RESUMEN
The prominent local myotoxic effects induced by Bothrops snake venom are due, in part, to myotoxins. This effect is not neutralized by antivenom, which is the main therapy for victims of snakebite. Two basic myotoxins named MjTX-I and MjTX-II were isolated from Bothrops moojeni venom. Both myotoxins have a Lys-49 phospholipase A2 structure devoid of enzymatic activity, but are highly myonecrotic and edema-inducing. In this study, we analyzed the effect of a low-level laser (LLL) at 685 nm, an energy density of 2.2 J cm(-2), and the irradiation time of 15 s, and a light emitting diode (LED) at 635 or 945 nm at energy densities of 4 and 3.8 J cm(-2), and irradiation times of 41 and 38 s, respectively, applied 30 min and 3 h after edema formation in mice caused by MjTX-I or MjTX-II. MjTX-I or MjTX-II caused a significant edema formation in envenomed paws. LLL and LED irradiation significantly reduced the edema formation by both myotoxins from 1 up to 6 hours after the injection. Both LLL and LEDs were similar in reducing the edema formation induced by myotoxins. The combined photobiostimulation with antivenom had the same effect in reducing edema as treatment with the LLL or LEDs alone. In conclusion, the results of this study indicate that photobiostimulation could be used in association with antivenom therapy for treatment of local effects of Bothrops species venom.
Asunto(s)
Bothrops/metabolismo , Edema/inducido químicamente , Fosfolipasas A/toxicidad , Ponzoñas/metabolismo , Animales , Edema/radioterapia , Terapia por Luz de Baja Intensidad , Masculino , Ratones , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/metabolismoRESUMEN
We genetically modified Eclipta alba using Agrobacterium rhizogenes LBA 9402, with the aim of producing secondary metabolites with pharmacological properties against phospholipase A(2) and the myotoxic activities of snake venom. Extracts from in natura aerial parts and roots, both native and genetically modified (in vitro), were prepared and analysed by high-performance liquid chromatography. In natura materials showed the coumestan wedelolactone at higher concentration in the aerial parts, while demethylwedelolactone appeared at higher concentration in roots. Among the modified roots, clone 19 showed higher concentrations of these coumestans. Our results show that the in natura extracts of plants collected from Botucatu and Ribeirão Preto were efficient in inhibiting snake venom phospholipase A(2) activity. Regarding in vitro material, the best effect against Crotalus durissus terrificus venom was that of clone 19. Clone 19 and isolated coumestans (wedelolactone and demethylwedelolactone) inhibited the myotoxic activity induced by basic phospholipases A(2) isolated from the venoms of Crotalus durissus terrificus (CB) and Bothrops jararacussu (BthTX-I and II). The search for antivenom is justified by the need of finding active principles that are more efficient in neutralizing snake venoms and also as an attempt to complement serum therapy.
Asunto(s)
Venenos de Crotálidos/antagonistas & inhibidores , Eclipta/química , Fosfolipasas A/antagonistas & inhibidores , Extractos Vegetales/farmacología , Animales , Bothrops , Brasil , Cumarinas/aislamiento & purificación , Cumarinas/farmacología , Venenos de Crotálidos/enzimología , Crotalus , Eclipta/genética , Masculino , Ratones , Fosfolipasas A/aislamiento & purificación , Componentes Aéreos de las Plantas , Extractos Vegetales/genética , Raíces de Plantas , Plantas Modificadas Genéticamente/química , Rhizobium/genéticaRESUMEN
BmTX-I, an Asp49 phospholipase A(2), was purified from Bothrops moojeni venom after only one chromatographic step using reverse-phase HPLC on mu-Bondapak C-18 column. A molecular mass of 14238.71Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BmTX-I PLA(2) had a sequence of 121 residues of amino acids: DLWQFNKMIK KEVGKLPFPF YGAYGCYCGW GGRGEKPKDG TDRCCFVHDC CYKKLTGCPK WDDRYSYSWK DITIVCGEDL PCEEICECDR AAAVCFYENL GTYNKKYMKH LKPCKKADYP C and pI value 7.84, and showed a high degree of homology with basic Asp49 PLA(2) myotoxins from other Bothrops venoms. BmTX-I presented PLA(2) activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45 degrees C. Maximum PLA(2) activity required Ca(2+) and in the presence of Mg(2+), Cd(2+) and Mn(2+) it was reduced in presence or absence of Ca(2+). Crotapotin from Crotalus durissus colillineatus rattlesnake venom has significantly inhibited (P<0.05) the enzymatic activity of BmTX-I. In vitro, the whole venom and BmTX-I caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other bothrops species. In mice, BmTX-I and the whole venom-induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Edema-forming activity was also analyzed through injection of the venom and the purified BmTX-I into the subplantar region of the right footpad. Since BmTX-I exert a strong proinflammatory effect; the enzymatic phospholipids hydrolysis might be relevant for these phenomena.
Asunto(s)
Bothrops , Venenos de Crotálidos/química , Neurotoxinas/química , Fosfolipasas A/química , Secuencia de Aminoácidos , Animales , Fraccionamiento Químico , Pollos/fisiología , Cromatografía Líquida de Alta Presión , Venenos de Crotálidos/enzimología , Venenos de Crotálidos/farmacología , Crotalus/metabolismo , Crotoxina/aislamiento & purificación , Crotoxina/farmacología , Cinética , Masculino , Ratones , Datos de Secuencia Molecular , Bloqueo Neuromuscular , Neurotoxinas/aislamiento & purificación , Neurotoxinas/farmacología , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/farmacología , Alineación de Secuencia , Análisis de Secuencia de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
BaTX PLA(2), a K49 phospholipase A(2) homologue was purified from Bothrops alternatus venom after two chromatographic steps, molecular exclusion on Superdex 75 and reverse phase HPLC on mu-Bondapack C-18. A molecular mass of 13898.71 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that BaTX has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). The complete amino acid sequence of BaTX PLA(2) contains 121 residues, resulting in a calculated pI value of 8.63. This sequence shows high identity values when compared to other K49 PLA(2)s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA(2)s. The sequence was SLFELGKMIL QETGKNPAKS YGAYYCYCGW GGQGQPKDAT DRCCYVHKCC YKKLTGCNPK KDRYSYSWKD KTIVCGENNS CLKELCECDK AVAICLRENL NTYNKKYRYY LKPLCKKADA C. In mice, BaTX induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. The LD(50) of BaTX was 7 mug/g body weight, by intravenous route. In vitro, the toxin caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparations. The blockage 50% was achieved at a concentration of 0.03 microM: 40+/-0.4 min and 0.07 microM: 35+/-0.3 min. Moreover, this protein induced a rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. Thus, the combined structural and functional information obtained identify BaTX as a new member of the K49 PLA(2) family, which presents the typical bioactivities described for such proteins.
Asunto(s)
Bothrops/metabolismo , Venenos de Crotálidos/enzimología , Fosfolipasas A/química , Fosfolipasas A/toxicidad , Secuencia de Aminoácidos , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Pollos , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Edema/inducido químicamente , Técnicas In Vitro , Isoenzimas/química , Dosificación Letal Mediana , Lisina , Ratones , Datos de Secuencia Molecular , Peso Molecular , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Mioblastos Esqueléticos/efectos de los fármacos , Necrosis , Unión Neuromuscular/efectos de los fármacos , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A2 , Conformación Proteica , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de TiempoRESUMEN
Cdr-12 and Cdr-13 isoforms of PLA2, a D49 protein, were purified from Crotalus durissus ruruima venom after one chromatographic step, reverse phase HPLC on micro-Bondapack C-18. The molecular mass by SDS-PAGE of Cdr-12 and Cdr-13 isoforms of PLA2 was 14333.49 Da and 14296.42 Da, respectively and confirmed by MALDI-TOF mass spectrometry. The amino acid composition showed that both isoforms Cdr-12 and Cdr-13 have a high content of Lys, Tyr, Gly, Arg, and 14 half-Cys residues, typical of a basic PLA2. The isoforms Cdr-12 and Cdr-13 had a sequence of amino acids of 122 amino acid residues, being Cdr-12: SLLQFNKMIK FETRKNAIPF YAFYGCYCGW GGQGRPKDAT DRCCIVHDCC YGKLAKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGYMIY PDSRCREPSE TC and pI value 8.37 and Cdr-13: SLVQFEKMIK EETGKNAVPF YAFYGCYCGW GGRGRPKDAT DRCCIVHDCC YEKLVKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGKMIY PDSRCREPSE TC with a pI value of 8.13 This sequence shows high identity values when compared to other D49 PLA2s isolated from venoms of crotalics snakes. Skeletal muscle preparations from the young chicken have been previously used in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. In mice, the PLA2 isoforms Cdr-12 and Cdr-13 induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. In vitro, Cdr-12 and Cdr-13 isoforms of PLA2, caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and produced cytotoxicity in murine C2C12 skeletal muscle myotubes and lack cytolytic activity upon myoblasts in vitro. Thus, the combined structural and functional information obtained identify Cdr-12 and Cdr-13 isoforms as members of the PLA2 family, which presents the typical bioactivities described for such proteins.
Asunto(s)
Venenos de Crotálidos/enzimología , Fosfolipasas A/química , Fosfolipasas A/toxicidad , Secuencia de Aminoácidos , Animales , Células Cultivadas , Fraccionamiento Químico , Pollos , Venenos de Crotálidos/química , Venenos de Crotálidos/aislamiento & purificación , Venenos de Crotálidos/toxicidad , Crotalus , Diafragma/efectos de los fármacos , Punto Isoeléctrico , Isoenzimas , Ratones , Peso Molecular , Músculo Esquelético/patología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Necrosis/patología , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A2 , Nervio Frénico/efectos de los fármacos , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.
Asunto(s)
Bothrops/metabolismo , Fosfolipasas A/metabolismo , Venenos de Serpiente/enzimología , Secuencia de Aminoácidos , Animales , Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Calcio/metabolismo , Carcinoma de Ehrlich/inducido químicamente , Carcinoma de Ehrlich/enzimología , Carcinoma de Ehrlich/patología , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Ensayo de Inmunoadsorción Enzimática , Hemoglobinas/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Células K562 , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Peso Molecular , Fosfolipasas A/química , Fosfolipasas A/aislamiento & purificación , Agregación Plaquetaria/efectos de los fármacos , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Venenos de Serpiente/farmacologíaRESUMEN
Cr 5 PLA(2) homologous (K49) was isolated from Calloselasma rhodostoma venom in only one chromatographic step in reverse phase HPLC (RP-HPLC) (on mu-Bondapack C-18). A molecular mass of 13.965 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that Cr 5 had a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical residues of a basic PLA(2). The complete amino acid sequence of Cr 5 PLA(2) contains 120 residues, resulting in a calculated pI value of 5.55. This sequence shows high identity values when compared to other K49 PLA(2)s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA(2)s. The sequence found was SLVELGKMIL QETGKNPAKS YGAYGCNCGV LGRHKPKDAT DRCCFVHKCC YKKLTGCDPK KDRYSYSWKD KTIVCGENNP CLKEMCECDK AVAICLRENL DTYNKKYRYL KPFCKKADDC. In mice, Cr 5 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD(50) of Cr 5 was 0.070 mg/kg of the animal weight, by intracerebroventricular (i.c.v.) route. In vitro, the toxin caused rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. The isolation of this PLA(2) and the combined structural and functional information obtained classify Cr 5 as a new member of the K49 PLA(2) family, since it presents typical features from such proteins.
Asunto(s)
Venenos de Crotálidos/química , Fosfolipasas A/aislamiento & purificación , Viperidae/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Edema/inducido químicamente , Ratones , Datos de Secuencia Molecular , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Mioblastos/citología , Mioblastos/efectos de los fármacos , Fosfolipasas A/química , Fosfolipasas A/toxicidad , Fosfolipasas A2 , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
For the first time, a complete X-ray diffraction data set has been collected from a myotoxic Asp49-phospholipase A2 (Asp49-PLA2) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) and a molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxin Asp49-PLA2 PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA2s. In contrast, the oligomeric structure of BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA2 from B. jararacussu). Thus, comparison between these structures should add insight into the catalytic and myotoxic activities of bothropic PLA2s.
Asunto(s)
Asparagina , Bothrops , Venenos de Crotálidos/toxicidad , Fosfolipasas A/toxicidad , Animales , Catálisis , Venenos de Crotálidos/química , Venenos de Crotálidos/metabolismo , Cristalografía por Rayos X , Cinética , Fosfolipasas A/química , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Conformación ProteicaRESUMEN
In this paper we reported the purification, the biological characterization and the amino acid sequence of two new isoforms basic 6-1 (Bj-IV) and 6-2 (Bj-V) PLA(2) D49 purified from the Bothrops jararacussu venom. The isoforms 6-1 and 6-2 had a sequence of amino acids of 121 amino acid residues 6-1: DLFEWGQMIL KETGKNPFPY YGAYGCYCGW GGRGKPKDKD TDRCCYVHDC CYKKLTGCPK TDDRYSYSWL DLTIVCGEDD PCKELCECDK AIAVCFRENL GTYNKKYRYH LKPCKKADKP C and pI value 7.83 and 6-2: DLWQFGQMIL KETGKIPFPY YGAYGCYCGW GGRGGKPKDG TDRCCYVHDC CYKKLTGCPK TDDRYSYSWL DLTIVCGEDD PCKELCECDK AIAVCFRENL GTYNKKYRYH LKPCKKADKP C with a pI value of 7.99. Skeletal muscle preparations from the young chicken have been used previously in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. Both isoforms have produced neuromuscular blockade in young chicken biventer cervicis nerve-muscle preparations in presence or absence of crotapotin crotalic (F3 and F4) indicating that catalytic activity was not essential for neuromuscular action in this preparation.
Asunto(s)
Bothrops , Venenos de Crotálidos/enzimología , Unión Neuromuscular/efectos de los fármacos , Fosfolipasas A/química , Fosfolipasas A/toxicidad , Secuencia de Aminoácidos , Animales , Pollos , Venenos de Crotálidos/toxicidad , Crotoxina/farmacología , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Isoenzimas/toxicidad , Datos de Secuencia Molecular , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Homología de Secuencia de AminoácidoRESUMEN
A novel acidic Asp49 phospholipase A(2) was isolated from Bothrops erythromelas (jararaca malha-de-cascavel) snake venom by four chromatographic steps. BE-I-PLA2 present a molecular weight of 13,649.57 Da as estimated by mass spectrometry. N-terminal and four internal peptides were sequenced, covering around one-third of the complete toxin sequence. The complete BE-I-PLA2 cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 457 bp and encodes a protein with significant sequence similarity to many other phospholipase A(2) from snake venoms. When tested in platelet rich plasma, the enzyme showed a potent inhibitory effect on aggregation induced by arachidonic acid and collagen, but not ADP. On the other hand, BE-I-PLA2 did not modify aggregation in washed platelet. Furthermore, no action of BE-I-PLA2 on the principal platelets receptors was observed. Chemical modification with p-bromophenacyl bromide abolished the enzymatic activity of BE-I-PLA2, but its anti-platelet activity was only partially inhibited. In human umbilical-cord veins endothelial cells, BE-I-PLA2 was neither apoptotic nor proliferative but stimulated endothelial cells to release prostaglandin I(2), suggesting an increase of its potential anti-platelet activity in vivo. Further studies are required in order to determine the exact mechanism of action of BE-I-PLA2 in the inhibition of platelet aggregation.
Asunto(s)
Bothrops/genética , Venenos de Crotálidos/genética , Venenos de Crotálidos/aislamiento & purificación , Células Endoteliales/metabolismo , Epoprostenol/metabolismo , Fosfolipasas A/genética , Fosfolipasas A/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Clonación Molecular , Venenos de Crotálidos/química , Venenos de Crotálidos/metabolismo , Venenos de Crotálidos/farmacología , ADN Complementario/química , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Epoprostenol/biosíntesis , Femenino , Fosfolipasas A2 Grupo II , Fosfolipasas A2 Grupo IV , Humanos , Masculino , Datos de Secuencia Molecular , Fosfolipasas A/metabolismo , Fosfolipasas A/farmacología , Fosfolipasas A2 , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Proteínas de Reptiles , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA2, crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 microM/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA2, thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.
Asunto(s)
Venenos de Crotálidos/enzimología , Crotoxina/química , Factor de Activación Plaquetaria/química , Serina Endopeptidasas/química , Secuencia de Aminoácidos , Animales , Plaquetas/efectos de los fármacos , Crotalus/metabolismo , Crotoxina/aislamiento & purificación , Fibrinógeno/efectos de los fármacos , Datos de Secuencia Molecular , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A2 , Factor de Activación Plaquetaria/aislamiento & purificación , Factor de Activación Plaquetaria/farmacología , Agregación Plaquetaria , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/farmacología , Inhibidores de Serina Proteinasa/farmacología , Trombina/aislamiento & purificación , Trombina/farmacología , Clorometilcetona Tosilisina/farmacologíaRESUMEN
The whole venom of Lachesis muta muta is preponderantly neurotoxic but moderately myotoxic on the chick biventer cervicis preparation (BCp). We have now examined these toxic activities of a basic phospholipase A(2), LmTX-I, isolated from the whole venom. LmTX-I caused a significant concentration-dependent neuromuscular blockade in the BCp. The time to produce 50% neuromuscular blockade was 14.7+/-0.75 min (30 microg/ml), 23.6+/-0.9 min (10 microg/ml), 34+/-1.7 min (2.5 microg/ml) and 39.2+/-3.6 min (1 microg/ml), (n=5/concentration; p<0.05). Complete blockade with all tested concentrations was not accompanied by inhibition of the response to ACh. At the highest concentration, LmTX-I (30 microg/ml) significantly reduced contractures elicited by exogenous KCl (20mM), increased the release of creatine kinase (1542.5+/-183.9 IU/L vs 442.7+/-39.8 IU/L for controls after 120 min, p<0.05), and induced the appearance of degenerating muscle fibers ( approximately 15%). Quantification of myonecrosis indicated 14.8+/-0.8 and 2.0+/-0.4%, with 30 and 10 microg/mlvenom concentration, respectively, against 1.07+/-0.4% for control preparations. The findings indicate that the basic PLA(2) present on venom from L. m. muta (LmTX-I) possesses a dominant neurotoxic action on isolated chick nerve-muscle preparations, whereas myotoxicity was mainly observed at the highest concentration used (30 microg/ml). These effects of LmTX-I closely reproduce the effects of the whole venom of L. m. muta in chick neuromuscular preparations.
Asunto(s)
Venenos de Crotálidos/enzimología , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/farmacología , Viperidae/fisiología , Acetilcolina , Animales , Pollos , Venenos de Crotálidos/química , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Bloqueantes Neuromusculares/química , Bloqueantes Neuromusculares/aislamiento & purificación , Bloqueantes Neuromusculares/farmacología , Fosfolipasas A/química , Fosfolipasas A2 , Cloruro de PotasioRESUMEN
Lonomia obliqua caterpillar bristle extract induces both direct and indirect hemolytic activity on human and rat washed erythrocytes, and provokes intravascular hemolysis in Wistar rats. Indirect hemolytic activity is assumed to be caused by a phospholipase A(2) (PLA(2)) present in this extract, and this investigation was initiated in order to characterize this enzyme. Phospholipase A(2) activity of crude extract was inhibited by both a PLA(2)-specific inhibitor (pBpb) and the metal ion chelator EDTA. L. obliqua PLA(2) was purified by liquid chromatography from the crude bristle extract and had a molecular mass of 15kDa and a pI of 5.9; its N-terminal sequence showed high homology to a sequence of a putative PLA(2) obtained from a cDNA library of L. obliqua bristles, and it is tentatively placed among Group III phospholipases A(2). This enzyme was stable at 4 degrees C, sensitive to higher temperatures, and its maximum catalytic activity was at pH 8.0. L. obliqua PLA(2) induced hemolysis only when incubated with exogenous lecithin. Thus, the PLA(2) purified herein appears to be responsible for the indirect hemolytic activity of the crude bristle extract.
Asunto(s)
Cabello/enzimología , Lepidópteros/enzimología , Fosfolipasas A/química , Fosfolipasas A/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Activación Enzimática , Humanos , Datos de Secuencia Molecular , Peso Molecular , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Homología de Secuencia de AminoácidoRESUMEN
Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 microg) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d t. venom (4 microg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms.
Asunto(s)
Antivenenos/inmunología , Crotalus/inmunología , Crotoxina/antagonistas & inhibidores , Inmunoglobulina G/inmunología , Pruebas de Neutralización/métodos , Fosfolipasas A/inmunología , Animales , Especificidad de Anticuerpos , Antivenenos/biosíntesis , Antivenenos/farmacología , Tampones (Química) , Hemólisis/inmunología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/farmacología , Masculino , Ratones , Bloqueo Neuromuscular , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A2 , ConejosRESUMEN
Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 Ag) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 Ag) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms (AU)
El veneno de Crotalus durissus terrificus (C.d.t.) (Cascabel de Sud América) posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2) y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75). Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 Ag de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del antisuero se analizó en ratones por inoculación con diluciones de veneno entero preincubado con un volumen adecuado de anticuerpos IgG anti-PLA2. Se inocularon ratones controles con 0.5 ml i.p. de veneno (4 Ag.ml-1). El número de muertes fue contabilizado a las 24 y 48 h posteriores a la inoculación, demostrándose que la capacidad neutralizante de los anticuerpos IgG anti-PLA2 fue superior a la obtenida con el antiveneno crotálico. Los resultados obtenidos demuestran la potencial aplicación de antivenenos constituidos por anticuerpos específicos contra PLA2, y/o la inclusión de estos anticuerpos como suplementos en antivenenos polivalentes (AU)
Asunto(s)
Animales , Masculino , Ratones , Conejos , Crotalus/inmunología , Crotoxina/inmunología , Fosfolipasas A/inmunología , Antivenenos/inmunología , Inmunoglobulina G/inmunología , Pruebas de Neutralización/métodos , Crotoxina/toxicidad , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/toxicidad , Antivenenos/biosíntesis , Antivenenos/farmacología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/farmacología , Bloqueo Neuromuscular , Immunoblotting , Inmunoelectroforesis , Ensayo de Inmunoadsorción Enzimática , Especificidad de Anticuerpos , Cromatografía en Agarosa , Tampones (Química) , Hemólisis/inmunología , Modelos Animales de EnfermedadRESUMEN
Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 Ag) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 Ag) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms (AU)
El veneno de Crotalus durissus terrificus (C.d.t.) (Cascabel de Sud América) posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2) y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75). Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 Ag de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del antisuero se analizó en ratones por inoculación con diluciones de veneno entero preincubado con un volumen adecuado de anticuerpos IgG anti-PLA2. Se inocularon ratones controles con 0.5 ml i.p. de veneno (4 Ag.ml-1). El número de muertes fue contabilizado a las 24 y 48 h posteriores a la inoculación, demostrándose que la capacidad neutralizante de los anticuerpos IgG anti-PLA2 fue superior a la obtenida con el antiveneno crotálico. Los resultados obtenidos demuestran la potencial aplicación de antivenenos constituidos por anticuerpos específicos contra PLA2, y/o la inclusión de estos anticuerpos como suplementos en antivenenos polivalentes (AU)
Asunto(s)
Animales , Masculino , Ratones , Conejos , Crotalus/inmunología , Crotoxina/inmunología , Fosfolipasas A/inmunología , Antivenenos/inmunología , Inmunoglobulina G/inmunología , Pruebas de Neutralización/métodos , Crotoxina/toxicidad , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/toxicidad , Antivenenos/biosíntesis , Antivenenos/farmacología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/farmacología , Bloqueo Neuromuscular , Immunoblotting , Inmunoelectroforesis , Ensayo de Inmunoadsorción Enzimática , Especificidad de Anticuerpos , Cromatografía en Agarosa , Tampones (Química) , Hemólisis/inmunología , Modelos Animales de EnfermedadRESUMEN
Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 µg) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 µg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms
El veneno de Crotalus durissus terrificus (C.d.t.) (Cascabel de Sud América) posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2) y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75). Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 µg de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del antisuero se analizó en ratones por inoculación con diluciones de veneno entero preincubado con un volumen adecuado de anticuerpos IgG anti-PLA2. Se inocularon ratones controles con 0.5 ml i.p. de veneno (4 µg.ml-1). El número de muertes fue contabilizado a las 24 y 48 h posteriores a la inoculación, demostrándose que la capacidad neutralizante de los anticuerpos IgG anti-PLA2 fue superior a la obtenida con el antiveneno crotálico. Los resultados obtenidos demuestran la potencial aplicación de antivenenos constituidos por anticuerpos específicos contra PLA2, y/o la inclusión de estos anticuerpos como suplementos en antivenenos polivalentes
Asunto(s)
Animales , Masculino , Ratones , Conejos , Antivenenos/inmunología , Crotalus/inmunología , Crotoxina/inmunología , Inmunoglobulina G/inmunología , Pruebas de Neutralización/métodos , Fosfolipasas A/inmunología , Especificidad de Anticuerpos , Antivenenos/biosíntesis , Antivenenos/farmacología , Tampones (Química) , Cromatografía en Agarosa , Crotoxina/toxicidad , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Hemólisis/inmunología , Immunoblotting , Inmunoelectroforesis , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/farmacología , Bloqueo Neuromuscular , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/toxicidadRESUMEN
A new PLA2 (F16) was purified from Crotalus durissus terrificus venom by molecular exclusion chromatography followed by analytical reverse phase HPLC. The PLA2 (14.86 kDa by MALDI-TOF mass spectrometry) had an amino acid sequence of SLLQFNKMIKFETRKNAVPFYAFYGCYCGWGGRRRPKDATDRCCFVHDCCYEKVTKCNTKWDIYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNGYMFYPDSRCRGPSETC, and showed highly conserved Ca2+-binding and catalytic sites. F16 showed allosteric behavior with 10 mM Ca2+ and had temperature and pH optima of 25 degrees C and 7.9, respectively. F16 (10 microg/ml) produced neuromuscular blockade in chick biventer cervicis preparations in the absence and presence of crotapotin, indicating that crotapotin was not essential for neuromuscular action in this preparation. In contrast, in mouse phrenic nerve-diaphragm preparations, the neuromuscular blockade produced by the same concentration of toxin was dependent on crotapotin. Pre-incubation with heparin markedly reduced the neurotoxicity of F16. These results show that the biochemical and structural properties of F16 are similar to those of the PLA2 isoforms F15 and F17, but that the neurotoxicity and the requirement for crotapotin to form the crotoxin complex varies according to the neuromuscular preparation.
Asunto(s)
Venenos de Crotálidos/enzimología , Fosfolipasas A/química , Fosfolipasas A/farmacología , Secuencia de Aminoácidos , Animales , Pollos , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Crotalus , Diafragma/efectos de los fármacos , Masculino , Ratones , Datos de Secuencia Molecular , Unión Neuromuscular/efectos de los fármacos , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Nervio Frénico/efectos de los fármacos , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
A new Phospholipase A(2) (PLA(2)) from Micrurus dumerilii carinicauda venom was isolated and its primary structure determined. This new PLA(2) showed a low enzymatic activity when compared with other PLA(2)s and it is moderately basic with an isoelectric point of 8.0. Its amino acid sequence showed the presence of 120 amino acid residues and its sequence was: NLIQFLNMIQCTTPGREPLVAFANYGCYCGRGGSGTPVDELDRCCQVHDNCYDTAKKVFGCSPYFTMYSYDCSEGKLTCKDNNTKCKAAVCNCDRTAALCFAKAPYNDKNYKIDLTKRCQ. The structural model of MIDCA1, when compared with other strong neurotoxic PLA(2)s, such as Naja naja, showed significant differences in the beta-wing and neurotoxic sites, despite the high level of amino acid sequence similarity. These observations indicate a dissociation between the biological and catalytic activity of this new PLA(2), supporting the view that other regions of the protein are involved in the biological effects.
Asunto(s)
Secuencia de Aminoácidos , Venenos Elapídicos/enzimología , Fosfolipasas A/genética , Animales , Elapidae , Modelos Moleculares , Datos de Secuencia Molecular , Fosfolipasas A/química , Fosfolipasas A/aislamiento & purificación , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Bothrops jararacussu myotoxin I (BthTx-I; Lys 49) and II (BthTX-II; Asp 49) were purified by ion-exchange chromatography and reverse phase HPLC. In this work we used the isolated perfused rat kidney method to evaluate the renal effects of B. jararacussu myotoxins I (Lys49 PLA2) and II (Asp49 PLA2) and their possible blockage by indomethacin. BthTX-I (5 microg/ml) and BthTX-II (5 microg/ml) increased perfusion pressure (PP; ct120=110.28+/-3.70 mmHg; BthTX I=171.28+/-6.30*mmHg; BthTX II=175.50+/-7.20*mmHg), renal vascular resistance (RVR; ct120=5.49+/-0.54 mmHg/ml.g(-1)min(-1); BthTX I=8.62+/-0.37*mmHg/ml g(-1)min(-1); BthTX II=8.9+/-0.36*mmHg/ml g(-1)min(-1)), urinary flow (UF; ct(120)=0.14+/-0.01ml g(-1)min(-1); BthTX I=0.32+/-0.05*ml g(-1)min(-1); BthTX II=0.37+/-0.01*ml g(-1)min(-1)) and glomerular filtration rate (GFR; ct120=0.72+/-0.10 ml g(-1)min(-1); BthTX I=0.85+/-0.13*ml g(-1)min(-1); BthTX II=1.22+/-0.28*ml g(-1)min(-1)). In contrast decreased the percent of sodium tubular transport (%TNa(+); ct(120)=79,76+/-0.56; BthTX I=62.23+/-4.12*; BthTX II=70.96+/-2.93*) and percent of potassium tubular transport (%TK(+);ct120=66.80+/-3.69; BthTX I=55.76+/-5.57*; BthTX II=50.86+/-6.16*). Indomethacin antagonized the vascular, glomerular and tubular effects promoted by BthTX I and it's partially blocked the effects of BthTX II. In this work also evaluated the antibacterial effects of BthTx-I and BthTx-II against Xanthomonas axonopodis. pv. passiflorae (Gram-negative bacteria) and we observed that both PLA2 showed antibacterial activity. Also we observed that proteins Also we observed that proteins chemically modified with 4-bromophenacyl bromide (rho-BPB) decrease significantly the antibacterial effect of both PLA2. In conclusion, BthTx I and BthTX II caused renal alteration and presented activity antimicrobial. The indomethacin was able to antagonize totally the renal effects induced by BthTx I and partially the effects promoted by BthTx II, suggesting involvement of inflammatory mediators in the renal effects caused by myotoxins. In the other hand, other effects could be independently of the enzymatic activity of the BthTX II and the C-terminal domain could be involved in both effects promoted for PLA2.