Lethal, oedema, haemorrhagic activity of spotted butterfish (Scatophagus argus, Linn) sting extract and its neutralization by antiserum and pharmacological antagonists

An attempt has been made in this communication to develop antiserum in rabbit against Scatophagus. argus sting extract. Antiserum did not neutralized the sting extract induced proinflammatory and haemorrhagic activity but successfully neutralized lethality upto 2LD50. Cyproheptadine, indomethacin and BW 755C pretreatment significantly reduced sting extract induced proinflammatory activity. The haemorrhagic activity of sting extract was significantly inhibited by temperature, UV-exposure, EDTA, cyproheptadine, indomethacin and BW 755C pretreatment. The results conclude that the local effects of S.argus venom is likely to be mediated through release of mediators and may be encountered by pharmacological antagonists better than the antiserum.     

Comparative detoxification and protection of scorpion (Heterometrus bengalensis) venom by toxoid antiserum

Comparative detoxification of scorpion venom by using different chemical agents was investigated. Detoxification by formalin showed the best optimum detoxifying agent. The formalin treatment resulted in 2.3% protein loss with 6-fold detoxification. This formal toxoid was immunogenic in rabbit giving high neutralizing antibodies as revealed from indirect haemagglutination test. Toxoid antiserum protected mice against the lethal action of venom. It also effectively antagonized the smooth muscle contractile response of venom, and venom-induced neuromuscular paralysis. This toxoid antiserum also protected the venom-induced cardiac arrest. The possibility of using this formal toxoid for antisera production and immunization for therapeutic use needs to be explored.     

Regulation of Na, K-ATPase activity by monovalent cations]

A deviation from optimal conditions of the Na, K-ATPase reaction results in a drastic change in the plot: enzyme activity versus Na/K ratio. Acidification of the medium and a decrease in Mg2+ concentration and temperature results in two peaks on the curve at Na/K ratio of about 1 and at Na/K ratio greater than 4. The enhancement of pH of the medium and increase in Mg2+ concentration decreases the first peak and increases the second one. A comparison of these curves for hydrolysis of ATP, UTP and p-nitrophenylphosphate and temperature dependence of the hydrolysis of the substrates suggest that the anomalies observed may be accounted for the Na+ effect on the K-sites or K+ effect on the Na-sites under conditions when cation-binding sites are heterogeneous.     

Purification and characterization of an organ specific haemorrhagic toxin from Vipera russelli russelli (Russell's viper) venom

A haemorrhagic toxin (VRR-12) from Vipera russelli russelli (Russell's viper) venom has been purified by ion-exchange chromatography on CM-Sephadex C-50 followed by size-exclusion HPLC to electrophoretically homogeneous state. It is a 12 kDa single polypeptide having 1 mole of Zn+2 ion. This toxin induces intense intestinal haemorrhage and to a lesser extent skeletal muscle haemorrhage in mice. It does not show detectable proteolytic and esterolytic activity with selected substrates under specified conditions, haemolytic and phospholipase activity. When VRR-12, preincubated with bivalent antiserum against Saw-scaled and Russell's viper venom or EDTA was injected, haemorrhagic activity was not reduced, on the other hand preincubation with phenylmethyl sulphonyl fluoride reduced the activity markedly. Biodistribution studies with 125I VRR-12 show that haemorrhagic manifestation by this toxin is not a direct function of the fraction of the totally administered toxin distributed to that tissue.     

The activity and reaction specificity of tyrosine phenol-lyase regulated by monovalent cations

This work was aimed at studying the effect of monovalent inorganic cations (Li+, Na+, K+, Rb+, Cs+, NH+4) on the catalytic and spectral characteristics of tyrosine phenol-lyase from Citrobacter intermedius. These cations were shown to influence the proportion of the beta-elimination reaction rate to the rate of side transamination reaction. Most of the monovalent cations are non-competitive activators of the beta-elimination reaction; Li+ exerts no effect on the enzyme activity in this reaction; Na+ is an inhibitor of the beta-elimination reaction. The activation of tyrosine phenol-lyase by monovalent cations stems from the creation of an active holoenzyme form (lambda max 420 nm) due to conformational rearrangements of the protein molecule.     

The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide

The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1-60 microM and 50-370 microM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca2(+)-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca2(+)-binding affinity was 1.5-2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca2(+)-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca2(+)-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca2(+)-ATPase activity of permeabilized erythrocytes. At Ca2+ concentrations below 0.7 microM, ouabain (0.5-1 mM) activated whereas at higher Ca2+ concentrations it inhibited the Ca2(+)-ATPase activity. Taking this observation into account the Na+/K(+)-ATPase was determined as the difference of between the ATPase activities in the presence of Na+ and K+ and in the presence of K+ alone. At physiological concentration of Mg2+ (370 microM), the addition of 0.3-1 microM Ca2+ increased Na+/K(+)-ATPase activity by 1.5-3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg2+ concentration (e.g., 50 microM) only Na+/K(+)-ATPase inhibition by Ca2+ was seen. It was found that at [NaCl] less than 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca2(+)-free media. This activating effect of furosemide was enhanced with a diminution of [Na+] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na+/K(+)-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb+ influx into intact erythrocytes.     

Production of a monovalent staphylococcal enterotoxic antiserum type A and its use for typing staphylococcal enterotoxins

A monovalent specific staphylococcal antiserum, type A, was obtained by means of the isolated and purified preparation of type A staphylococcal enterotoxin. This antiserum was proved to be identical to antiserum of the same type, manufactured by Serva Feinbiochemica GmbH & Co. (West Germany). The titer of the newly obtained antiserum in the precipitation test was 1 : 16, and its use allowed one to detect enterotoxin of the above-mentioned type at a concentration of 0.004 mg/ml. The study of 320 staphylococcal strains with the use of this antiserum revealed that 25 strains (7.8%) produced type A enterotoxin.     

Population Pharmacokinetics of an Indian F(ab')2 Snake Antivenom in Patients with Russell's Viper (Daboia russelii) Bites

Background

There is limited information on antivenom pharmacokinetics. This study aimed to investigate the pharmacokinetics of an Indian snake antivenom in humans with Russell’s viper bites.

Methods/Principal Findings

Patient data and serial blood samples were collected from patients with Russell’s viper (Daboia russelii) envenoming in Sri Lanka. All patients received Indian F(ab’)₂ snake antivenom manufactured by VINS Bioproducts Ltd. Antivenom concentrations were measured with sandwich enzyme immunoassays. Timed antivenom concentrations were analysed using MONOLIXvs4.2. One, two and three compartment models with zero order input and first order elimination kinetics were assessed. Models were parameterized with clearance(CL), intercompartmental clearance(Q), central compartment volume(V) and peripheral compartment volume(V_P). Between-subject-variability (BSV) on relative bioavailability (F) was included to account for dose variations. Covariates effects (age, sex, weight, antivenom batch, pre-antivenom concentrations) were explored by visual inspection and in model building. There were 75 patients, median age 57 years (40-70y) and 64 (85%) were male. 411 antivenom concentration data points were analysed. A two compartment model with zero order input, linear elimination kinetics and a combined error model best described the data. Inclusion of BSV on F and weight as a covariate on V improved the model. Inclusion of pre-antivenom concentrations or different batches on BSV of F did not. Final model parameter estimates were CL,0.078 Lh^-1, V,2.2L, Q,0.178Lh^-1 and V_P,8.33L. The median half-life of distribution was 4.6h (10-90%iles:2.6-7.1h) and half-life of elimination, 140h (10^th-90^th percentilesx:95-223h).

Conclusion

Indian F(ab’)₂ snake antivenom displayed biexponential disposition pharmacokinetics, with a rapid distribution half-life and more prolonged elimination half-life.     

Detection of Venom after Antivenom Is Not Associated with Persistent Coagulopathy in a Prospective Cohort of Russell's Viper (Daboia russelii) Envenomings

Background

Venom recurrence or persistence in the circulation after antivenom treatment has been documented many times in viper envenoming. However, it has not been associated with clinical recurrence for many snakes, including Russell''s viper (Daboia spp.). We compare the recovery of coagulopathy to the recurrence or persistence of venom in patients with Russell''s viper envenoming.

Methodology/Principal Findings

The study included patients with Russell''s viper (D. russelii) envenoming presenting over a 30 month period who had Russell''s viper venom detected by enzyme immunoassay. Demographics, information on the snake bite, and clinical effects were collected for all patients. All patients had serum collected for venom specific enzyme immunoassay and citrate plasma to measure fibrinogen levels and prothrombin time (international normalised ratio; INR). Patients with venom recurrence/persistence were compared to those with no detectable recurrence of venom. There were 55 patients with confirmed Russell''s viper envenoming and coagulopathy with low fibrinogen concentrations: 31 with venom recurrence/persistence, and 24 with no venom detected post-antivenom. Fibrinogen concentrations increased and INR decreased after antivenom in both the recurrence and non-recurrence patients. Clinical features, laboratory parameters, antivenom dose and length of hospital were similar for both groups. Pre-antivenom venom concentrations were higher in patients with venom recurrence/persistence with a median venom concentration of 385 ng/mL (16–1521 ng/mL) compared to 128 ng/mL (14–1492 ng/mL; p = 0.008).

Conclusion

Recurrence of Russell''s viper venom was not associated with a recurrence of coagulopathy and length of hospital stay. Further work is required to determine if the detection of venom recurrence is due to the venom specific enzyme immunoassay detecting both venom-antivenom complexes as well as free venom.     

Neutralizing activity of polyvalent Gb3, Gb2 and galacto-trehalose models against Shiga toxins

Shiga toxin (Stx) is one of the most critical factors in the development of hemolytic uremic syndrome and other systemic complications following enterohemorrhagic Escherichia coli (EHEC) infection. Substances neutralizing Stx by interfering with toxin-receptor binding have been explored as therapeutic candidates for EHEC infection. In this study, we examined globotriaosyl (Gb3), galabiosyl (Gb2) and galacto-trehalose, each of which was synthetically conjugated with a polyacrylamide backbone, for Stxneutralizing activity. Galacto-trehalose was designed as a Gb2 mimicking, unnatural Stx-ligand that was expected to show tolerance to enzymatic degradation in vivo. Galacto-trehalose copolymer showed neutralizing activity against Stx-1 but not Stx-2 in a HeLa cell cytotoxicity assay. It was thought that galactotrehalose copolymer could be a lead compound for the treatment of Stx-mediated diseases, although it requires modification to show neutralizing activity to Stx-2. The Gb3 copolymer with high sugar unit density showed stronger neutralizing activity against Stx-2 than those with lower density. However, the density-dependency of the neutralizing activity was less obvious against Stx-1. Intravenous administration of the Gb3 copolymer prevented death in mice lethally infected with Stx-1- and Stx-2-producing E. coli O157:H7. Thus, we demonstrated that the artificial Gb3 copolymer could neutralize Stx-1 and the more clinically relevant Stx-2 in vitro and effectively inhibit Stx toxicity in vivo.     

Inhibition of movement and ATP-ase activity of demembranated sea urchin spermatozoa by anti-dynein antiserum.

Anti-dynein antiserum was prepared in rabbits against a tryptic fragment (Fragment A) of dynein ATPase. The antiserum inhibited the ATP-induced movement of demembranated sea urchin spermatozoa. The complete inhibition of progressive movement occurred at relatively low concentrations of the antiserum, which affected the ATPase activity to a rather small extent. The anti-dynein antiserum seems to inhibit not only ATPase activity but also bending-wave formation, in some way.     

Modulation of SHP‐1 phosphatase activity by monovalent and bivalent SH2 phosphopeptide ligands

A sequence derived from the epithelial receptor tyrosine kinase Ros (pY2267) represents a high‐affinity binding partner for protein tyrosine phosphatase SHP‐1 and was recently used as lead structure to analyze the recognition requirements for the enzyme's N‐SH2 domain. Here, we focused on a set of peptides comprising C‐terminally extended linear and conformationally constrained side chain‐bridged cyclic N‐SH2 ligands based on the consensus sequence LxpYhxh(h/b)(h/b) (x = any amino acid, h = hydrophobic, and b = basic residue). Furthermore, the bivalent peptides described were designed to modulate the activity of SHP‐1 through binding to both, the N‐SH2 domain as well as an independent binding site on the surface of the catalytic domain (PTP domain). Consistent with previous experimental findings, surface plasmon resonance experiments revealed dissociation constants of most compounds in the low micromolar range. One peptide, EGLNpYc[KVD]MFPAPEEE? NH₂, displayed favorable binding affinity, but reduced ability to stimulate SHP‐1. Docking experiments revealed that the binding of this ligand occurs in binding mode I, recently described to lead to an inhibited activation of SHP‐1. In summary, results presented in this study suggest that inhibitory N‐SH2 ligands of SHP‐1 may be obtained by designing bivalent compounds that associate with the N‐SH2 domain and simultaneously occupy a specific binding site on the PTP domain. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 102–112, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Effects of prolonged poisoning by Russell's viper venom on blood coagulation, platelets and fibrinolysis

The effects of Russell's viper venom on blood coagulation, platelets and the fibrinolytic enzyme system were studied in rabbits after injecting repeated doses of 0.05 MLD of the venom. Thrombocytopenia was the earliest change to appear. It was followed by rise in serum fibrinogen degradation products and prolongation of prothrombin time, activated partial thromboplastin time and thrombin time indicating a progressive consumption coagulopathy and activation of fibrinolysis. Red blood cell morphology was unchanged during the first three weeks; whereas the fragmentation appeared after the fourth week and it increased in severity with further envenomations, i.e. when chronic DIC was established.