Profiling the proteome complement of the secretion from hypopharyngeal gland of Africanized nurse-honeybees (Apis mellifera L.)

The protein complement of the secretion from hypopharyngeal gland of nurse-bees (Apis mellifera L.) was partially identified by using a combination of 2D-PAGE, peptide sequencing by MALDI-PSD/MS and a protein engine identification tool applied to the honeybee genome. The proteins identified were compared to those proteins already identified in the proteome complement of the royal jelly of the honey bees. The 2D gel electrophoresis demonstrated this protein complement is constituted of 61 different polypepides, from which 34 were identified as follows: 27 proteins belonged to MRJPs family, 5 proteins were related to the metabolism of carbohydrates and to the oxido-reduction metabolism of energetic Substrates, I protein was related to the accumulation of iron in honeybee bodies and I protein may be a regulator of MRJP-1 oligomerization. The proteins directly involved with the carbohydrates and energetic metabolisms were: alpha glucosidase, glucose oxidase and alpha amylase, whose are members of the same family of enzymes, catalyzing the hydrolysis of the glucosidic linkages of starch; alcohol dehydrogenase and aldehyde dehydrogenase, whose are constituents of the energetic metabolism. The results of the present manuscript support the hypothesis that the most of these proteins are produced in the hypoharyngeal gland of nurse-bees and secreted into the RJ. (C) 2004 Elsevier Ltd. All rights reserved.

Differential proteomic analysis of Acidithiobacillus ferrooxidans cells maintained in contact with bornite or chalcopyrite: Proteins involved with the early bacterial response

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Expressão gênica diferencial em palmitos de cana-de-açúcar submetida a diferentes períodos de estresse hídrico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise proteômica diferencial de Acidithiobacillus ferrooxidans em resposta aos sulfetos minerais calcopirita e bornita e efeito de uma proteína recombinante de A. ferrooxidans LR contendo cisteína na oxidação bacteriana da calcopirita

Pós-graduação em Biotecnologia - IQ

Ethanol modulates the synthesis and catabolism of retinoic acid in the rat prostate

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

Metabolismo do malondialdeído em peixes: implicações na avaliação da peroxidação lipídica como biomarcador de contaminação aquática

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

In vitro and in situ activity of carboxymethyl cellulase and glutamate dehydrogenase according to supplementation with different nitrogenous compounds

Two experiments were carried out to evaluate the effect of supplementation with different nitrogenous compounds on the activities of carboxymethil cellulase (CMCase) and glutamate dehydrogenase (GDH). In the first experiment, four treatments were evaluated in vitro: cellulose, cellulose with casein, cellulose with urea, and cellulose with casamino acids. After 6, 12 and 24 hours of incubation, CMCase and GDH activity, pH, and concentrations of ammonia nitrogen (AN) and microbial protein were measured. In the three incubation periods, the concentration of AN was higher when urea was used as a supplemental source of nitrogen. The activity of CMCase was higher with the addition of urea and casamino acids when compared with the control and the casein treatment. Supplementation with casamino acids provided higher GDH activity when compared with the control at 6 hours of incubation. At 12 hours of incubation, the GHD activity was also stimulated by casein. At 24 hours, there was no difference in GHD activity among treatments. In the second experiment, three rumen-fistulated bulls were used for in situ evaluation. Animals were fed Tifton hay (Cynodon sp.) ad libitum. The treatments consisted of control (no supplementation), supplementation with non-protein nitrogenous compounds (urea and ammonium sulphate, 9:1) and supplementation with protein (albumin). In treatments with nitrogenous compound supplementation, 1 g of crude protein/kg of body weight was supplied. The experiment was conducted in a 3 × 3 Latin square design. The measurements were performed at 6, 12 and 24 hours after supplementation. No difference in GDH activity was observed among treatments. The control treatment showed higher CMCase activity when compared with the treatments containing supplemental sources of nitrogen. However, urea supplementation provided higher CMCase activity compared to albumin.

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.

Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

Os autores padronizaram métodos para a avaliação da atividade da glicose-6-fosfato desidrogenase e glutationa redutase. O princípio geral do primeiro método baseou-se na formação de metahemoglobina pelo nitrito de sódio, seguido da estimulação da via das pentoses pelo azul de metileno. Foram estudados 46 indivíduos adultos, sendo 23 do sexo masculino e 23 do feminino, não deficientes em glicose-6-fosfato desidrogenase (G6PD), com idades variando entre 20 e 30 anos. Os resultados revelaram que a redução da metahemoglobina pelo azul de metileno para sangue total, foram de 154.50 e 139.90 mg/min (p<0.05) respectivamente para o sexo masculino e feminino. Para hemácias lavadas os valores foram de 221.10 e 207.85 mg/min (n.s.) respectivamente. Estas observações permitiram concluir que ao se empregar hemácias lavadas e 0.7 g% de concentração de nitrito de sódio, por um lado não houve diferença entre os sexos e por outro, abreviou o tempo de leitura da quantidade residual de metahemoglobina para 90 minutos. A avaliação da atividade da glutationa redutase foi feita baseado no fato de que a cistamina (agente tiol) liga-se aos grupos SH da hemoglobina formando complexos. Estes complexos são revertidos pela ação da glutationa redutase, ocorrendo conjuntamente nesta reação a redução da metahemoglobina. Foram estudados 32 indivíduos adultos, sendo 16 do sexo masculino e 16 do feminino, não deficientes em G6PD, com idades variando entre 20 e 30 anos. Os resultados revelaram valores de redução da metahemoglobina pela cistamina de 81.27 e 91.13 mg/min (p<0.01) respectivamente para o sexo masculino e feminino. Estas observações permitiram concluir que o emprego de hemácias lavadas e 0.1 molar de concentração de cistamina torna possível a leitura da quantidade residual de metahemoglobina aos 180 minutos de incubação. A atividade da glutationa redutase avaliada por meio da redução da metahemoglobina pela cistamina, foi estudada em 14 indivíduos do sexo feminino antes e após o tratamento com 10 mg por dia de riboflavina durante 8 dias. Os resultados foram de 73.69 e 94.26 mg/min (p<0.01) antes e após o tratamento. Estas observações permitiram concluir que a oferta de riboflavina, mesmo para indivíduos normais, aumenta a atividade da glutationa redutase. Foram ainda avaliados 3 indivíduos da raça negra e deficientes em G6PD, sendo 2 do sexo masculino e 1 do feminino. Houve ativação parcial da G6PD e glutationa redutase, sendo estas alterações mais intensas nos indivíduos do sexo masculino. Considerando-se a raça e as características laboratoriais observadas, foi possível sugerir que a deficiência em G6PD verificada é do tipo Africano, bem como, permitiu considerar os indivíduos do sexo feminino coin o sendo heterozigoto para esta deficiência. Por fim, a análise dos resultados em seu conjunto permitiu concluir que os métodos propostos se mostraram eficientes para avaliar a atividade da G6PD e glutationa redutase. Esta última é dependente da via das pentoses, geradora de NADPH e da riboflavina, vitamina precursora de FAD.

Dihydrolipoyl dehydrogenase as a source of reactive oxygen species inhibited by caloric restriction and involved in Saccharomyces cerevisiae aging

Replicative life span in Saccharomyces cerevisiae is increased by glucose (G1c) limitation [ calorie restriction (CR)] and by augmented NAD(+). Increased survival promoted by CR was attributed previously to the NAD(+)-dependent histone deacetylase activity of sirtuin family protein Sir2p but not to changes in redox state. Here we show that strains defective in NAD(+) synthesis and salvage pathways (pnc1 Delta, npt1 Delta, and bna6 Delta) exhibit decreased oxygen consumption and increased mitochondrial H2O2 release, reversed over time by CR. These null mutant strains also present decreased chronological longevity in a manner rescued by CR. Furthermore, we observed that changes in mitochondrial H2O2 release alter cellular redox state, as attested by measurements of total, oxidized, and reduced glutathione. Surprisingly, our results indicate that matrix-soluble dihydrolipoyl-dehydrogenases are an important source of CR-preventable mitochondrial reactive oxygen species (ROS). Indeed, deletion of the LPD1 gene prevented oxidative stress in npt1 Delta and bna6 Delta mutants. Furthermore, pyruvate and alpha-ketoglutarate, substrates for dihydrolipoyl dehydrogenase-containing enzymes, promoted pronounced reactive oxygen release in permeabilized wild-type mitochondria. Altogether, these results substantiate the concept that mitochondrial ROS can be limited by caloric restriction and play an important role in S. cerevisiae senescence. Furthermore, these findings uncover dihydrolipoyl dehydrogenase as an important and novel source of ROS leading to life span limitation.

Alcohol dehydrogenase 3 genotype as a risk factor for upper aerodigestive tract cancers

Objective: To assess alcohol dehydrogenase 3 (ADH3) polymorphism at position Ile349Val as indicator of risk factor for upper aerodigestive tract (UADT) cancer to verify its association with UADT cancer in nonalcoholic or nonsmoking individuals.Design: Cross-sectional study.Setting: Primary care or referral center.Patients: the study group consisted of 141 consecutive patients with newly diagnosed squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx, or larynx admitted for surgical treatment. The comparison group consisted of 94 inpatients without cancer from the A. C. Camargo or other São Paulo (Brazil) hospital and 40 healthy individuals.Intervention: All participants were interviewed and data were collected using a structured questionnaire. After written informed consent was obtained, 20 mL of blood was collected in heparinized tubes.Main Outcome Measures: Odds ratio for ADH3 genotypes using logistic regression models.Results: After adjustment for sex, age, tobacco use, and history of cancer in first-degree family relatives, a significantly higher odds ratio for UADT cancer was observed among individuals with AA genotype and low cumulative alcohol consumption (:5 100 kg of ethanol) (odds ratio=3.8 [95% confidence interval, 1.5-9.7]). A 4-fold increase in odds ratio for UADT cancer among individuals with AA genotype and low tobacco consumption (:525 pack-years) was also found in the adjusted model.Conclusions: These results suggest that genotype AA may be a risk factor for UADT cancer, especially in individuals with low alcohol or tobacco consumption. However, further epidemiological case-control or cohort studies, preferably prospective, are needed to establish the exact role of ADH3 polymorphism and its association with the development of UADT cancers.

Molecular, kinetic, thermodynamic, and structural analyses of Mycobacterium tuberculosis hisD-encoded metal-dependent dimeric histidinol dehydrogenase (EC 1.1.1.23)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Functional shikimate dehydrogenase from Mycobacterium tuberculosis H37Rv: Purification and characterization

Tuberculosis (TB) poses a major worldwide public health problem. The increasing prevalence of TB, the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, the causative agent of TB, and the devastating effect of co-infection with HIV have highlighted the urgent need for the development of new antimycobacterial agents. Analysis of the complete genome sequence of M. tuberculosis shows the presence of genes involved in the aromatic amino acid biosynthetic pathway. Experimental evidence that this pathway is essential for M. tuberculosis has been reported. The genes and pathways that are essential for the growth of the microorganisms make them attractive drug targets since inhibiting their function may kill the bacilli. We have previously cloned and expressed in the soluble form the fourth shikimate pathway enzyme of the M. tuberculosis, the aroE-encoded shikimate dehydrogenase (mtSD). Here, we present the purification of active recombinant aroE-encoded M. tuberculosis shikimate dehydrogenase (mtSD) to homogeneity, N-terminal sequencing, mass spectrometry, assessment of the oligomeric state by gel filtration chromatography, determination of apparent steady-state kinetic parameters for both the forward and reverse directions, apparent equilibrium constant, thermal stability, and energy of activation for the enzyme-catalyzed chemical reaction. These results pave the way for structural and kinetic studies, which should aid in the rational design of mtSD inhibitors to be tested as antimycobacterial agents. (c) 2005 Elsevier B.V. All rights reserved.

Structural studies of shikimate 5-dehydrogenase from Mycobacterium tuberculosis

Tuberculosis (TB) remains the leading cause of mortality due to a single bacterial pathogen, Mycobacterium tuberculosis. The reemergence of TB as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, the proliferation of multi-drug-resistant strains (MDR-TB) and, more recently, of extensively drug resistant isolates (XDR-TB) have created a need for the development of new antimycobacterial agents. Amongst the several proteins and/or enzymes to be studied as potential targets to develop novel drugs against M. tuberculosis, the enzymes of the shikimate pathway are attractive targets because they are essential in algae, higher plants, bacteria, and fungi, but absent from mammals. The mycobacterial shikimate pathway leads to the biosynthesis of chorismate, which is a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Here we report the structural studies by homology modeling and circular dichroism spectroscopy of the shikimate dehydrogenase from M. tuberculosis (MtSDH), which catalyses the fourth step of the shikimate pathway. Our structural models show that the MtSDH has similar structure to other shikimate dehydrogenase structures previously reported either in presence or absence of NADP, despite the low amino acid sequence identity. The circular dichroism spectra corroborate the secondary structure content observed in the MtSDH models developed. The enzyme was stable up to 50 degrees C presenting a cooperative unfolding profile with the midpoint of the unfolding temperature value of similar to 63-64 degrees C, as observed in the unfolding experiment followed by circular dichroism. Our MtSDH structural models and circular dichroism data showed small conformational changes induced by NADP binding. We hope that the data presented here will assist the rational design of antitubercular agents.