Daboxin P,a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity

In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A₂ activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca⁺² binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A₂ enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity.     

Silicium-Stoffwechsel bei Mikroorganismen

Zusammenfassung Untersuchungen zur Mobilität der aufgenommenen Kieselsäure in Bakterien zeigen, daß die Kieselsäure in Abwesenheit von Glucose rasch wieder gegen Phosphat ausgetauscht werden kann. Wenn jedoch die Aufnahme der Kieselsäure aus glucosehaltigen Si-Lösungen erfolgte, so ergibt sich, daß nur ein geringer Teil dieser Kieselsäure durch Inkubation in Phosphatlösungen wieder ausgetauscht werden kann. Dieser Si-Anteil wird jedoch bei Inkubation der Zellen in Carbonatlösung vollständig ausgeschieden. Die Möglichkeit, daß diese Kieselsäure in Form von C–Si-Bindungen in der Zelle vorliegt, wird diskutiert. Weitere Untersuchungen zu diesen Fragen sind im Gange.

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Summary Studies on the mobility of silicon in bacterial cells show, that Si is readily excanged against phosphate if the cells are incubated in absence of glucose. However if the bacteria are preincubated in silicate solutions in the presence of glucose, the silicon taken up is not mobile any more during incubation in phosphate solutions. The element is extruded completely by incubating the cells in carbonate solutions. Evidence has been made that silicon is deposited within the cells as organic compound in which C–Si-bonds are formed.

     

Control of the bifunctional O2-sensor kinase NreB of Staphylococcus carnosus by the nitrate sensor NreA: Switching from kinase to phosphatase state

The NreB–NreC two-component system of Staphylococcus carnosus for O₂ sensing cooperates with the accessory nitrate sensor NreA in the NreA–NreB–NreC system for coordinated sensing and regulation of nitrate respiration by O₂ and nitrate. ApoNreA (NreA in the absence of nitrate) interacts with NreB and inhibits NreB autophosphorylation (and activation). NreB contains the phosphatase motif DxxxQ. The present study shows that NreB on its own was inactive for the dephosphorylation of the phosphorylated response regulator NreC (NreC-P), but co-incubation with NreB and NreA stimulated NreC-P dephosphorylation. Either the presence of instead of apoNreA or mutation of the phosphatase motif (D160 or Q164) of NreB abrogated phosphatase activity of NreB. Phosphatase activity was observed for anoxic (active) NreB as well as oxic NreB, therefore the functional state of NreB is not relevant for phosphatase activity. Thus, NreB is a bifunctional sensor kinase with an integral cryptic phosphatase activity. Activation of phosphatase activity and dephosphorylation of NreC-P requires NreA as a cofactor. Accordingly, NreA and nitrate have major and dual roles in NreA–NreB–NreC regulation by (i) inhibiting NreB phosphorylation and (ii) triggering a kinase/phosphatase switch of NreB when present as apoNreA.     

Phylogeny of snakes (Serpentes): Combining morphological and molecular data in likelihood,Bayesian and parsimony analyses

Abstract

The phylogeny of living and fossil snakes is assessed using likelihood and parsimony approaches and a dataset combining 263 morphological characters with mitochondrial (2693 bp) and nuclear (1092 bp) gene sequences. The ‘no common mechanism’ (NCMr) and ‘Markovian’ (Mkv) models were employed for the morphological partition in likelihood analyses; likelihood scores in the NCMr model were more closely correlated with parsimony tree lengths. Both models accorded relatively less weight to the molecular data than did parsimony, with the effect being milder in the NCMr model. Partitioned branch and likelihood support values indicate that the mtDNA and nuclear gene partitions agree more closely with each other than with morphology. Despite differences between data partitions in phylogenetic signal, analytic models, and relative weighting, the parsimony and likelihood analyses all retrieved the following widely accepted groups: scolecophidians, alethinophidians, cylindrophiines, macrostomatans (sensu lato) and caenophidians. Anilius alone emerged as the most basal alethinophidian; the combined analyses resulted in a novel and stable position of uropeltines and cylindrophiines as the second‐most basal clade of alethinophidians. The limbed marine pachyophiids, along with Dinilysia and Wonambi, were always basal to all living snakes. Other results stable in all combined analyses include: Xenopeltis and Loxocemus were sister taxa (fide morphology) but clustered with pythonines (fide molecules), and Ungaliophis clustered with a boine‐erycine clade (fide molecules). Tropidophis remains enigmatic; it emerges as a basal alethinophidian in the parsimony analyses (fide molecules) but a derived form in the likelihood analyses (fide morphology), largely due to the different relative weighting accorded to data partitions.