Trypanosoma cruzi: Isolation and characterization of aspartyl proteases

Two aspartyl proteases activities were identified and isolated from Trypanosoma cruzi epimastigotes: cruzipsin-I (CZP-I) and cruzipsin-II (CZP-II). One was isolated from a soluble fraction (CZP-II) and the other was solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate(CZP-I). The molecular mass of both proteases was estimated to be 120 kDa by HPLC gel filtration and the activity of the enzymes was detected in a doublet of bands (56 and 48 kDa) by substrate-sodium dodecyl sulphate-polyacrylamide-gelatin gel electrophoresis. Substrate specificity studies indicated that the enzymes consistently hydrolyze the cathepsin D substrate Phe-Ala-Ala-Phe (4-NO(2))-Phe-Val-Leu-O(4)MP but failed to hydrolyze serine and other protease substrates. Both proteases activities were strongly inhibited by the classic inhibitor pepstatin-A (>= 68%) and the aspartic active site labeling agent, 1,2-epoxy-3-(phenyl-nitrophenoxy) propane (>= 80%). These findings show that both proteases are novel T. cruzi acidic proteases. The physiological function of these enzymes in T. cruzi has under investigation. (c) 2009 Elsevier Inc. All rights reserved.

Salinity influences glutathione S-transferase activity and lipid peroxidation responses in the Crassostrea gigas oyster exposed to diesel oil

Biochemical responses in bivalve mollusks are commonly employed in environmental studies as biomarkers of aquatic contamination. The present study evaluated the possible influence of salinity (35, 25,15 and 9 ppt) in the biomarker responses of Crassostrea gigas oysters exposed to diesel at different nominal concentrations (0.01, 0.1 and 1 mLL(-1)) using a semi-static exposure system. Salinity alone did not resulted in major changes in the gill`s catalase activity (CAT), glutathione S-transferase activity (GST) and lipid peroxidation levels (measured as malondialdehyde. MDA), but influenced diesel related responses. At 25 ppt salinity, but not at the other salinity levels, oysters exposed to diesel showed a strikingly positive concentration-dependent GST response. At 25 ppt and 1 mLL(-1) diesel, the GST activity in the gills remained elevated, even after one week of depuration in clean water. The increased MDA levels in the oysters exposed to diesel comparing to control groups at 9, 15 and 35 ppt salinities suggest the occurrence of lipid peroxidation in those salinities, but not at 25 ppt salinity. The MDA quickly returned to basal levels after 24 h of depuration. CAT activity was unaltered by the treatments employed. High toxicity for 1 mLL(-1) diesel was observed only at 35 ppt salinity, but not in the other salinities. Results from this study strongly suggest that salinity influences the diesel related biomarker responses and toxicity in C. gigas, and that some of those responses remain altered even after depuration. (C) 2011 Elsevier B.V. All rights reserved.

Isolation, characterization and structural determination of a unique type of arabinogalactan from an immunostimulatory extract of Chlorella pyrenoidosa

An arabinogalactan was isolated from a hot water extract of freeze-dried cells of the green microalga, Chlorella pyrenoidosa. This hot water extract is a proprietary immunomodulator, with the trademark Respondin™ (ONC-107). The arabinogalactan was recovered from the ethanol-soluble fraction of the supernatant resulting from a process that involved controlled ethanol precipitation followed by size exclusion chromatography on Sephadex G-100, then Cetavlon precipitation. Sugar analyses, GC–MS data for (S)-2-octyl glycosides, and 1D and 2D NMR experiments established unambiguously that the repeating unit was →2)-α-l-Araf-(1→3)-[α-l-Araf-(1→4)]-β-d-Galp-(1→. This structure does not fit into any of the known classes of arabinogalactans. SEC/MALS experiments gave a molecular mass for the arabinogalactan isolated as 47 ± 4 kDa but the original structure was probably larger.

Parâmetros ruminais e desaparecimento da MS, PB e FDN da forragem em bovinos suplementados em pastagem na estação seca

O trabalho foi conduzido com o objetivo de determinar o efeito de suplementos concentrados com diferentes degradabilidades da proteína (alta-70%, média-50% e baixa-30%) e o efeito da quantidade dos mesmos (0,5, 1,0 e 1,5 kg de MS/dia) sobre os parâmetros ruminais (pH e N-NH3) e o desaparecimento da MS, PB e FDN da forragem em bovinos pastejando Brachiaria brizantha cv. Marandu no período da seca. Foram utilizados 10 novilhos canulados no rúmen com peso médio de 354 kg em um esquema fatorial com três repetições (blocos). Não houve influência da degradabilidade protéica e/ou quantidade de suplemento sobre os valores de pH ruminal, que variaram de 6,38 a 6,91. As concentrações de N-NH3 ruminal foram crescentes com o aumento da degradabilidade do suplemento e quantidade de suplementação, sendo maiores uma hora após o fornecimento do suplemento e decrescendo até cinco horas. O tratamento-controle apresentou concentrações de N-NH3 consideradas adequadas para boa atividade microbiana. Não houve efeito da degradabilidade protéica do suplemento ou quantidade na degradação ruminal da MS, PB e da FDN da forragem, não diferindo do tratamento-controle. Quanto aos parâmetros de degradação ruminal da forrageira, os valores médios foram de 29% para a fração solúvel da PB e de de 47% para a insolúvel potencialmente degradável, com taxa de degradação de 4,88%. Para FDN, a fração potencialmente degradável foi de 56% e a taxa de degradação, de 4,33%.

Efeito do emurchecimento e da adição de fubá na degradabilidade in situ da silagem de alfafa (Medicago sativa L.)

O objetivo deste ensaio foi avaliar o efeito do emurchecimento e da adição de fubá durante o processo de ensilagem da alfafa sobre a degradabilidade in situ da matéria seca (MS), da fibra em detergente neutro (FDN) e da proteína bruta (PB). Foram utilizados três bovinos adultos mestiços fistulados no rúmen, nos quais foram incubados seis tipos de silagens (1-alfafa fresca; 2-alfafa fresca +5% de fubá; 3-alfafa fresca +10% de fubá; 4-alfafa emurchecida; 5-alfafa emurchecida + 5% de fubá ; 6-alfafa emurchecida + 10% de fubá) durante três tempos de incubação (6, 24, 96 horas). O delineamento experimental foi em parcelas subdivididas, com tratamentos em parcelas, tempos em subparcelas e animais como blocos. A adição de fubá à silagem aumentou (P<0,05) a degradação da MS e FDN com o tempo de incubação. O emurchecimento proporcionou diminuição (P<0,05) na degradabilidade dos nutrientes, entretanto esse efeito foi ligeiramente contornado pela adição de 10% de fubá. Os tratamentos com a adição de fubá e o emurchecimento proporcionaram maior taxa de degradabilidade da MS de fração b, entretanto o emurchecimento diminuiu o potencial de degradação a, enquanto que a adição de níveis de fubá não alterou a degradabilidade da MS dessa fração. A degradação da fração b da FDN aumentou com a adição de fubá e diminuiu com o emurchecimento, entretanto houve aumento dessa fração quando se adicionou fubá à forragem emurchecida. A degradação da fração a da PB aumentou com a adição de fubá e diminuiu com o emurchecimento; para a fração b ocorreu o inverso. Concluiu-se que a prática do emurchecimento, por reduzir a fração solúvel da forragem, diminui a degradabilidade dos nutrientes da silagem de alfafa, que no entanto pode ser melhorada com a adição de fubá.

Expression, purification, and circular dichroism analysis of human CDK9

The human cyclin-dependent kinase 9 (CDK9) protein was expressed in E coli BL21 using the pET23a vector at 30 degrees C. Several milligrams of protein were purified from soluble fraction using ionic exchange and ATP-affinity chromatography. The structural quality of recombinant CDK9 and the estimation of its secondary structure were obtained by circular dichroism. Structural models of CDK9 presented 26% of helices in agreement with the spectra by circular dichroism analysis. This is the first report on human CDK9 expression in Escherichia coli and structure analysis and provides the first step for the development of CDK9 inhibitors. (c) 2006 Elsevier B.V. All rights reserved.

Efeito de Diferentes Níveis de bicarbonato de sódio sobre a degradação in situ do bagaço de cana-de-açucar auto-hidrolisado

The objective of this work was to evaluate the buffering effects, using sodium bicarbonate (NaHCO3) at the levels: 0; 0.7; 1.4; and 2.1% of dry matter, on the in situ degradation of autohidrolised sugar-cane bagasse. A diet was used with 60% autohidrolised sugar bagasse (AHB) and 40% of concentrate, plus urea, minerals and limestone. The rations was calculated to allow 300g of daily gain. After 20 days adaptation to the treatment (level of NaHCO3), 5 g of AWE was incubated on rumen of four bovines for 3, 6, 12, 24 and 48 hours, using naylon bag measuring 7,5 x 17,5 cm with pores of 36 micras. It was used a randomized blocks design with four treatments (NaHCO3) and four replications(animals). For the calculations of the protein degradation, it was considered the soluble fraction in water plus the degraded fraction in the same proportion as for the neutral detergent fiber (NDF). It was observed that the buffer did not affect the degradability in situ of AHB, whose averages in 48 hours of incubation for dry matter, organic matter, crude protein and neutral detergente fiber (NDF) were 34.83; 36.90; 55.40; and 25.56%, respectively.

Expression and purification of human respiratory syncytial virus recombinant fusion protein

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

Avaliação das silagens de capim-elefante aditivadas com nabo forrageiro, pinhão manso e tremoço, pela técnica de produção de gases

Objetivou-se, na condução deste trabalho, a avaliação das silagens de capim-elefante aditivadas com tortas de nabo forrageiro, pinhão manso e tremoço pela técnica de produção de gás. O experimento foi desenvolvido no Laboratório de Nutrição Animal do Centro de Energia Nuclear na Agricultura da Universidade de São Paulo (LANA/CENA/USP). Como doadores de líquido de rúmen, foram utilizados 2 ovinos da raça Santa Inês, machos, adultos, castrados e providos de cânula ruminal permanente. A alimentação dos animais doadores foi constituída de forragem de gramínea cultivada e uma suplementação, ao final do dia, com feno de Tifton, concentrado comercial e sal mineral à vontade. Os substratos foram secos a 60ºC, moídos em moinho do tipo Willey, provido de peneira com perfurações de 2 mm. Os gases produzidos durante os diferentes períodos de fermentação (0, 4, 8, 12, 24, 36, 48, 72 e 96 h) foram medidos com um transducer - medidor de pressão.O experimento foi instalado segundo um delineamento de blocos ao acaso em que os tratamentos foram arranjados em um esquema de parcelas subdivididas no tempo. Os maiores valores de produção de gás observados para os tratamentos em que adicionou-se torta de tremoço quando comparados com as outras tortas, decorreu do fato da torta de tremoço apresentar menor teor de fibras, propiciando assim, uma maior fermentação ruminal e, consequentemente, maior produção de gás em relação a outros alimentos com maior proporção de carboidratos estruturais (parede celular).As taxas de degradação da fração solúvel da matéria seca foi menor para NF 8% e PM 11% em relação às outras silagens estudadas. Foram encontradas diferenças significativas para as TNF, TPM e TT, nos diferentes níveis, em relação ao volume de gases em 96 h de incubação (P<0,05). As silagens contendo torta de tremoço apresentaram maior produção de gases quando comparadas Às outras tortas. em todos os tratamentos, exceto naqueles em que adicionou-se TT, houve diminuição (P<0,05) nos valores médios de degradabilidade da matéria seca às 96 horas, à medida que aumentou-se o nível de inclusão das tortas. As silagens de capim-elefante adicionadas de tortas de nabo forrageiro ou tremoço, nos diferentes níveis, apresentaram maiores taxas de degradação e maiores produções de gases que as adicionadas de torta de pinhão manso.

Efeitos de dietas com polpa cítrica em substituição ao milho em grãos no concentrado sobre a degradabilidade e a fauna ruminal em bubalinos

Quatro búfalos com cânulas ruminais foram distribuídos aleatoriamente em quadrado latino 4 x 4, compreendendo quatro rações com feno de capim-coastcross (Cynodon dactylon) (50% MS) e níveis crescentes de polpa cítrica peletizada em substituição ao milho em grão moído no concentrado (0, 33, 66 e 100% MS). Foram avaliadas a degradabilidade dos nutrientes e a população de protozoários no rúmen. Houve efeito quadrático da fração solúvel dos nutrientes entre os tratamentos, com valores maiores nos dois extremos. A taxa de degradação da PB do feno e da MS da polpa cítrica apresentou-se linear com aumento da polpa cítrica na ração. As rações contendo polpa cítrica promoveram menores concentrações de Entodinium e número total de ciliados/mL que a dieta só com milho. Houve predominância dos Entodinium nas dietas contendo somente milho ou somente polpa cítrica, enquanto em mistura predominaram os ciliados da subfamília Diplodiniinae. Observou-se efeito de interação da polpa cítrica com o milho, promovendo modificações no metabolismo ruminal, especialmente na solubilidade dos nutrientes, na curva do pH e na composição da fauna.

Isolation and in vitro hydrolysis of lentil protein fractions by trypsin

The albumin and globulin fractions from lentil seeds were isolated and characterised by gel filtration. The latter was shown to be homogeneous and the former heterogeneous on PAGE. The aminoacid analysis revealed high values of amidic amino acids for both fractions with great differences in the sulphur-containing amino acids. Native albumin, globulin and salt-soluble proteins were markedly resistant to trypsin hydrolysis compared to casein. The SDS-PAGE of native salt-soluble proteins indicated that the globulin fragments (20 to 30 kD) were slowly digested in the presence of albumin. The heating increased the hydrolysis of the proteins in the order: salt-soluble, albumin and globulin. The facilitated hydrolysis of the heated salt-soluble fraction seemed to be due to protein-protein interactions induced by heat.

Secretion of metalloendopeptidase 24.15 (EC 3.4.24.15)

The metalloendopeptidase EP24.15 (EC3.4.24.15) is a neuropeptide-metabolizing enzyme present in neural and endocrine tissues, presumably functioning extracellularly, Because the majority of the EP24.15 activity is identified in the soluble fraction of cellular homogenates, suggesting that the enzyme is primarily an intracellular protein, we addressed the issue of how EP24.15 arrives in the extracellular environment, We utilized a model system of neuroendocrine secretion, the AtT20 cell, According to both enzymatic activity and immunologic assays, EP24.15 was synthesized in and released from AtT20 cells. Under basal conditions and after stimulation by corticotropin-releasing hormone or the calcium ionophore A23187, EP24.15 activity accumulated in the culture medium. This secretion was not attributable to cell damage, as judged by the absence of release of cytosolic enzyme markers and the ability to exclude trypan blue dye. Pulse-chase analysis and subcellular fractionation of AtT20 cell extracts suggested that the mechanism of EP24.15 secretion is not solely via classical secretory pathways, Additionally, drugs which disrupt the classical secretory pathway, such as Brefeldin A and nocodazole, blocked A23187-stimulated EP24.15 release yet had no effect on basal EP24.15 release, suggesting differences in the basal and stimulated pathways of secretion for EP24.15. In summary, EP24.15 appears to be secreted from AtT20 pituitary cells into the extracellular milieu, where the enzyme can participate in the physiologic metabolism of neuropeptides.

Evaluation of in vitro gas production of roughages and their mixtures using the curves subtraction method

The in vitro gas production of four single roughages and their paired combinations (1:1 on dry matter basis) were evaluated. Two roughage samples (100 mg) per treatment were fermented with ruminal fluid during a 48 h incubation period. Total 48 h gas volumes of fermentation dry matter (DM), neutral detergent fiber (NDF) and soluble compounds in neutral detergent (NDS) were for sugarcane = 16.8, 11.2, 6.9 mL; sugarcane + corn silage = 20.1, 12.6, 9.1 mL; sugarcane + 60-day elephantgrass = 16.5, 17.6 mL; sugarcane + 180-day elephantgrass = 13.8, 8.2, 5.9 mL; corn silage = 18.8, 16.8, 4.7 mL; corn silage + 60-day elephantgrass = 16.3, 15.4, 2.4 mL; corn silage + 180-day elephantgrass = 16.1, 11.8, 4.2 mL; 60-day elephantgrass = 16.9, 19.0 mL and 180-day elephantgrass = fermented 10.7, 12.2 mL, respectively. The NDS gas production was not possible to estimate for sugarcane + 60-day elephantgrass, 60-day elephantgrass and 180-day elephantgrass. The present data shows that the curves subtraction method can be an option to evaluate the contribution of the soluble fractions in roughages to digestion kinetics. However, this method underestimates the NDS gas contribution when roughages are low in crude protein and soluble carbohydrates. It is advisable to directly apply the two-compartmental mathematical model to the digestion curves for roughage DM, when determining the NDS gas volume and the digestion rate. This method is more straightforward and accurate when compared to the curve subtraction method. Non-structural carbohydrates combined with fiber and protein promoted a positive associative effect in sugarcane + corn silage (50:50) mixture. Therefore, it can be concluded that the soluble fraction of roughages greatly contributes to gas production. (C) 2004 Elsevier B.V. All rights reserved.

Degradação ruminal de silagem de capim-elefante com adição de vagem de algaroba triturada

This research was carried out to evaluate the ruminal degradation of dry matter (DM), crude protein (CP) and neutral detergent fiber (NDF) of silages of elephant grass (Pennisetum purpureum) cutting in 70; 90 and 110 days after regrowth with inclusion of 0; 5; 10 and 15% of mesquite (Prosopis juliflora) meal, based on natural matter in a completely randomized design, in split plot arrangement. Samples of silages were incubated in the rumen of two Jersey cows for 3; 6; 12; 24; 48; 72 and 96 h, and the bags at time "zero" were only washed with water to determine the soluble fraction. There was not interaction (P> 0.05) incubation time x inclusion of mesquite pods x cutting age of the grass for DM degradability, there was only interaction (P <0.05) between these factors for CP and NDF degradability. The most effective DM degradability (42.54%) was observed for 15% inclusion of mesquite pods. The effective CP degradability was higher (69.04%) for elephant grass silage with 70 days after regrowth with 15% of mesquite pods. The inclusion of mesquite pods in elephant grass silages improve DM, CP and NDF degradability, while increment of the age after regrowth result in reduction of this parameters.

Polysiloxane-poly(propylene oxide) hybrid discs as solid phase in anti-HCV detection using a recombinant core protein

In this work, siloxane-poly(propylene oxide) discs (PPO disc) prepared using the sol-gel process were used as solid phase in enzyme-linked immunosorbent assays (ELISA) for the detection of anti-hepatitis C virus (HCV) antibodies. The HCV RNA from serum (genotype 1b) was submitted to the RT-PCR technique and subsequent amplification of the HCV core 408 pb. This fragment was cloned into expression vector pET42a and expressed in Escherichia coli as recombinant protein with glutathione S-transferase (GST). Cell cultures were grown and induced having a final concentration of 0.4 x 10(-3) mol L-1 of IPTG. After induction, the cells were harvested and the soluble fraction was analyzed using polyacrilamide gel 15% showing a band with an approximate molecular weight of 44 kDa, the expected size for this GST-fused recombinant protein. The recombinant protein was purified and continued by immunological detection using HCV-positive serum and showed no cross-reactivity with positive samples for other infectious diseases. An ELISA was established using 1.25 ng of recombinant protein per PPO disc, a dilution of 1: 10,000 and 1:40 for a peroxidase conjugate and serum, respectively, and solutions of hydrogen peroxide and 3,3',5,5'-tetra-methylbenzidine in a ratio of 1: 1. The proposed methodology was compared with the ELISA conventional polystyrene-plate procedure and the performance of the PPO discs as a matrix for immunodetection gave an easy synthesis, good performance and reproducibility for commercial application. (c) 2007 Elsevier B.V. All rights reserved.