Experimental osteoporosis induced in female albino rats and its antagonism by Indian black scorpion (Heterometrus bengalensis C.L.Koch) venom

The present study was designed to explore the antiosteoporosis activity of the Indian black scorpion (Heterometrus bengalensis) venom on experimental osteoporosis female albino rats. Sham operated control rats were designated as Gr I, Gr II animals served as osteoporosis control, Gr III osteoporosis rats were treated with SV (1/25th of MLD), Gr IV osteoporosis rats were treated with 1/50th of MLD of SV and Gr V osteoporosis rats were treated with standard (calcium and vit-D3). As compared with the Gr I rats, the Gr II rats showed typical osteoporosis changes in increased of urinary Ca²⁺, PO₄³⁻, CRE, OH-P levels, serum/plasma Ca²⁺, PO₄³⁻, TRAP, IL1, IL6, TNFα and PTH level, bone Ca²⁺, PO₄³⁻, Mg²⁺, Zn²⁺ and decreased level of serum/plasma ALP, EST and PTH, bone Na⁺. In Gr III, Gr IV and Gr V rats, the osteoporosis changes of urine, serum and bone, were significantly restored as compared with the Gr II rats. The bone dimensions, morphology and histological changes observed in Gr II rats were restored in Gr III, Gr IV and Gr V rats. This study confirms that the Indian black scorpion venom may influence bone remodeling process by stimulating bone formation and reducing bone resorption process of osteogenesis.     

Fractionation and lethality of venom from the scorpion Buthus minax (L. Koch)

The venom from the scorpion Buthus minax was fractionated into seven components by cellulose acetate electrophoresis, using acetate buffer pH 4·2. Two of the electrophoretic fractions were lethal to mice, while the crude venom and one of the lethal fractions increased the amplitude of the twitches of the rat phrenic nerve-diaphragm preparation; the other lethal fraction produced a blocking effect. 5-Hydroxytryptamine was not demonstrated in the venom; however, paper chromatography of the dialysate from, or the acetone extract of the crude venom revealed the presence of 18 ninhydrin stainable spots and two u.v. fluorescent spots. Thirteen of the ninhydrin spots were identified as amino acids.     

Immunological studies of scorpion (Buthus minax, L. Koch) venom

An antivenom was prepared for Buthus minax venom by hyper-immunizing rabbits. The antivenom protected the rats against the hypertensive effect of the venom but it did not prevent the respiratory arrest. The antivenom also protected the rats against several times the lethal dose. In mice the antivenom completely protected the animals against doses of the venom equal to the ld₅₀ but no such protection was achieved when doses equal to 5 times the ld₅₀ were injected.Using the immunodiffusion and immunoelectrophoresis techniques, four precipitin bands were revealed with B. minax venom but only one band was formed with Leiurus quinquestriatus venom and it was not identical with any of the bands formed with B. minax. A cross-reaction occurred between the band formed with L. quinquestriatus venom and one of the bands formed with B. minax venom. The antivenom did not prevent the hypertensive or the lethal actions of L. quinquestriatus venom.In view of the abundance of B. minax in the Sudan and other neighbouring countries and the absence of an antivenom for B. minax in the polyvalent antiscorpion venoms, it is suggested that the B. minax antivenom ought to be included in the polyvalent scorpion antivenin.     

Pharmacological studies of the venom from the scorpion Buthus minax (L. Koch)

The venom from the scorpion Buthus minax produced a positive inotropic effect on isolated rabbit and guinea pig hearts but no alteration in rate. The cardiac stimulant action was blocked by propranolol and was absent in reserpinized hearts, indicating the possibility of an indirect action of the venom, probably through the release of catecholamines. In the reserpinized hearts, and in normal hearts after propranolol treatment, the venom produced bradycardia which was blocked by atropine. The venom produced a marked hypertensive effect in cats, dogs, rats and guinea pigs, which was blocked by tolazoline and phenoxy-benzamine. In cats and rats the hypertensive effect was preceded by brief hypotension which was partially blocked by atropine. The venom markedly decreased the rate of flow in the perfused hindquarter of the rat and moderately increased capillary permeability. It stimulated the rabbit intestine and guinea pig ileum, an action which was largely blocked by atropine. It increased the size of twitches in the isolated, indirectly stimulated, phrenic hemidiaphragm of the rat and contracted the rectus abdominis muscle of the frog. The latter effect was blocked by tubocurarine. The venom decreased the respiration rate in cats and dogs but caused a slight increase in depth. The decrease in respiration rate was blocked by carotid sinus denervation. It is postulated that the venom stimulates both the sympathetic and parasympathetic systems.     

Purification and properties of hyaluronidase from Palamneus gravimanus (Indian black scorpion) venom.

Scorpion venoms are a rich source of enzymes. Some of the enzymes such as phospholipase A2, proteolytic enzymes and phosphodiesterase are well characterized. However, hyaluronidase has not been studied extensively. In this paper we describe the purification and characterization of hyaluronidase (Hyaluronate lyase, E.C.3.2.1.35) from the Palamneus gravimanus scorpion venom by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose. The optimal pH and temperature for its maximum activity of the isolated enzyme were 4.5 and 37 degrees C, respectively, and its K(m) was 47.61 microg/ml at 37 degrees C and its specific activity was 6411.7 +/- 117TRU/min per mg against 250 +/- 4.0 TRU/min per mg for the whole desiccated venom suggesting 25-fold purification. The molecular weight of the isolated enzyme was 52 +/- 1 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography on Sephadex G-75. The enzyme was stable for 30 days in the presence of NaCl; no loss of activity was observed up to 37 degrees degrees C and showed a sharp decrease in its activity at 40 degrees C. Heparin inhibited the enzyme activity.     

Electrocardiographic studies with scorpion (Buthus minax, L. Koch) venom.

The intravenous injection of Buthus minax venom into rabbits caused an initial brief bradycardia followed by tachycardia and then a prolonged bradycardia. The initial bradycardia is probably due to central vagal stimulation, while the tachycardia is possibly due to sympathetic stimulation and release of tissue catecholamines. The venom caused depression of the ST segment and symmetrical inversion of the T wave possibly due to coronary vasoconstriction leading to anterior wall infarction. Some of the electrocardiographic effects of the venom appeared to be mediated through hyperkalemia and hypocalcemia as evidenced from the tall, slender and peaked T wave, the wide QRS complex and the prolonged ST segment. The effect of the venom in causing sympathetic stimulation and release of tissue catecholamines seemed to mask the effects of electrolytes, since more pronounced effects were shown after blocking the B-adrenergic receptors with propranolol or depleting the catecholamine stores with reserpine. Treatment with atropine, aminophylline or the specific antivenin failed to protect the rabbits against the electrocardiographic effects of the venom.     

Glycemic effect of venom from the scorpion Buthus minax (L. Koch)

The venom from the scorpion Buthus minax caused hyperglycemia in rats. Tolazoline and specific antivenom prevented or abolished this effect while propranolol did not modify the hyperglycemic response. Liver glycogenolysis seems to be the major contributing factor for this rise in blood glucose.     

Dose-dependent serum biochemical alterations in Wistar albino rats after Palamneus gravimanus (Indian black scorpion) envenomation

Palamneus gravimanus envenomated rats showed dose-dependent alterations in serum biochemical parameters. Sub lethal doses of 100, 200, and 400 microg/kg of P. gravimanus venom were injected intramuscularly into rats. Blood samples were collected by heart puncture before and 4 h after crude venom administration. Serum was analyzed for glucose, blood urea nitrogen (BUN), uric acid, total protein, cholesterol, sodium, potassium, inorganic phosphorus, alkaline phosphatase, aspartate aminotransferase (AST-SGOT), alanine amino-transferase (ALT-SGPT), lactate dehydrogenase (LDH), and creatinine phosphokinase (CPK). Statistically significant increases in serum levels of glucose, creatinine, AST, ALT, BUN, CPK, and LDH and significant decreases in serum levels of total protein, uric acid, cholesterol, calcium, and potassium 4 h after venom administration could be due to the toxic action of P. gravimanus venom on certain organs in rats.     

Purification and characterization of Heteroscorpine-1 (HS-1) toxin from Heterometrus laoticus scorpion venom.

Crude venom from Thai giant scorpion, Heterometrus laoticus, most commonly found in the northeastern area of Thailand, was evaluated for PD50 (paralytic dose 50) activities from abdominal injection to cricket (Gryllus sp.) and activities against various kinds of microorganisms. It exhibited good results in disc diffusion assay for Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. After purification, toxin showed acceptable results for PD50 determination in entrapped cricket as well as inhibitory activity against B. subtilis, K. pneumoniae, and P. aeruginosa with activities over 300 times higher than that of the crude venom. The purified fraction was showed to contain a single band in SDS-PAGE. MALDI-TOF-MS/MS analysis showed one peak of major protein with 8293Da. Partial amino acid sequence show high homology to Scorpine-a polypeptide toxin family with potassium channel blocking and defensin activity. The novel toxin was named "Heteroscorpine-1" (HS-1) as the first Scorpine from genus Heterometrus. After full length determination by PCR, HS-1 gene was found to be composed of two exons, separated by an intron. Deduction revealed 95 amino acid residues with 19 residues as the leading sequence. It showed about 80% similarity to Panscorpine and Opiscorpine.     

Effect of Buthus minax (L. Koch) scorpion venom on plasma and urinary electrolyte levels

B. minax venom was injected into rats in a total dose equal to the ld₅₀ and divided into five doses (days 0, 1, 3, 4, 5). The venom caused a significant decrease in urine volume in days 1, 2, 4, 5 and 6 with an increase on day 3. The oliguria was suggested to be due to the release of the antidiuretic hormone secondary to acetylcholine release by the venom and anticholinesterase activity of the venom. Urinary sodium and potassium were significantly decreased the first day of venom injection possibly due to stress and secondary shock resulting from acute adrenal insufficiency. In the subsequent days of venom injection urinary sodium was significantly decreased and that of potassium significantly increased. The effect was suggested to be due to secretion of aldosterone. Urinary calcium was significantly decreased and phosphate significantly increased. A parallelism was found between the urinary excretion of sodium and calcium. The effect was suggested to be partly due to decreased calcium absorption from the intestine as the venom significantly decreased the in vitro absorption of calcium through the everted rat duodenum.     

Scorpion (Buthus minax, L. Koch) venom fractions with anticholinesterase activity

M. F. El-Asmar, M. Ismail, Kh. Ghoneim and O. H. Osman. Scorpion (Buthis minax, L. Koch) venom fractions with anticholinesterase activity. Toxicon15, 63–69, 1977.—Anticholinesterase activity was demonstrated in crude scorpion (Buthus minax) venom. Venom fractions with similar activities were separated by cellulose acetate electrophoresis at pH 8·4 and by Biogel P 100 fractionation. Anticholinesterase activity was demonstrated towards both acylcholine-acyl-hydrolase (EC 3.1.1.8) and acetycholine-acetyl-hydrolase (EC 3.1.1.7). Both the crude venom and the fractions containing anticholinesterase activity increased the height of twitches of the rat phrenic nerve-hemidiaphragm preparation. The crude venom but not the fractions produced contracture of the cat tibialis anterior muscle preparation, followed by complete blockade of twitch activity. The crude venom markedly potentiated the contraction of the frog rectus abdominis muscle induced by acetylcholine, while none of the fractions showed such activity. Neither the crude venom nor the various fractions showed any effect on the superior cervical ganglion preparation of the cat nictitating membrane.     

A heat stable protein toxin (drCT-I) from the Indian Viper (Daboia russelli russelli) venom having antiproliferative, cytotoxic and apoptotic activities.

A heat stable 7.2kDa protein toxin (drCT-I) has been purified and crystallized from Indian Daboia russelli russelli venom (Roy Choudhury et al., 2006. Acta Cryst. F Struct Biol Cryst Commun, 62(Pt. 3), 292). The N-terminal (first 20) amino acid sequence of drCT-I was LKCNKLVPLFYKTCPAGKNL, which showed sequence homology to cytotoxins isolated from Naja venom. drCT-I has been evaluated for anticancer activity against EAC cells in vivo and human leukemic cells (U937, K562) in vitro. drCT-I (125 microg/kg, i.p/day for 10 days) significantly decreased EAC cell count, cell viability (p<0.001) and significantly increased the survival time of tumour bearing mice (T/C% 178.64, p<0.01) in comparison to untreated tumour bearing control. drCT-I, produced dose and time-dependent inhibition of U937 and K562 cell growth and had an IC50 of 8.9 and 6.7 microg/ml respectively after 24h treatment. The reduced MTT values after drCT-I treatment indicated its cytotoxic nature, which supported its antiproliferative action. Scanning electron microscopy and confocal microscopy in U937 and K562 cells after drCT-I treatment indicated certain features of apoptosis such as membrane blebbing, perforations, nuclear fragmentation. The induction of apoptosis was further confirmed by phosphatidylserine externalization observed using annexinV-FITC/PI staining and flow cytometric analysis. drCT-I brought about apoptosis by G1 phase arrest of the cell cycle. The effect of drCT-I on normal human peripheral blood mononuclear cell (PBMNC) viability and cytotoxicity was studied in culture and was found to be lower than that on U937 and K562 cells. Thus both in vivo and in vitro experimental results suggested that drCT-I possessed anticancer potential.     

Identification of high molecular weight serine-proteases in Loxosceles intermedia (brown spider) venom.

High molecular weight serine-proteases have been identified in Loxosceles intermedia (brown spider) venom. The mechanism by which Loxosceles spp venoms cause dermonecrotic injury (a hallmark of loxoscelism) is currently under investigation, but it seems to be molecularly complex and in some instance proteases might be expected to play a role in this skin lesion. In the present investigation, when we submitted L. intermedia venom to linear gradient 3-20% SDS-PAGE stained by a monochromatic silver method we detected a heterogeneous protein profile in molecular weight, ranging from 850- to 5-kDa. In an attempt to detect zymogen molecules of proteolytic enzymes, venom aliquots were treated with several exogenous proteases. Among them, trypsin activated two gelatinolytic molecules of 85- and 95-kDa in the venom. In experiments of hydrolysis inactivation using different protease inhibitors for four major class of proteases, we detected that only serine-type protease inhibitors were able to inactivate the 85- and 95-kDa enzymes in the venom. An examination of the 85- and 95-kDa gelatinolytic activities as a function of pH showed that these proteases had no apparent activities at pH below 5.0 and higher than 9.0 and displayed little activity at pH 6.0. with the optimal pH for their activities ranging from 7.0 to 8.0. Evaluation of the functional specificities of the 85- and 95-kDa venom proteases showed that these proteases efficiently degrade gelatin (denatured collagen) but have no proteolytic activity on hemoglobin, immunoglobulin, albumin, librinogen or laminin, suggesting specificity of their proteolytic actions. We describe here two serine-proteases activities in L. intermedia venom probably involved in the harmful effects of the venom.     

Identification of a novel pulmonary oedema producing toxin from Indian red scorpion (Mesobuthus tamulus) venom.

The experiments were conducted to identify the toxin that produces pulmonary oedema in Mesobuthus tamulus (BT) envenomed animals. Crude BT venom was subjected to Sephadex gel filtration (G-75) and the fractions were screened for optical density (OD), neurotoxicity (prolongation of compound action potential in frog sciatic nerve) and lethality. All these parameters exhibited a peak between 54-94 ml eluates. Fractions of this peak were pooled (SP) and loaded on to carboxymethyl cellulose column. The column was then eluted with increasing buffer concentrations at constant pH and temperature. Eluates were screened for neurotoxicity and OD. Four peaks of neurotoxic activity (T1-T4) were detected. T2 and T3 were lethal whereas T1 and T4 were non-lethal. T2 exhibited mainly neurotoxicity and failed to augment phenyldiguanide (PDG)-induced reflex response or to produce pulmonary oedema. T3 was having minimal neurotoxic actions but augmented PDG-reflex and produced pulmonary oedema. The effects of T3 persisted even after dialysis with 8 kDa cut-off filter but not those of T2. The T3 effects resembled toxic manifestations of BT venom and were blocked by aprotinin pre-treatment. T3 demonstrated a band at approximately 100 kDa in SDS-PAGE. The results demonstrate the presence of a lethal, high molecular weight, pulmonary oedema producing toxin in BT venom.     

A novel low molecular weight hemorrhagic toxin from Trimeresurus flavoviridis (Vorojima) venom

A low molecular weight hemorrhagic toxin, LMHT, was isolated from the venom of Trimeresurus flavoviridis from Yorojima using Q-Sepharose column chromatography.The purified hemorrhagic toxin was homogeneous by disc polyacrylamide gel electrophoresis at pH 8.3, and Ouchterlony immunodiffusion. LMHT has a molecular weight of 16,500 and possesses hemorrhagic activity. It did not show casein, azocasein, azoalbumin, or TAME (tosyl-L-arginine methyl ester hydrochloride) hydrolytic activities. Hemorrhagic activity was inhibited by EDTA, TEP (tetraethylenepentamine), p-APMSF(p-amidinophenylmethanesulfonylfluoride HCl), and N-bromosuccinimide. The minimum hemorrhagic dose of this hemorrhagic toxin was 7.1 microg/mouse. LMHT induced hydrolysis of the Aa and Bbeta chains of bovine fibrinogen.     

A lethal cardiotoxic-cytotoxic protein from the Indian monocellate cobra (Naja kaouthia) venom

A lethal cardiotoxic-cytotoxic protein (mol. wt. 6.76 kDa) has been purified from the Indian monocellate cobra (Naja kaouthia) venom by ion-exchange chromatography and HPLC. CD spectra indicated the presence of 23% α helix, 19% β sheets and 35% coil. Complete amino acid sequence was determined by MALDI, which showed similar homology with cardiotoxins/cytotoxins isolated from venom of other Naja species. Intraperitoneal LD₅₀ was 2.5 mg kg⁻¹ in BalbC male mice. In vitro cardiotoxicity studies on isolated guinea pig auricle showed that the molecule produced auricular blockade that was abolished after trypsin treatment. Cytotoxicity studies on human leukemic U937 and K562 cells showed that it significantly inhibited cell proliferation in a dose and time dependent manner, as observed by trypan blue exclusion method and tetrazolium bromide reduction assay. IC₅₀ on U937 and K562 cells were 3.5 μg/ml and 1.1 μg/ml respectively. Morphometry and cell sorting studies indicated apoptosis induction in toxin treated leukemic cells. Apoptosis was caspase 3 and 9 dependent and the treated leukemic cells were arrested in sub-G1 stage. There was an increase in Bax-Bcl2 ratio, decrease in HSP (Heat shock protein) 70 and HSP90 and induction of PARP cleavage after NK-CT1 treatment. The toxin showed low cytotoxic effect on normal human leukocytes as compared with imatinib mesylate. Further detailed cytotoxic and cardiotoxic effects at the molecular level are in progress.     

Occurrence of a unique protein toxin from the Indian King Cobra (Ophiophagus hannah) venom.

A unique (lethal-cardiotoxic-hemorrhagic) protein toxin (Toxin CM55) was isolated and purified from Indian King Cobra (Ophiophagus hannah) venom by CM-sephadex ion exchange chromatography and reverse phase HPLC. The purified toxin had an SDS-molecular weight of 22 +/- 0.5 kD. UV absorption spectra of Toxin CM55 showed a peak at 280 nm, whereas when excited at 280 nm fluorescence, Toxin CM55 showed an E(max) at 333.4 nm. Toxin CM55 had an LD(50) of 28.28 microg/20 g (i. v.) in albino mice. The cardiotoxic action of the toxin was established on isolated guinea pig/rabbit heart and guinea pig auricle. In rats, Toxin CM55 caused ECG abnormalities including widened QRS complex and monomorphic ventricular tachycardia suggesting that the possible site of action of Toxin CM55 was the ventricle. Toxin CM55 produced significant vasoconstriction on peripheral blood vessels. It produced significant contraction of isolated guinea pig ileum, rat fundus and rat uterus, which was completely antagonised by methysergide. The toxin was found to release a significant amount of serotonin from rabbit platelets. Toxin CM55 produced cutaneous hemorrhage in albino mice, which was also produced in reserpine and p-chloro phenylalanine pretreated animals. Rabbit antiserum was raised against Toxin CM55, which gave prominent bands in immunogel diffusion and immunoelectrophoresis. The antiserum provided 2 LD(50) protection against Toxin CM55-induced lethality in mice and also neutralised 3 MHD hemorrhagic dose of the toxin.     

Effect of scorpion (Buthus minax, L. koch) venom on succinic dehydrogenase and cholinesterase activity of mouse tissue

The histochemical activities of succinic dehydrogenase and cholinesterase in mouse tissues after administration of scorpion (Buthus minax) venom have been studied. There were obvious changes in the enzyme activities, of the chronically treated animals, in the kidney, heart and liver. The decreases in cholinesterase activity were greatest in the white matter of the brain, in the portal tract and in some liver cells, while the spleen showed increased activity. A decrease in succinic dehydrogenase activity was observed in the kidney, heart, and liver of the chronically treated mice, while the spleen showed no change in the activity. The striated muscles of the acutely treated mice were fragmented and vacuolated with a loss of their striation. The intramuscular nerve axons became beaded and the subneural apparatus and their terminal expansions were smaller.