gamma responses were identified for male and female mice. Understanding the cellular and molecular basis of gender-determined immune responses will be important for optimizing induction of anti-HIV-1 mucosal immune responses in both males and females.     

The genome sequence of a reassortant bluetongue virus serotype 3 from India

All 10 genome segments (Seg-1 to 10-a total of 19,188 bp) were sequenced from a strain of bluetongue virus serotype 3 (BTV-3) from India (strain IND2003/08). Sequence comparisons showed that nine of the genome segments from this virus group with other eastern topotype strains. Genome Seg-2 and Seg-6 group with eastern BTV-3 strains from Japan. However, Seg-5 (the NS1 gene) from IND2003/08 belongs to a western lineage, demonstrating that IND2003/08 is a reassortant between eastern and western topotype bluetongue viruses. This confirms that western BTV strains have been imported and are circulating within the subcontinent.