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J R Giglio

Publications and source records attributed to J R Giglio.

At least 19 recordsLinked to original sources
PubMed1992

What is tityustoxin?

Tityus serrulatus scorpion venom was fractionated by gel filtration affording two heterogeneous toxic fractions, T1 and T2; the latter was further fractionated by ion-exchange chromatography. The fraction of T2 eluted with 0.15 M ammonium acetate buffer, originally named 'tityustoxin', was shown to be a pool of several proteins. One of them, TsTX, as well as T1, was also named 'tityustoxin'. The major and perhaps most potent toxin of the venom, gamma-toxin, was eluted with 0.30 M buffer as a highly purified protein and shown to be different from TsTX. gamma-Toxin is contained in both T1 and T2.

Chromatography, Gel↗
PubMed1992

Effects induced by bothropstoxin, a component from Bothrops jararacussu snake venom, on mouse and chick muscle preparations.

Bothropstoxin, a 13,700 mol. wt myotoxic phospholipase homologue isolated from the venom of Bothrops jararacussu and devoid of PLA2, proteolytic or hemolytic activities, inhibited muscle twitch tension, evoked either directly or indirectly through stimulation of the motor nerve in the mouse phrenic-diaphragm preparations. The compound action potential of the muscle was also abolished with a similar time course. In addition, the toxin (0.7 mM) evoked membrane depolarization which was inhibited in the presence of 10 mM Ca2+. In chick biventer cervicis muscle, the toxin (2 mM) induced a contracture that reached its maximum amplitude in 44.8 +/- 15.6 min (n = 6) and was not blocked by either d-tubocurarine or tetrodotoxin. The time to maximum amplitude was reduced to 5.5 +/- 1.0 min (n = 4) in nominally Ca(2+)-free Krebs solution and was completely abolished in Ca(2+)-free Krebs solution containing 1 mM EGTA.

Action Potentials↗
PubMed1991

Skeletal muscle degeneration and regeneration after injection of bothropstoxin-II, a phospholipase A2 isolated from the venom of the snake Bothrops jararacussu.

A myotoxic phospholipase A2, named bothropstoxin II (BthTX-II), was isolated from the venom of the South American snake Bothrops jararacussu and the pathogenesis of myonecrosis induced by this toxin was studied in mice. BthTX-II induced a rapid increase in plasma creatine kinase levels. Histological and ultrastructural observations demonstrate that this toxin affects muscle fibers by first disrupting the integrity of plasma membrane, as "delta lesions" were the earliest morphological alteration and since the plasma membrane was interrupted or absent in many portions. In agreement with this hypothesis, BthTX-II released peroxidase entrapped in negatively charged multilamellar liposomes and behaved as an amphiphilic protein in charge shift electrophoresis, an indication that its mechanism of action might be based on the interaction and disorganization of plasma membrane phospholipids. Membrane damage was followed by a complex series of morphological alterations in intracellular structures, most of which are probably related to an increase in cytosolic calcium levels. Myofilaments became hypercontracted into dense clumps which alternated with cellular spaces devoid of myofibrillar material. Later on, myofilaments changed to a hyaline appearance with a more uniform distribution. Mitochondria were drastically affected, showing high amplitude swelling, vesiculation of cristae, formation of flocculent densities, and membrane disruption. By 24 hr, abundant polymorphonuclear leucocytes and macrophages were observed in the interstitial space as well as inside necrotic fibers. Muscle regeneration proceeded normally, as abundant myotubes and regenerating myofibers were observed 7 days after BthTX-II injection. By 28 days regenerating fibers had a diameter similar to that of adult muscle fibers, although they presented two distinctive features: central location of nuclei and some fiber splitting. This good regenerative response may be explained by the observation that BthTX-II does not affect blood vessels, nerves, or basal laminae.

Actin Cytoskeleton↗
PubMed1991

Further characterization of toxins T1IV (TsTX-III) and T2IV from Tityus serrulatus scorpion venom.

Toxins T1IV (TsTX-III) and T2IV have been purified to homogeneity from Tityus serrulatus scorpion venom and further characterized. Their amino acid composition and SDS-PAGE reveal an approximate mol. wt of 7000. Their intracisternal LD50 (micrograms/kg) in mice were 12.9 +/- 1.6 and 3.0 +/- 0.5, while their N-terminal amino acid sequences were K-E-G-Y-A-M-D-H-E-G-C-K-F-S- and K-E-G-Y-L-M-D-H-E-G-C-K-L-S-C-F-I-R-P-S-G-Y-C-G-R-E-, respectively. This sequence of T2IV, its amino acid composition and its chromatographic and electrophoretic behaviour identify it as toxin gamma (TsTX-I), which is the major toxin from this venom. TsTX-III (13 to 102 micrograms/kg) produced a long lasting enhancement of the hypertensive effect of noradrenaline and a slight decrease of the hypotensive effect of acetylcholine, while T2IV (115 micrograms/kg) induced a prolonged hypotensive effect on the anesthetized rat. On the isolated guinea-pig vas deferens, TsTX-III (2.1 and 3.0 micrograms/ml) produced a horizontal shift of the dose-response curve for noradrenaline to the left with no change of the maximal response. At a concentration of 1.43 microM, it induced a prolongation of the duration of the B component of the compound action potential. This prolongation was strongly reduced after addition of tetrodotoxin.

Acetylcholine↗
PubMed1991

The hypotensive activity of Crotalus atrox (western diamondback rattlesnake) venom: identification of its origin.

Viperidae snakes belonging to the genus Crotalus, subfamily Crotalinae, include the species durissus terrificus, durissus durissus, adamanteus, atrox, cerastes, horridus, molossus, scutulatus, viridis and others. All of them, except for the first 2, are found in North America. Crotalus atrox, or diamondback rattlesnakes, live in the southwest United States and in Mexico. This paper describes the fractionation of C. atrox venom in order to isolate and identify its hypotensive agents.

Amino Acid Sequence↗
PubMed1990

The complete amino acid sequence of toxin TsTX-VI isolated from the venom of the scorpion Tityus serrulatus.

The complete sequence of the toxin TsTX-VI from the venom of the scorpion Tityus serrulatus Lutz and Mello is presented. The sequence has been determined by automated Edman analysis of the reduced and carboxymethylated protein as well as of the resulting peptides, obtained from S. aureus protease and tryptic digestions. TsTX-VI is composed of 62 residues and has a calculated molecular weight of 6717. Homology studies with other scorpion toxins show that TsTX-VI is more similar to the Old World than to the North American scorpion toxins. The hydropathic index indicates that TsTX-VI is more hydrophobic than Ts-gamma. Toxicity studies carried out in mice demonstrate that i.v. injection of TsTX-VI is unable to evoke the usual symptoms induced by the typical neurotoxins of this venom, but only a generalized allergic reaction. These properties are important in clarifying the relationship between primary structure and biological function of scorpion toxins.

Amino Acid Sequence↗
PubMed1990

Primary structure and biological activity of bradykinin potentiating peptides from Bothrops insularis snake venom.

Several bradykinin potentiating peptides (BPPs) were isolated from the venom of the Brazilian arboricole snake Bothrops insularis by gel filtration on Sephadex G-150-120, followed by sequencial high-voltage paper electrophoreses at pH 3.5, 6.5, and 2.1. The BPPs were assayed by their ability to potentiate the contractile activity, on the isolated guinea pig ileum, and the hypotensive activity, on anesthetized rats, of bradykinin. Eight BPPs, containing 3-13 amino acid residues, were sequenced and their primary structures were shown to have a marked degree of homology with those of several BPPs from other venoms.

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of hemorrhagic, myonecrotic and edema-inducing toxins from Bothrops insularis (jararaca ilhoa) snake venom.

Bothrops insularis snake venom was fractionated by gel filtration on Sephadex G-150 followed by ion-exchange chromatography on SP-Sephadex C-25. Two active fractions were purified to homogeneity: (1) SIII-SpI, approximate mol. wt 32,000 and N-terminal amino acid residue Val. This fraction showed esterase activity on TAME, edema-inducing activity on the rat hind paw and contractile activity on the isolated guinea pig ileum. The latter two activities were antagonized by benadryl plus methysergide; (2) SIII-SpVI, a myonecrotic and edema-inducing phospholipase, approximate mol. wt 29,000, N-terminal amino acid residue pyro-Glu, consisting of two chains of approximately 15,000 mol. wt each linked by disulphide bridge(s). The induction of edema by this fraction was not antagonized by benadryl plus methysergide, indomethacin, BW755C or BN52021, but it was antagonized by dexamethasone. Three highly purified hemorrhagic heterodimeric fractions, SIII-SpIII-3, SIII-SpIII-4 and SIII-SpIII-5, of approximate mol. wts 26,000, 29,000 and 26,000, and having N-terminal residues of Asx, Asx and Gly, respectively, were further isolated by preparative polyacrylamide slab gel electrophoresis. SIII-SpIII-4 and SIII-SpIII-5 increased the recalcification time of citrated rat plasma. None of the five isolated fractions showed any proteolytic (on casein) or kininogenase activity.

Animals↗
PubMed1990

Isolation and characterization of toxic proteins from the venom of the Venezuelan scorpion Tityus discrepans (Karsch).

Four toxic, electrophoretically homogeneous proteins were isolated by ion-exchange chromatography on CM-cellulose-52 from the venom of the scorpion Tityus discrepans (range North Central Venezuela), named TdIV, TdV, TdVIII and TdIX. Component TdVIII, with 56 amino acid residues and mol. wt 6140 was the most toxic by i.p. injections into mice and had an intracisternal LD50 of 7.9 micrograms protein/kg body weight. Amino acid compositions of components TdIV and TdV were very similar, suggesting that they could be highly homologous proteins, although presumably contaminated one by the other. A fifth component, named TdIII, non-toxic by i.p. injections, was also isolated in homogeneous form. The i.v. and intracisternal LD50 values of the whole T. discrepans venom were 2.5 mg/kg and 16.0 micrograms/kg, respectively.

Amino Acids↗
PubMed1990

Interchain disulfide bonds in crotamine self-association.

Crotamine, a basic neurotoxic protein, was isolated from the venom of the Southern Brazilian rattlesnake (Crotalus durissus terrificus) by gel filtration. The isolated protein showed a single band on PAGE at pH 4.5 and 7% (w/v) gel concentration, but two or more bands at 14% gel concentration, even in the presence of 4 M urea. After reduction and carboxymethylation, however, a single band was again detected. SDS-PAGE as well as ultracentrifugal analysis of the native (NC) and of the reduced and carboxymethylated (RCC) crotamine revealed a molecular weight of 4,500-5,000 for RCC and 9,000-10,000 for NC. Both components of a two-band crotamine preparation were isolated by preparative PAGE and characterized. Their particular electrophoretic mobility was retained. Their amino acid composition. N-terminal residue, and apparent toxicity were the same as those of the original sample. It was concluded that crotamine is able to form a dimer of 9,760 Da with two identical polypeptide chains crosslinked by interchain disulfide bonds and a shape not very far from spherical, which covalently binds extra subunits of 4,880 Da each.

Animals↗
PubMed1989

A proton nuclear magnetic resonance study on the solution structure of crotamine.

Proton nuclear magnetic resonance (NMR) spectra of crotamine, a myotoxic protein from a Brazilian rattlesnake (Crotalus durissus terrificus), have been analyzed. All the aromatic proton resonances have been assigned to amino acid types, and those from Tyr-1, Phe-12, and Phe-25 to the individual residues. The pH dependence of the chemical shifts of the aromatic proton resonances indicates that Tyr-1 and one of the two histidines (His-5 or His-10) are in close proximity. A conformational transition takes place at acidic pH, together with immobilization of Met-28 and His-5 or His-10. Two sets of proton resonances have been observed for Ile-17 and His-5 or His-10, which suggests the presence of two structural states for the crotamine molecule in solution.

Amino Acid Sequence↗
PubMed1989

A simplified procedure for the fractionation of Tityus serrulatus venom: isolation and partial characterization of TsTX-IV, a new neurotoxin.

Five toxins from the venom of the Brazilian scorpion Tityus serrulatus were purified to homogeneity by a combination of ion exchange chromatography with ammonium bicarbonate buffer (pH 7.8) on CM-cellulose-52 and rechromatography on the same resin equilibrated with ammonium acetate buffer (pH 4.7). Four of these proteins, obtained in one or two steps in high yield and lethality (named toxins IX3, IX5, and X4 and XIII) were shown to be identical with other toxins already described. A fifth one, TsTX-IV, is reported as a new toxin. Except for IX3, which showed Gly as the sole N-terminal residue, the other four toxins showed Lys. TsTX-IV has an approximate mol. wt of 6880, an i.v. LD50, in mice, of 826 +/- 156 micrograms/kg and an intracisternal LD50 of 11 +/- 9 micrograms/kg, compared to 375 +/- 45 and 4.9 +/- 0.8, respectively, for the whole venom extract. It has 61 amino acid residues and an amino acid composition different from that of any other toxin from Tityus serrulatus venom so far described. Toxins IX5, TsTX-IV and XIII induced a prejunctional type of supersensitivity on the guinea pig vas deferens, probably due to an increased release of noradrenaline.

Amino Acids↗
PubMed1989

Effects of the venom of the Brazilian scorpion Tityus serrulatus and two of its fractions on the isolated diaphragm of the rat.

1. The effects of Tityus serrulatus venom and of two of its toxic fractions, toxin gamma (Tx gamma) and T2III1, on the rat isolated diaphragm were examined. 2. The crude venom (5 ng) facilitated the neuromuscular transmission and increased the twitch tension evoked by retrograde injection of Ach. 3. Tx gamma (25-100 ng) and fraction T2III1 (2.5 ng) also facilitated the neuromuscular transmission but only fraction T2III1 increased the twitch tension evoked by retrograde injection of Ach. 4. Tx gamma (50 ng) and fraction T2III1 (2.5 ng) produced a tetrodotoxin-sensitive increase in the frequency of miniature endplate potentials (m.e.p.p.) and a transitory reduction of the resting potential. The latter effect of the fractions was prevented by treating muscles with tetrodotoxin or D-tubocurarine. Fraction T2III1 also produced a tetrodotoxin-resistance increase of m.e.p.p. amplitude. 5. These results suggest that Tx gamma enhances Ach output through the activation of Na+ channels in the motor nerve terminals. Fraction T2III1 produced effects similar to those induced by Tx gamma but also acted at postjunctional sites, probably by increasing subsynaptic membrane sensitivity to the neurotransmitter.

Animals↗
PubMed1989

Convulxin does not induce convulsions when injected into the rat dorsal hippocampus.

The search for new and selective neurotoxins has attracted several efforts in the recent years. Convulxin, a neurotoxin isolated from the Crotalus durissus terrificus venom, was reported to elicit convulsions upon intravenous injection in mice and cats. Therefore we have studied the behavioral, electroencephalographic and neuropathological effects of convulxin when microinjected into the dorsal hippocampus of rats. To test the hypothesis that substances released by convulxin from platelets and not convulxin itself is the convulsive agent, we injected a mixture of convulxin and platelet rich plasma. Results demonstrated that neither convulxin nor convulxin/platelet rich plasma exhibited neurotoxic effects upon intrahippocampal injection.

Animals↗
PubMed1988

Fractionation of Bothrops jararacussu snake venom: partial chemical characterization and biological activity of bothropstoxin.

A myotoxin, bothropstoxin (BthTX), showing no detectable phospholipase A2 activity, was purified to homogeneity from the venom of the Brazilian snake Bothrops jararacussu by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on SP-Sephadex C-25. Four phospholipases (Sm-SP1 to Sm-SPIV) were also isolated, the latter showing, similarly to BthTX (Sm-SPv) myonecrotic activity. Approximate mol. wts, as determined by SDS-PAGE, and pI of Sm-SPI to Sm-SPIV are: 22,400-4.2; 15,500-4.8; 13,800-6.9; and 13,200-7.7, respectively. BthTX is a single chain protein, approximate mol. wt 13,000, with 16 half-cystine residues, pI = 8.2 and LD50 = 7.5 mg/kg (i.p.) and 4.8 mg/kg (i.v.) for 20 g mice. The ten first N-terminal amino acid residues show a significant homology to other toxins with phospholipase structure. BthTX is specifically myotoxic, contrary to crude B. jararacussu venom which, although also myotoxic, affects intramuscular arteries and veins leading to thrombosis. BthTX and Sm-SPIV also differ from toxins isolated from the venom of other Brazilian snakes which are strongly hemorrhagic.

Amino Acid Sequence↗
PubMed1987

Isolation and characterization of a thrombin-like enzyme from the venom of the snake Bothrops insularis (jararaca ilhoa).

A thrombin-like enzyme (I-SII-R) from the venom of the Brazilian snake Bothrops insularis (jararaca ilhoa) was purified to homogeneity by gel filtration on Sephadex G-150 followed by precipitation of contaminating proteins with half-saturated NaCl and two further gel filtrations under the same conditions. I-SII-R showed specific activities of 820 clotting units/mg protein and 530 TAME units/mg protein (a 23-fold purification) and a single precipitation band against antibothropic horse antiserum by immunoelectrophoresis, as well as a single band by PAGE and by SDS-PAGE with beta-mercaptoethanol. The estimated molecular weight was 45,000, of which the neutral sugars account for 9900, or 22%. Its amino acid composition, which accounts for a minimum mol. wt of 33,900 is as follows: Asx35, Thr18, Ser17, Glx15, Pro29, Gly22, Ala27, Val21, Cys18, Met7, Ile19, Leu25, Tyr11, Phe11, His7, Lys8, Arg17, Trp5. When compared to thrombin, I-SII-R is relatively stable at 45 degrees C and 60 degrees C, pH 7.4, but is inhibited by heparin at a higher rate than thrombin. Both enzymes hydrolyze the alpha and beta chains of fibrinogen and lose more than 95% of their clotting and esterase activities when treated with phenylmethylsulphonyl fluoride. In contrast to thrombin, I-SII-R does not activate factor XIII.

Amino Acids↗
PubMed1986

Toxin gamma of the scorpion Tityus serrulatus modifies both activation and inactivation of sodium permeability of nerve membrane.

The effects of the major neurotoxic fraction isolated from scorpion venom of Tityus serrulatus, TiTx gamma, on peripheral nerve membrane of Xenopus laevis were studied under current- and voltage-clamp conditions. 700 nmol/l TiTx gamma depolarized the membrane and induced spontaneous activity (150 s-1, maximum value), which ceased within a few minutes. It reduced the amplitude of the action potentials from 109 mV to 52 mV and increased their duration from 1.25 ms to 4.5 ms. 440 nmol/l TiTx gamma induced inward Na current flow at resting potential. The descending branch of the Na current-voltage curve was flattened and shifted approximately 10 mV to more negative potentials. Maximum Na permeability was reduced to about 20%. Both development of and recovery from inactivation of Na permeability were slowed. The steepness of the steady-state inactivation curve was decreased, but the mid-potential changed only insignificantly. No prepulse was necessary to elicit either a shift of activation or an inward current at resting potential. Expressing the toxin effect either in terms of the decrease of Na peak current or of the slowing of inactivation, half-maximum effects were found with 0.3 +/- 0.1 and 3.7 +/- 0.7 mumol/l TiTx gamma, respectively.

Action Potentials↗