Recombinant expression and characterization of a Xylella fastidiosa cysteine protease differentially expressed in a nonpathogenic strain

Xylella fastidiosa is a xylem-limited, Gram-negative bacterium responsible for citrus variegated chlorosis (CVC) in sweet oranges. In the present study, we present the recombinant expression, purification and characterization of an X. fastidiosa cysteine protease (dubbed Xylellain). The recombinant Xylellain ((HIS)Xylellain) was able to hydrolyze carbobenzoxy-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-MCA) and carbobenzoxy-Arg-Arg-7-amido-4-methylcoumarin (Z-RR-MCA) with similar catalytic efficiencies, suggesting that this enzyme presents substrate specificity requirements similar to cathepsin B. The immunization of mice with (HIS)Xylellain provided us with antibodies, which recognized a protein of c. 31 kDa in the X. fastidiosa pathogenic strains 9a5c, and X. fastidiosa isolated from coffee plants. However, these antibodies recognized no protein in the nonpathogenic X. fastidiosa J1a12, suggesting the absence or low expression of this protein in the strain. These findings enabled us to identify Xylellain as a putative target for combating CVC and other diseases caused by X. fastidiosa strains.

Identification of phospholipase C zeta in normospermic and teratospermic domestic cat sperm

In mammalian species, oocyte activation is initiated by oscillations in the intracellular concentration of free calcium ([Ca2+]i), which are also essential to allow embryonic development. To date, evidence supporting the hypothesis that a sperm factor is responsible for initiating oocyte activation has been presented in various mammalian species. Among the possible candidates to be the active sperm factor is the novel sperm-specific phospholipase C ζ (PLCζ), which besides its testis-specific expression is capable of initiating [Ca2+]i oscillations. In this study, we investigated the presence of PLCζ in the sperm of the domestic cat and whether normospermic and teratospermic cats differ in their PLCζ expression. Immunoblotting with anti-PLCζ antibodies confirmed the presence of an immunoreactive band of ~70 kDa in whole sperm lysates of domestic cat as well as in both soluble and insoluble fractions from this sperm. Additional immunoreactive bands, probably C- and N-terminal truncated versions of PLCζ, were also visualized in the soluble sperm fractions. Interestingly, immunoreactivity of PLCζ was detectable in teratospermic sperm, although with slightly less intensity than in normospermic sperm. In conclusion, domestic cat sperm express PLCζ in both cytosolic and high-pH fractions, which is consistent with data in other mammals. Sperm from teratospermic cats also express PLCζ, albeit at reduced concentrations, which may affect the fertility of these males. © 2013 Elsevier Inc..

Complementação do seqüenciamento do genoma do Southern bean mosaic virus, isolado São Paulo, expressão da porção C-terminal da polimerase e produção de anti-soro policlonal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Expressão em Escherichia coli e produção de antissoro policlonal para proteína P1 do Southern bean mosaic virus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Obtenção de mutantes de Bacillus thuringiensis por inserção do transposon Tn-5 e avaliação da toxicidade frente a larvas de Spodoptera frugiperda

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Reação em cadeia da polimerase e proteinograma no diagnóstico da leishmaniose ocular canina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção dos fragmentos de anticorpos recombinantes scFv-N e scFv-S1 e suas aplicações na detecção e diferenciação do Vírus da Bronquite Infecciosa

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Caracterização e ocorrência de bidens mosaic virus (bimv) em regiões produtoras de alface no Estado de São Paulo

Pós-graduação em Agronomia (Proteção de Plantas) - FCA

Peptídeos derivados da toxina ParE: síntese, estrutura e estudos de inibição de DNA topoisomerases

Pós-graduação em Biotecnologia - IQ

Produção de fitase por Rhizopus microsporus var. microsporus: purificação, caracterização bioquímica e aplicação

Pós-graduação em Biotecnologia - IQ

Eletroforese das proteínas séricas e urinárias de cães com erliquiose subclínica

Pós-graduação em Medicina Veterinária - FCAV

Grãos secos de destilaria com solúveis em dietas para tilápia-do-Nilo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Processo de obtenção da enzima lacase por fungo marinho, enzima lacase e uso da mesma

A presente invenção está relacionada com a otimização das condições de cultivo para produção de lacase por um 5 fungo derivado de ambiente marinho. Tais condições permitiram a obtenção de quantidades significativas de lacase após a purificação (1.300 U/L) com atividade enzimática ótima em PH 3,0 e 60ºC e com peso molecular na faixa de 60 a 70 KDa. Nas condições de cultivo otimizadas a 10 enzima apresentou eficiente estabilidade frente a diferentes valores de pH e temperatura, com manutenção de 100% da estabilidade térmica após 1 h mesmo em temperatura de 62ºC, e da estabilidade frente a diferentes PHs após 48h, além da resistência a íons metálicos.

The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

Background: Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. Methods. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. Results: In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role in YFV replication. Conclusions: Although the precise function of eIF3L on interactions with viral proteins is not entirely understood, these results indicate an interaction of eIF3L with YF NS5 and that eIF3L overexpression facilitates translation, which has potential implications for virus replication. © 2013 Morais et al.; licensee BioMed Central Ltd.

Hepatic concentration of heat shock protein 70 kD (Hsp70) in broilers subjected to different thermal treatments

1. The relationship between repeated thermal treatments and hepatic synthesis of Hsp 70 was studied in broiler chickens.2. Sixty broilers were submitted to 5 different treatments (12 birds each) from day 1 to day 42. Four groups were kept in a thermoneutral environment and subjected to 0, 1, 2 and 3 heat stress episodes at 35 degrees C for 4 h per week (TN-0, TN-1, TN-2 and TN-3, respectively). The last group (HT-35) was reared at a room temperature of 35 degrees C.3. From 39 to 42 old, the birds experienced acute heat stress at 41 degrees C. Resistance to heat stress was evaluated by the time taken for rectal temperature to increase by 3 degrees C above the pre-treatment value. Livers were collected (before and after heat stress) and Hsp70 was determined using Western Blot analysis with monoclonal anti-Hsp70 antibody.4. Resistance to heat stress and concentration of Hsp70 were higher in those birds subjected to more heat stress episodes during the experimental period (TN-3) and HT-35. A positive correlation was observed between Hsp70 concentration and the time taken for a 3 degrees C increase in rectal temperature (r=0.42; P<0.01).5. Exposing birds to episodes of heat stress (35 degrees C) during rearing may improve their resistance to acute heat stress, but the previous thermal history did not seem to influence the hepatocyte Hsp70 content after exposure to more severe heat stress (41 degrees C).