Effects of chelating calcium in cryopreservation extender on frozen-thawed dog semen / [Efeitos da quelação de cálcio no meio de criopreservação no sêmen descongelado de cão]

We evaluated the effect of reducing free calcium in the cryopreservation medium, using the calcium chelator ethylene diamine tetracetic acid (EDTA) at 0.3% and 0.5% concentrations. Three male mixed breed dogs were subjected to semen collection by digital manipulation (n=16). Each ejaculate was divided in three aliquots, and each one was diluted in TRIS-glucose-egg yolk extender with 6% glycerol and 0.5% Equex STM Paste® (TGE, control); and added with 0.3% EDTA (EDTA 0.3) or 0.5% EDTA (EDTA 0.5). Calcium concentration reduced in EDTA 0.3 and all the calcium ions were chelated in EDTA 0.5. The EDTA addition did not affect sperm morphology or plasma membrane integrity; however, by removing all free calcium (EDTA 0.5), the sperm motility reduced (64.7% in TGE and 45% in EDTA 0.5; p<0.05). Acrosome integrity and sperm binding ability were not improved by calcium chelation. The failure to prevent the premature AR may explain why sperm longevity was not affected by calcium removal. Thus, the partial or complete calcium removal, through EDTA addition, is not able to prevent acrosomal damage or premature acrosomal reaction, and therefore does not improve the dog sperm binding ability.(AU)

Avaliou-se o efeito da redução do cálcio livre no meio de congelamento, usando-se o quelante de cálcio etilenodiaminotetracético (EDTA) a 0,3% e 0,5%. Três cães machos sem raça definida foram submetidos à coleta de sêmen por manipulação digital (n=16). Cada ejaculado foi diluído em diluidor controle com TRIS-glicose - gema de ovo (TGE, controle), ou em diluidor TGE enriquecido com 0,3% (EDTA 0,3) ou 0,5% de EDTA (EDTA 0,5). A concentração de cálcio reduziu no meio EDTA 0,3, e todos os íons de cálcio foram quelados no meio EDTA 0,5. A adição do EDTA e a consequente quelação do cálcio não afetaram a morfologia espermática ou a integridade da membrana plasmática, no entanto, ao remover todo o cálcio do meio (EDTA 0,5), a motilidade espermática se reduziu (64,7% no TGE e 45% no EDTA 0,5; P<0,05). A integridade do acrossoma e a capacidade de ligação do espermatozoide não melhoraram com a quelação do cálcio. Apesar da influência da concentração de cálcio sobre a motilidade espermática após o descongelamento, a falha em prever a reação acrossomal prematura pode explicar por que a longevidade espermática não foi afetada pela remoção do cálcio no meio. Dessa forma, a remoção parcial ou total do cálcio, por meio da adição de EDTA, não é capaz de prevenir o dano no acrossoma ou a reação acrossomal prematura e, portanto, não aumenta a capacidade do espermatozoide de se ligar ao oócito.(AU)

Desordens hematológicas em onças-pardas (Puma concolor; Linnaeus, 1771) infectadas por Citauxzoon felis / [Hematological disorders in Brown jaguars (Puma concolor; Linnaeus 1771) infected by Citauxzoon felis]

Este estudo objetivou descrever o aspecto hematológico de seis onças-pardas (Puma concolor) infectadas pelo Cytauxzoon felis. Os seis casos de infecção foram identificados durante o manejo sanitário de 11 animais de um centro de reabilitação de animais silvestres. Estruturas compatíveis com piroplasmídeos foram observadas durante a avaliação do esfregaço sanguíneo e confirmadas como Cytauxzoon felis pela técnica de PCR. A análise estatística demonstrou diferença significativa (P<0,05) no número absoluto dos linfócitos entre os grupos dos animais infectados e não infectados. Assim, expressivas alterações hematológicas e bioquímicas entre os grupos investigados alertam para a dificuldade de identificação de onças-pardas infectadas por C. felis, apoiada apenas em exames de rotina, bem como para o risco, sobretudo, da reintrodução desses animais na natureza.(AU)

This Cytauxzoon felis by the PCR technique. Statistical analysis showed a significant difference is study aimed to describe the hematological appearance of six puma (puma concolor) infected with cytauxzoon felis. The six cases of infection were identified during the sanitary management of 11 animals from a wild animal rehabilitation center. Piroplasmid compatible structures were observed during the blood smear evaluation and confirmed as (P<0.05) in the absolute number of lymphocytes between the groups of infected and uninfected animals. Thus expressive hematological and biochemical alterations between the groups investigated alert to the difficulty of identifying infected brown jaguars by C. felis, supported only by routine examinations, and the risk especially when aiming at the reintroduction of these animals in the wild.(AU)

Effects of two protease inhibitors from Bauhinia bauhinoides with different specificity towards gut enzymes of Nasutitermes corniger and its survival.

Two recombinant protease inhibitors from Bauhinia bauhinioides, rBbKI (kallikrein inhibitor) and rBbCI (cruzipain inhibitor) were evaluated for insecticidal activity against workers and soldiers of Nasutitermes corniger (order: Isoptera; family: Termitidae) through the inhibitors' effect on the insect's gut enzymes. The inhibitor rBbKI was more effective than rBbCI in inhibiting the termite's gut enzymes. The kallikrein inhibitor showed termiticidal activity in workers with an LC50 of 0.9 mg mL-1 after 4 days. Conversely, rBbKI did not affect the survival of soldiers and rBbCI did not show termiticidal activity against N. corniger. The two inhibitors showed different specificity towards the termite's gut enzymes, representing interesting tools to characterize N. corniger enzymes. The different effects of rBbKI and rBbCI on the termite's enzymes and survival may be linked to slight structural differences between these inhibitors.

The ambient intelligence for the sake of accessibility in residential projects: a proper study to the Brazilian scene.

The concept of Ambient Intelligence consists of intuitive interfaces embedded in everyday objects that interact with the user and function in an integrated and discrete generating and transmitting information. This article has as objective to present applications that when focused on accessibility criteria, can contribute to the work of architects during the design of residential projects. In Brazil, pioneering projects are already being developed and the use of Ambient Intelligence systems are already widespread, both by private and public, however much care technologies developed in other countries.

Baupain, a plant cysteine proteinase that hinders thrombin-induced human platelet aggregation.

Bauninia forficata is trivially known as cow paw, and popularly used in Brazil for treatment of diabetes mellitus. Denominated baupain a cysteine proteinase was purified from B. forficata leaves. In this study, we investigated the baupain effect on aggregation of isolated human platelets in vitro and the results show that baupain hinders thrombin - but not ADP- and collagen- induced platelet aggregation. With synthetic quenched-fluorescent peptides, the kinetics of the cleavage site of human proteinase-activated receptor 1 / 2 / 3 and 4 [PAR-1 / 2 / 3 and 4] by baupain was determined. In conclusion, similar to bromelain and papain, baupain hinders human platelets aggregation, probably through an unspecific cleavage in the Phe-Leu bond of PAR1.

Anti-Trypanosoma cruzi activity of Pterodon pubescens seed oil: geranylgeraniol as the major bioactive component.

In the search for new therapeutic agents for Chagas' disease, we screened extracts obtained from the Brazilian plant Pterodon pubescens found commercially in the medicinal flora market. We investigated the potential trypanocidal effect of the oleaginous ethanolic extract of P. pubescens seeds and its fractions (PF1, PF1.1, PF1.2, and PF1.3) and of geranylgeraniol (GG-OH), the sole component of the hexane fraction (PF1.2). In experiments with bloodstream trypomastigotes of Trypanosoma cruzi, performed at 37 degrees C in culture medium, PF1.2 and GG-OH showed similar potency, while the oleaginous extract from P. pubescens seeds and the other fractions were about three times less active. GG-OH inhibited the proliferation of intracellular amastigotes, at concentrations which do not affect the mammalian host cell. Transmission electron microscopy and flow cytometry analysis indicate the mitochondrion, an organelle that plays a central role in apoptosis, of both epimastigotes and of trypomastigotes as the major target of GG-OH. On the other hand, the ultrastructural images of the endoplasmic reticulum profiles, myelin-like figures, and concentric membranous arrangements inside damaged mitochondrion are suggestive of an autophagic pathway leading to parasite death. Because the different forms of cell death share some morphological features such as mitochondrial collapse, further studies are needed to disclose the trypanocidal action of GG-OH.

Avaliação da qualidade microbiológica do queijo-de-minas artesanal do Serro-MG / Microbiological quality of artisanal minas cheese, manufactured in the region of Serro-MG

Avaliaram-se a qualidade microbiológica do queijo-de-minas artesanal do Serro e a variação da microbiota do queijo recém-fabricado e no último dia de validade, após estocagem sob as condições recomendadas no rótulo. Foram analisadas 40 amostras de queijo, sendo 20 analisadas frescas, imediatamente após a coleta, e 20 no último dia de validade, após estocagem em câmara fria a 10ºC. As amostras foram analisadas para coliformes a 35ºC e 45ºC, Staphylococcus coagulase positiva, Salmonella spp. e Listeria monocytogenes. Trinta e sete amostras (92,5 por cento) encontraram-se impróprias para o consumo humano, de acordo com os parâmetros estabelecidos pela resolução: RDC ANVISA nº12/01, sendo a principal causa de condenação a contagem de Staphylococcus coagulase positiva. Nenhuma das amostras analisadas apresentou contaminação por Salmonella sp. ou Listeria monocytogenes. As contagens de Staphylococcus coagulase positiva dos queijos frescos foram mais altas (P<0,01) que as dos queijos analisados no último dia da validade

The microbiological quality of artisanal minas cheese produced in the region of Serro-MG was evaluated. Cheeses were analyzed fresh and at the end of the shelf life, under specific storage conditions (30 days, 10ºC). Forty samples of minas cheese were analyzed, twenty still fresh, and 20 after storage at 10ºC. The microbiological analyses were counts of fecal and total coliforms, and Staphylococcus coagulase positive; and search of Salmonella sp. and Listeria monocytogenes. Twenty-seven samples (92.5 percent) were in disagreement with the Brazilian Standard, Resolution nº 12 of January 12, 2001, from ANVISA (National Health Surveillance Agency), and Staphylococcus coagulase positive was the major contaminant. No sample was contaminated by Salmonella sp. or Listeria monocytogenes. During the storage, there was a decrease of countings in the population of Staphylococcus coagulase positive (P<0.01)

Acute and topic anti-edematogenic fractions isolated from the seeds of Pterodon pubescens.

We previously demonstrated that alcoholic extracts from Pterodon pubescens Benth. (Sucupira branca, Leguminosae) seeds exhibit anti-arthritic activity. In the present work we show that the oleaginous extract obtained from P. pubescens seeds (OEP) exhibits acute or topic anti-edematogenic activity when tested in carrageenan-induced paw edema or in croton oil-induced ear edema assays, respectively. Four fractions were obtained from OEP by sequential liquid-liquid extraction. The anti-edematogenic properties were predominant in the hexanic fraction, which was further fractionated by HPLC, yielding three sub-fractions (PF1.1, PF1.2 and PF1.3). PF1.1 and PF1.3 showed potent acute and topic anti-edematogenic activity. The PF1.2 sub-fraction, although not active in the carrageenan assay, exhibited a potent anti-edematogenic activity in the croton oil-induced ear edema. This sub-fraction shows a maximum efficacy similar to indometacin in a lower dose. The PF1.1 sub-fraction presented a complex mixture containing furane diterpene derivatives of vouacapan. PF1.2 consists of a single substance, geranylgeraniol, as determined by GC/MS and NMR, while PF1.3 contains farnesol.

Hematological, hemostatic and clinical chemistry disturbances induced by Crotalus durissus terrificus snake venom in dogs.

The aim of this study was to investigate the hematological, hemostatic and biochemical disturbances induced by the injection of Crotalus durissus terrificus venom in dogs under controlled conditions. For this purpose three groups of animals were used: an experimental group (E), which was injected i.m. with C. durissus terrificus venom (1 mg/kg); and two control groups--antivenom (AV) and control (C)--which were injected i.m. with 150 mM NaCl. Groups E and AV were treated i.v. with Crotalus antivenom 2 hours after the first injection. Serum levels of alkaline phosphatase and alanine aminotransferase were increased in groups E and AV at 24 and 48 hours after serumtherapy, respectively. The increased serum levels of myoglobin, creatine kinase and aspartate aminotransferase demonstrated that animals developed rhabdomyolysis. A persistent neutrophilic leukocytosis was already noticeable at 2 hours after envenomation and lasted even after serumtherapy. The animals of groups E and AV presented eosinopenia 24 hours after serumtherapy, and collagen-induced platelet hypoaggregation was observed without thrombocytopenia. Increased levels of fibrinogen/fibrin degradation products (FnDP/FgDP), hypofibrinogenemia, and alpha2-antiplasmin consumption were observed at 2 hours after envenomation, indicating secondary activation of fibrinolysis. Our data suggest that the biochemical and hemostatic disturbances induced by C. durissus terrificus venom in dogs are related to its myotoxic and thrombin-like activities.

Contribution of crotoxin for the inhibitory effect of Crotalus durissus terrificus snake venom on macrophage function.

Previous work of our group demonstrated that Crotalus durissus terrificus venom has a dual effect on macrophage function: it inhibits spreading and phagocytosis and stimulates hydrogen peroxide and nitric oxide production, antimicrobial activity and glucose and glutamine metabolism of these cells. Crotalid venom also induces analgesia and this effect is mediated by opioid receptors. The involvement of opioidergic mechanism and the determination of the active component responsible for the inhibitory effect of crotalid venom on macrophage function were investigated. The venom reduced the spreading and phagocytic activities of peritoneal macrophages. This effect was observed in vitro, 2 h after incubation of resident peritoneal macrophages with the venom, and in vivo, 2 h after subcutaneous injection of the venom. The inhibition of phagocytosis was not modified by naloxone, an antagonist of opioid receptors. Venom neutralization with crotalid antivenom abolished the inhibitory effect of the venom, indicating that venom toxins are involved in this effect. Crotoxin, the main toxin of crotalid venom, s.c. injected to rats or added to the medium of peritoneal cell incubation, inhibited macrophage function in a similar manner to that observed for crude venom. The present results suggest that crotoxin causes a direct inhibition of macrophage spreading and phagocytic activities and may contribute to the inhibitory effect of crotalid venom on macrophage function.

Biological and immunochemical characterization of Micrurus altirostris venom and serum neutralization of its toxic activities.

Micrurus altirostris venom from Rio Grande do Sul State, Brazil, was characterized by its biological activities, immunochemical properties and electrophoretic pattern. The results showed a high edematogenic activity, whose peak was observed after 30min of venom injection, as well as a high indirect hemolytic activity. This venom was myotoxic, as shown by a peak of CK release at 6h after injection, and also by the appearance of muscular lesions characterized by necrosis, loss of striated muscle fibers, and the presence of vacuolization, edema and inflammatory infiltrate. This venom showed minimum proteolytic activity and no hemorrhagic, dermonecrotic or coagulant activities. Nonetheless, M. altirostris venom presented high lethal activity. Electrophoretic patterns of Micrurus frontalis and M. altirostris venoms showed different protein bands. Anti-elapidic serum could recognize M. frontalis (homologous) and M. altirostris (heterologous) venoms by Western blotting, and both venoms presented similar titers when assayed by ELISA. The results observed on neutralization tests showed that the anti-elapidic serum produced by Instituto Butantan neutralized myotoxic and hemolytic activities. However, this antivenom could not neutralize the lethal activity of M. altirostris venom. Thus, these data suggest that M. altirostris venom presents different biological, enzymatic and immunological characteristics from other Micrurus venoms, and some activities are not neutralized by the commercial anti-elapidic serum produced in Brazil.

Mortalidade por "septicemia dos peixes tropicais" em tilápias criadas em consorciação com suínos

A rural property was observed for one year to obtain seasonal observations of Edwardsiella septicemia in tilapias (Oreochromis sp.) from an integrated fish farm (using pig excrements as food). Fifty tilapias on summer, forty-five on autumn, fifty-five on winter and one hundred and five on spring were collected and evaluated microbiologically for the presence of Edwardsiella tarda. Samples of the external surface (skin, gills and fin), intestines and muscle were analyzed. Fishes were transported alive to the laboratory, and maintained for five days for observation. The external signs observed were opacity of cornea, respiratory difficulty, disordered swimming, nodules on gills, hemorrhagic lesions under the skin and fins and incoordination of the posterior part of the body. In general, after three days of observation, the tilapias died. At necropsy, ascites with the distension of the celomatic cavity, hemorrhagic and necrotic lesions in the liver, spleen and kidneys were found. Hemorrhagic enteritis was observed in the gut tube. Edwardsiella tarda isolated presented the following biochemical profile: negative urea, sucrose, phenylalanine desaminase and malonate utilization, and positive indole, methyl red, H2S, motility, glucose with gas production and lysine decarboxylase. The mortality rates were 69.9% on spring, 63.6% on winter, 48.0% on summer and 40.0% on autumn, highest rates occurring on spring and winter (P 0,05).

Com o objetivo de estudar a variação sazonal da septicemia dos peixes tropicais provocada por Edwardsiella tarda foram observadas 50 tilápias (Oreochromis sp.) no verão, 45 no outono, 55 no inverno e 105 na primavera. Os peixes doentes apresentavam opacidade de córnea, dificuldade respiratória, nado desordenado, nódulos nas brânquias e lesões hemorrágicas na pele e nadadeiras. Após três dias de observação as tilápias morriam naturalmente. Nas necrópsias foi encontrado ascite com distensão da cavidade celomática e lesões hemorrágicas e necróticas no fígado, baço e rins. No interior do tubo digestivo observou-se enterite hemorrágica. Isolou-se Edwardsiella tarda a partir de provas positivas para H2S, lisina, indol, motilidade e glicose com gás. Os índices de mortalidade foram: na primavera 69,9%, no inverno 63,6%, no verão 48,0% e no outono 40,0%.

Hematological changes induced by Bothrops jararaca venom in dogs

The mechanism of consumption coagulopathy observed in cases of human envenomation by Bothrops jararaca is well established. However, this mechanism may vary according to the animal species studied. In order to study both the clinical and laboratory aspcts of bothropic envenomation in dogs, a sublethal defibrinating dose of venom (100 µg/kg) was given intravenously. A coagulopathy similar to that observed in humans - including fibronogen depletion, consumption of factors II, X, V and antithrombin III, and moderate thrombocytopenia -was observed. The presence of circulatin activated platelets was also noted. Neutrophilic leukocytosis, lymphopenia, and monocytosis occurred at different times. Erythrocytic values remained normal in dogs treated with B. jararaca venom compared with those treated with saline alone. The erythrocyte sedimentation rate fell rapidly after venom administration and this fall was correlated logarithmically with fibrinogen concentration. Since the effect of envenomation in dogs is similar to that in humans, it was concluded that the dog can be used as a good animal model for studying human venom-induced coagulopathy