Enzymatic Degradation of PrPSc by a Protease Secreted from Aeropyrum pernix K1

BACKGROUND: An R30 fraction from the growth medium of Aeropyrum pernix was analyzed for the protease that can digest the pathological prion protein isoform (PrP(Sc)) from different species (human, bovine, deer and mouse). METHODOLOGY/PRINCIPAL FINDINGS: Degradation of the PrP(Sc) isoform by the R30 fraction and the purified protease was evaluated using the 6H4 anti-PrP monoclonal antibody. Fragments from the N-terminal and C-terminal of PrP(Sc) were also monitored by Western blotting using the EB8 anti-PrP monoclonal antibody, and by dot blotting using the C7/5 anti-PrP monoclonal antibody, respectively. For detection of smaller peptides from incomplete digestion of PrP(Sc), the EB8 monoclonal antibody was used after precipitation with sodium phosphotungstate. Characterization of the purified active protease from the R30 fraction was achieved, through purification by fast protein liquid chromatography, and identification by tandem mass spectrometry the serine metalloprotease pernisine. SDS-PAGE and zymography show the purified pernisine plus its proregion with a molecular weight of ca. 45 kDa, and the mature purified pernisine as ca. 23 kDa. The purified pernisine was active between 58 °C and 99 °C, and between pH 3.5 and 8.0. The temperature and pH optima of the enzymatic activity of the purified pernisine in the presence of 1 mM CaCl(2) were 105 °C ± 0.5 °C and pH 6.5 ± 0.2, respectively. CONCLUSIONS/SIGNIFICANCE: Our study has identified and characterized pernisine as a thermostable serine metalloprotease that is secreted from A. pernix and that can digest the pathological prion protein PrP(Sc)

Do common infections trigger disease-onset or -severity in CTLA-4 insufficiency?

PurposeHeterozygous mutations in CTLA4 lead to an inborn error of immunity characterized by immune dysregulation and immunodeficiency, known as CTLA-4 insufficiency. Cohort studies on CTLA4 mutation carriers showed a reduced penetrance (around 70%) and variable disease expressivity, suggesting the presence of modifying factors. It is well studied that infections can trigger autoimmunity in humans, especially in combination with a genetic predisposition.MethodsTo investigate whether specific infections or the presence of specific persisting pathogens are associated with disease onset or severity in CTLA-4 insufficiency, we have examined the humoral immune response in 13 CTLA4 mutation carriers, seven without clinical manifestation and six with autoimmune manifestations, but without immunoglobulin replacement therapy against cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus 1/2 (HSV 1/2), parvovirus B19 and Toxoplasma gondii. Additionally, we have measured FcγRIII/CD16A activation by EBV-specific IgG antibodies to examine the functional capabilities of immunoglobulins produced by CTLA4 mutation carriers.ResultsThe seroprevalence between affected and unaffected CTLA4 mutation carriers did not differ significantly for the examined pathogens. Additionally, we show here that CTLA4 mutation carriers produce EBV-specific IgG, which are unimpaired in activating FcγRIII/CD16A.ConclusionsOur results show that the investigated pathogens are very unlikely to trigger the disease onset in CTLA-4-insufficient individuals, and their prevalence is not correlated with disease severity or expressivity

Effects of soil preparation systems and Efficient Microorganisms (EM) on soil physical properties, and improvement of potato yield and quality

As propriedades físicas do solo são preponderantes para a qualidade e produtividade da cultura da batata. Assim, o objetivo do presente trabalho foi avaliar algumas propriedades físicas do solo e a produtividade e qualidade dos tubérculos de batata. O experimento foi instalado em áreas de produção de batata na Chapada Diamantina (BA), de abril a agosto de 2005. O solo foi preparado pelo sistema convencional e pelo sistema alternativo MAFES, ambos associados ou não com a aplicação de Microrganismos Eficazes (EM). Foram avaliadas: a densidade do solo, a estabilidade de agregados, a resistência do solo à penetração, a taxa de infiltração de água e a retenção de água no solo. Também foi avaliada a alteração dessas propriedades do solo em relação à área antes do preparo, que estava sob pastagem de Brachiaria brizantha. A massa de torrões e touceiras colhidas juntamente com os tubérculos, bem como os danos mecânicos verificados durante a colheita mecanizada e a porcentagem de tubérculos com danos causados por larva alfinete, sarna e podridões foram quantificados. A produtividade nos diferentes tratamentos foi avaliada dentro da classificação comercial. Em relação ao pasto, os preparos convencional e MAFES alteraram a maioria das propriedades físicas avaliadas. Na área preparada pelo sistema MAFES foi observado, em relação ao preparo convencional, menor resistência à penetração em profundidades de 20 a 45 cm e entre os camalhões. A taxa de infiltração de água foi semelhante entre as áreas preparadas pelos dois sistemas, porém no sistema convencional a infiltração é mais superficial ocorrendo de forma lateral ao longo do perfil. No sistema MAFES a infiltração ocorreu verticalmente no perfil do solo. Menor porcentagem de tubérculos com danos mecânicos foi obtida na área preparada pelo sistema MAFES, devido, principalmente, a menor massa de torrões e touceiras colhidas juntamente com os tubérculos nessa área. A aplicação de EM teve pouca influência nas propriedades físicas do solo, mas propiciou condições que favoreceram o ataque de larva alfinete e sarna aos tubérculos. Apesar da utilização de EM ter reduzido a produção de tubérculos na área preparada pelo sistema MAFES, a produtividade e qualidade dos tubérculos nessa área foi superior a da área preparada pelo sistema convencional.The physical soil properties have a relevant role on the yield and quality of potato crop. Thus, the purpose of the present research was to evaluate the effects on some soil physical properties on productivity and quality of potato tubers. The trial was set up in a commercial potato production field located in the Chapada Diamantina, BA, from April to August, 2000. Two distinct soil preparation systems (conventional and MAFES system), either associated or not with application of EM, have been trialed. The following soil properties were evaluated: bulk density, aggregate stability, soil penetration resistance, soil water retention and water infiltration rate. In addition, were evaluated the changes of such soil properties in comparison with the area cultivated with the grass Brachiaria brizantha, prior soil preparation. Other evaluations were performed such as the clod mass and clump of roots harvested together with potato tubers, the level of damage to the tubers caused by mechanical harvesting, and the amount of tubers affected by potato tuber moth, rots, and common scab. The yields of the different treatments were evaluated and tubers graded according to commercial patterns. Compared to the grass, both conventional and MAFES soil preparation systems have changed the physical soil properties studied. MAFES system outperformed the conventional preparation giving lesser resistance to penetration at 20 to 45 cm deep and between hills. The water infiltration rate was similar for both soil preparation systems; however for the conventional system the infiltration pattern is superficial occurring laterally in the soil profile. Regarding MAFES systems, the infiltration occurred vertically. The area prepared MAFES system also gave a fewer percentage of tubers exhibiting mechanical damage, due to smaller clod mass and clump of roots harvested together with potato tubers. EM application did not benefit physical soil properties, however influenced a higher attack of potato tuber moth and common scab. Although the yield decrease in the area prepared with MAFES system when the EM was applied, in this area the productivity and tuber quality outperformed the conventional system

Aspectos morfofuncionais das células de Sertoli de peixes teleósteos

The literature was reviewed around the Sertoli cells in teleost fish, regarding their morphological and functional aspects, importance, terminology and interaction with other testis cells. In the 76 reports searched, it was observed that the Sertoli cells have these functions: phagocytose, nutrition of germinative cells, take part in development of germinative cists, development of the hemotesticular barrier and efferent ducts, and formation of the specialized structures to carry the cluster of sperm in the female oviduct of teleost species that have internal fertilization. In spite of the morphological differences and localization of the Sertoli cells of mammals, the cell found in the teleost was considered homologs to this one, because its similarity in functionsA literatura foi revisada em relação à célula de Sertoli de peixes teleósteos, levando em conta seus aspectos morfofuncionais, sua importância, a terminologia e a interação entre ela e outras células do testículo. No universo de 76 trabalhos pesquisados, foi notado que as células de Sertoli têm função de fagocitose, nutrição das células germinativas, esteroidogênese, participa na formação do cisto de células germinativas, e na formação da barreira hemotesticular. Além dessas funções, nos peixes teleósteos com fertilização interna a célula de Sertoli participa na formação dos dúctulos eferentes e das estruturas especializadas para levar grupos de espermatozóides para dentro do oviduto. A despeito da diferença entre a morfologia e a localização das células de Sertoli de mamíferos, a célula encontrada nos teleósteos foi considerada homóloga à mesma, devido à similaridade de funçõe

Occurrence of an Affinity Site apart from the Active Site on the Raw-Starch-Digesting but Non-Raw-Starch-Adsorbable Bacillus subtilis 65 α-Amylase

α-Cyclodextrin specifically inhibited raw starch digestion by Bacillus subtilis 65 α-amylase. The raw starch digestibility and α-cyclodextrin-Sepharose 6B adsorbability of this α-amylase were simultaneously lost when the specific domain corresponding to the affinity site essential for raw starch digestion was deleted by proteolysis. Occurrence of the affinity site on raw-starch-digesting enzymes was proven also with bacterial amylase