Endocytosis of prion protein is required for ERK1/2 signaling induced by stress-inducible protein 1

The secreted cochaperone STI1 triggers activation of protein kinase A (PKA) and ERK1/2 signaling by interacting with the cellular prion (PrPC) at the cell surface, resulting in neuroprotection and increased neuritogenesis. Here, we investigated whether STI1 triggers PrPC trafficking and tested whether this process controls PrPC-dependent signaling. We found that STI1, but not a STI1 mutant unable to bind PrPC, induced PrPC endocytosis. STI1-induced signaling did not occur in cells devoid of endogenous PrPC; however, heterologous expression of PrPC reconstituted both PKA and ERK1/2 activation. In contrast, a PrPC mutant lacking endocytic activity was unable to promote ERK1/2 activation induced by STI1, whereas it reconstituted PKA activity in the same condition, suggesting a key role of endocytosis in the former process. The activation of ERK1/2 by STI1 was transient and appeared to depend on the interaction of the two proteins at the cell surface or shortly after internalization. Moreover, inhibition of dynamin activity by expression of a dominant-negative mutant caused the accumulation and colocalization of these proteins at the plasma membrane, suggesting that both proteins use a dynamin-dependent internalization pathway. These results show that PrPC endocytosis is a necessary step to modulate STI1-dependent ERK1/2 signaling involved in neuritogenesis.

Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge

Strategies for the development of new vaccines against Streptococcus pneumoniae infections try to overcome problems such as serotype coverage and high costs, present in currently available vaccines. Formulations based on protein candidates that can induce protection in animal models have been pointed as good alternatives. Among them, the Pneumococcal Surface Protein A (PspA) plays an important role during systemic infection at least in part through the inhibition of complement deposition on the pneumococcal surface, a mechanism of evasion from the immune system. Antigen delivery systems based on live recombinant lactic acid bacteria (LAB) represents a promising strategy for mucosal vaccination, since they are generally regarded as safe bacteria able to elicit both systemic and mucosal immune responses. In this work, the N-terminal region of clade I PspA was constitutively expressed in Lactobacillus casei and the recombinant bacteria was tested as a mucosal vaccine in mice. Nasal immunization with L. casei-PspA 1 induced anti-PspA antibodies that were able to bind to pneumococcal strains carrying both clade 1 and clade 2 PspAs and to induce complement deposition on the surface of the bacteria. In addition, an increase in survival of immunized mice after a systemic challenge with a virulent pneumococcal strain was observed. (C) 2008 Elsevier Masson SAS. All rights reserved.

Functional Expression of Chemoreceptors with the Help of a Guanine Nucleotide Exchange Factor

Odorant receptors and other chemoreceptors are usually poorly expressed in the plasma membrane of heterologous cells. A key point of regulation in G protein-mediated signaling is the interconversion between the active GTP-bound and inactive GDP-bound states of the G alpha subunit, which regulatory proteins, such as guanine nucleotide exchange factors (GEFs), can control. GEFs stimulate formation of the GTP-bound state of G alpha and therefore are considered to work as positive regulators of G protein-coupled receptor signaling. Ric-8B, a GEF that is specifically expressed in olfactory sensory neurons, promotes functional expression of odorant receptors in HEK293T cells because it amplifies the initially low receptor signaling through G alpha olf. This same strategy could be used to functionally express other types of chemoreceptors.

alpha-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization

Xylella fastidiosa is a xylem-restricted plant pathogen that causes a range of diseases in several and important crops. Through comparative genomic sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. The experimental determination of the primary sequence of some markedly expressed proteins for X fastidiosa and the comparison with the nucleic acids sequence of genome identified one of them as being SCJ21.16 (XFa0032) gene product. The comparative analysis of this protein against SWISSPROT database, in special, resulted in similarity with a-hydroxynitrile lyase enzyme (HNL) from Arabidopsis thaliana, causing interest for being one of the most abundant proteins both in the whole cell extract as well as in the extracellular protein fraction. It is known that HNL enzyme are involved in a process termed ""cyanogenesis"", which catalyzes the dissociation of alpha-hydroxinitrile into carbonyle and HCN when plant tissue is damaged. Although the complete genome sequences of X.fastidiosa are available and the cyanogenesis process is well known, the biological role of this protein in this organism is not yet functionally characterized. In this study we presented the cloning, expression, characterization of recombinant HNL from X fastidiosa, and its probable function in the cellular metabolism. The successful cloning and heterologous expression in Escherichia coli resulted in a satisfactory amount of the recombinant HNL expressed in a soluble, and active form giving convenient access to pure enzyme for biochemical and structural studies. Finally, our results confirmed that the product of the gene XFa0032 can be positively assigned as FAD-independent HNLs. (C) 2009 Elsevier Ltd. All rights reserved.

Detection of infectious bronchitis virus and specific anti-viral antibodies using a concanavalin A-Sandwich-ELISA

Concanavalin A-Sandwich ELISA (Con A-S-ELISA) was developed for the detection of infectious bronchitis virus (IBV) or chicken specific anti-viral antibodies. The antigen detection limit for the Con A-S-ELISA was 10(5,1) EID50/mL. Three homologous and four heterologous IBV strains were similarly detected. This assay was highly effective in detecting the virus after infected tissue homogenates were passed once in embryonated chicken eggs, showing a good agreement with virus isolation technique. The Con A-S-ELISA was also used to measure anti-IBV chicken antibodies and showed a high coefficient of correlation (r = 0.85) and an agreement of k = 0.80 with the commercially available Indirect-ELISA. The relative sensitivity and specificity between these two tests were, respectively, 92.86% and 95.65% with an accuracy of 93.39%. Thus, the Con A-S-ELISA proved to be able to detect alternatively homologous and heterologous IBV strains or specific chicken anti-IBV antibodies, using the Con A as capture reagent of this assay.

Repair of lamellar scleral lesions in dogs with preserved equine renal capsule: short report

OBJETIVO: Avaliar o uso da cápsula renal de eqüino preservada em glicerina 98% no reparo de lesões lamelares esclerais em cães. MÉTODOS: Foram utilizados 12 cães, machos e fêmeas, com peso médio de 12kg. Foram realizadas avaliações clínica e morfológica aos 1, 3, 7, 15, 30 e 60 dias de pós-operatório. Após anestesia geral e procedimentos padrões de preparo do campo operatório, foi realizada cantotomia temporal, seguida de incisão conjutival e escleral com área de 0,5x0,5 cm na posição de 1hora, próxima ao limbo. em seguida, um fragmento de mesma dimensão de cápsula renal de eqüino preservada em glicerina, previamente hidratado em solução salina, foi aplicado ao defeito escleral criado sendo fixado com pontos simples isolados com vicryl 7-0®. RESULTADOS: A avaliação clínica revelou blefaroespasmo/fotofobia até o sétimo dia de pós-operatório. Foi observado edema conjuntival até o quinto dia, acompanhado de secreção ocular mucóide, que persistiu até o décimo dia de pós-operatório. Não foram observados sinais clínicos de rejeição do enxerto em todos os animais, em todos os períodos avaliados. Os segmentos anterior e posterior do bulbo ocular não apresentaram sinais de inflamação. A análise morfológica revelou exsudação inflamatória aguda nos períodos precoces e intermediários da avaliação e inflamação crônica nos períodos tardios da observação. Houve incorporação do enxerto ao leito receptor. CONCLUSÃO: Os resultados sugerem que a cápsula renal de eqüino preservada pode ser mais uma alternativa de membrana biológica para o reparo de lesões esclerais lamelares em cães e no homem.

Evaluation of the counterimmunoelectrophoresis reaction as a serodiagnosis test for swine leptospirosis

Avaliou-se a reação de contraimunoeletroforese (CIE) como teste gênero-específico para diagnóstico da leptospirose suína, usando-se três extratos solúveis de Leptospira sp, sorovares pomona, icterohaemorrhagiae e patoc, obtidos pelo tratamento com Triton X-100 a quente e aplicados a amostras de soro de suínos subdivididos em três grupos: Grupo 1, 10 suínos experimentalmente infectados com estirpe Pomona; Grupo 2, 50 suínos naturalmente infectados e Grupo 3, controle. As amostras de soros foram submetidas à reação de CIE e os resultados comparados aos da Soroaglutinação Microscópica (SAM), técnica de referência pela WHO. Os Grupos 1 e 3 foram monitorados por 93 dias após a inoculação (p.i.). Pela SAM a soroconversão do Grupo 1 ocorreu por volta do 10º dia p.i., enquanto pela CIE, empregando-se qualquer extrato antigênico, foi anterior à SAM. Quando a CIE foi realizada frente a antigeno homólogo à infecção, seus resultados foram equivalentes aos da SAM, não se verificando o mesmo frente aos antígenos heterólogos. Neste aspecto, os Grupos 1 e 3 mostraram comportamento diferente pois não houve diferença significativa entre os resultados da CIE frente aos três antígenos, o que poderia significar serem independentes do sorovar responsável pelo surto ou infectante. Embora a CIE seja segura, rápida, de fácil execução, de baixo custo e ideal para análise em grande escala de amostras, revelou-se de limitada capacidade gênero-específica, o que não é desejavel para testes de triagem de campo; mas poderia ser útil na detecção precoce de resposta sorológica em relação à SAM.

Immunoprophylactic strategies against enterotoxemia caused by Clostridium perfringens type D in goats

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

Construção de uma biblioteca de fragmentos de anticorpos monoclonais de galinhas com cadeia única (scFv) por phage display com reatividade cruzada para estirpes heterólogas do vírus da bronquite infecciosa aviária

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

Conditions affecting production of functional muscle recombinant alpha-tropomyosin in Saccharomyces cerevisiae

Yeasts are attractive hosts for heterologous protein production as they follow the general eukaryotic post-translational modification pattern. The well-known Saccharomyces cerevisiae has been used to produce a large variety of foreign proteins. The proper function of muscle tropomyosin depends on a specific modification at its N-terminus. Although tropomyosin has been produced in different expression systems, only the recombinant protein produced in the yeast Pichia pastoris has native-like functional properties. In this paper we describe the production of functional skeletal muscle tropomyosin in the yeast S. cerevisiae. The recombinant protein was produced in high amounts and production was strongly affected by genetic and environmental factors, including plasmid copy number, promoter strength, and growth media composition. (C) 2003 Elsevier B.V. (USA). All rights reserved.

Transferability of microsatellite loci from exotic Cervidae to Brazilian brocket deer (Mazama spp, Mammalia: Cervidae)

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

Recombinant expression of glycerol-3-phosphate dehydrogenase using the Pichia pastoris system

In the present study, the GPD2 gene from Saccharomyces cerevisiae, which codifies for the enzyme glycerol-3-phosphate dehydrogenase (GPDH), was cloned from the pPICZ-alpha expression vector and used with the purpose of inducing the extracellular expression of the glycerol-3-phosphate dehydrogenase under the control of the methanol-regulated AOX promoter. The presence of the GPD2 insert was confirmed by PCR analysis. Pichia pastoris X-33 (Mut(+)) was transformed with linearized plasmids by electroporation and transformants were selected on YPDS plates containing 100 mu g/mL of zeocin. Several clones were selected and the functionality of this enzyme obtained in a culture medium was assayed. Among the mutants tested, one exhibited 3.1 x 10(-2) U/mg of maximal activity. Maximal enzyme activity was achieved at 6 days of growth. Medium composition and pre-induction osmotic stress influenced protein production. Pre-induction osmotic stress (culturing cells in medium with either 0.35 M sodium chloride or 1.0 M sorbitol for 4h prior to induction) led to an increase in cell growth with sorbitol and resulted in a significant increase in GPDH productivity with sodium chloride in 24h of induction approximately fivefold greater than under standard conditions (without pre-induction). (C) 2010 Elsevier B.V. All rights reserved.

Detection of IgG in cerebrospinal fluid for diagnosis of neurocysticercosis: evaluation of saline and SDS extracts from Taenia solium and Taenia crassiceps metacestodes by ELISA and immunoblot assay

We compared saline (S) and sodium dodecyl sulphate (SDS) extracts from Taenia solium (homologous species - HO) and Taenia crassiceps (heterologous species - HE) metacestodes in order to detect Ige by ELISA and immunoblot assay (IBA) in cerebrospinal fluid (CSF) for the diagnosis of human neurocysticercosis (NC). CSF samples were obtained from 93 patients. of these, 40 had NC, five had a diagnosis of probable NC, nine had central nervous system schistosomiasis or strongyloidiasis and 39 had other neurological alterations. Samples were analysed by ELISA and the results were compared with IBA in all samples with confirmed and probable NC diagnosis, in all samples with other central nervous system parasitic infection, and in 10 of those with another neurological alterations. ELISA sensitivity was 100%, 85%, 95% and 87.5% for the S-HO, S-HE, SDS-HO and SDS-HE extracts, respectively, and ELISA specificity was 100% for S-HO, S-HE, SDS-HO extracts and 97.9% for SDS-HE antigen. Immunodominant peptides detected by IBA were, by decreasing percentage of recognition: 64-68 and 45 kDa for S-HO; 108-114, 92-95, 64-68, 83 and 88 kDa for S-HE; 64-68, 108-114, 77 and 86 kDa for SDS-HO; and 108-114, 88 and 92-95 kDa for SDS-HE. Overall the homologous antigenic extracts showed higher sensitivity than the heterologous extracts in the diagnosis of NC in CSF samples. The heterologous extracts contained most of the immunodominant peptides presented in the homologous extracts, which are recognized by Ige antibodies in CSF samples.

Toxoplasma gondii and Leishmania spp. infection in captive crab-eating foxes, Cerdocyon thous (Carnivora, Canidae) from Brazil

The zoonoses toxoplasmosis and leishmaniasis are important worldwide and also affect wild animals. Thus, the present study aimed to assess the prevalence of Leishmania spp. and Toxoplasma gondii antibodies in 52 serum samples from captive crab-eating foxes (Cerdocyon thous) kept in 17 zoos in São Paulo State, Brazil. Modified agglutination test (MAT, for toxoplasmosis) and indirect fluorescent antibody test (IFAT, for toxoplasmosis and leishmaniasis) were employed with heterologous anti-dog immunoglobulin. Antibodies to T. gondii were found in 19.2% animals, with an almost perfect concordance (kappa = 0.86; standard error = 9.31%; CI95% = 68.25-104.76%; P < 0.0001) and a strong correlation coefficient (rs = 0.87; P < 0.0001), which allows the use of heterologous anti-dog immunoglobulin to perform IFAT for toxoplasmosis in crab-eating foxes. No sample was positive for Leishmania spp. Toxoplasmosis infection occurs in wild animals from the studied Brazilian zoos, which indicates a probable environmental contamination, highlighting the importance of appropriate zoo management and the action of the parasite as a sentinel to human infection. (C) 2009 Elsevier B.V. All rights reserved.

Penetration of zona-free hamster, bovine and equine oocytes by stallion and bull spermatozoa pretreated with equine follicular fluid, dilauroylphosphatidylcholine or calcium ionophore A23187

Experiments evaluated the ability of follicular fluid (FF), dilauroylphosphatidylcholine (PC12) and the calcium ionophore A23187 (A23187) to induce capacitation in stallion and bull spermatozoa, determined by the ability of the spermatozoa to penetrate zona-free hamster, bovine and equine oocytes. Spermatozoa suspensions were incubated at 37 degreesC in one of the following treatments: 1) a modified Tyrode's medium (BGM3) alone, 2) BGM3 + FF; 3) BGM3 + PC12; 4) BGM3 + FF + PC12; 5) BGM3 + A23187; and 6) BGM3 + FF + A23187. Treated spermatozoa were incubated with zona-free hamster, bovine and equine oocytes for 3 h, after which oocytes were stained to assess spermatozoa penetration. The number of hamster oocytes penetrated by spermatozoa incubated in BGM3 alone (1/30) or in presence of FF (2/31) was significantly lower (P < 0.05) than by spermatozoa treated with PC12 or A23187 (16/30 and 17/30, respectively). Processing stallion spermatozoa either by a swim-up procedure or by centrifugation through a Percoll gradient increased the percentages of motile spermatozoa in the final sample, and spermatozoa collected by both processes penetrated similar numbers of zona-free hamster oocytes (P > 0.05). Although treating spermatozoa with PC12 or A23187 enabled both stallion and bull spermatozoa to penetrate oocytes, higher numbers of bovine oocytes were penetrated by bull spermatozoa (25/30) than by stallion spermatozoa (4/30) regardless of spermatozoal treatment. However, the number of zona-free hamster and equine oocytes penetrated by bull spermatozoa (25/30 and 12/18 respectively) and stallion spermatozoa (17/30 and 15/21 respectively) were similar (P > 0.05). We conclude that both PC12 and A23187 capacitate stallion and bull spermatozoa sufficiently to permit the acrosome reaction to occur, enabling spermatozoa to penetrate homologous and heterologous zona-free oocytes. (C) 2001 by Elsevier B.V.