PubMed Health⌕ Search

Biomedical subjects

B Lomonte

Publications and source records attributed to B Lomonte.

At least 91 records · Page 5Linked to original sources

Production and partial characterization of monoclonal antibodies to Bothrops asper (terciopelo) myotoxin.

Seven murine monoclonal antibodies against Bothrops asper myotoxin were produced and partially characterized. They revealed the presence of at least four cross-reacting basic components in crude venom, with a common subunit mol. wt of 16,000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis, but slight differences in charge. These myotoxin-related components might be isoforms of the toxin. By Western blotting and enzyme-immunoassay binding techniques, differences in the reactivities with basic venom fractions were observed among monoclonal antibodies, suggesting differences in epitope specificities. Three antibodies cross-reacted with B. nummifer crude venom. Two monoclonal antibodies were utilized to develop a two-site enzyme-immunoassay for myotoxin detection at the nanogram level. Three of the antibodies (one IgM and two IgG1) showed ability to neutralize myotoxicity of purified myotoxin in preincubation type experiments.

Animals↗

Antibody neutralization of a myotoxin from the venom of Bothrops asper (terciopelo).

Neutralization of biological activities of B. asper myotoxin by a monospecific anti-myotoxin rabbit serum and polyvalent antivenom was studied. Both antisera neutralized myotoxic, phospholipase A and lethal activities of myotoxin, whereas edema-forming activity of myotoxin was not neutralized by these antisera. Anti-myotoxin rabbit serum neutralized most of the myotoxic activity of B. asper venom, but did not neutralize phospholipase A activity of this venom. Neutralization curves showed that myotoxicity induced by myotoxin persisted at antivenom/toxin or antiserum/toxin ratios at which phospholipase A activity was completely neutralized. This suggests that myotoxicity does not depend upon enzymatic activity of the toxin. Antiserum to myotoxin neutralized approximately 70% of myonecrosis induced by crude B. asper venom. This demonstrates that myotoxin is the main factor responsible for the development of myonecrosis in envenomations by B. asper.

Animals↗

Effects of a myotoxic phospholipase A2 isolated from Bothrops asper venom on skeletal muscle sarcoplasmic reticulum.

The myotoxin from B. asper snake venom inhibited Ca-ATPase activity of rabbit sarcoplasmic reticulum after incubation in vitro. Inhibition was non-competitive and albumin enhanced the effect of the toxin. Furthermore, B. asper myotoxin hydrolyzed sarcoplasmic reticulum phospholipids and induced a dose-dependent release of horseradish peroxidase that had been trapped in sarcoplasmic reticulum vesicles. Binding studies indicated that myotoxin does not bind to any particular protein of this membrane, suggesting that the toxin might interact with phospholipids. Inhibition of Ca-ATPase is probably a consequence of an alteration in sarcoplasmic reticulum phospholipids.

Animals↗

Detection of proteins antigenically related to Bothrops asper myotoxin in crotaline snake venoms.

The presence of components antigenically related to Bothrops asper myotoxin was investigated by Western blotting and immunoelectrophoretic techniques. B. asper myotoxin is a non-glycosylated monomeric phospholipase A with a molecular weight by SDS-PAGE of 16,000 and isoelectric point of pH 9.8-10.0. Results showed that proteins in the venoms of B. nummifer, B. godmani, B. schlegelii, B. picadoi, and Agkistrodon bilineatus were recognized by monospecific antibodies to B. asper myotoxin raised in rabbit and sheep. Western blotting indicated that cross-reacting proteins have a molecular weight of 16,000, with the exception of that of B. picadoi, which is of 24,000 mol. wt. However, immunoelectrophoresis indicated that these components are highly heterogeneous in charge, ranging from basic to acidic proteins. The cross-reacting component(s) present in newborn B. asper venom has a different charge from that of the 'adult-type'. Venoms from newborn specimens showed an additional cross-reacting band of 18,000 mol. wt. Myotoxin is an abundant component in adult B. asper venom. Myotoxin-antimyotoxin complexes had different electrophoretic mobilities in rocket immunoelectrophoresis depending upon the species in which monospecific immune sera were produced.

Animals↗

Immunohistochemical demonstration of the binding of Bothrops asper myotoxin to skeletal muscle sarcolemma.

The binding of Bothrops asper myotoxin to mouse skeletal muscle was studied at both the light and electron microscope levels using the peroxidase anti-peroxidase technique. The toxin binds to muscle cell sarcolemma, and there are no intracellular binding sites. This study corroborates previous observations which suggested that the sarcolemma is the first site of action of B. asper myotoxin.

Animals↗

Isolation and partial characterization of a myotoxin from the venom of the snake Bothrops nummifer.

A myotoxin from the venom of the snake Bothrops nummifer was purified to homogeneity by ion-exchange chromatography on CM-Sephadex. The toxin is a basic dimer with a subunit molecular weight of 16,000, as estimated by SDS-polyacrylamide gel electrophoresis. The toxin lacks phospholipase A2 activity when tested on egg yolk lecithin and skeletal muscle homogenates. It induces skeletal muscle damage both in vivo and in vitro. When injected i.m. it promotes a drastic increase in serum creatine kinase levels; the isozyme CK-MM is responsible for this increment. A rapid release of creatine kinase was observed when mouse gastrocnemius muscle was incubated with the toxin, suggesting that it induces the formation of relatively large 'lesions' in the plasma membrane of muscle cells. Moreover, analysis of the dose-response data indicated that the myotoxin affects muscle sarcolemma by a 'one hit' mechanism. Skeletal muscle cells are affected by the toxin when calcium is eliminated from the medium. The myotoxin has an i.v. LD50 of 3.9 mg/kg body weight in mice, and induces edema when injected in the foot pad. On the other hand, it is not directly hemolytic, anticoagulant, hemorrhagic nor cytotoxic for lymphocytes. The myotoxin shows partial immunologic identity with a myotoxic phospholipase A2 isolated from Bothrops asper venom. The polyvalent antivenom produced in Costa Rica forms a precipitation arc against B. nummifer myotoxin on immunoelectrophoresis.

Animals↗

Comparative study of the edema-forming activity of Costa Rican snake venoms and its neutralization by a polyvalent antivenom.

The edema-forming activity of eight Costa Rican crotaline snake venoms and its neutralization by a polyvalent antivenom were studied using the mouse footpad test. All of the venoms induced edema, the highest activity being present in the venoms of Bothrops lateralis and Bothrops picadoi. When experiments were performed with preincubation of venom and antivenom, neutralization of edema was poor. Moreover, it was observed that, with some venoms, edema increased when large doses of antivenom were used. This effect was also observed when some venoms were incubated with coral snake antivenom, suggesting that venoms may release some pharmacologically active component(s) from antivenom, since the latter contains traces of alpha-2 and beta globulins. Based on these findings, an alternative approach to the study of the neutralization of edema was used; in this new method, antivenom was injected i.v. before venom administration, thereby avoiding preincubation. With this technique, a much better neutralization of edema was observed, although with some venoms it was still poor. Venoms contain low molecular weight factors which induce edema, suggesting that lack of immunogenicity of some components may cause a poor neutralization. However, such components are responsible for only a minor portion of the edema induced by crude venoms. It is suggested that experiments in which venom and antivenom are preincubated preincubated in testing the neutralization of edema should be avoided, and that a more adequate approach may be an independent inoculation of venom and antivenom.

Animals↗

Pharmacological activities of a toxic phospholipase A isolated from the venom of the snake Bothrops asper.

A toxic phospholipase A was isolated from the venom of Bothrops asper. It induced skeletal muscle damage, anticoagulant effects and edema in the foot pad. The toxin had an intravenous LD50 of 95 micrograms/16-18 g mouse body wt and an intraventricular LD50 of 0.42 micrograms/16-18 g mouse body wt. Upon intramuscular and intravenous injections, the toxin induced a prominent increase in serum creatine kinase (CK) levels; only the CK-MM isozyme increased markedly. The toxin induced CK and creatine release from skeletal muscle incubated in vitro. The rate of efflux of creatine was higher than that of CK, although both markers were partially released as early as 15 min after incubation. The toxin also induced elevation of serum levels of lactic dehydrogenase isozymes. However, histological examination of skeletal muscle, kidneys, heart and lungs revealed cell damage only in skeletal muscle. The toxin was not cytotoxic to erythrocytes, lymphocytes or macrophages. In addition, it did not induce a mitogenic response on lymphocytes. In the absence of albumin in the medium, there was no significant difference between myotoxic activities in Ca2+-free and Ca2+-containing bathing solutions. However, when albumin was added, there was a significantly higher myotoxic effect in the presence of Ca2+. Thus, although phospholipolytic activity of the toxin plays a role in muscle damage when albumin is present, the toxin induces muscle damage even when phospholipase A activity is inhibited.

Animals↗

Pathogenesis of myonecrosis induced by coral snake (Micrurus nigrocinctus) venom in mice.

The mode by which coral snake (Micrurus nigrocinctus) venom affects skeletal muscle was studied using a combined approach. The venom induced early functional and structural alterations in the plasma membrane of muscle cells, suggesting that sarcolemma is the primary site of action of this venom. This was shown by the presence of wedge-shaped ('delta') lesions at the periphery of the cells, as well as by focal disruptions in the continuity of plasma membrane as early as 15 min after envenomation. After this initial alteration the rest of the organelles were severely affected. Myofilaments were hypercontracted leaving, as a consequence, areas of overstretched myofibrils as well as empty spaces. Eventually, myofilaments formed dense, clumped masses in which the striated structure was totally lost. At 24 h, myofilaments were still disorganized but they presented a more hyaline and homogeneous appearance. As early as 15 and 30 min mitochondria were swollen; later, by I, 3 and 24 h, they showed further alterations such as the presence of dense intracristal spaces and vesiculated cristae, as well as disruption in the integrity of their membranes. Sarcoplasmic reticulum was dilated and disorganized into many small vesicles randomly distributed throughout the cellular space. Moreover, the venom induced a rapid decrease in muscle levels of creatine and creatine-kinase (CK) and a calcium influx. Since the rates of efflux of creatine and CK were similar, it is suggested that the lesions produced in the membrane are large enough to allow the escape of these two molecules. As corroboration of the severe myotoxic effect, envenomated mice excreted reddish urine containing large quantities of myoglobin. Skeletal muscle cells are more susceptible to the action of the venom than erythrocytes, since coral snake venom induced only a mild direct haemolytic effect in vitro and haemolysis is not a significant effect in vivo. M. nigrocinctus venom induced a drastic increase in plasma levels of lactate dehydrogenase. Isozymes LDH-3, LDH-4, and LDH-5 increased markedly, suggesting that the systemic pathology of coral snake envenoming may be more complex than previously thought.

Animals↗

Isolation from a polyvalent antivenom of antibodies to a myotoxin in Bothrops asper snake venom.

Antibodies against Bothrops asper myotoxin were purified from a polyvalent antivenom by affinity chromatography. These antibodies neutralized most of the myotoxic activity of crude B. asper venom, as judged by histologic evaluation of skeletal muscle and determination of plasma creatine kinase levels in mice. When tested by immunoelectrophoresis, purified antibodies formed two superimposed bands of precipitation against an homogeneous (by SDS-PAGE analysis) preparation of B. asper myotoxin, as well as against crude B. asper venom. Ouchterlony immunodiffusion analysis of purified antibodies showed two precipitation bands with a pattern of complete immunologic identity between samples of crude B. asper venoms from specimens collected in the Atlantic and Pacific regions of Costa Rica. In addition, these antibodies precipitated when reacted against venom of newborn B. asper specimens from the Pacific region, but not against venom of newborn specimens from the Atlantic region. Purified antibodies were tested by immunodiffusion against eleven different snake venoms from Costa Rica. Only the venom of B. schlegelii cross-reacted, indicating the presence in this venom of components immunologically related to B. asper myotoxin. Analysis of purified antibodies to B. asper myotoxin by agarose electrophoresis and by SDS-PAGE suggests the presence of both IgG and IgM on the basis of electrophoretic position and molecular weight of the bands. Results obtained in neutralization experiments suggest that this myotoxin is a major factor in the development of local myonecrosis induced by crude B. asper venom.

Animals↗

Edema-forming activity of bushmaster (Lachesis muta stenophrys) and Central American rattlesnake (Crotalus durissus durissus) venoms and neutralization by a polyvalent antivenom.

The edema effect induced in mice by venoms of Crotalus durissus durissus and Lachesis muta stenophrys was studied. Minimum edema-forming doses were 11 and 5 micrograms, respectively. Edema developed very rapidly after injections of both venoms and reached a maximum at 6 hr. Neutralizing activity was tested by preincubation of the venoms with polyvalent antivenom. The edema induced by the venom of L. m. stenophrys was partially neutralized, whereas that induced by the venom of C. d. durissus was not neutralized.

Animals↗

[Changes in the electrophoretic pattern of the venom of the bushmaster (Lachesis muta stenophrys) stored under various conditions].

Liquid and lyophilized samples of Lachesis muta venom were stored at different temperatures and for different periods of time, and analyzed by polyacrylamide gel electrophoresis (PAGE) and immunoelectrophoresis. Only slight variations were evident when three pools of freeze-dried venom, that had been kept at -30 degrees C for several months were compared with fresh venom. These results suggest that L. muta venom is not altered drastically when stored under these conditions.

Animals↗

Local effects induced by coral snake venoms: evidence of myonecrosis after experimental inoculations of venoms from five species.

The local effects induced by intramuscular inoculations of venoms from six species of coral snakes were studied in mice. Venoms of Micrurus nigrocinctus nigrocinctus, M. n. mosquitensis, M. alleni, M. frontalis, M. carinicauda and M. surinamensis induced prominent myonecrosis which was observed histologically. From a morphological point of view all these venoms induced a similar pattern of myonecrosis, characterized by a conspicuous alteration of the intracellular structure. This myotoxic activity was corroborated by an increase in plasma creatine kinase levels 3 hr after i.m. injection of M. n. nigrocinctus, M. n. mosquitensis, M. frontalis and M. carinicauda venoms. M. mipartitus venom did not induce myonecrosis. None of the venoms induced edema or hemorrhage at the site of injection.

Animals↗

[Comparative study of venoms of newborn and adult rattlesnakes (Crotalus durissus durissus)].

Venoms from adult and newborn Central American rattlesnakes (Crotalus durissus durissus) were compared for lethal, proteolytic, hemorrhagic, myonecrotic, edematigenous and in vitro hemolytic activities. Electrophoretic and immunoelectrophoretic patterns showed some differences between these venoms. Venom from newborn snakes was devoid of hemorrhagic and edematigenous activities, whereas the venom from adult specimens induced these effects. On the other hand, newborn snake venom showed higher lethality and indirect hemolytic activity, and lower proteolytic activity, than venom from adult specimens. Both types of venoms induced only slight myonecrosis in mice, as judged by histological observation. The ED50 of an antivenom, in terms of absolute weight neutralized per ml of serum, was lower for the newborn specimens venom than for adult's venom, however, for each venom the number of LD50 neutralized was similar.

Age Factors↗

[Proteolytic activity of snake venoms of Costa Rica on casein].

Experimental conditions for the comparative measurement of proteolytic activity of Costa Rican snake venoms on casein are established, and the activity of 13 different venoms is described. Venoms showed marked differences in activity not only between species, but between specimens of the same species captured in different geographic regions of Costa Rica.

Caseins↗

Neutralization of local effects of the terciopelo (Bothrops asper) venom by blood serum of the colubrid snake Clelia clelia.

The neutralizing capacity of the blood serum of the non-venomous snake Clelia clelia against the hemorrhagic, edematous and myonecrotic effects of Bothrops asper venom in white mice was tested using in vitro preincubation experiments. Untreated serum neutralized to a different extent all local effects evaluated, but showed toxicity to mice, evidenced by edema and myonecrosis. Serum heated at 56 degrees C for 30 min lost its toxic properties but also its neutralizing capacity against hemorrhagic and edematous effects of the venom. The myonecrotic effect was neutralized by large quantities of heated serum. The serum of C. clelia formed a single precipitation arc in immunoelectrophoretic slides against B. asper venom, corresponding to the slow anodic proteins of the serum.

Animals↗