Gamma Radiation Induced Intestinal Proteomic Modulation in Mice: A Two Dimensional Electrophoretic Analysis

Exposure to high doses of radiation causes serious injuries in gastrointestinal tract, by affecting biomolecules of the tissue. To demonstrate the modulation of intestinal proteome by ionising radiations, we analysed changes in protein expression in 9 Gy irradiated C57BL/6 mice at 24 h and 72 h by using two dimensional electrophoresis technique. A total of 19 protein spots with statistical significance (fold change>1.5 and P<0.05) were found to be differentially expressed. Of these 07 spots were identified by MALDI-TOF MS and peptide mass fingerprinting techniques which matched with the known proteins documented in the online database. These proteins belong to biological-functional categories like cytoskeleton system, molecular chaperones, DNA damage response, and stress response. These identified radiation induced proteins can help in understanding the mechanisms behind the intestinal injuries and thus can become potential targets for therapeutics and also aid in drug development

Two acidic, anticoagulant PLA2 isoenzymes purified from the venom of monocled cobra Naja kaouthia exhibit different potency to inhibit thrombin and factor Xa via phospholipids independent, non-enzymatic mechanism.

BackgroundThe monocled cobra (Naja kaouthia) is responsible for snakebite fatality in Indian subcontinent and in south-western China. Phospholipase A2 (PLA2; EC 3.1.1.4) is one of the toxic components of snake venom. The present study explores the mechanism and rationale(s) for the differences in anticoagulant potency of two acidic PLA2 isoenzymes, Nk-PLA2α (13463.91 Da) and Nk-PLA2β (13282.38 Da) purified from the venom of N. kaouthia.Principal findingsBy LC-MS/MS analysis, these PLA2s showed highest similarity (23.5% sequence coverage) with PLA2 III isolated from monocled cobra venom. The catalytic activity of Nk-PLA2β exceeds that of Nk-PLA2α. Heparin differentially regulated the catalytic and anticoagulant activities of these Nk-PLA2 isoenzymes. The anticoagulant potency of Nk-PLA2α was comparable to commercial anticoagulants warfarin, and heparin/antithrombin-III albeit Nk-PLA2β demonstrated highest anticoagulant activity. The anticoagulant action of these PLA2s was partially contributed by a small but specific hydrolysis of plasma phospholipids. The strong anticoagulant effect of Nk-PLA2α and Nk-PLA2β was achieved via preferential, non-enzymatic inhibition of FXa (Ki = 43 nM) and thrombin (Ki = 8.3 nM), respectively. Kinetics study suggests that the Nk-PLA2 isoenzymes inhibit their "pharmacological target(s)" by uncompetitive mechanism without the requirement of phospholipids/Ca(2+). The anticoagulant potency of Nk-PLA2β which is higher than that of Nk-PLA2α is corroborated by its superior catalytic activity, its higher capacity for binding to phosphatidylcholine, and its greater strength of thrombin inhibition. These PLA2 isoenzymes thus have evolved to affect haemostasis by different mechanisms. The Nk-PLA2β partially inhibited the thrombin-induced aggregation of mammalian platelets suggesting its therapeutic application in the prevention of unwanted clot formation.Conclusion/significanceIn order to develop peptide-based superior anticoagulant therapeutics, future application of Nk-PLA2α and Nk-PLA2β for the treatment and/or prevention of cardiovascular disorders are proposed

Current Insights in the Mechanisms of Cobra Venom Cytotoxins and Their Complexes in Inducing Toxicity: Implications in Antivenom Therapy

Cytotoxins (CTXs), an essential class of the non-enzymatic three-finger toxin family, are ubiquitously present in cobra venoms. These low-molecular-mass toxins, contributing to about 40 to 60% of the cobra venom proteome, play a significant role in cobra venom-induced toxicity, more prominently in dermonecrosis. Structurally, CTXs contain the conserved three-finger hydrophobic loops; however, they also exhibit a certain degree of structural diversity that dictates their biological activities. In their mechanism, CTXs mediate toxicity by affecting cell membrane structures and membrane-bound proteins and activating apoptotic and necrotic cell death pathways. Notably, some CTXs are also responsible for depolarizing neurons and heart muscle membranes, thereby contributing to the cardiac failure frequently observed in cobra-envenomed victims. Consequently, they are also known as cardiotoxins (CdTx). Studies have shown that cobra venom CTXs form cognate complexes with other components that potentiate the toxic effects of the venom’s individual component. This review focuses on the pharmacological mechanism of cobra venom CTXs and their complexes, highlighting their significance in cobra venom-induced pathophysiology and toxicity. Furthermore, the potency of commercial antivenoms in reversing the adverse effects of cobra venom CTXs and their complexes in envenomed victims has also been discussed

Effects of Nk-PLA₂ isoenzymes on inhibition of amidolytic activity of FXa.

<p>(<b>a</b>)The Nk-PLA₂α (1.0 µg) or Nk-PLA₂β (1.0 µg) was pre-incubated with FXa (0.15 µM) against its chromogenic substrate F3301 (0.2 mM) for 60 min at 37°C, pH 7.4 before the amidolytic activity assay. A control (FXa treated with buffer) was also run in parallel. The values are mean of triplicate determinations. (<b>b</b>) Michaelis-Menten plot to determine the inhibitory constant (K_i) of Nk-PLA₂α (50 nM and 100 nM) on amidolytic activity of FXa (0.15 µM) at 37°C, pH 7.4.</p

Inhibition of prothrombin activation by FXa pre-treated with NkPLA₂ isoenzymes.

<p>(<b>a</b>) The FXa (20 nM) was pre-incubated with Nk-PLA₂α (1.0 µg), or Nk-PLA₂β (1.0 µg), or buffer (control) for 60 min at 37°C, pH 7.4. The prothrombin (1.4 µM) activation by FXa (treated or control) was determined by formation of thrombin by using its chromogenic substrate T1637 (0.2 mM). (<b>b</b>) SDS-PAGE (15%) analysis (reduced conditions) of affect of Nk-PLA₂α and Nk-PLA₂β on inhibition of prothrombin activation by FXa. Before addition of prothrombin (10.0 µg), FXa (20 nM) was pre-incubated with Nk-PLA₂α or Nk-PLA₂β for 60 min at 37°C, pH 7.4. Lane 1, prothrombin treated with FXa for 60 min at 37°C (control); lane 2, prothrombin treated with FXa (pre-incubated with 2.0 µg of Nk-PLA₂β) for 60 min at 37°C; lanes 3 and 4, prothrombin treated with FXa (pre-incubated with 1 and 2 µg of Nk-PLA₂α, respectively) for 60 min at 37°C. The experiment was repeated three times to assure the reproducibility.</p

Comparison of anticoagulant activity and plasma phospholipids hydrolytic activity of Nk-PLA₂α and Nk-PLA₂β.

<p>(<b>a</b>) A comparison of dose-dependent anticoagulant activity (Ca-clotting time of citrated PPP) of Nk-PLA₂α, Nk-PLA₂β, heparin and warfarin. Unit is defined as 1s increase in clotting of PPP in presence anticoagulants compared to the clotting time of control PPP. (<b>b</b>) Effect of PPP/PLA₂ pre-incubation time on anticoagulant activity. (<b>c</b>) Effect of PPP/PLA₂ pre-incubation on release of free fatty acids from plasma phospholipids. Values are mean ± SD of triplicate determination.</p

A summary of the purification of PLA₂ isoenzymes from the venom of <i>N. kaouthia</i>.

<p>The PLA₂ activity was assayed by egg-yolk phospholipids hydrolysis method using 0.1 M Tris-HCl, pH 8.0 buffer. Data are from a typical experiment.</p>a<p>One unit of PLA₂ activity is defined as decrease of 0.01 absorbance at 740 nm per 10 min at 25°C.</p

Unraveling the Proteome Composition and Immuno-profiling of Western India Russell’s Viper Venom for In-Depth Understanding of Its Pharmacological Properties, Clinical Manifestations, and Effective Antivenom Treatment

The proteome composition of western India (WI) Russell’s viper venom (RVV) was correlated with pharmacological properties and pathological manifestations of RV envenomation. Proteins in the 5–19 and 100–110 kDa mass ranges were the most predominate (∼35.1%) and least abundant (∼3.4%) components, respectively, of WI RVV. Non-reduced SDS-PAGE indicated the occurrence of multiple subunits, non-covalent oligomers, self-aggregation, and/or interactions among the RVV proteins. A total of 55 proteins belonging to 13 distinct snake venom families were unambiguously identified by ESI-LC-MS/MS analysis. Phospholipase A₂ (32.5%) and Kunitz-type serine protease inhibitors (12.5%) represented the most abundant enzymatic and non-enzymatic proteins, respectively. However, ATPase, ADPase, and hyaluronidase, detected by enzyme assays, were not identified by proteomic analysis owing to limitations in protein database deposition. Several biochemical and pharmacological properties of WI RVV were also investigated. Neurological symptoms exhibited by some RV-bite patients in WI may be correlated to the presence of neurotoxic phospholipase A₂ enzymes and Kunitz-type serine protease inhibitor complex in this venom. Monovalent antivenom was found to be better than polyvalent antivenom in immuno-recognition and neutralization of the tested pharmacological properties and enzyme activities of WI RVV; nevertheless, both antivenoms demonstrated poor cross-reactivity and neutralization of pharmacological activities shown by low-molecular-mass proteins (<18 kDa) of this venom

Spectrofluorometric assay of the interaction of Nk-PLA₂ isoenzymes with FXa, thrombin and PC.

<p>(<b>a</b>) FXa incubated with Nk-PLA₂α or Nk-PLA₂β (at 1∶10 ratio), (<b>b</b>) interaction of thrombin with Nk-PLA₂α or Nk-PLA₂β (at 1∶10 ratio), (<b>c</b>) PC with Nk-PLA₂α/Nk-PLA₂β (at 10∶1 ratio) in presence of 0.5 mM EDTA (to prevent the phospholipids hydrolysis). The data shown above represent a typical experiment; however, the experiments were repeated three times to assure the reproducibility.</p