Biochemical and biological evaluation of gyroxin isolated from Crotalus durissus terrificus venom

Gyroxin, a thrombin-like enzyme isolated from Crotalus durissus terrificus venom and capable of converting fibrinogen into fibrin, presents coagulant and neurotoxic activities. The aim of the present study was to evaluate such coagulant and toxic properties. Gyroxin was isolated using only two chromatographic steps - namely gel filtration (Sephadex G-75) and affinity (Benzamidine Sepharose 6B) - resulting in a sample of high purity, as evaluated by RP-HPLC C2/C18 and electrophoretic analysis that showed a molecular mass of 30 kDa. Gyroxin hydrolyzed specific chromogenic substrates, which caused it to be classified as a serine proteinase and thrombin-like enzyme. It was stable from pH 5.5 to 8.5 and inhibited by Mn²+, Cu²+, PMSF and benzamidine. Human plasma coagulation was more efficient at pH 6.0. An in vivo toxicity test showed that only behavioral alterations occurred, with no barrel rotation. Gyroxin was not able to block neuromuscular contraction in vitro, which suggests that its action, at the studied concentrations, has no effect on the peripheral nervous system.

Purification and n-terminal sequencing of two presynaptic neurotoxic PLA2, neuwieditoxin-I and neuwieditoxin-II, from Bothrops neuwiedi pauloensis (jararaca pintada) venom

Two presynaptic phospholipases A2 (PLA2), neuwieditoxin-I (NeuTX-I) and neuwieditoxin-II (NeuTX-II), were isolated from the venom of Bothrops neuwiedi pauloensis (BNP). The venom was fractionated using molecular exclusion HPLC (Protein-Pak 300SW column), followed by reverse phase HPLC (æBondapak C18 column). Tricine-SDS-PAGE in the presence or absence of dithiothreitol showed that NeuTX-I and NeuTX-II had a molecular mass of approximately 14 kDa and 28kDa, respectively. At 10æg/ml, both toxins produced complete neuromuscular blockade in indirectly stimulated chick biventer cervicis isolated preparation without inhibiting the response to acetylcholine, but NeuTX-II reduced the response to KCl by 67.0±8.0 percent (n=3; p<0.05). NeuTX-I and NeuTX-II are probably responsible for the presynaptic neurotoxicity of BNP venom in vitro. In fact, using loose patch clamp technique for mouse phrenic nerve-diaphragm preparation, NeuTX-I produced a calcium-dependent blockade of acetylcholine release and caused appearance of giant miniature end-plate potentials (mepps), indicating a pure presynaptic action. The N-terminal sequence of NeuTX-I was DLVQFGQMILKVAGRSLPKSYGAYGCYCGWGGRGK (71 percent homology with bothropstoxin-II and 54 percent homology with caudoxin) and that of NeuTX-II was SLFEFAKMILEETKRLPFPYYGAYGCYCGWGGQGQPKDAT (92 percent homology with Basp-III and 62 percent homology with crotoxin PLA2). The fact that NeuTX-I has Q-4 (Gln-4) and both toxins have F-5 (Phe-5) and Y-28 (Tyr-28) strongly suggests that NeuTX-I and NeuTX-II are Asp49 PLA2.

Inhibition of L-glutamate and GABA synaptosome uptake by crotoxin, the major neurotoxin from Crotalus durissus terrificus venom

This paper describes a brief study on the crotoxin mechanism of action, regarding the transport of GABA and L-glutamate in rats cortico-cerebral synaptosomes and in heterologous systems, such as COS-7 cells expressing gabaergic transporters, and C6 glioma cells and Xenopus oocytes expressing glutamatergic transporters. Crotoxin concentrations over 1 µM caused an inhibitory effect of ³H-L-glutamate and ³H-GABA, and reversibly inhibited L-glutamate uptake by C6 glioma cells. When COS-7 cells were assayed, no inhibition of the ³H-GABA transport could be evidenced. Crotoxin kept its inhibitory effect on neurotransmitters uptake even when Ca2+ ions were removed from the medium, therefore, independently of its PLA2 activity. In addition, high concentrations (2 mM) of BPB did not avoid the action of crotoxin on the neurotransmitters uptake. Crotoxin also inhibited ³H-L-glutamate, independently on Na+ channel blockade by TTX. In addition, an evaluation of the lactic dehydrogenase activity indicated that uptake inhibition does not involve a hydrolytic action of crotoxin upon the membrane. We may also suggest that crotoxin acts, at least partially, altering the electrogenic equilibrium, as evidenced by confocal microscopy, when a fluorescent probe was used to verify cell permeability on C6 glioma cells in presence of crotoxin.

Rabbit antivenom efficacy against myotoxic and neurotoxic activities of Bothrops jararacussu venom and Bothropstoxin-I

Bothrops jararacussu venom and its major toxin bothropstoxin-I (BthTX-I) possess myotoxic and neurotoxic properties. The efficacy of a rabbit antivenom raised against B. jararacussu venom in the neutralization of physiological, biochemical, and morphological changes induced by the venom and its major toxin BthTX-I was studied in mouse isolated phrenic nerve-diaphragm (PND) and extensor digitorum longus (EDL) preparations. The times required for 50 per cent neuromuscular blockade in PND and EDL preparations for venom were 70ñ11.5 (S.E.M., n=5) min and 58ñ8 (n=16) (50 µ/mL), and for BthTX-I 31ñ6 (n=3) min and 30ñ3 (n=5) min (20 µg/mL), respectively. After 120 min incubation, creatine kinase (CK) concentrations in solution containing the EDL preparations were 3464ñ346 U/L after exposure to venom (50 µg/mL, n=5) and 3422ñ135 U/L to BthTX-I (20µg/mL, n=4), respectively. Rabbit antivenom dose-dependently neutralized venom and toxin-induced neuromuscular blockade in both preparations and effectively prevented venom and toxin-induced CK release from EDL. Histological analysis showed that rabbit antivenom neutralized morphological damage caused by B.jararacussu venom and BthTX-I in EDL preparations. these results indicate that rabbit antivenom effectively neutralized the biological activities of B.jararacussu venom and BthTX-I.

Intraspecific variation in neurotoxic and myotoxic activities of Bothrops neuwiedii venoms

Snake venoms frequently vary in composition. In this work, we compared the neurotoxic and myotoxic activities of 16 lots of Bothrops neuwiedii venoms from different regions of Brazil, using chick biventer cervicis preparations. The neuromuscular blockade varied from 2 per cent to 100 per cent after 120 min incubation with venoms (50µg/ml). In all cases, this blockade was irreversible and concentration-dependent; at low concentrations (10-20 µg/ml), 15 of the 16 venom lots failed to abolish responses to acetylcholine (110µM), but blocked responses to KCI (13.4mM), and induced contracture. At 5-20µg/ml, the most active venom totally blocked twitch-tension without affecting responses to acetylcholine and KCI. Polyacrylamine gel electrophoresis for basic proteins showed that the most active samples contained a band that was absent in the less active venoms. These results indicate that there may be considerable intraspecific variation in the neurotoxic activity of B. ineuwiedii venoms, whereas myotoxic activity is less variable.

Enfermedad de Paget mamaria / Paget's disease of breast

Presentamos un caso de enfermedad de Paget mamario con estudio de los aspectos clínicos, histopatológicos, radiológicos y el tratamiento quirúrgico con terapia sistémica adyuvante. Se presenta esta patología poco frecuente para revisar su diagnóstico diferencial y el procedimiento adecuado para enfrentarlo y mejorar la calidad de vida del paciente.

Polyacrylamide gel electrophoresis as a tool for the taxonomic identification of snakes from the Elapidae and Viperidae families

Polyacrylamide gel electrophoresis (PAGE) for basic proteins may be a useful toll for the characterization of whole snake venoms and for the taxonomic classification of snakes of the Elapidae and Viperidae families. However, due to the close proximity of PAGE was not able to provide an efficient differentiation. This article reports the electrophoretic analysis of several venoms from the genera Micrurus, Bothrops, Bothriopsis, Crotalus and Lachesis and shows a typical and distinctive electrophoretic profile for each species, with intraspecific and geographic variation. Even in cases in which extreme morphological similarities were present, such as between B. jararacussu and B. pirajai ("Bahia jararacussu"), differentiation could be evidenced by PAGE. This simple and sensitive procedure may be applied to similar cases involving basic toxins.

Methodological care in the evaluation of the LD50 and of the neutralization of the lethal effect of Crotalus terrificus venom by the plant Peschiera fuchsiaefolia (Apocynaceae)

In the present study, we demonstrate that the volumes in which a given protein mass of Crotalus durissus terrificus venom or of a lyophilized stabilized aqueous extract (LSAE) of Peschiera fuchsiaefolia, an antivenom agent injected intramuscularly, have a decisive influence on the results. The LD50 of C. d. terrificus venom injected i.m. in a final volume of 200µg (2µl/g) (saline solution, 0.9 per cent NaCl) was 180µg/100g rat body weight (p<0.05, 161 to 202µ/100g body weight) and the LD50 of the venom injected i.m. in a volume of 50µl (0.5µl/g) was 120µg/100g body weight (p<0.05, 107 to 134µg/100g body weight). The reduction of the final volume injected i.m. also required a reduced mass of LSAE necessary to neutralize the lethal effect of C. d. terrificus venom. The dose of 60mg LSAE/100g rat body weight in a final volume of 200µl administered i.m., 20 seconds after venom injection, and that of 40mg LSAE/100g body weight/200µl mixed and incubated with the venom for 1h at 25º before i.m. injection were able to neutralize the lethal activity of 2LD50. However, the LSAE doses that neutralized the 2LD50 were reduced to 20mg LSAE/100g body weight in a final volume of 50µl when administered i.m. 20 seconds after venom injection and to 2.5mg LSAE/100g body weight/50µl when mixed and incubated for 1h at 25º with the venom i.m. injection. The LD50 of C. d. terrificus venom and the doses of P. fuchsiaefolia LSAE that neutralized the venom lethal activity were, therefore, significantly lower when the final volume injected i.m. was reduced.

Aspectos psicossociais mais relevantes evidenciados em anamneses de pacientes acometidos de artrite reumatóide / Relevant psychosocial aspects detected in the anammesis of patients with rheumatoid arthritis

Este trabalho focaliza aspectos psicossociais mais relevantes evidenciados nas anammeses de 59 pacientes acometidos de artrite reumatóide, tratados no ambulatório de Reumatologia do HC da Faculdade de Ciências Médicas da Universidade Estadual de Campinas. Säo salientados tópicos ligados a desestruturaçöes de muitos lares na infância, devido a mortes precoces da mäe, e também vínculos conflituais de dependência afetiva por parte destes enfermos. Aborda a situaçäo psicossocial da mulher, que constitui quase 80% desta populaçäo de estudo. Os autores enfatizam aspectos ligados às repercussöes da doença à vida destes pacientes, particularmente no que tange à cronicidade da moléstia

Eventos estressantes de vida e ocorrência de artrite reumatóide / Life stressing events and occurrence of rheumatoid arthritis

Faz-se um estudo de aspectos psicológicos e sociais de 59 pacientes de artrite reumatóide tratados no Ambulatório de Reumatologia da Faculdade de Ciências Médicas da Universidade Estadual de Campinas - UNICAMP, no período compreendido entre fevereiro e outubro de 1984. Dentre os aspectos focalizados, ressaltam-se eventos estressantes de vida, responsáveis por distúrbios afetivoemocionais nos 12 meses que antecedem o aparecimento da AR e que podem ter colaborado nos fatores causais e/ou agravantes da mesma

Características de personalidade de pacientes artríticos reumatóides, tratados num ambulatório de um hospital-escola / Personality characteristics of patients with rheumatoid arthritis, treated in a outpatient clinic of a teaching hospital

Relata-se um estudo sobre as características de personalidade pré-mórbida de pacientes acometidos de artrite reumatóide, tratados no ambulatório de Reumatologia do HC da FMC da Unicamp. As altas freqüências de respostas positivas encontradas para características obsessivas de personalidade säo salientadas, fato este comprovado também através das análises estatísticas das correlaçöes positivas e negativas entre estas respostas