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P Gopalakrishnakone

Publications and source records attributed to P Gopalakrishnakone.

At least 55 records · Page 3Linked to original sources
PubMed1998

Four new postsynaptic neurotoxins from Naja naja sputatrix venom: cDNA cloning, protein expression, and phylogenetic analysis.

cDNAs encoding 4 short chain alpha-neurotoxins from Malayan spitting cobra (Naja naja sputatrix) venom were cloned and expressed in Escherichia coli. The recombinant toxins possessed identical amino acid sequences to alpha-neurotoxins. This is the first report on cloning and expression of isoforms of neurotoxins from a species of spitting cobra. Two of these isoforms were also identified in the crude venom by reverse phase-high performance liquid chromatography (RP-HPLC), capillary electrophoresis followed by mass spectrometry and characterized by protein sequencing. Based on the variable amino acid residues, the neurotoxins in N. n. sputatrix could be assigned to 2 major groups, 10E11T and 10Q11A, which could be further subdivided into 10E11T28S: 10E11T28G and 10Q11A28S; 10Q11A28G respectively. These substitutions were also found to be unique to N. n. sputatrix neurotoxins. Phylogenetic analysis based on molecular properties of the toxins provided further support for the classification of N. n. sputatrix neurotoxin into 2 fundamental groups.

Amino Acid Sequence↗
PubMed1998

Snake envenomation and protective natural endogenous proteins: a mini review of the recent developments (1991-1997).

The properties of several factors--antihaemorrhagic, antineurotoxic, antimyotoxic--isolated from the blood serum or plasma of different animals are described with more emphasis placed on the structural differences and similarities among the factors of the snake (Trimeresurus flavoviridis) and mammals (Didelphis marsupials and Herpestes edwardsii). Classification of antihaemorrhagic factors of snake and mammals according to structural homologies, and their effectiveness in neutralizing venom haemorrhagic activities in comparison with that of commercial antivenoms are also reviewed. The antineurotoxic factors isolated so far from the sera of viperid (Vipera palestinae, Daboia r. siamensis), crotalid (Crotalus d. terrificus, T. flavoviridis, Agkistrodon b. siniticus) and elapid (Naja naja atra) snakes, as reviewed, are inhibitors of phospholipase A2, and the amino acid sequences, particularly of those inhibitors from the sera of crotalid snakes, do not share significant sequence homology even within the same family Crotalidae. The amino acid sequences of antineurotoxic factors of the snake (Crotalus d. terrificus) also are not homologous to those of the antihaemorrhagic factors from the blood of the snake (T. flavoviridis) or mammals (Didelphis virginiana, Herpestes edwardsii). The mechanism of action of antihaemorrhagic and antineurotoxic factors is briefly discussed as well as the possibility that crotalids and viperids might possess both of those endogenous neutralizing factors in their blood. Some recent findings on the antimyotoxic factors from the snake serum or plasma with inhibition properties against PLA2 activity and myotoxicity of venoms or toxins are also shortly reviewed.

Amino Acid Sequence↗
PubMed1997

Makatoxin I, a novel toxin isolated from the venom of the scorpion Buthus martensi Karsch, exhibits nitrergic actions.

Buthus martensi Karsch venom exhibits nitrergic action in rat anococcygeus muscle (ACM). We have purified a novel toxin, makatoxin I (MkTx I), which exhibits nitrergic action, to homogeneity from this venom by a combination of gel-filtration, cation-exchange chromatography, and reverse-phase chromatography. Its purity was assessed by capillary electrophoresis and mass spectrometry. Its molecular weight was found to be 7031.71 +/- 2.88 as calculated from electrospray mass spectrographic data. The complete amino acid sequence was elucidated by sequencing of reduced and S-pyridylethylated toxin and a carboxyl-terminal peptide, P55-64, generated by the cleavage of toxin with endoproteinase Lys-C. The complete sequence of MkTx I is GRDAYIADSENCTYTCALNPYCNDLCTKNGAKSGYCQWAGRYGNACWCIDLPDKVPIRISGSCR. This toxin is composed of 64 amino acid residues and contains 8 half-cystine residues. Structurally, MkTx I has high similarity to Bot I and Bot II when compared with toxins from other scorpion species. The effects of MkTx I on nitrergic responses were investigated using the rat isolated ACM mounted in Krebs solution (37 degrees C, 5% CO2 in O2). MkTx I (2 microg/ml) markedly relaxed the carbachol-precontracted ACM; the relaxation was inhibited by the stereoselective inhibitor of nitric oxide synthase, Nomega-nitro-L-arginine methyl ester (50 microM). Thus, MkTx I is the first alpha-toxin that can mediate nitrergic responses in the rat isolated ACM.

Amino Acid Sequence↗
PubMed1997

Complete amino acid sequence of an acidic, cardiotoxic phospholipase A2 from the venom of Ophiophagus hannah (King Cobra): a novel cobra venom enzyme with "pancreatic loop".

A phospholipase A2 (OHV A-PLA2) from the venom of Ophiophagus hannah (King cobra) is an acidic protein exhibiting cardiotoxicity, myotoxicity, and antiplatelet activity. The complete amino acid sequence of OHV A-PLA2 has been determined using a combination of Edman degradation and mass spectrometric techniques. OHV A-PLA2 is composed of a single chain of 124 amino acid residues with 14 cysteines and a calculated molecular weight of 13719 Da. It contains the loop of residues (62-66) found in pancreatic PLA2s and hence belongs to class IB enzymes. This pancreatic loop is between two proline residues (Pro 59 and Pro 68) and contains several hydrophilic amino acids (Ser and Asp). This region has high degree of conformational flexibility and is on the surface of the molecule, and hence it may be a potential protein-protein interaction site. A relatively low sequence homology is found between OHV A-PLA2 and other known cardiotoxic PLA2s, and hence a contiguous segment could not be identified as a site responsible for the cardiotoxic activity.

Amino Acid Sequence↗
PubMed1997

Role of enzymatic activity in the antiplatelet effects of a phospholipase A2 from Ophiophagus hannah snake venom.

A phospholipase A2 (OHV A-PLA2) from the venom of Ophiophagus hannah (King cobra) is an acidic protein exhibiting antiplatelet activity. In in vitro tests, OHV APLA2 showed a marked inhibitory effect on platelet aggregation induced by ADP, collagen and arachidonic acid in both human whole blood and platelet-rich plasma in a dose-dependent manner. The antiplatelet effects of OHV A-PLA2 did not increase when preincubation times of platelets and OHV A-PLA2 were prolonged indicating phospholipid hydrolysis did not significantly contribute to the antiplatelet effects. Alkylation of active site His residue using p-bromophenacyl bromide resulted in the complete loss of enzymatic activity, but the modified enzyme retained more than 30% of its antiplatelet effects. These results indicate that the antiplatelet effects of OHV A-PLA2 appear to be independent of its enzymatic activity, and there are separate sites responsible for the catalytic and antiplatelet activities.

Animals↗
PubMed1997

Cloning and characterization of cDNAs encoding three isoforms of phospholipase A2 in Malayan spitting cobra (Naja naja sputatrix) venom.

cDNAs encoding three phospholipase A2 (PLA2) isoforms in Naja naja sputatrix were cloned and characterized. One of them encoded an acidic PLA2 (APLA) while the others encoded neutral PLA2 (NPLA-1 and NPLA-2). The specific characteristics of APLA and NPLA were attributed to mutations at nt139 and nt328 from G to C and G to A, respectively, resulting in amino acid substitutions from Asp20 and 83 in APLA to His20 and Asn83 in NPLA. Amino acid sequencing of purified protein also showed the presence of this Asp20 and His20 in APLA and NPLA, respectively. The cDNA encoding one of the PLA2 (NAJPLA-2A), when expressed in Escherichia coli, yielded a protein that exhibited PLA2 activity.

Amino Acid Sequence↗
PubMed1997

Renal lesions in rhabdomyolysis caused by Pseudechis australis snake myotoxin.

The renal lesions at various time intervals after i.m. injection of Pseudechis australis myotoxin (PA myotoxin) causing myoglobinuria in mice was studied. Biochemical assay of serum creatine phosphokinase (CK) and lactate dehydrogenase (LDH) showed marked elevations [7166 +/- 2064 IU and 1626 +/- 211 Berger-Broida U/ml (B-B U/ml)] six hours after injection, indicative of rhabdomyolysis. Serum creatinine (1.6 +/- 0.39) and urea (147 +/- 40) showed significant rise by 48 hours indicative of acute renal failure (ARF). Immunodiffusion showed the presence of myoglobin in the urine (myoglobinuria) of experimental animals. Light microscopic (LM) and scanning electron microscopic (SEM) studies of the urinary sediments from experimental mice revealed granular casts of varying size and shape. LM of kidney showed casts from one hour and tubulopathy with degenerated tubular epithelial cell from 12 hours onwards. Focal glomerular changes, such as dilated Bowman's space with poorly stained substance and reduction in number of glomerular tufts were observed. Immunofluorescence microscopy for myoglobin showed fluorescence of the casts in the tubules. Transmission electron microscopy (TEM) showed electron dense casts occupying the entire lumen of the distal convoluted tubules (DCT). The proximal convoluted tubules (PCT) showed features of proximal tubular necrosis (PTN). There was reduction in the basal infolding with activation of lysosomal system in the PCT. The glomeruli showed changes in the visceral epithelium that included intracellular edema, vesiculation and occasional fusion of the podocytes. Numerous granular materials were observed in the Bowman's space as well as in the lumen of the capillaries from 1 to 24 hours. Electron dense deposits of the glomerular basement membrane (GBM) capillaries were observed from 1 to 24 hours. SEM study revealed loss of microvilli of the PCT and some tubular lumen were filled with cast like material. Some glomeruli displayed a relatively flattened podocytes with thickened major foot processes. Regeneration of the tubules were seen from three weeks onwards.

Acute Kidney Injury↗
PubMed1997

Adrenergic and nitrergic responses of the rat isolated anococcygeus muscle to a new toxin (makatoxin I) from the venom of the scorpion Buthus martensi Karsch.

1. Makatoxin I (MkTx I) is a new toxin purified from the venom of the scorpion Buthus martensi Karsch. Contractile (excitatory, adrenergic) and relaxant (inhibitory, nitrergic) responses of the rat isolated anococcygeus muscle (Acm) to MkTx I were investigated. 2. MkTx I (0.28 microM) produced a rapid and very marked rise in the tone of the Acm which then gradually wanted to the control baseline. Phentolamine (5 microM), guanethidine (5 microM), tetrodotoxin (2 microM) and reserpine pretreatment in vivo (5 mg kg-1 s.c. at 24 hr and 5 mg kg-1 i.p. at 3 h) completely blocked the contractile responses of the Acm to MkTx I. The responses to noradrenaline (NA) were blocked by phentolamine, but were potentiated by guanethidine. 3. MkTx I (0.28 microM) also marked and rapidly relaxed the tone of the carbachol (CCh; 3 microM), precontracted Acm. The addition of sodium nitroprusside (SNP; 1 microM) also produced a marked and rapid relaxation of the Acm. TTx (2 microM) or NG-nitro-L-arginine methylester (L-NAME, 50 microM) markedly inhibited the relaxant responses of the Acm to field stimulation (FS) as well as to MkTx I, but not the responses to SNP. 4. Therefore, the contractile responses of the rat anococcygeus muscle to MkTx I can be attributed to the release of transmitter NA on postjunctional alpha-adrenoceptors, whereas the relaxant responses of the Acm to MkTx I involve the release of nitric oxide as the neurotransmitter which, presumably, results in the activation of the enzyme guanylate cyclase leading to relaxation of the muscle.

Amino Acid Sequence↗
PubMed1996

Pathological changes induced by an acidic phospholipase A2 from Ophiophagus hannah venom on heart and skeletal muscle of mice after systemic injection.

An acidic phospholipase A2 (OHV A-PLA2) isolated from the venom of the king cobra (Ophiophagus hannah) was tested for its ability to cause pathological changes to myocardium, skeletal muscle and cardiac ganglia. White mice were injected intravenously with dose of 8 mg/kg or 4 mg/kg of OHV A-PLA2 and tissue samples were taken at 6 or 24 hr. Light microscopic examination failed to show significant changes in cardiac muscle and ganglia. Skeletal muscle showed myofibre degeneration and necrosis. Electron microscopic study revealed myodegeneration in cardiac and skeletal muscles, and reduction in synaptic vesicle population of preganglionic nerve terminals in cardiac ganglia. Ultrastructural changes in tissues were dose related. The lower dose (4 mg/kg) of OHV A-PLA2 produced mild myocardial changes, the myofilaments were intact but contracted, and the A band and I band were skewed. OHV A-PLA2 caused myocardial degeneration at a higher dose of 8 mg/kg. The changes included dissolution of actin and myosin filaments, dilatation and disorganization of sarcoplasmic reticulum and degeneration of mitochondria. The skeletal muscle lesions were more severe than the myocardial changes. Some of the myofibrils were severely disorganized and lack typical striated appearance, sarcomeres disrupted, most of mitochondria were vesiculated and destroyed.

Animals↗
PubMed1996

Synaptosomal binding of 125I-labelled daboiatoxin, a new PLA2 neurotoxin from the venom of Daboia russelli siamensis.

Daboiatoxin (DbTx), the PLA2 neurotoxin from Daboia russelli siamensis venom, was shown to bind specifically and saturably to rat cerebrocortical synaptosomes and synaptic membrane fragments. Two families of binding sites were detected by equilibrium binding analysis in the presence and absence of Ca2+. Scatchard analysis of biphasic plateaus revealed Kdl 5 nM and Bmax1, 6 pmoles/mg protein, and Kd2 80 nM and Bmax2 20 pmoles/mg protein, respectively, for the high- and low-affinity binding sites. The binding of 125I-DbTx to synaptosomes did not show marked dependence on Ca2+, Mg2+, Co2+ and Sr2+. Native DbTx was the only strong competitor to 125I-DbTx synaptosomal binding (IC50 12.5 nM, KI 5.5 nM). Two other crotalid PLA2 neurotoxins, crotoxin CB and mojave toxin basic subunit, and nontoxic C. Atrox PLA2 enzyme, were relatively weaker inhibitors, while two viperid PLA2 neurotoxins, ammodytoxin A and VRV PL V, were very weak inhibitors. Crotoxin CA was a poor inhibitor even at microM concentrations, whereas no inhibitory effect at all was observed with crotoxin CACB, ammodytoxin C, VRV PL VIIIa, taipoxin, beta-bungarotoxin, or with PLA2 enzymes from N. naja venom, E. schistosa venom, bee venom and porcine pancreas. All other pharmacologically active ligands examined (epinephrine, norepinephrine, histamine, choline, dopamine, serotonin, GABA, naloxone, WB-4101, atropine, hexamethonium and alpha-bun-garotoxin) also failed to interfere with 125I-DbTx binding. As those competitors that showed partial inhibition were effective only at microM concentration range compared to the Kd (5 nM) of 125I-DbTx synaptosomal binding, DbTx could well recognize a different neuronal binding site. Rabbit anti-DbTx polyclonal antisera completely blocked the specific binding. When a range of Ca2+ and K+ channels modulators were examined, Ca2+ channel blockers (omega-conotoxins GVIA and MVIIC, taicatoxin, calciseptine and nitrendiprene) did not affect the binding even at high concentrations, while charybdotoxin was the only K+ channel effector that could partially displace 125I-DbTx synaptosomal binding amongst the K+ channel blockers tested (apamin, dendrotoxin-I, iberiotoxin, MCD-peptide, 4-aminopyridine and tetraethylammonium), suggesting that neither K+ nor Ca2+ channels are associated with DbTx binding sites.

Animals↗
PubMed1996

Establishment of an animal model for myoglobinuria by use of a myotoxin from Pseudechis australis (king brown snake) venom in mice.

A new laboratory animal model for studying the pathologic mechanisms of myoglobinuria in mice after envenomation with Pseudechis australis snake venom or its myotoxin has been established. The experimental mice (Swiss albino) had myoglobinuria 60 min after administration of the venom, as indicated by red or dark-brown urine. Light microscopic studies revealed myonecrosis of the locally injected soleus muscle 30 min after exposure to the myotoxin, followed by regeneration in 7 to 10 days. Electron microscopic studies of the soleus muscle revealed fragmentation and dissolution of the Z disk, followed by degeneration of the sarcomere. Light microscopy of the kidneys revealed numerous pigmented casts filling the lumen of the tubules; some tubules had features of acute tubular necrosis. Immunohistochemical localization of myoglobin by the immunoperoxidase method confirmed myoglobin casts in the renal tubules. Electron microscopy of the kidneys also revealed intratubular casts composed of markedly electron-dense material filling the lumen. These results indicate that rhabdomyolysis caused by the venom or toxin is followed by myoglobinuria, with renal manifestations in the form of myoglobin cast nephropathy and tubulopathy. This mouse model of experimental snake venom-induced myoglobinuria is an ideal model for investigating the entire sequence of myoglobinuria and related cast nephropathy.

Animals↗
PubMed1995

A major lethal factor of the venom of Burmese Russell's viper (Daboia russelli siamensis): isolation, N-terminal sequencing and biological activities of daboiatoxin.

A major lethal factor, daboiatoxin (DbTx), showing strong PLA2 activity (specific activity 91.7 nmoles/min/mg), was purified to homogeneity from the venom of Burmese Russell's viper (Daboia r. siamensis) by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on CM-Sephadex C-25, followed by purification on high-performance gel filtration Shim-pack Diol-150 column. DbTx is a single-chain PLA2 toxin with approximate mol. wt 15,000 as determined by HPLC gel filtration and SDS-PAGE. It constitutes 12% of total venom protein and is the main lethal component of Burmese Russell's viper venom with an LD50 i.p. (0.05 mg/kg) 12-fold greater than that of the whole venom (LD50 i.p. 0.6 mg/kg). DbTx produces neurotoxic symptoms in mice and exhibits potent oedema-inducing activity (minimum oedema dose 0.05 microgram), indirect haemolytic activity and a strong myonecrotic activity, but no haemorrhagic activity. DbTx is cytotoxic to HeLa cells causing cytolysis of the cells 24 hr post-exposure to toxin (50 micrograms/ml). The first 20 N-terminal amino acid sequence (NFFQF AEMIV KMTGK EAVHS) shows a significant resemblance to those of the PLA2s from the venoms of Bulgarian viper (V. a. ammodytes) and Taiwan Russell's viper (V. r. formosensis).

Amino Acid Sequence↗
PubMed1995

Buthus martensi karsch venom: prejunctional adrenergic activity in the rat isolated anococcygeus muscle.

The effect of Buthus martensi Karsch venom (MKV) on adrenergic responses was investigated using the rat isolated anococcygeus muscle (Acm), since several scorpion venoms can cause peripheral sympathetic nerve stimulation with enhanced adrenergic responses. The effects of phentolamine (5 microM), guanethidine (5 microM), tetrodotoxin (2 microM), desipramine (1.5 microM) and reserpine pretreatment in vivo (5 mg/kg s.c. x 24 hr and 5 mg/kg i.p. x 3 hr) on contractile responses of the rat Acm to field stimulation, noradrenaline (3 microM), tyramine (10 microM), crude MKV (2 micrograms/ml), carbachol (3 microM) and potassium chloride (50 mM) were compared. Phentolamine, guanethidine, tetrodotoxin and reserpine pretreatment completely blocked the contractile responses of the Acm to MKV and to field stimulation but desipramine potentiated the responses. The responses to NA were completely blocked by phentolamine, but were potentiated by guanethidine, desipramine and reserpine pretreatment. The contractile responses to tyramine were completely blocked by phentolamine, desipramine and reserpine pretreatment. The low doses (0.1 microgram/ml x 3) of MKV, which did not produce any observable increase in tone of the anococcygeus muscle, potentiated the contractile responses to field stimulation, but not the responses to exogenous NA. Thus, the adrenergic agonist action of MKV in the rat isolated anococcygeus muscle is mediated by some prejunctional mechanism(s) of action, presumably stimulating the release of the neurotransmitter noradrenaline.

Animals↗
PubMed1995

Involvement of the L-arginine-nitric oxide synthase pathway in the relaxant responses of the rat isolated anococcygeus muscle to a scorpion (Leiurus quinquestriatus quinquestriatus) venom.

Venom from several species of scorpions can cause generalized depolarization of peripheral nerves with enhancement of neurotransmitter release. The effects of the venom (LQV) from the scorpion Leiurus quinquestriatus quinquestriatus were investigated using the rat isolated carbachol (CCh) precontracted anococcygeus muscle (Acm) mounted in Krebs solution containing phentolamine (5 microM). LQV (0.2 microgram/ml and 1.5 micrograms/ml) markedly relaxed the tone of the CCh precontracted (non-stimulated) Acm by 31.5 +/- 5.1% and 35.5 +/- 4.7%, respectively; the onset was immediate after the high dose, followed by a slow and gradual return of the muscle tone to 88.7 +/- 1.8% of the initial peak tension in 50.2 +/- 4 min. Subsequent doses of LQV produced essentially no appreciable changes in muscle tone, but the addition of L-arginine (250 microM) or, especially sodium nitroprusside (SNP; 1 microM) produced marked and rapid relaxations of the Acm. Similar results were obtained with LQV during electrical field stimulation (EFS) of the precotracted Acm; however, during the gradual return of the muscle tone from the relaxed state induced by LQV, the EFS-induced relaxant (NANC) responses were also progressively inhibited by 84.9 +/- 3.4% and 64.3 +/- 4.5 by LQV 0.2 microgram/ml and 1.5 micrograms/ml, respectively. Tetrodotoxin (TTx; 2 microM) or NG-nitro-L-arginine methylester (L-NAME; 50 microM) markedly inhibited the relaxant responses of the Acm to EFS as well as to LQV but not the responses to SNP: L-arginine (250 microM) partially restored the relaxant (NANC) responses of the Acm to EFS. Thus, the L-arginie-nitric oxide synthase-nitric oxide pathway is involved in mediating the marked relaxant responses of the CCh precontracted Acm to LQO.

Animals↗
PubMed1995

Anticoagulant effects of Pseudechis australis (Australian king brown snake) venom on human blood: a computerized thromboelastography study.

The crude venom of Pseudechis australis exhibited a dose-dependent anticoagulant action on human blood in vitro using computerized thromboelastography. Clot progress parameters (K and alpha) were affected at low dose levels which had no effect on onset of coagulation parameters (SP, R). At high dose levels there was total anticoagulant effect, but in all cases there was no evidence of fibrinolytic activity. These results generally agree with the known effects of this venom on coagulation in vivo, and further support our earlier suggestion that TEG may be a useful, one-step tool in the diagnosis and monitoring of the progress of envenomation patients.

Anticoagulants↗
PubMed1995

Morphological changes induced by a generalized myotoxin (myoglobinuria-inducing toxin) from the venom of Pseudechis australis (king brown snake) in skeletal muscle and kidney of mice.

A myotoxin causing myoglobinuria was isolated from the venom of Pseudechis australis (PA myotoxin). Myoglobinuria was observed in mice 60 min post-injection (4.5 mg/kg i.m.) into calf muscles. Light microscopic observation revealed hypercontraction of muscle fibres with delta lesions and vacuolation. Severe necrosis was observed as early as 30 min. Infiltration of the muscle fibres with macrophages was seen by 3 hr with peak infiltration by 12-48 hr. Electron microscopic study showed pathological changes in skeletal muscle as early as 5 min. Electron microscopic study showed disruption of the sarcolemma with dissolution and degeneration of the Z-band. Degeneration of the I-band was followed by degenerative changes in the A-band. Regeneration of muscle was evident by 3-5 days by the presence of many myotubes containing central nuclei. Regeneration was almost complete by 3 weeks. Contralateral soleus muscle which was not injected with toxin also showed degeneration followed by regeneration with central nuclei. Light microscopic studies of kidney showed myoglobin casts in both proximal and distal tubules, collecting ducts and loops of Henle. We conclude that this myotoxin probably acts on the Z-disc structures and also causes renal damage due to 'myoglobin cast nephropathy'.

Animals↗