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This study compares the allergenic potential of Isosoy®, a soy germen commercialised as isoflavone for dietary supplements, with crude soy extract (CSE) and purified isoflavone. Specific antibodies (IgG, IgE and IgG1) generated in Swiss mice by oral and subcutaneous route with Isosoy® also recognised proteins of crude soy extract, but no reaction against purified isoflavone, like clearly demonstrated by ELISA, Western blot and immunoprecipitation assay. These results are in agreement with the same protein profile between Isosoy® and CSE observed by polyacrylamide gel electrophoresis (SDS-PAGE). The immunoprecipitation assay demonstrates that Isosoy® boiling cannot inactivate the interaction with specific antibodies generated by oral route with unboiled Isosoy®. Results suggest that Isosoy® could lead to allergic reactions even after heat treatment.

In Brazil, shrimp farming has been developed most intensely in the Northeast Region. Recently, however, exporters have become concerned over the appearance of Infectious Myonecrosis (IMN), the etiological agent of which is a virus called Infectious Myonecrosis Virus (IMNV). Although IMNV has been characterized extensively, purification methods are complicated to reproduce and very expensive. The objective of this study was to purify the IMNV virus using an easy reproductive method and to produce anti-IMNV antibodies to be used in diagnostic methods. Shrimp samples showing symptoms of IMN obtained from two aquaculture farms in Ceará were used for this purpose.IMNV-positive shrimps were macerated in phosphate buffer, pH 7.5, enriched with antioxidants, clarified with chloroform and the supernatant was submitted to differential centrifugation, precipitated using PEG and NaCl and finally loaded on a discontinuous gradient of sucrose. Purified IMNV was submitted to RT-PCR and electrophoresis either in agarose gel or SDS-PAGE, which revealed RNA and protein bands, characteristic of IMNV. IMNV induced humoral immune response in Swiss mice when administered subcutaneously. Anti-IMNV antibodies were identified by ELISA (enzyme-linked immunosorbent assay) and Western blotting methods and produced a response against purified IMNV and the crude extract obtained from the infected shrimp. However, antibodies specific to the crude extract obtained from uninfected shrimp were not detected. This is the first report of IMNV having been purified in Brazil and the first time that specific antibodies against IMNV proteins have been produced. These results suggest that easy methods can be developed to produce specific antiserum for viral diagnosis on a large scale.

Caprine arthritis-encephalitis virus (CAEV) belongs to Retroviridae family, Lentivirus genus. This virus infects caprine all over the world causing arthritis, encephalitis, mammitis and progressive emaciating. This research showed chimera’s building made by mixing up CAEV p28, with glutaraldehyde, and CPSMV, purified through the chromotography in biogel and sephadex (G-150). After that, some measures in a spectrophometric were developed to absorbance at 280nm. Peaks, which contained chimera, were collected and submitted to SDS-PAGE, evidencing the band relative to itself. Groups of swiss mice were immunized with chimeric virus, purified CPSMV and with p28 protein using incomplete Freund Adjuvant. CPSMV and p28 specific antibodies recognized chimeric protein in Western Blotting and ELISA showing the efficacy of the method. The results showed the covalent coupling between CPSMV and CAEV p28 was successfully archieved, originating a stable molecule, which no disestablished the capside from CPSMV. Besides, it showed that chimeric virus presents more immunogenicity than protein p28 isolated. It’s suggesting CPSMV can be used as a carrier molecule in the production of vaccines to the virus, which infect animals. O vírus da Artrite-encefalite caprina (CAEV) pertence à família Retroviridae, gênero Lentivirus. O CAEV infecta caprinos do mundo inteiro causando artrite, encefalite, mamite, pneumonia e emagrecimento rogressivo. Este trabalho mostra a formação de uma quimera construída através da mistura da p28 do CAEV com glutaraldeído e CPSMV, purificada por meio de cromatografia em biogel e sephadex G-150. As cromatografias foram monitoradas através de leituras em espectrofotômetro no comprimento de onda de 280nm, dos líquidos coletados nos tubos. Os picos contendo a quimera foram coletados e submetidos à eletroforese (SDS-PAGE), sendo assim evidenciada a banda correspondente à mesma. Grupos de camundongos swiss foram imunizados com o vírus quimérico (CPSMV + p28), com o vírus CPSMV purificado e com a proteína p28 do CAEV, utilizando o adjuvante de Freund incompleto. Os anticorpos específicos produzidos contra o CPSMV e p28 reconheceram a proteína quimérica em Western Blotting e em teste de ELISA. Os anticorpos contra o vírus quimérico apresentaram títulos mais elevados do que os anticorpos produzidos contra a p28, demonstrando que o vírus quimérico apresenta maior imunogenicidade do que a proteína p28 sozinha. Os resultados mostraram que o acoplamento covalente entre o CPSMV e a p28 do CAEV foi obtido com sucesso, originando uma molécula estável não comprometendo a estrutura do capsídeo do CPSMV. Desta forma, sugere-se que o CPSMV possa ser utilizado como molécula carreadora na produção de vacinas para vírus que infectam animais.

Summary Salt modulation of the tonoplast H+-pumping V-ATPase and H+-PPase was evaluated in hypocotyls ofVigna unguiculata seedlings after 3 and 7 days of treatment. In 3-day-old seedlings, treatment with 100 mmol/L NaCl decreased the proton transport and hydrolytic activities of both the V-ATPase and the H+-PPase. After 7 days, the proton transport and hydrolysis activities of the V-ATPase were higher, while the H+-PPase activities were lower in seedlings. Western blot analysis of A- and B-subunits of V-ATPase revealed that the protein content of the two subunits varied in parallel with their activities, i.e. to a higher activity corresponded a higher protein content of the subunits and vice versa. Contrarily, Western blot analysis of H+-PPase levels failed to show any correlation with PPase activity, suggesting a partial enzyme inactivation. The results indicate that salt stress induces V-ATPase expression inV. unguiculata with concomitant enhancement of its activity as a homeostatic mechanism to cope with salt stress. Under the same conditions PPase is inhibited.

Vita 3 and Vita 5 are two Vigna unguiculata cultivars that differ in their capacities for survival in saline environments; Vita 3 is more tolerant and Vita 5 more sensitive. Both cultivars were submitted to salt stress with 0.1 M NaCl. After 8 days, root and shoot growth from both cultivars was reduced but reduction was more pronounced in Vita 5. Furthermore, leaf area was also reduced in this cultivar. Chlorophyll content and chlorophyll fluorescence parameters were not affected by salt stress, but the specific activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) decreased in Vita 3 and increased in Vita 5. The use of immunological techniques also revealed that the Rubisco content from Vita 3 decreased while that of Vita 5 increased. The discussion of these results is aimed at reaching a better understanding of the differences between these cultivars in relation to salt stress. Vita 3 e Vita 5 são cultivares de Vigna unguiculata que diferem na capacidade de sobrevivência em ambiente salino: Vita 3 é mais tolerante e, Vita 5, mais suscetível. Ambas as cultivares foram submetidas a um tratamento salino com NaCl 0,1 M. Após 8 dias da germinação, o crescimento das raízes e das partes aéreas foram reduzidas, sendo a redução mais acentuada na cultivar Vita 5. Além do mais, houve uma diminuição na sua área foliar. Os conteúdos de clorofila, bem como seus parâmetros de fluorescência não foram afetados pelo estresse salino, mas as atividades da ribulose-1,5-bisfosfato carboxilase/oxigenase (Rubisco) diminuiu na Vita 3 e aumentou na Vita 5. Uma abordagem imunológica mostrou que o conteúdo da proteína Rubisco diminuiu para a cultivar Vita 3 e aumentou para a cultivar Vita 5. Esses resultados são discutidos para obter melhor entendimento das diferenças entre essas cultivares em relação ao estresse salino.