Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.

Purification of a membrane-bound trypsin-like enzyme from the gut of the velvetbean caterpillar (Anticarsia gemmatalis Hübner) / Purificação de uma enzima "tipo tripsina" não-solúvel do intestino da lagarta da soja (Anticarsia gemmatalis Hübner)

Disruption of protein digestion in insects by specific endoprotease inhibitors is being regarded as an alternative to conventional insecticides for pest control. To optimize the effectiveness of this strategy, the understanding of the endoprotease diversity of the target insect is crucial. In this sense, a membrane-bound trypsin-like enzyme from the gut of Anticarsia gemmatalis fifth-instar larvae was purified. Non-soluble fraction of the gut extract was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and subjected to a p-aminobenzamidine affinity chromatography followed by anion-exchange chromatography. The yield of the purified enzyme was 11% with a purification factor of 143 and a final specific activity of 18.6 µM min.-1 mg-1 protein using N-α-benzoyl-L- Arg-p-nitroanilide (L-BApNA) as substrate. The purified sample showed a single band with proteolytic activity active and apparent molecular mass of 25 kDa on SDS-PAGE. Molecular mass determined by MALDI-TOF mass spectrometry was 28,632 ± 26 Da. Although the low recovery and the difficulties in purifying large enzyme amounts limited its further characterization, the results contribute for the understanding of the proteases present on A. gemmatalis gut, which are potential targets for natural or specifically designed protease inhibitors.

Comprometer a digestão de proteínas dos insetos pelo uso de inibidores específicos de endoproteases tem sido amplamente estudado como um método de controle de pragas alternativo ao uso dos inseticidas convencionais. No processo de otimização desta estratégia, o conhecimento da diversidade das endoproteases do inseto alvo torna-se crucial. Neste sentido, uma enzima "tipo-tripsina" ligada à membrana obtida do intestino de larvas do 5° instar de A. gemmatalis foi purificada. A fração insolúvel do extrato do intestino foi solubilizada com 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) e submetida à uma cromatografia de afinidade em uma coluna de p-aminobenzamidina, seguida por uma cromatografia de troca-aniônica. O rendimento da enzima purificada foi de 11% com fator de purificação de 143 e uma atividade específica final de 18.6 µM min.-1 mg-1 de proteína usando N-α-benzoyl-L- Arg-p-nitroanilide (L-BApNA) como substrato. Após a separação da amostra purificada por SDS-PAGE e incubação subsequente com caseína, uma única banda ativa com massa molecular aparente de 25 kDa foi observada. A massa molecular determinada por espectrometria de massa (MALDI-TOF) foi de 28,632 ± 26 Da. O baixo rendimento e as dificuldades em purificar grandes quantidades da enzima limitaram caracterização complementar. A observação desta enzima, no entanto, é mais uma etapa no processo de conhecer as endoproteases presentes no intestino de A. gemmatalis, alvos potenciais de inibidores de proteases naturais ou especificamente projetados.

Determination of optimal glucose concentration for microcalorimetric metabolic evaluation of equine spermatozoa

The heat conduction microcalorimeter can be used to evaluate the metabolic rates of the sperm cell. Two ejaculates of four stallions were cooled to +5ºC and checked for sperm motility (bright field microscopy), viability (eosin 3 percent), functional membrane integrity (hyposmotic swelling test), and heat production (microcalorimetry). Glucose and sperm cell concentrations were determined in order to measure the heat outputs resulting from sperm metabolism. Sperm viability, membrane integrity and sperm motility did not differ among the different glucose concentrations tested. Nevertheless, the highest heat output detected by the microcalorimeter was obtained with 6 mM glucose and 10(8) spermatozoa/mL. Since conduction microcalorimetry offered additional information on equine sperm metabolism, it could be used as a method to study equine semen preservation.

O microcalorímetro de condução pode ser usado para avaliar as taxas metabólicas do espermatozóide eqüino. Dois ejaculados de quatro garanhões foram avaliados quanto à motilidade progressiva pela microscopia, viabilidade espermática (eosina 3 por cento), integridade funcional da membrana (teste hiposmótico) e produção de calor (microcalorimetria). Concentrações ótimas de glicose e de células espermáticas foram determinadas, para mensurar o calor liberado resultante do metabolismo espermático em relação à capacidade de detecção do calor pelo microcalorímetro. Não foi observada diferença da motilidade, viabilidade e integridade funcional de membrana espermática quando adicionada glicose nas três concentrações estudadas. No entanto a avaliação por microcalorimetria ressaltou um maior fluxo de calor a uma concentração de 6 mM de glicose e uma concentração espermática de 10(8) espermatozóides/mL. Portanto, a técnica de microcalorimetria oferece informações adicionais sobre o metabolismo tornando-se uma ferramenta importante no estudo do processo de preservação do sêmen eqüino.

Improved purification process of β- and α-trypsin isoforms by ion-exchange chromatography

The purpose of this work was to improve the separation and yield of pure β- and α-trypsin isoforms by ion-exchange chromatography and to characterize some physical-chemical properties of these isoforms. Purification of trypsin isoforms was performed by ion-exchange chromatography in 0.1 mol/L tris-HC buffer, pH 7.10 at 4ºC. The sample loading, salt concentration, flow rate and pH of mobile phase were varied to determine their effects on the resolution of the separation. The resolution was optimized mainly between β- and α-trypsin. Pure isoforms were obtained by chromatographying 100 mg of commercial trypsin during seven days, yielding 51 mg of high purity β-trypsin and 13 mg of α-trypsin partially pure, with small amounts of contaminating of ψ-trypsin. Thus, time and resolution of purification were optimized yielding large amounts of pure active enzymes that are useful for several research areas and biotechnology.

O propósito deste trabalho foi melhorar a separação e o rendimento das isoformas puras β- e α-tripsina por meio de cromatografia de troca iônica e caracterizar algumas propriedades físico-químicas dessas isoformas. A purificação de isoformas de tripsina foi realizada em SE Sephadex, com tampão tris-HCl, pH 7,10 a 4ºC. A quantidade de amostra, a concentração salina, o fluxo e o pH da fase móvel foram variados para determinar o efeito sobre a resolução da separação. A resolução foi otimizada principalmente entre β- e α-tripsina, utilizando o pH 7,10 a 4ºC. Isoformas puras foram obtidas a partir de 100 mg de tripsina comercial bovina depois de sete dias de cromatografia, fornecendo 51,0 mg de β-tripsina totalmente pura e 13,0 mg de α-tripsina parcialmente pura, com quantidades pequenas de contaminação por ψ-Tripsina. Assim, tempo e resolução da purificação foram otimizados redendo grandes quantidades de enzimas puras e ativas que são úteis em várias áreas de pesquisa e ciências biotecnológicas.

Production of specific polyclonal antibodies anti-human factor IX

In this work, polyclonal antibodies anti-human Factor IX were produced in New Zealand rabbits by immunization with commercial pure human FIX (hFIX) (Octanyne®, Octapharma, USA). The serum containing immunoglobulins anti-hFIX was useful to detect hFIX antigen in human plasma fractions submitted to anionic exchange chromatographic process and with a large yield. Immunoassays (ELISA) using bovine serum albumin, trypsin and peptides generated by cleavage assays with trypsin as digestion enzyme was performed and revealed adequate specificity of the polyclonal antibodies produced.

Neste trabalho foram produzidos anticorpos policlonais anti-fator IX humano em coelhos New Zealand imunizados com FIX humano (hFIX) comercial puro (Octanyne®, Octapharma, EUA). O soro contendo as imunoglobulinas anti-hFIX foi útil para a detecção do antígeno hFIX em frações do plasma humano submetido a cromatografia de troca iônica. Imunoensaios (ELISA) usando soro-albumina bovina, tripsina e peptídeos gerados por ensaios de clivagem com tripsina com enzima de digestão foram realizados e revelaram especificidade adequada dos anticorpos policlonais produzidos.

Hidrolisados enzimáticos de leite em pó desnatado como fonte de oligopeptídeos para formulações dietéticas / Enzymatic hydrolysates from skimmed powder milk as source of oligopeptides for dietary formulations

Seis hidrolisados de leite em pó desnatado foram preparados visando a produção de hidrolisados protéicos para uso em formulações dietéticas para fenilcetonúricos. Para isso utilizaram-se uma protease do Aspergillus oryzae (AO), isoladamente ou em associação com a papaína (PA), em diferentes tempos de reação e relação enzima:substrato (E:S). O interesse deste trabalho está associado ao estudo da distribuição dos peptídios nestas amostras, de acordo com o tamanho da cadeia, como critério de avaliação de sua qualidade nutricional. Inicialmente, os hidrolisados foram fracionados por cromatografia líquida de alta eficiência de exclusão molecular (SE-HPLC) e, para a quantificação dos componentes das frações cromatográficas, empregou-se o método rápido da Área Corrigida da Fração (ACF)...

Alpha-glycerophosphate dehydrogenase activity in flight muscles of triatomine bugs Panstrongylus megistus and Triatoma sordida

The alpha-glycerophosphate dehydrogenase (alpha-GPDH) activity in flight muscles of Panstrongylus megistus and Triatoma sordida, vectors of Chagas disease in Brazil, was studied. Both species showed higher enzymatic activities in fliers than in non-fliers insects. T. sordida exhibited a higher proportion of flier insects than P. megistus. A possible role of alpha-GPDH on triatomines flight is discussed.

Protective immunity induced in mice by F8.1 and F8.2 antigens purified from Schistosoma mansoni eggs

Schistosoma mansoni soluble egg antigens (SEA) were fractionated by isolectric focusing, resulting in 20 components, characterized by pH, absorbance and protein concentration. The higher absorbance fractions were submitted to electrophoresis, and fraction 8 (F8) presented a specific pattern of bands on its isoelectric point. Protein 3 was observed only on F8, and so, it was utilized to rabbit immunization, in order to evaluate its capacity of inducing protective immunity. IgG antibodies from rabbit anti-F8 serum were coupled to Sepharose, and used to obtain the specific antigen by affinity chromatography. This antigen, submitted to electrophoresis, presented two proteic bands (F8.1 and F8.2), which were transferred to nitrocellulose membrane (PVDF) and sequenciated. The homology of F8.2 to known proteins was determined using the Basic Local Alignment Search Tool program (BLASTp). Significant homologies were obtained for the rabbit cytosolic Ca2+ uptake inhibitor, and for the bird a1-proteinase inhibitor. Immunization of mice with F8.1 and F8.2, in the presence of Corynebacterium parvum and Al(OH)3 as adjuvant, induced a significant protection degree against challenge infection, as observed by the decrease on worm burden recovered from portal system.