Biomedical subjects

L D Possani

Publications and source records attributed to L D Possani.

At least 109 records · Page 6Linked to original sources

PubMed1992

Amino acid sequence and immunological characterization with monoclonal antibodies of two toxins from the venom of the scorpion Centruroides noxius Hoffmann.

Two toxins, which we propose to call toxins 2 and 3, were purified to homogeneity from the venom of the scorpion Centruroides noxius Hoffmann. The full primary structures of both peptides (66 amino acid residues each) was determined. Sequence comparison indicates that the two new toxins display 79% identity and present a high similarity to previously characterized Centruroides toxins, the most similar toxins being Centruroides suffusus toxin 2 and Centruroides limpidus tecomanus toxin 1. Six monoclonal antibodies (mAb) directed against purified fraction II-9.2 (which contains toxins 2 and 3) were isolated in order to carry out the immunochemical characterization of these toxins. mAb BCF2, BCF3, BCF7 and BCF9 reacted only with toxin 2, whereas BCF1 and BCF8 reacted with both toxins 2 and 3 with the same affinity. Simultaneous binding of mAb pairs to the toxin and cross-reactivity of the venoms of different scorpions with the mAb were examined. The results of these experiments showed that the mAb define four different epitopes (A-D). Epitope A (BCF8) is topographically unrelated to epitopes B (BCF2 and BCF7), C (BCF3) and D (BCF9) but the latter three appear to be more closely related or in close proximity to each other. Epitope A was found in all Centruroides venoms tested as well as on four different purified toxins of C. noxius, and thus seems to correspond to a highly conserved structure. Based on the cross-reactivity of their venoms with the mAb, Centruroides species could be classified in the following order: Centruroides elegans, Centruroides suffusus suffusus = Centruroides infamatus infamatus, Centruroides limpidus tecomanus, Centruroides limpidus limpidus, and Centruroides limpidus acatlanensis, according to increasing immunochemical relatedness of their toxins to those of Centruroides noxius. All six mAb inhibited the binding of toxin 2 to rat brain synaptosomal membranes, but only mAb BCF2, which belongs to the IgG2a subclass, displayed a clear neutralizing activity in vivo.

Amino Acid Sequence↗

PubMed1992

Isolation and physiological characterization of taicatoxin, a complex toxin with specific effects on calcium channels.

Taicatoxin is a new complex oligomeric toxin that was isolated from the venom of the Australian taipan snake Oxyuranus scutellatus scutellatus. It is composed of three different molecular entities: an alpha-neurotoxin-like peptide of mol. wt 8000, a neurotoxic phospholipase of mol. wt of 16,000 and a serine protease inhibitor of mol. wt 7000, linked by non-covalent bonds, at an approximate stoichiometry of 1:1:4. The most active form of the complex was isolated by ion exchange chromatography through DE-Cellulose followed by two steps of CM-Cellulose chromatography at pH 4.7 and pH 6.0, respectively. At this stage the complex migrates as a single component in beta-alanine-acetate-urea gel electrophoresis and is very toxic to mice (1 or 2 micrograms of the complex protein kills a mouse of 20 g within 2 hr). It blocks the high threshold calcium channel current of excitable membranes in heart and does not affect the low threshold calcium channel current. The block occurs at a site that is accessible extracellularly but not intracellularly. The block is selective for calcium channels, reversible, does not affect single channel conductance but only changes channel gating, and is voltage dependent with higher affinity for inactivated channels. The phospholipase activity of the complex toxin can be separated by affinity-chromatography using a phospholipid analog (PC-Sepharose). The resulting complex contains only alpha-neurotoxin and protease inhibitor and is still capable of blocking calcium channels, although with less potency than the native oligomeric form. Sephadex G-50 gel filtration chromatography in the presence of high salt (1M NaCl) at alkaline pH (8.2), separates the alpha-neurotoxin-like peptide from the protease inhibitor, but at this stage the resulting peptides lose physiological activity towards the calcium channels. The amino acid sequence of the protease inhibitor was determined by automatic Edman degradation. The alpha-neurotoxin-like peptide and two isosubunits displaying phospholipase activity were sequenced at the N-terminal part of the molecule.

Alkylation↗

PubMed1992

Dendrotoxin-like effects of noxiustoxin.

Noxiustoxin from the Mexican scorpion (Centruroides noxius Hoffmann) is known to block neuronal K+ channels. Noxiustoxin facilitated acetylcholine release in chick biventer cervicis nerve-muscle preparations, but not in mouse phrenic nerve-hemidiaphragm preparations. Noxiustoxin displaced binding of a radiolabelled dendrotoxin from synaptosomal membranes from rat brain, with a Ki of 10(-10) M. It is concluded that noxiustoxin shares some pharmacological properties with the K+ channel blocking dendrotoxins.

Animals↗

PubMed1992

Noxiustoxin and leiurutoxin III, two homologous peptide toxins with binding properties to synaptosomal membrane K+ channels.

Noxiustoxin (NTX), a short-chain peptide toxin from the scorpion C. noxius, binds to a single class of non-interacting binding sites in brain synaptosomal membranes with a KD = 160-300 nM and a Bmax = 2.2 pmols.mg-1 protein. The K+ channel blocking agents tetraethylammonium, 4-aminopyridine and Charybdotoxin partially inhibit the binding of [125I]NTX, while a variety of toxins targeted against Ca2+ and Na+ channels have no effect, suggesting that NTX interacts with a bona fide K+ channel protein. NTX totally displaces the binding of [125I]Leiurutoxin III, a novel peptide from L. quinquestriatus venom with N-terminal amino acid sequence homologous to that of NTX. These results suggest that both toxins bind to a common receptor site and are promising tools to dissect the molecular mechanisms of channel-toxin interaction.

4-Aminopyridine↗

PubMed1992

Characteristics of pancreatic exocrine secretion produced by venom from the Brazilian scorpion, Tityus serrulatus.

The influence of venom (TSV) from the Brazilian scorpion, Tityus serrulatus, on exocrine pancreatic secretion was studied in relation to known cholinergic and peptidergic secretagogue activity. Pulse-labeling followed by chase incubation in the presence of secretagogues and various pharmacological agents revealed unique physiological characteristics of TSV in guinea pig pancreatic lobules. Exocytotic discharge of newly synthesized 3H-labeled proteins during a 3-h chase incubation showed a marked increase over basal discharge levels using logarithmic TSV doses of 0.10 to 100 micrograms/ml. This stimulation was comparable to maximal values elicited by carbachol, cholecystokinin-octapeptide (CCK-8) or caerulein and discharge kinetics were similar. TSV-mediated secretion was ATP and calcium dependent and partially inhibited by atropine. Only tetrodotoxin completely blocked TSV stimulation of newly synthesized protein discharge. Both botulinum toxin and curare had no effect on venom stimulation, indicating that TSV interaction with exocrine pancreatic cells occurs postsynaptically. Verapamil, a calcium channel antagonist, produced a moderate inhibition of TSV stimulation. When antagonists to the cholecystokinin (CCK) receptor were incubated with TSV, no change in secretory activity occurred. Therefore, TSV does not bind to CCK receptors and probably operates through its own receptor which may be an ion channel. Additionally, morphological studies in vitro revealed a high level of pancreatic secretory activity as evidenced by dense secretory acinar luminal content, reduction in zymogen granule (ZG) population, and development of exocytotic images.

Amylases↗

PubMed1992

Use of synthetic peptides corresponding to sequences of Mycobacterium leprae proteins to study delayed-type hypersensitivity response in sensitized guinea pigs.

In this work we report the synthesis of 10 peptides (P1-P10) corresponding to one or several segments of the amino acid sequence of proteins from Mycobacterium leprae: 65 kDa, 28 kDa, 18 kDa, and 28 kDa superoxide dismutase, recently renamed antigens 2L, 9L, 12L, and 14L, respectively. They were assayed in the guinea pig model for the induction of a delayed-type hypersensitivity response in M. leprae and BCG-sensitized animals. To sensitize the animals two schemes were used: either a single dose of 5 x 10(9) irradiated or autoclaved whole bacilli, or four weekly intramuscular injections each containing 500 micrograms of soluble extract of M. leprae (MLSE) in incomplete Freund's adjuvant. Because the second scheme used far too much antigen, we decided to use the first scheme for the experiments we report here. DTH reactions of sensitized animals were induced after 30 days with intradermal injections of 5 micrograms of MLSE and with each of the 10 peptides at three different concentrations: 250 micrograms, 100 micrograms, and 0.05 micrograms. All M. leprae-sensitized guinea pigs gave indurations of 10 mm or more with MLSE, which indicates that the animals were sensitized. None of them gave DTH indurations with 250 micrograms or 100 micrograms, but some of them had positive DTH reactions with the 0.05 micrograms doses of the synthetic peptides. This is most likely due to the fact that we have used an outbred strain of guinea pigs. The peptides were also tested at 0.05 micrograms in animals sensitized with BCG. P7 and P10 seem to be nonspecific peptides; the remaining peptides only induced DTH in the M. leprae-sensitized guinea pigs. P3 (segments 65-85 of the 65-kDa protein) induced a positive DTH in 58% of the tested animals. In other experiments, guinea pigs were sensitized with a single injection (500 micrograms) of each of the synthetic peptides. All animals, except those sensitized with P4 and P8, had positive DTH responses when the homologous peptide was used. Those sensitized with P2, P4, P5, P7, and P8 were able to produce indurations when MLSE was used for the induction of the DTH reaction.

Amino Acid Sequence↗

PubMed1991

Discharge effect on pancreatic exocrine secretion produced by toxins purified from Tityus serrulatus scorpion venom.

Three toxic polypeptides were purified from the venom of the Brazilian scorpion Tityus serrulatus by means of gel filtration in Sephadex G-50 and ion-exchange chromatography in carboxymethylcellulose. The peptides are basic molecules with molecular weights in the range of 7000 for which the amino acid compositions and sequences were determined. The effect of the purified peptides on pancreatic exocrine secretion in the guinea pig was studied. Biochemical measurements show that the cells are stimulated by these peptides to discharge their zymogen granules. Light and electron microscopic images confirm the biochemical measurements. At the light microscope level, acinar cells show dramatically fewer zymogen granules than in control pancreas with the appearance of large vacuoles and some loss of morphological integrity. Electron micrographs display apical regions devoid of zymogen granules and condensing vacuoles whereas acinar lumina contain crystalline secretory material. The secretory effect observed in vitro is comparable to that of carbamylcholine and that of the peptidergic secretagogue cholecystokinin-pancreozymin.

Amino Acid Sequence↗

PubMed1990

Bradykinin-induced potassium current in cultured bovine aortic endothelial cells.

Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca2+ concentration, [Ca2+]i, accompanied by an increase in surface membrane K+ permeability. In this study, electrophysiological measurement of K+ current was combined with 86Rb+ efflux measurements to characterize the K+ flux pathway in BAECs. Bradykinin- and Ca2(+)-activated K+ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin, noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca2+ activation is in the range of 10 to 100 nM [Ca2+]i. The whole-cell current measurements show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K+ current present in control conditions, an inwardly rectifying K+ current, was blocked by Ba2+ and was not affected by noxiustoxin or tetrabutylammonium chloride. Efflux of 86Rb+ from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and by noxiustoxin, but not by apamin or furosemide. Thus, 86Rb+ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca2(+)-activated membrane currents. The results confirm that bradykinin-stimulated 86Rb+ efflux occurs via Ca2(+)-activated K+ channels. The blocking agents identified may provide a means for interpreting the role of the Ca2(+)-activated K+ current in the response of BAECs to bradykinin.

Animals↗

PubMed1990

Characterization of the venom from Crotalus molossus nigrescens Gloyd (black tail rattlesnake): isolation of two proteases.

The venom from Crotalus molossus nigrescens contains many activities including: hyde powder azure proteinase; N-benzoyl-arginine-ethyl-ester hydrolase; phospholipase; phosphodiesterase; desoxyribonuclease; fibrinogen coagulase; collagenase, fibrinolytic activity, and hemorrhagic factors. The venom, assayed with amounts of venom up to 50 micrograms protein per assay, does not contain acetylcholinesterase, phosphatase, amylase, ribonuclease, tyrosyl-ester hydrolase or hyaluronidase activities. The venom is lethal to mice with an i.p. LD50 of 2.35 mg/kg mouse. Fractionation of soluble venom by Sephadex G-75 separates at least five families of components. Fractions I-III contains all the enzymes, and fraction V have six small peptides. Further separation of fractions II-III on diethyl-amino-ethyl-cellulose columns at pH 8.0 and 8.3 gave pure proteinase E with a mol. wt of 21,390 and the following N-terminal amino acid sequence; Phe-Ala-Lys-Arg-Tyr-Val-Glx-Leu-Val-Ile-Val-Ala. A thrombin-like enzyme with a mol. wt of 75,000 was also purified from this venom by means of affinity and ion exchange chromatographies.

Amino Acid Sequence↗

PubMed1990

Isolation and characterization of helothermine, a novel toxin from Heloderma horridum horridum (Mexican beaded lizard) venom.

A protein toxic to mice was isolated from the venom of the Mexican beaded lizard Heloderma horridum horridum by a combination of gel filtration (Sephadex G-75) and ion exchange chromatography (both diethylaminoethyl-cellulose [DE-cellulose] and carboxymethyl-cellulose [CM-cellulose]). The purified polypeptide component has an apparent mol. wt of 25,500 and is composed of approximately 220 amino acid residues. It has an isoelectric point (pI) of 6.8 and its N-terminal amino acid sequence was shown to be: Glu-Ala-Ser-Pro-Lys-Leu-Pro-Gly-Leu-Met-Thr-Ser-Asn-Pro-Asp-Gln-Gln-Thr- Glu-Ile. The sequence has no significant similarity with any other protein previously reported in the literature. Enzymatic activities such as phospholipase, hyaluronidase and proteinase, commonly present in venoms, could not be demonstrated in this protein. Patch-clamp experiments conducted with excitable membranes show no effects on Na+, K+ or Ca2+ ion channels. Among the constant physiological effects observed in mice injected with this toxin are lethargy, partial paralysis of rear limbs and lowering of body temperature, suggesting that it might be a hypothermic toxin. We propose calling this toxin Helothermine.

Amino Acid Sequence↗

PubMed1989

Purification and reconstitution of potassium channel proteins from squid axon membranes.

Voltage-dependent K+ channels are responsible for repolarization of the cell membrane during the late phase of the action potential. Here we report the purification of proteins from squid axon membranes which bind the K+-channel blocker noxiustoxin (NTX), and their subsequent functional reconstitution in planar bilayers. The NTX-affinity purified proteins had Mr values of 60,000 +/- 6,000, 160,000 +/- 15,000 and 220,000 +/- 20,000. Their incorporation into bilayers resulted in single-channel currents with three conductances, the most frequent one of 11 pS in 300/100 mM KCl (cis/trans). The voltage dependence, reversal potential and bursting behavior suggest that these are the K+ channels involved in the squid axon action potential.

Animals↗

PubMed1989

Synthetic peptides corresponding to the sequence of noxiustoxin indicate that the active site of this K+ channel blocker is located on its amino-terminal portion.

A nonapeptide Thr-Ile-Ile-Asn-Val-Lys-Cys-Thr-Ser (NTX1-9) and a decapeptide Met-Asn-Gly-Lys-Cys-Lys-Cys-Tyr-Asn-Asn (NTX30-39) corresponding to the N-terminal and C-terminal sequences respectively of Noxiustoxin (NTX) were synthesized by the solid phase method of Merrifield (1963). The first synthetic peptide (NTX1-9) was shown to be toxic to mice independently of the route of administration: intraperitoneally, subcutaneously or intraventricularly (100-200 micrograms/20 g mouse weight). The second (NTX30-39) was not toxic even at higher dose (400 micrograms/20 g mouse). When the effects of the peptide NTX1-9 and of the authentic toxin (Noxiustoxin) were studied on the liberation of [3H] 4-aminobutyric acid (3H-GABA) from mouse synaptosomes, both gave essentially the same results, except that peptide NTX1-9 was needed at higher concentration. Synthetic peptide NTX30-39 had no effect in the same preparation at even higher doses. The GABA release produced by toxic peptide NTX1-9 was not affected by tetrodotoxin but was completely abolished by the presence of the K+ ionophore valinomycin, mimicking the effect of native NTX in the same system (Sitges et al., 1986). These results indicate that the toxic active site of Noxiustoxin is possibly located in or near the N-terminal amino acid portion of the molecule.

Amino Acid Sequence↗

PubMed1989

Modification of Na channel gating by an alpha scorpion toxin from Tityus serrulatus.

The effects of TsIV-5, a toxin isolated from the Brazilian scorpion Tityus serrulatus, on whole-cell and single-channel Na currents were determined in N18 neuroblastoma cells. In whole-cell records at a test potential of -10 mV, external application of 500 nM TsIV-5 slowed inactivation 20-fold and increased peak current by about one-third without changing time-to-peak. Both the steady-state activation and inactivation curves were shifted to more negative potentials. Other alpha scorpion toxins produce similar effects but the single-channel mechanism is not known. TsIV-5 caused a voltage-dependent prolongation of mean single-channel open time such that at a test potential of -60 mV no change was observed, whereas at -20 mV mean open time increased about threefold and prolonged bursting was observed. Macroscopic current reconstructed from summed single-channel records showed a characteristic toxin-induced potentiation of peak current and a 20-fold slowing of the decay phase. TsIV-5 does not discriminate between tissue-specific Na channel subtypes. Prolonged open times and bursting were also observed in toxin-treated Na channels from rat ventricular myocytes, rat cortical neurons, and mouse skeletal muscle. The toxin effects are shown to be consistent with a kinetic model in which TsIV-5 selectively interferes with the ability of the channel to reach the inactivated state.

Animals↗

PubMed1988

Charybdotoxin and noxiustoxin, two homologous peptide inhibitors of the K+ (Ca2+) channel.

We show that noxiustoxin (NTX), like charybdotoxin (CTX) described by others, affects Ca2+-activated K+ channels of skeletal muscle (K+(Ca2+) channels). Chemical characterization of CTX shows that it is similar to NTX. Although the amino-terminal amino acid of CTX is not readily available, the molecule was partially sequenced after CNBr cleavage. A decapeptide corresponding to the C-terminal region of NTX shows 60% homology to that of CTX, maintaining the cysteine residues at the same positions. While CTX blocks the K+ (Ca2+) channels with a Kd of 1-3 nM, for NTX it is approx. 450 nM. Both peptides can interact simultaneously with the same channel. NTX and CTX promise to be good tools for channel isolation.

Animals↗

PubMed1988

Isolation of several toxins from the venom of the scorpion Centruroides limpidus tecomanus Hoffmann.

The soluble venom of the scorpion of Colima, Mexico, was fractionated by Sephadex G-50 gel filtration followed by separation of the toxic fraction (number II) with carboxymethyl-cellulose columns in 20 mM ammonium acetate buffer, pH 4.7. Of 24 fractions five were toxic to mice and were further separated in 50 mM phosphate buffer, pH 6.0 using the same ion exchange resin. Final separation included chromatography of the toxic subfractions in the same resin, but in 75 mM ammonium acetate buffer, pH 5.0. By this procedure at least five distinct toxins were obtained in pure form according to gel electrophoresis analysis. Amino acid composition of toxin 1 (component II.20.3.4) and toxic component II.22.5 is included. The N-terminal amino acid sequence of component II.22.5 was shown to be: Lys-Glu-Gly-Tyr-Ile-Val-Asn-Tyr-His-Thr-Gly-Cys-Lys-Tyr-Thr-Cys-Ala-Lys- Leu-Gly - Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Lys-.

Amino Acid Sequence↗

PubMed1988

Amino acid sequence and physiological characterization of toxins from the venom of the scorpion Centruroides limpidus tecomanus Hoffmann.

The complete amino acid sequence of the major toxic component (II.20.3.4), named toxin 1, from the venom of the Mexican scorpion C. l. tecomanus is reported. The sequence (66 amino acids) was obtained by direct Edman degradation of reduced and alkylated toxin, followed by sequence determination of selected peptides separated after enzymatic cleavage with S. aureus V8 protease. In cultured chick dorsal root ganglion cells, 0.5 microM toxin 1 slowed down specifically the time course of Na+ current inactivation, while Ca2+ currents from the same preparation were little affected. In neonatal rat ventricular heart cells, toxin 1, at concentrations between 0.1 and 0.5 microM, reduced Na+ currents without changing the kinetics and Ca2+ currents were unaffected. Comparative analysis of the primary structure of this toxin with other scorpion toxins shows a high degree of similarity with the north American scorpion toxins. This analysis suggests that the 'fine tuning' of the molecular mechanism of action of these toxins is related to variations in the primary structure as well as to the type of membrane under study (tissue specificity).

Action Potentials↗

PubMed1988

Isolation and characterization of two toxins from the Mexican scorpion Centruroides limpidus limpidus Karsch.

1. Several toxic polypeptides were found in the venom of the scorpion Centruroides limpidus limpidus. Comparative studies of the potency of the venom in different strains of mice were conducted. 2. A new type of toxin (component II.9), specific for crustaceans (crayfish and isopods), was isolated from this scorpion and was shown to have the following N-terminal amino acid sequence: Lys-Lys-Asp-Gly-Tyr-Leu-Val-Asn-Lys-Tyr-Thr-Gly-Cys-Lys-Val-Asn-Cys- Tyr-Lys-Leu-Gly-Glu-Asn-Lys-Phe-Cys-Asn-Arg-Glu-. 3. A polypeptide toxic to mice (component II.6) from this venom was shown to have the following N-terminal sequence: Lys-Glu-Gly-Tyr-Leu-Val-Asn-His-Ser-Thr-Gly-Cys-Lys-Tyr- Glu-Cys-Tyr-Lys-Leu-Gly-Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Lys-. 4. In cultured chick dorsal root ganglion cells, 1 microM of toxin II.6 was shown to reduce the size of sodium currents and to slow-down their activation-inactivation kinetics. The toxin had also a depressive action on the classical Ca2+ current activated at high membrane potentials (greater than 0 mV).

Amino Acid Sequence↗

PubMed1988

Effects of New World scorpion toxins on single-channel and whole cell cardiac sodium currents.

Purified toxins from a North American scorpion, Centruroides noxius (Cn II-10), and a South American scorpion, Tityus serrulatus (Ts-gamma), were tested on cardiac sodium channels using patch-clamp methods to record whole cell and single-channel currents. The two toxins produced similar effects on sodium currents; potassium and calcium currents were not affected. Macroscopic sodium current amplitudes, measured at test potentials greater than -20 mV where the opening probability was high, decreased in a concentration-dependent manner with a half maximum inhibitory concentration of 6 X 10(-8) M. Block was unchanged by repetitive depolarizing pulses. In the presence of scorpion toxin, the currents were rapidly blocked by tetrodotoxin (3 X 10(-5) M). Both toxins shifted the voltage dependence of sodium channel inactivation to more negative potentials. At test potentials between -50 and -70 mV, where the sodium channel opening probability is normally low, both toxins produced an increase in sodium current and slowed the rates of activation and inactivation. At intermediate potentials between -50 and -20 mV the currents in the presence of toxins crossed over the control currents. At a test potential of -20 mV, the toxins decreased single-channel activity and increased the latency to first opening. At a test potential of -60 mV, the toxins significantly prolonged channel open time. The unitary current amplitudes were unchanged at either potential. We conclude that New World scorpion toxins produce apparently complex effects on whole cell currents primarily by retarding activation gating of cardiac sodium channels.

Animals↗