The alpha 1-proteinase inhibitors of trypsin, Spi1, Spi3A, and Spi3B, in bronchoalveolar lavage fluid (BALF) and serum of horses were separated by electrophoresis, and their proportions were quantified in 12 control horses and 12 with chronic obstructive pulmonary disease (COPD). A significantly lower proportion of Spi3B (P < 0.05) and higher proportion of Spi1 (P < 0.02 to P < 0.01) were detected in BALF, compared with serum, in control and COPD-affected horses and appeared to be attributable to reduced Spi3 activity in BALF. There was no significant difference between the control and COPD groups in this respect, indicating that the decrease in Spi3 may be a physiologic phenomenon. The differences observed may be associated with proteolytic damage to or preferential complex formation by Spi3.

The proteins of the bovine erythrocyte membrane were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and their relations to the slow sedimentation rate of bovine erythrocytes were investigated by treating the erythrocytes with trypsin. The erythrocyte sedimentation rates of bovine erythrocytes from Holstein and Korean native cattle were very slow compared with the human one (1/7 as slow as the human one) as reported previously. However, when human and Holstein erythrocytes were treated with trypsin (0.2 and 0.5 mg/ml) for 1 hour at 37deg C, their sedimentation rates were markedly accelerated while the sedimentation rate of Korean native cattle's erythrocytes were not affected. Although the general protein profiles of the bovine erythrocyte membranes were almost similar to that of human, bovine erythrocyte membranes showed one additional protein band called band Q in this study, which migrated electrophoretically to the mid-position between band 2 and band 3 in human erythrocyte membranes. Treatment of Holstein and human erythrocytes with trypsin caused a decrease or disappearance of the band Q from the erythrocyte membrane. Although the band Q in Korean native cattle's erythrocyte membrane was decreased by trypsin treatment of the erythrocytes, the magnitude of the decrement was not so pronounced as in the case of human and Holstein erythrocytes. The glycoprotein profiles of the bovine erythrocyte membranes revealed by periodic acid-Schiff stain showed a marked difference from that of human. The PAS-1 (glycophorin) and PAS-2 (sialoglycoprotein) present in human erythrocyte membrane were almost absent from the bovine erythrocyte membranes. Instead, the bovine erythrocyte membranes showed a strong PAS-positive band near the origin of the electrophorograms, which is named as PAS-B in this study

For the laboratory diagnosis of Sarcocystis infections especially in domesticated food animals, several antificial digestion techniques were applied for the musculature specimens and several staining techniques wes applied for the bradyzoites of Sarcocystis species isolated. The digestion technique using trypsin (0.5%) and sodium chloride (0.85%) mixed solution was regarded as the most valuable for the detection of asexual stages of Sarcocystis in bovine musculature specimens. Optimal time for digestion was approximately one to four hours. The trypsin digestion technique with Giemsa's stain could be helpful for the detection of Sarcocystis proliferative forms and for the observation of the nucleus of the parasite. A systematic detection was also performed in an autopsy for a bovine carcass naturally infected with Sarcocystis species, and the asexual stages such as metrocytes and bradyzoites were observed in the specific organs, respectively

Os efeitos das enzimas hialuronidase e tripsina foram avaliados quanto à liquefação, motilidade, vigor e integridade do acrossoma, no sêmen de seis macacos pregos (Cebus apella), mantidos na Fundação Parque Zoológico de São Paulo. O sêmen foi colhido por eletroejaculação, após anestesia geral, e a fração líquida foi imediatamente avaliada. A fração coagulada do sêmen foi tratada com as enzimas hialuronidase e tripsina, na dose de 1mg/ml, diluídas em meio 199 (Nutricel, Campinas/SP, Brasil), na proporção 1:4 e examinada após 5 e 15 minutos. Test t de Student foi utilizado para comparar os tratamentos. Não houve diferença significativa quanto a motilidade, vigor e integridade de acrossoma (p &gt;; 0.05), entre a fração de sêmen coagulado diluído em hialuronidase e tripsina, após cinco ou quinze minutos. No entanto, houve diferença significativa quanto a motilidade e vigor entre a fração líquida e a coagulada do sêmen (p &lt; 0.05), após quinze minutos. Não houve diferença significativa com relação à integridade de acrossoma (p &gt;; 0.05) entre a fração líquida e coagulada do sêmen, após 15 minutos. De acordo com os resultados, podemos concluir que não houve efeito aparente na fração coagulada do sêmen tratado com as enzimas hialuronidase e tripsina com relação a motilidade, vigor e integridade do acrossoma. No entanto, houve diferença significativa entre a fração líquida e coagulada do sêmen com relação a motilidade e vigor, porém não quanto à integridade do acrossoma. De maneira geral, em ambos os tratamentos, não houve a completa dissolução do coágulo. | The effect of the enzymes hyaluronidase and trypsin were recorded on the motility, vigor and acrosome integrity in the semen of capuchin monkey (Cebus apella). The animals (n=6) were maintained at Fundação Parque Zoológico de São Paulo. Under anesthesia semen samples were collected by electroejaculation. Immediately after the ejaculation, the semen liquid fraction was analyzed for volume (ml), pH, motility (%), vigor (0-5), concentration (cells/ml), defects (%) and percentage of intact acrosome (%). The coagulated fraction was treated with a solution of hyaluronidase or trypsin, 1mg/ml in commercial medium (199-Nutricel, Campinas/SP, Brazil) in a proportion of 1:4 and the samples were examined after 5 and 15 minutes. The Student T Test (95%) was used to compare the treat-ments. There was no significant difference in the motility, vigor or acrossome integrity (p &gt;; 0.05) between coagulated fraction diluted ei-ther in trypsin or in hyaluronidase, after 5 or 15 minutes. However, there was significant difference in motility and vigor between liquid and coagulated fraction, after 15 minutes, for both treatments (p; 0.05). In conclusion, there were no apparent effects in the coagulum for both treatments regarding motility, vigor and acrosome integrity. There were significant differences between liquid and coagulated fractions regarding motility and vigor, but not for acrosome integrity. In both enzyme treatments there were no complete dissolution of the coagulum.

<p>The alpha₂-macroglobulin (alpha₂M) protease inhibitor was purified from bovine plasma. The alpha₂M preparations at various purification steps were identified by immunodiffusion and crossed immunoelectrophoresis with anti-human alpha₂ serum. Anti-bovine alpha₂M serum was prepared for the quantitative determinations. The purest alpha₂M preparation was obtained by affinity chromatography and used as primary standard in radial immunodiffusion. Alpha₂M preparations were submitted to binding tests with p' - NPGB (p‘ - nitrophenyl-p-guanidinebenzoate HCL) titrated trypsin and plasmin. Alpha₂M protected 35% of the esterolytic activity of trypsin and 50% of the amidolytic activity of plasmin.</p> | <p>Alpha₂ Macroglobulina, uma proteína inibidora de proteases, foi isolada do plasma bovino. O processo de purificação foi monitorado por imunodifusão e imunoeletroforese cruzada com soro anti alpha₂M-humana. Para as determinações quantitativas foi preparado um soro anti alpha₂M bovino. A preparação mais pura de alpha₂M foi obtida por cromatografia de afinidade e usada como padrão primário na imunodifusão radial de Mancini. Preparações de alpha₂M foram usadas em testes de ligação com tripsina e plasmina (tituladas com NPGB). Nos testes de ligação 50% de plasmina e 35% de tripsina foram "protegidas” pela alpha₂M. Não foi possível determinar se houve ineficiência na ligação ou se a perda de atividade ocorreu por alterações na afinidade do complexo alpha₂M-protease, em relação aos substratos usados.</p>

<p>The fitness of formalin - treated materials for use in rapid rabies diagnosis was evaluated through Fluorescent Antibody (FA) test, using the digestion technique of pepsin and trypsin and the impression method of slide preparation. To achievc this proposal, brain fragments of experimentally rabid mice were submitted to different treatments of brain preservation by either using. pH adjusted formalin solutions or refrigeration, and the FA tests were run at ten experimental phases for a test period of 28 days. The results of FA reactivity ranged from 90.0% to 58.0%, depending on the treatments submitted; under the condition of the experiment, brain specimens should be maintained at refrigerating temperature until 96 hours for suitable FA examination, over this period the results should be irregular due to tissue degradation. The preservation of brain fragments in formalin, with further enzymatic digestion of pepsin and trypsin and the use of impression method did not mask or alter the virus antigenicity for adequate identification through FA technique, although the procedures should never substitute the existing methods of rapid rabies diagnosis.</p> | <p>Avaliou-se a adequação do emprego de cérebros preservados em formol para o estabelecimento rápido do diagnóstico da raiva pela reação de imunofluorescência direta, utilizando a técnica de digestão enzimática de pepsina e tripsina e método de impressão para o preparo de lâminas. O delineamento proposto contou com fragmentos de cérebros de camundongos experimentalmente infectados submetidos a diferentes tratamentos de conservação, com o uso de soluções de formol com pH corrigidos, ou submetidos à refrigeração; os testes de imunofluorescência foram realizados em 10 fases experimentais, por um período de 28 dias. Os resultados da prova de imunofluorescência variaram de 58.0% a 90.0% de positividade, dependendo dos tratamentos dispensados. Nas condições do experimento, os materiais destinados à prova de imunofluorescência podem ser conservados em temperatura de refrigeração por até 96 horas; após este período aumentam os resultados irregulares devido à degradação tissular. Nos tecidos mantidos em formol e após digestão enzimática, com a aplicação do método de impressão, observou-se o fenômeno de restauração da antigenicidade do vírus rábico, permitindo uma adequada identificação através da prova de imunofluorescência; no entanto, estes procedimentos não devem substituir os métodos atualmente empregados para o diagnóstico rápido da raiva.</p>