Biomedical subjects

C R Carlini

Publications and source records attributed to C R Carlini.

At least 37 records · Page 2Linked to original sources

PubMed1993

Purification, physicochemical characterization and N-terminal-amino acid sequence of a phospholipase A2 from Bothrops jararaca venom.

Snake venoms usually contain multiple molecular forms of phospholipase A2 enzymes (phosphatide acyl hydrolase, E.C. 3.1.1.4; PLA2). Phospholipases A2 induce a wide range of pharmacological effects which may depend or not on the hydrolysis of phospholipids. In this study, a PLA2 from Bothrops jararaca venom was purified to homogeneity by gel filtration on a Sephacryl S-200 column, followed by FPLC reverse-phase chromatography on a Pep-RPC HR 5/5 column (yield 1.63% of venom protein). The PLA2 activity of the fractions was determined by indirect hemolysis using hen's egg yolk lecithin as substrate. The enzyme is an acidic protein with PI 4.5 and an apparent molecular weight of 14,200, as estimated by gel filtration on a Superose 12 FPLC column. Similar properties have been described for PLA2 from other snake venoms. The N-terminal-sequence of the purified protein was NLMQFETMIMXXAGQ. These partial sequence data show a high degree of homology between the B. jararaca PLA2 and the enzymes from other snake venoms as well as bovine pancreatic PLA2.

Amino Acid Sequence↗

PubMed1993

Partial purification and some physicochemical properties of phospholipases A2 from the venom of the bushmaster snake (Lachesis muta).

Screening of the biochemical-pharmacological properties of the crude venom from the snake Lachesis muta indicated the presence of phospholipase A2 (PLA2; 5260 U/mg protein), procoagulant (2630 U/mg protein), platelet aggregating (43 U/mg protein) and caseinolytic activities (6670 U/mg protein). These activities were separated by filtration of the crude venom on Sephacryl S-200. The material containing PLA2 activity was further fractioned by DEAE-cellulose ion exchange chromatography into four active fractions (F-I to F-IV, containing 1.7, 1.2, 0.3, and 0.05% of the crude venom protein, respectively) by stepwise elution with buffers of increasing ionic strength. All fractions presented a molecular weight of approximately 15,000 and isoelectric points in the range pH 4.6-6.0. In addition to their indirect hemolytic activity, the partially purified fractions inhibited platelet aggregation induced either by collagen or thrombin. p-Bromophenacyl bromide-treated fractions lost both phospholipase A2 activity and their inhibitory effect on collagen-induced platelet aggregation.

Animals↗

PubMed1992

Role of resident macrophages in canatoxin-induced in vivo neutrophil migration.

Canatoxin (Cntx), a toxic protein purified from Canavalia ensiformis seeds, was shown to have lipoxygenase-mediated effects either in vivo or in vitro. Data here show that Cntx induced a dose-dependent migration of neutrophils and mononuclear cells when injected into rat peritoneal cavities. Furthermore, Cntx was able to induce neutrophil migration into pleural cavities and into air pouches. These effects were inhibited by dexamethasone but not by inhibitors of arachidonic acid metabolism (indomethacin, NDGA, and BW-755c) or by a PAF antagonist (BN 52021). In the peritoneal cavity Cntx caused an increase in vascular permeability inhibited by dexamethasone and BW-755c. Neutrophil migration induced by this toxin was dependent on the number of resident macrophages, since the migratory effect was enhanced by increasing the peritoneal macrophage population with thioglycollate pretreatment and was diminished when this population was reduced by peritoneal wash. It was also observed that Cntx induced release of a chemotactic factor from macrophage monolayers in vitro. Dexamethasone blocked this release but did not affect in vivo neutrophil recruitment induced by that factor. These data suggest that Cntx-induced neutrophil migration may be mediated by the same macrophage-derived neutrophil chemotactic factor released by other stimuli such as LPS, IL-1, and INF-gamma.

Animals↗

PubMed1992

Pharmacological characterization of rat paw edema induced by canatoxin, the toxic protein from Canavalia ensiformis (jack bean) seeds.

Intraplantar injection of canatoxin (CNTX) induced a dose-dependent rat hind-paw edema which was distinguished by two phases. In the first phase, observed between 0 and 2 hr after CNTX injection, there was an increase in paw volume, with no apparent involvement of inflammatory phagocytic cells. The second phase, beginning at about the 3rd hr, was characterized by an intense cellular infiltration at the site of administration which was coincident with a further increase in paw swelling. The edema was maximum at 6 hr after injection and disappeared within 48 hr at doses of 50-100 micrograms, while at doses of 200-300 micrograms edema was present in excess of 48 hr. The pharmacological studies suggested that CNTX-induced edema is a multi-mediated phenomenon. Histamine, serotonin, PAF and prostaglandins are likely involved in the first phase. Lipoxygenase metabolites, probably leukotrienes, seem to play a major role in the second phase, and may account for the development of cellular infiltration in the inflammatory site.

Animals↗

PubMed1992

Effects of canatoxin on the Ca(2+)-ATPase of sarcoplasmic reticulum membranes.

Canatoxin, a toxic protein isolated from the seeds of Canavalia ensiformis, was studied for its effects on the sarcoplasmic reticulum vesicles obtained from rabbit skeletal muscle. Canatoxin inhibited Ca2+ accumulation catalysed by the Ca(2+)-ATPase, without affecting the hydrolytic activity of this enzyme or membrane permeability to Ca2+. The effects of canatoxin were dose dependent, but not time dependent. It is concluded that canatoxin interacts with the Ca2+ pump and uncouples Ca2+ uptake from Ca(2+)-dependent ATP hydrolysis.

Adenosine Triphosphate↗

PubMed1992

Canatoxin induces activation on mice peritoneal macrophages.

Canatoxin (CNTX), the toxic protein from Canavalia ensiformis seeds, injected into the peritoneal cavities of mice (10 micrograms/cavity) induced a significant neutrophil migration (10.5 +/- 0.5 x 10(6) cells/cavity) after 4 h. A later migratory effect (48 h) on mononuclear cells, predominantly macrophages, was also observed (controls: 7 +/- 0.9; CNTX: 17 +/- 2.0 x 10(6) cells/cavity). These CNTX-elicited macrophages, when compared to resident cells (R) or cells elicited by thioglycollate (TG), had an increased content of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (R: 4.5 +/- 0.5; TG: 7.2 +/- 1.0; CNTX: 20.2 +/- 3.0 mU/10(6) cells) and a greater (> or = 100%) phagocytic activity. The data suggest that CNTX-stimulated macrophages presented some characteristics of activated cells.

Acetylglucosaminidase↗

PubMed1991

Lipoxygenase-mediated secretory effect of canatoxin the toxic protein from Canavalia ensiformis seeds.

Canatoxin was shown to induce serotonin release from rabbit platelets and rat brain synaptosomes, as well as to release insulin from isolated pancreatic islets. All these effects were dose-dependent and were inhibited by lipoxygenase inhibitors, such as nordihydroguaiaretic acid and esculetin, but not by indomethacin, a cyclooxygenase inhibitor. The data suggest that canatoxin-induced secretory effect results from the activation of the lipoxygenase pathway which would elicit exocytosis. Thus, canatoxin might be a useful tool for the study of biological events that involve lipoxygenase mediation.

Animals↗

PubMed1991

Plant and microbial toxic proteins as hemilectins: emphasis on canatoxin.

Ribosome-inactivating plant toxic proteins and ADP-ribosylating microbial toxins share a common structural organization. These proteins present domains displaying different biological properties: a target cell membrane-binding component (B-subunit or haptomer) and an enzymatically active component (A-subunit or effectomer). Interactions of these toxins with the target cells are mediated by the hemilectin-like haptomer, which recognizes and specifically binds to a given glycoderivative present at the cell surface. After binding the holoprotein is internalized via endocytosis. Inside the endocytic compartment the toxin is processed to release its effectomer moiety which catalytically modifies a cytoplasmic component, and this step accounts for its toxic effect. The structural relationships between toxic hemilectins and plant lectins are discussed, with emphasis on the example of canatoxin and concanavalin A, both present in the seeds of the jack bean Canavalia ensiformis. Contrary to other plant toxic proteins, which inhibit protein synthesis, canatoxin-induced toxicity includes central nervous system-mediated effects. In vivo as well as in vitro canatoxin acts as lipoxygenase-mediated secretagogue in several types of cells: blood platelets, mast cells, pancreatic islets and synaptosomes. Elucidation of structure vs biological activity relationships of canatoxin and other toxic proteins may provide data for their utilization as pharmacological tools and as therapeutic agents.

Animals↗

PubMed1991

Effect of canatoxin on cell cultures.

Canatoxin, a toxic protein isolated from Canavalia ensiformis, was shown to inhibit DNA synthesis and to produce a cytolytic effect when added in vitro to various cells, in doses ranging from 50 to 500 nM. In this case no selectivity was found for a certain cell type, as both normal and transformed cells could be affected by the toxic protein. The cytostatic effect was irreversible upon removal of the toxic protein from the medium and could be fully attained after exposing the cells to canatoxin for only 30 min. The use of an in vitro cell culture system may allow for a better insight on the mode of action of canatoxin.

Animals↗

PubMed1991

Canatoxin, a plant protein, induces insulin release from isolated pancreatic islets.

Canatoxin (CNTX), the toxic protein purified from Canavalia ensiformis, has been shown to induce secretion from different cellular systems through a mechanism involving a lipoxygenase-mediated pathway. Here it is shown that CNTX causes insulin release from isolated rat pancreatic islets. This effect is time and dose dependent, occurs in the absence as well in the presence of glucose, and is markedly reduced at lower temperatures (15 C). At 1-2 microM, the insulinotropic effect of CNTX is equivalent to that of 20 mM glucose, and the two responses are not additive. The stimulatory effect of CNTX is not caused by a toxic or lytic effect of the toxin on the islets, since islets once exposed to CNTX are able to respond a new insulinotropic stimulus. The phospholipase inhibitor mepacrine impairs insulin release induced by either CNTX or glucose. Indomethacin, an inhibitor of the cyclooxygenation of arachidonic acid, fails to affect insulin release, but two lipoxygenase inhibitors block it, and epinephrine reduces it. These data suggest that CNTX may act on islets through the same pathway as that used by glucose, with both effects being mediated by lipoxygenases.

Animals↗

PubMed1990

Bothrops jararaca snake venom: effects on platelet aggregation.

Crude venom from Bothrops jararaca has procoagulant, platelet aggregating and phospholipase A2 (PLA2) activities. By chromatographic fractionation of the venom on Sephacryl S-200 it was possible to separate these three activities and to show that distinct protein components are involved. The procoagulant activity appears to involve the synergistic action of several components and was not further studied in the present work. The aggregating activity results from the action of two components: 1) a serino-proteinase PMSF-inhibitable similar to thrombocytin and 2) a PMSF-resistant, calcium- and plasma-dependent factor distinct from other previously described aggregating principles. Fractions possessing PLA2 activity were also able to inhibit platelet aggregation induced by collagen and accelerated the slow reversal of aggregation induced by ADP. Both PLA2 activity and inhibition of collagen-induced platelet aggregation displayed by these fractions were abolished by reaction with p-bromophenacyl bromide and 2-mercapto-ethanol. These results indicate that in B. jararaca venom the PLA2 activity and the factor inhibiting platelet aggregation may be related to the same protein molecule.

Amides↗

PubMed1989

Effect of canatoxin on the circulating levels of gonadotropins and prolactin in rats.

1. The effects of canatoxin, the toxic principle from Canavalia ensiformis seeds which has lipoxygenase-activating properties, were evaluated in rats using radioimmunoassay techniques to measure plasma levels of prolactin (PRL), progesterone, follicle stimulating (FSH) and luteinizing (LH) hormones. 2. The chronic administration of canatoxin (50, 100 or 200 micrograms/kg daily for 12 days, ip) to female rats induced a sharp rise in plasma LH and FSH concentrations with no changes in progesterone level. A fall in circulating PRL was also observed. The frequency of proestrus and weight gain increased in rats treated with the highest dose of toxin used, but there was no alteration in weight of uterus or ovaries. 3. The increases in gonadotropin levels with canatoxin are consistent with the lipoxygenase-activating properties of the toxin, but do not explain why plasma PRL concentrations decreased in canatoxin-treated rats. 4. Since the animals in the control group had high PRL and low LH levels and since canatoxin increased LH and decreased PRL in the circulation, a possible stress-prevention effect is discussed for the toxin. 5. This study supports previous suggestions of central actions for canatoxin, and indicates the hypophysis and/or hypothalamus as one of the target sites for the toxin in the central nervous system.

Animals↗

PubMed1989

Convulsions induced by canatoxin in rats are probably a consequence of hypoxia.

Canatoxin, a convulsant neurotoxin from the seeds of Canavalia ensiformis, induces lipoxygenase-dependent hypoxia and sex-related alterations of carbohydrate metabolism in rats which are blocked by glucose, diazepam and hexamethonium. The present study analyzes the possible casual relationship between the convulsant action of canatoxin and its effects on carbohydrate metabolism. The incidence of canatoxin-induced convulsions was greater in male than in female rats. Pretreatment of male rats with drugs that block hypoxia, such as glucose (2.5 g/kg, iv, 15 min), diazepam (5 mg/kg, ip, at 48 h, 24 h and 15 min), hexamethonium (4 mg/kg, ip, 15 min) and NDGA (125 mg/kg, ip, 1 h), also protected the animals against convulsions, respiratory distress and death. These results suggest that canatoxin-induced convulsions are probably the consequence of hypoxia and both effects are mediated by lipoxygenase activation.

Animals↗

PubMed1989

Alterations in rat carbohydrate metabolism induced by canatoxin as a probable consequence of primary hypoxia.

1. Canatoxin, a protein displaying lipoxygenase-activating properties isolated from Canavalia ensiformis seeds, induces hypoxia and hyperglycemia in male rats. 2. Liver glycogen, blood glucose and lactate levels were measured in male and female rats after canatoxin (50 mU, iv) injection. Increased levels of serum glutamic oxaloacetic transaminase activity were used as an indicator of hepatic injury. 3. There was no sex-related difference observable during canatoxin-induced hypoxia but male and female rats did show different patterns of metabolic change and hepatic injury after toxin administration. Increased blood glucose and lactate levels, liver glycogenolysis and hepatic injury were observed in male rats while female rats showed only hypoglycemia and glycogenolysis. 4. Pretreatment of male rats with either glucose, diazepam or hexamethonium abolished both the hypoxia and hepatic injury and the metabolic alterations produced by toxin injection. 5. The results suggest that the metabolic alterations and hepatic injury detected after canatoxin injection may be a consequence of primary hypoxia.

Animals↗

PubMed1988

The secretory effect of canatoxin on rat brain synaptosomes involves a lipoxygenase-mediated pathway.

Canatoxin (CNTX), a neurotoxic protein, is known to activate platelet secretion and aggregation in vitro through a lipoxygenase-dependent pathway. This study shows that CNTX also induces time and dose-dependent serotonin secretion from rat brain synaptosomes. The secretory effect induced by 6 microM CNTX was similar to that elicited by 150 mM KCl. Nordihydroguaiaretic acid (500 microM) completely abolished CNTX-induced serotonin release while 150 microM indomethacin had no effect. These data suggest the involvement of the lipoxygenase pathway in neurotransmitter release elicited by CNTX as occurs in the platelet.

Animals↗

PubMed1988

Biochemical and pharmacological screening of snake (Bothrops) venoms: characterization of components acting on blood coagulation and platelet aggregation.

Six venoms from snakes of the genus Bothrops were tested for coagulation, platelet aggregation and phospholipase A2 (PLA2) activity. Almost all showed pro-coagulant and PLA2 activities while pro-aggregating properties were found only for some venoms. Bothrops jararaca venom showed different protein peaks associated with these activities. The pro-aggregating activity was inhibited by EDTA, leupeptin and mepacrine while the PLA2 activity was blocked by p-bromophenacyl bromide and 2-mercaptoethanol. Venom screening tests for clotting and platelet aggregation may represent a valuable tool for snake taxonomy and for monitoring the quality of antisera.

Blood Coagulation↗

PubMed1986

Blood glucose alterations induced in rats by canatoxin, a protein isolated from jack bean (Canavalia ensiformis) seeds.

Canatoxin, a lethal convulsant protein isolated from the seeds of Canatoxin ensiformis (jack bean), induced a biphasic alteration in blood glucose levels when injected i.v. into rats and mice. After administration of subconvulsant doses of canatoxin the rats showed initially hyperglycemia during the first 30 min, followed by a long-lasting hypoglycemia. Return to basal glucose levels was not seen up to 15 days. The hyperglycemic effect was dose-dependent and occurred in starved and well-fed animals. A parallelism between convulsant and hyperglycemic activity was observed throughout all canatoxin purification steps. Mice were about 20-fold less sensitive (on a weight basis) than rats to canatoxin-induced hyperglycemia. The hyperglycemic phase was not affected by pretreatment of the rats with alpha- or beta-adrenergic blockers or chlorpromazine, but was potentiated by reserpine and haloperidol. Diazepam and hexamethonium were able to block the hyperglycemic phase of canatoxin's effect. The results suggest that the hyperglycemic alterations induced by canatoxin in rats and mice are probably mediated via the central nervous system.

Animals↗

PubMed1985

Platelet release reaction and aggregation induced by canatoxin, a convulsant protein: evidence for the involvement of the platelet lipoxygenase pathway.

Canatoxin is a toxic protein isolated from Canavalia ensiformis seeds. It induces death preceded by convulsions of spinal cord origin and also produces in vitro aggregation of platelets in rabbit, human and guinea-pig plasma. The aggregating effect is dose-dependent at nanomolar concentrations. Rabbit platelets pretreated with canatoxin became refractory to a second exposure to this protein or to collagen, but were still responsive to ADP, Paf-acether or arachidonic acid. [14C]-5-hydroxytryptamine was released from pre-labelled platelets on stimulation with canatoxin. Washed rabbit platelets, but not thrombin-degranulated ones, aggregated on stimulation with canatoxin provided that fibrinogen was added before the toxin. Canatoxin's pro-aggregating activity was inhibited by mepacrine, EDTA, caffeine, prostacyclin, adenosine monophosphate and also by the ADP scavenger system, creatine phosphokinase/creatine phosphate. Furthermore, 3-amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline (BW 755C), eicosatetraynoic acid (ETYA) and nordihydroguaiaretic acid (NDGA) were potent inhibitors of canatoxin-induced aggregation. In contrast, no inhibition was seen with indomethacin. The data indicate that canatoxin is mainly a release-reaction-promoting agent, being devoid of any direct aggregating activity. Thus the aggregation is totally dependent on the release of ADP. Furthermore, canatoxin-induced platelet activation is probably dependent on platelet phospholipase A2 and lipoxygenase activity but is not dependent on cyclo-oxygenase products or the release of Paf-acether.

5,8,11,14-Eicosatetraynoic Acid↗