Protein structure of the venom in nine species of snake: from bio-compounds to possible healing agents

Due to its various structures in bio-compounds, snake venom is the indisputable result of evolutionary stages of molecules with an increasingly complex structure, high specificity, and of great importance for medicine because of their potential. The present study proposed an underpinning examination of venom composition from nine species of venomous snakes using a useful and replicable methodology. The objective was the extension of the evaluation of protein fractions in the field up to 230 kDa to permit possible identification of some fractions that are insufficiently studied. The gel capillary electrophoresis method on the chip was performed using an Agilent 2100 bioassay with the 80 and 230-LabChip Protein kits. Interpretation of electrophoresis was performed using the Protein 2100 expert (Agilent) test software as follows: a) Protein 80 (peak size scale): 1.60, 3.5, 6.50, 15.00, 28.00, 46.00, 63.00, 95.00 kDa; b) Protein 230 (peak size scale): 4.50, 7.00, 15.00, 28.00, 46.00, 63.00, 95.00, 150.00, 240.00 kDa. The screening revealed the presence of compounds with a molecular weight greater than 80 kDa, in the case of Vipera aspis and Vipera xantina palestinae. For V. aspis, a 125 kDa molecular weight pro-coagulant protein was identified, known as being involved in the reduction of plasma clotting time without any direct activity in the fibrinogen coagulation process. The samples examined on the Protein 230-LabChip electrophoresis chip can be considered as a novelty with possible uses in medicine, requiring further approaches by advanced proteomics techniques to confirm the intimate structural features and biological properties of snake venoms.

PEGylation strategy to the development of analytical and therapeutic proteins / Potencial da PEGuilação para o desenvolvimento de proteínas para fins terapêuticos e analíticos

Protein PEGylation is the covalent bonding of polyethylene glycol (PEG) polymers to amino acid residues of the protein and it is one of the most promising techniques for improving the therapeutic effect of biopharmaceuticals and long-term stability of protein-based biosensors. This chemical modification brings advantages to biopharmaceuticals, such as an increased half-life, enhanced stability, and reduced immunogenicity. Moreover, in the analytical field, PEGylation improves the multiple properties of protein-based biosensors including biocompatibility, thermal and long-term stability, and solubility in organic solvents. However, the use of PEGylated conjugates in the analytical and therapeutic fields has not been widely explored. The limited industrial application of PEGylated bioconjugates can be attributed to the fact that the reaction and separation steps are currently a challenge. The correct selection of the PEGylation reaction design and the purification process are important challenges in the field of bioconjugation. In this sense, the design and optimization of site-specific PEGylation reactions and application of aqueous biphasic systems (ABS) as purification platforms for PEGylated conjugates are the two main objectives of this thesis. Regarding the purification step, the efficient fractionation (i) of the PEGylated conjugates from the native protein and (ii) of the PEGylated conjugates based on their degree of PEGylation was studied. Centrifugal partition chromatography (CPC) was applied as a continuous regime platform based on ABS technology to efficiently purify the PEGylated proteins. The two proteins under study are L-asparaginase, an important biopharmaceutical applied in the treatment of acute lymphoblastic leukemia and cytochrome c, a promising biosensor. The current work developed in this thesis demonstrates the great potential of ABS in the fractionation of PEGylated proteins, under batch and continuous regime. In addition, in situ recovery of the PEGylated products through one-pot bioconjugation and ABS purification was successfully demonstrated for both enzymes studied. Although further research on scale-up is still required, the results presented show the relevance of ABS platforms for the development of separation processes of PEGylated proteins

A PEGuilação de proteínas é a ligação covalente de polímeros de polietilenoglicol (PEG) a resíduos de aminoácidos da proteína e é uma das técnicas mais promissoras para melhorar o efeito terapêutico dos biofármacos e a estabilidade a longo prazo de biossensores proteícos. Esta modificação química traz vantagens aos produtos biofarmacêuticos, como um aumento da meia-vida, maior estabilidade e imunogenicidade reduzida. Além disso, no campo analítico, a PEGuilação melhora as múltiplas propriedades dos biossensores baseados em proteínas, incluindo biocompatibilidade, estabilidade térmica e a longo prazo, e solubilidade em solventes orgânicos. No entanto, o uso de conjugados PEGuilados em campos analíticos e terapêuticos não tem sido amplamente explorado. A aplicação industrial limitada dos bioconjugados PEGuilados pode ser atribuída ao facto de as etapas de reacção e separação serem atualmente um desafio. A seleção correcta do design da reacção de PEGuilação e do processo de purificação são importantes desafios no campo da bioconjugação. Neste sentido, a concepção e otimização de reações de PEGuilação sítio-específicas e aplicação de sistemas aquosos bifásicos (ABS) como plataformas de purificação de conjugados PEGuilados são os dois principais objetivos desta tese. No que concerne à etapa de purificação foi estudado o eficiente fracionamento (i) dos conjugados PEGuilados, da proteína nativa e (ii) dos conjugados PEGuilados baseados no seu grau de PEGuilação. A cromatografia por partição centrífuga (CPC) foi aplicada como uma plataforma de regime contínuo baseada na tecnologia de ABS para purificar eficientemente as proteínas PEGuiladas. As duas proteínas em estudo são a L-asparaginase, importante biofármaco aplicado no tratamento da leucemia linfoblástica aguda e o citocromo c, um potencial biossensor. A partir dos trabalhos desenvolvidos, é possível confirmar o grande potencial dos ABS no fracionamento de proteínas PEGuiladas, em regime contínuo e descontínuo. Além disso, a recuperação in situ dos produtos PEGuilados através da integração em uma única etapa de bioconjugação e purificação por ABS foi comprovada com sucesso para ambas as enzimas estudadas. Embora ainda sejam necessários estudos adicionais sobre a viabilidade destes sistemas em larga escala, os resultados aqui apresentados demonstram a relevância dos ABS para o desenvolvimento de processos de separação de proteínas PEGuiladas

Cardiac protein expression patterns are associated with distinct inborn exercise capacity in non-selectively bred rats

In the present study, we successfully demonstrated for the first time the existence of cardiac proteomic differences between non-selectively bred rats with distinct intrinsic exercise capacities. A proteomic approach based on two-dimensional gel electrophoresis coupled to mass spectrometry was used to study the left ventricle (LV) tissue proteome of rats with distinct intrinsic exercise capacity. Low running performance (LRP) and high running performance (HRP) rats were categorized by a treadmill exercise test, according to distance run to exhaustion. The running capacity of HRPs was 3.5-fold greater than LRPs. Protein profiling revealed 29 differences between HRP and LRP rats (15 proteins were identified). We detected alterations in components involved in metabolism, antioxidant and stress response, microfibrillar and cytoskeletal proteins. Contractile proteins were upregulated in the LVs of HRP rats (α-myosin heavy chain-6, myosin light chain-1 and creatine kinase), whereas the LVs of LRP rats exhibited upregulation in proteins associated with stress response (aldehyde dehydrogenase 2, α-crystallin B chain and HSPβ-2). In addition, the cytoskeletal proteins desmin and α-actin were upregulated in LRPs. Taken together, our results suggest that the increased contractile protein levels in HRP rats partly accounted for their improved exercise capacity, and that proteins considered risk factors to the development of cardiovascular disease were expressed in higher amounts in LRP animals.

Avaliação de biodisponibilidade e mecanismos de ação hipocolesterolemizante de peptídeos do amaranto (Amaranthus cruentus L. BRS-Alegria) / Evaluation of bioavailability and hypocholesterolemic mechanisms of peptides from Amaranth (Amaranthus cruentus L. BRS-Alegria)

Introdução: Doenças cardiovasculares constituem importante causa de morte em todo mundo e a hipercolesterolemia está diretamente relacionada a este problema de saúde pública. A dieta desempenha papel importante neste processo e alguns alimentos, como o amaranto (Amaranthus cruentus L. BRSAlegria), têm mostrado capacidade de redução do colesterol plasmático. Estudos sugerem que este efeito está relacionado a peptídeos liberados durante a digestão das proteínas, os quais atuam na modulação do metabolismo lipídico. Considerando-se que os efeitos da digestão gastrointestinal e da absorção destes peptídeos são claramente complexos torna-se importante a realização de estudos visando avaliar bioacessibilidade e mecanismos de ação destes peptídeos nos locais alvo do organismo. Objetivo: Analisar a biodisponibilidade de peptídeos em modelos animais após ingestão de isolado proteico de amaranto e relacioná-la com parâmetros ligados ao metabolismo do colesterol. Métodos: O amaranto teve sua proteína isolada. Os peptídeos da proteína do amaranto foram analisados após digestão in vitro. Dois experimentos in vivo foram conduzidos: um de fase aguda e outro de média duração. No primeiro, o isolado proteico de amaranto foi administrado a ratos e os peptídeos no sangue foram monitorados por 2 horas para verificação de fragmentos que resistissem à digestão gastrointestinal. O experimento in vivo 2 consistiu na alimentação de 3 grupos de hamster, um com dieta recomendada pela AIN93 (grupo N) e dois com dietas hipercolesterolêmicas por 21 dias, contendo a proteína de amaranto como única proteína da ração (grupo I), comparada ao controle de caseína (grupo H). Neste experimento foram analisados no plasma: peptídeos, colesterol total e frações; nas fezes: colesterol total e ácidos biliares; no fígado: colesterol, lipídeos totais, ácidos graxos, atividade enzimática da Hmgcr, expressão de Hmgcr, Srebf2, Lxr, Abca1, Abcg8 e Ampk. Resultados e discussão: Foram identificados fragmentos peptídicos provenientes da digestão in vitro do isolado proteico de amaranto, e outras dezenas de sequencias peptídicas em ratos após administração aguda de amaranto foram analisadas. Destaca-se a identificação do peptídeo ALGV, presente em proteína do amaranto de acordo com banco de dados, e similar a fragmentos com ação hipocolesterolemizante. No sangue de hamsters foram encontrados seis peptídeos com 100 por cento de cobertura e similaridade a base de dados de proteínas de amaranto, merecendo investigação sobre seus efeitos. Verificou-se que o isolado proteico de amaranto foi capaz de suprimir a hipercolesterolemia quando a dieta hipercolesterolemizante foi introduzida em paralelo a este ingrediente, com valores inferiores em 72 por cento (triglicerídeos), 64 por cento (colesterol total), 80 por cento (LDL-c) do grupo I em relação ao grupo H. Foi observada ainda menor concentração de colesterol e lipídeos totais no fígado dos animais do grupo I em relação ao grupo H (177 x 464 mg de colesterol/100 g de tecido; 2,06 x 2,86 g de lipídeos/100 g de tecido, respectivamente). Parâmetros lipídicos do sangue, das fezes e do fígado foram similares aos do grupo N, cuja dieta seguiu a preconização para roedores. Foi observada maior excreção de colesterol total no grupo I em relação ao grupo H, mas não houve maior excreção de ácidos biliares nas fezes. Não houve mudança na expressão dos genes analisados neste estudo, mas o amaranto reduziu a atividade da enzima Hmgcr. Postulase que parâmetros como expressão de Ldlr e atividade da Acat sejam alterados pela ingestão de amaranto. O perfil de ácidos graxos também foi modificado de forma a se assimilar ao grupo N, porém deve-se verificar parâmetros inflamatórios devido à maior proporção de ácido araquidônico em relação aos demais grupos estudados. Conclusão: Verifica-se biodisponibilidade dos peptídeos do amaranto e ação hipocolesterolemizante e hipolipemiante em diversas vias metabólicas, promovendo proteção cardiovascular

Introduction: Cardiovascular diseases are important causes of death worldwide, and hypercholesterolemia is directly related to this public health problem. Diet plays an important role in this process and some foods such as amaranth (Amaranthus cruentus L. BRS-Alegria) have been shown to reduce plasma cholesterol. Studies suggest that this effect is related to peptides released during the digestion of proteins, which would play an important role in the modulation of lipid metabolism. Considering that the effects of gastrointestinal digestion and the absorption of these peptides are clearly complex, it is important to carry out studies aiming to evaluate their bioaccessibility and evaluation of the mechanisms of action of these peptides in the target sites of the organism. Objective: To analyze the bioavailability of peptides in animal models after ingestion of amaranth protein isolate and to relate it to parameters associated to cholesterol metabolism. Methods: The amaranth was crushed, the flour was defatted and its protein isolated. Amaranth peptides were analysed after in vitro digestion. Two in vivo experiments were conducted: one of acute phase and one of medium duration. In the first, the amaranth protein isolate was administered to rats and the peptides in the blood were monitored for 2 hours to check for fragments that resisted gastrointestinal digestion. The in vivo experiment 2 consisted of feeding three groups of hamsters, one with a diet recommended by AIN93 (group N) and two with hypercholesterolemic diets for 21 days, containing amaranth protein as the only dietary protein (group I), compared to casein control (group H). In this experiment were analyzed in the plasma: peptides, total cholesterol and fractions; In feces: total cholesterol and bile acids; In the liver: cholesterol, total lipids, fatty acids, Hmgcr enzymatic activity, Hmgcr expression, Srebf-2, Lxr, Abca1, Abcg8 and Ampk. Results and discussion: Peptide fragments from the in vitro digestion of amaranth protein isolate were identified and other dozens of peptide sequences were found in rats after acute amaranth administration. A higher number of peptides were found in the serum in relation to the plasma of the animals. Remarkably, ALGV peptide was found in serum of rats. This peptide is present in amaranth protein, according to databases, and is similar to fragments that present hypocholesterolemic action. In the blood of hamsters it could be found six peptides with 100 per cent coverage and similarity to the database of amaranth proteins, deserving investigation about their effects. Amaranth protein was able to suppress hypercholesterolemia when the hypercholesterolemic diet was introduced in parallel with this ingredient, with values lower for group I in 72 per cent (triglycerides), 64 per cent (total cholesterol), 80 per cent (LDL-c) in relation to the H group. A lower concentration of cholesterol and total lipids were observed in the liver of the group I compared to the H group (177 x 464 mg cholesterol / 100 g of tissue, 2.06 x 2,86 g lipids / 100 g of tissue, respectively). Lipid parameters of blood, faeces and liver were similar to those of group N, whose diet followed the recommendation for rodents. There was greater excretion of total cholesterol in group I in relation to group H, but there was no greater excretion of bile acids in feces, indicating that the effect of amaranth protein may be due to increased transintestinal cholesterol excretion, decreased micellar solubilization of cholesterol and / or modification in the expression of cholesterol transport related proteins in the intestine. There was no change in the expression of the genes analyzed in this study, but amaranth reduced the activity of the Hmgcr enzyme. It is postulated that parameters such as Ldlr expression and Acat activity are altered by amaranth intake. The fatty acid profile was also modified in order to assimilate to the N group, but inflammatory parameters related to amaranth intake should be verified due to the higher proportion of arachidonic acid in relation to the higher proportion of arachidonic acid in relation to the other groups studied. Conclusion: The bioavailability of amaranth peptides and hypocholesterolemic and hypolipidemic activity in several metabolic pathways is verified, therefore promoting cardiovascular protection

Teores de proteínas e lipídeos de Chlorella sp. cultivada em concentrado de dessalinização residual / Protein and lipid contents from Chlorella sp. Cultivated in residual concentrated desalination

A dessalinização é um método de obtenção de água potável limitado pelos problemas ambientais, causados pelo resíduo gerado em seu processo. O objetivo deste estudo foi avaliar o cultivo de Chlorella sp., em meio de cultura a base de concentrado de dessalinização, e determinar os teores de proteínas e lipídeos ao longo dos ciclos de cultivo. Os cultivos foram desenvolvidos em fotobiorreatores cônicos invertidos (4L) e mantidos durante 28 dias resultando em quatro ciclos de cultivo. Para cada ciclo de cultivo, os valores de biomassa (peso seco, g L-1) resultaram em 1º ciclo, 1,55; 2º ciclo, 0,96; 3º ciclo, 0,62, e 4º ciclo em 0,42. Os teores de proteínas e lipídeos variaram entre 45,2 a 48,8% e 8,5 a 11,4%, respectivamente. O primeiro ciclo de cultivo apresentou a maior produtividade em biomassa (PB = 200 mg L-1 dia-1) e produtividade lipídica (PL = 19,6 mg L-1 dia-1), bem como o maior teor de proteínas (48,8%). O maior teor de lipídeos (11,4%) foi obtido no segundo ciclo de cultivo. Foi observado, nos quatro ciclos de cultivo, que a produtividade em biomassa está diretamente correlacionada com a produtividade lipídica, indicando que quanto maior a PB maior será a PL. Os resultados da cultura de Chlorella sp. demonstraram que é possível utilizar o concentrado de dessalinização residual como meio de cultura alternativo e obter biomassa ao longo de quatro ciclos de cultivo, sem comprometer os teores de proteínas e lipídeos na célula microalgal.

Desalination process is a method of obtaining potable drinking water which is limited by environmental problems caused by the waste generated in the process. The aim of this study was to evaluate the cultivation of Chlorella sp. in a culture medium based on residual concentrated desalination and determines the protein and lipid contents along the cultivation cycles. The cultures were developed in a reverse conical photobioreactors (4L) during 28 days resulting in four cultivation cycles. For each cultivation cycle, the value of biomass (dry weight g L-1) resulted: 1st cycle, 1.55 g; 2nd cycle, 0.96; 3rd cycle, 0.62 and 4th cycle, 0.42. The protein and lipid levels ranged from 45.2 to 48.8% and 8.5 to 11.4%, respectively. The first cultivation cycle showed the highest biomass productivity (PB = 200 mg L-1 dia-1) and lipid productivity (PL = 19.6 mg L-1 dia-1), as well as the highest protein content (48.8%). The highest lipid content (11.4%) was observed on second cultivation cycle. It was observed during the four cultivation cycles that the biomass productivity is directly correlated to lipid productivity, indicating that higher PB higher will be PL. The results from this study showed that it is possible to growth Chlorella sp. in a culture medium based on residual concentrated desalination for biomass production along the four cultivation cycles and also, without compromise the protein and lipid contents into the microalgal cell.

Cloning, expression and characterization of SeM protein of Streptococcus equi subsp. equi and evaluation of its use as antigen in an indirect ELISA / Clonagem, expressão e caracterização de proteína SeM de Streptococcus equi subsp. equi e avaliação de sua utilização como antígeno em um ELISA indireto

Strangles is an economically important horse disease caused by Streptococcus equi subsp. equi. The diagnosis can be confirmed either directly by bacterial isolation and PCR or by ELISA, which is an indirect method based on the detection of serum antibodies. The aim of this study was to clone, express and characterize the SeM protein of Streptococcus equi subsp. equi, evaluate its use as antigen in indirect ELISA and determine its performance to distinguish sera of negative, vaccinated and positive animals. This was initially performed by cloning the gene encoding the SeM protein and its expression in Escherichia coli. Subsequently, the protein produced was characterized and used as antigen in ELISA. Serum samples for evaluation were taken from 40 negative foals, 46 horses vaccinated with a commercial vaccine against strangles and 46 horses diagnosed with the disease. The test showed high specificity and sensitivity, allowing discrimination between negative and positive, positive and vaccinated animals, and vaccinated animals and negative sera. Thus, it was concluded that the protein produced rSeM, which can be used as antigen for disease diagnosis, and the described ELISA might be helpful to evaluate the immune status of the herd...

A adenite equina é uma enfermidade economicamente importante de equinos, causada por Streptococcus equi subsp. equi. Seu diagnóstico pode ser confirmado de forma direta, por meio de isolamento bacteriano e de PCR, ou de forma indireta, por meio de ELISA, método baseado na detecção de anticorpos séricos. O objetivo deste estudo foi clonar, expressar e caracterizar a proteína SeM de Streptococcus equi subsp. equi, avaliar sua utilização como antígeno em um ELISA indireto e determinar a capacidade do teste de distinguir soros de animais negativos, vacinados e positivos. Para tal, foi inicialmente realizada a clonagem do gene que codifica para a proteína SeM e sua expressão em Escherichia coli. Posteriormente, a proteína produzida foi caracterizada e utilizada como antígeno em um teste de ELISA indireto. Para avaliação do teste, foram utilizadas amostras de soro de 40 potros negativos, de 46 equinos vacinados com uma vacina comercial contra adenite equina e de 46 equinos com diagnóstico da doença. O teste demonstrou alta sensibilidade e especificidade, permitindo discriminar entre soros negativos e positivos, positivos e de animais vacinados, e negativos e de animais vacinados. Assim, conclui-se que a proteína rSeM produzida pode ser usada como antígeno para o diagnóstico da enfermidade e que o ELISA descrito pode ser útil para avaliar o estado imunológico do rebanho...

[Comparison and evaluation of Echinococcus granulosus protoscoleces excretory/secretory proteins in PBS complemented with glucose, DMEM and RPMI culture media]

Preparation of proper antigens is an important issue in serology of hydatidosis. Investigators have been able to obtain excretory/secretory antigens [E/S Ags] by short-term culture of protoscoleces in a couple of culture media. However, no data are available about production rate of such antigens in different culture media. The present study was carried out to evaluate the production of E/S Ags [proteins] in PBS complemented with glucose, DMEM and RPMI culture media. To obtain E/S proteins, protoscoleces of echinococcus were cultured in PBS complemented with 10% glucose, RPMI and DMEM for 72 hours. Proteins secreted in culture media were concentrated and assayed. To characterize different components, proteins were electrophoresed on SDSPAGE. Data were analyzed using One-way ANOVA and Tukey HSD tests. The mean concentration of E/S proteins in PBS medium in 24 hours of culture was significantly higher than DMEM and RPMI [P<0.05]. However, such a difference was not observed between E/S proteins in DMEM and RPMI media. E/S proteins obtained from PBS medium were separated into 12 major bands and the two other media into 14 major bands within a range of molecular masses of 16 to 67 kDa. PBS complemented with glucose is more appropriate than the two other media for E/S proteins production. The best time to obtain E/S proteins is the first 48 hours of culture

Efeito da ingestão de proteína de amaranto no metabolismo do colesterol em ratos / Effect of amaranth protein isolate intake on cholesterol metabolism in rats

Introdução As doenças cardiovasculares estão entre as principais causas de morte no Brasil e no mundo. Evidências epidemiológicas e clínicas estabelecem associação entre dieta, dislipidemia e aumento do risco de morte. O consumo de proteína isolada de amaranto tem efeito hipocolesterolemizante e por isso pode reduzir, de modo significativo, os fatores de risco das doenças cardiovasculares. Objetivo Avaliar o efeito da ingestão do isolado protéico de amaranto, no perfil de lipoproteínas plasmáticas e na expressão de proteínas relacionadas à modulação da síntese do colesterol hepático. Métodos Vinte e oito ratos Wistar (Ratus novergicus) foram distribuidos em quatro grupos e receberam dietas diferenciadas pela fonte protéica. Os grupos experimentais (I e Icol) receberam dieta com 20por cento de proteína de amaranto e os grupos controle (C e Ccol) receberam dieta com 20por cento de caseína. As dietas col apresentavam 1por cento de colesterol. Ao grupo controle foi fornecida a média da quantidade de ração ingerida pelos grupos experimentais I e Icol (controle pair feeding). Para determinar o efeito da ingestão das dietas no metabolismo do colesterol, foram avaliadas as concentrações plasmáticas de triacilgliceróis, colesterol total e HDL-c, e as concentrações hepáticas de colesterol e lipídios totais. O efeito da ingestão da proteína de amaranto na regulação das vias de síntese do colesterol hepático foi investigado pela avaliação da expressão das proteínas nucleares:receptor X hepático alfa (LXR alfa), receptor ativado por proliferadores de peroxissoma alfa (PPAR alfa) e proteína ligadora do elemento regulado por esterol 2 (SREBP-2). Resultados A dieta Icol promoveu menor concentração plasmática de colesterol total e triacilgliceróis (36por cento e 47por cento , respectivamente) em comparação ao grupo Ccol...

Bt protein rhizosecreted from transgenic maize does not accumulate in soil

The persistence of CryIAb protein rhizosecreted in soil is important in the assessment of its environmental risk. Here we report that CryIAb protein from transgenic maize does not accumulate at high levels in soils. Levels of CryIAb protein rhizosecreted by three maize transgenic events (BT11, MON810 and 176) were studied in hydroponic cultures and found only in the MON810 and BT11 events but not in event 176 or control plants. Under field conditions, the cryIAb gene and a basal level of CryIAb protein was detected in soils from plots cultivated with transgenic and non-transgenic maize, possibly from Bacillus thuringiensis present in the soils.

Amplification of six putative RDI genes of Mycobacterium tuberculosis for cloning and expression in Escherichia coli and purifications of expressed proteins

To amplify, clone and express in Escherichia coli six open reading frames [ORFs] predicted in the RD1 DNA segment of Mycobacterium tuberculosis and purify the expressed proteins to homogeneity. DNA corresponding to the coding regions of six RD1 ORFs, i.e. ORF10 to ORF15, was amplified from genomic DNA of M. tuberculosis, cloned in the plasmid vector pPCR-Script and subcloned in expression plasmid vectors pET29a and/or pGEX-4T for expression in E. coli as fusion proteins. The recombinant fusion proteins were identified by sodium dodecyl polyacrylamide gel electrophoresis and Western immunoblotting. Attempts were made to obtain purified proteins, free of the fusion partner, using affinity and fast protein liquid chromatography. DNA corresponding to all six targeted RD1 ORFs was amplified from the genomic DNA of M. tuberculosis and five of the six ORFs, with the exception of ORF13, were cloned in the plasmid vectors and expressed in E. coli. Because of extensive degradation of ORF10 and ORF12 fusion proteins or nonbinding to the affinity columns of ORF15 fusion proteins, only ORF11 and ORF14 proteins were purified, free of the fusion partner, to homogeneity. All of the six targeted RD1 genes were amplified and five expressed using E. coli hosts, but only two of the expressed proteins were purified to homogeneity. Alternative expression systems are required to obtain all RD1 proteins for functional characterization

A simple and cost-effective protocol for DNA isolation from buccal epithelial cells

Buccal cells provide a convenient source of DNA for epidemiological studies. The goal of this study was to develop a convenient method to obtain buccal cells from mouthwash samples to be used as a source of DNA, and to evaluate the stability of the DNA in mouthwash solution over time. The procedures used in the method described in this paper avoid the use of any organic solvents. This is achieved by salting out the cellular proteins by dehydration and precipitation with a saturated ammonium acetate solution. The protocol described here is fast, simple to perform, sensitive, economical and several samples can be processed at the same time. The analyses provide consistent evidence that DNA extracted by this methodology is sufficient for several PCR amplifications. The total DNA yield ranged from 5 to 93 µg (median 15 µg, mean 20.71 µg). DNA can be extracted and PCR amplified after storage of mouthwash solution at room temperature for periods of up to 30 days.

Células bucais são fontes convenientes de DNA para diagnóstico e estudos epidemiológicos. O objetivo deste trabalho foi desenvolver um método simples e prático para obter células epiteliais, através de bochechos, a fim de serem usadas como fonte de DNA e avaliar a estabilidade do DNA na solução de bochecho no decorrer do tempo. Os procedimentos usados neste estudo evitam o uso de solventes orgânicos permitindo uma pratica laboratorial mais segura. Isto é alcançado pela remoção das proteínas celulares por desidratação e precipitação com uma solução saturada de acetato de amônio. Este protocolo permite a extração de maneira rápida, simples, econômica e garante o processamento de várias amostras ao mesmo tempo, agilizando assim os procedimentos laboratoriais. Nossas análises forneceram evidências consistentes de que o DNA extraído por esta metodologia é suficiente para diversas amplificações por PCR (polymerase chain reaction - reação em cadeia pela polimerase). O produto total de DNA variou de 5 a 93 µg (mediana 15 µg; média 20,71 µg). Além disso, o DNA mostrou-se eficientemente preservado na solução de bochecho, a qual pode ser estocada em temperatura ambiente por até trinta dias.

Isolation, identification and characterization of secretory proteins of IVMFC embryos and blood circulation of estrus and early pregnant goat.

The aim of the present study was to isolate, identify and characterize the secretory proteins of IVM oocytes and IVMFC embryos to evaluate its immunogenecity. and identify of such proteins if any, in blood circulation of estrus and early pregnant goats. Oocytes were matured in TCM-199 with 1 microg/ml, estradiol-17beta; 0.5 microg/ml, FSH; 100 IU/ml, LH and 10% FCS on granulosa cell monolayer. After 18 hr of maturation, oocytes were further cultured in maturation medium containing 3 mg/ml polyvinyl alcohol (PVA) without serum and BSA for 12 hr and medium was collected. The IVF embryos of 4-8 cell stage were cultured in medium containing PVA without serum and BSA. Embryo culture medium was collected after 24 hr of culture and was pooled. The proteins were analyzed on SDS-PAGE (12.5%). Four secretory proteins of oocytes with approximately molecular weight of 45, 55, 65 and 95 kDa and three secretory proteins of embryos 45, 55 and 65 kDa were obtained on SDS-PAGE in silver staining. The protein profile of midluteal, estrus and early pregnant goat serum was similar and no variation was observed among the proteins on SDS-PAGE. Two secretory proteins of 55 and 65 kDa of both IVM oocytes and IVMFC embryos were observed on Western analysis. None of such proteins was observed in midluteal, estrus and early pregnant goat serum on western blotting. It can be concluded that IVM oocytes and IVMFC embryos secrete proteins in medium and two of them can develop antibody. The proteins secreted from embryos till morula stage was similar to that of oocytes. None of these oocyte/embryo released proteins were observed in blood circulation of estrus and early pregnant goats.

Identification of glandular (preputial and clitoral) proteins in house rat (Rattus rattus) involved in pheromonal communication.

Proteins (18-20 kDa) belonging to lipocalin family have been reported to act as carriers for ligands binding to pheromones in mouse urine, pig saliva, hamster vaginal fluid and human sweat, that are involved in pheromonal communication. As the preputial gland is a major pheromonal source, the present study was aimed to detect the specific protein bands (around 18-20 kDa) in the preputial and clitoral glands of the house rat, R. rattus. The amount of protein was higher in preputial gland of the male than that of female (clitoral) gland. A 20 kDa protein was noted in male and female glands; however, the intensity of the band was much higher in male than in female. In addition, 70, 60, 35 kDa bands, identified in male preputial gland, were absent in females. The presence of higher concentration of glandular proteins in the male preputial gland suggests that male rats may depend more on these glandular proteins for the maintenance of reproductive and dominance behaviours. The results further suggest that these glandular proteins (20 kDa) may act as a carrier for ligand binding.

Manual de procedimientos de electroforesis para proteínas y ADN / Procedures Manual for protein and DNA electrophoresis

El presente manual de procedimientos es la recopilación de protocolos actualizados por los investigadores de la División de Biología Molecular del Instituto Nacional de Salud. En este material se incluye, además de los procedimientos técnicos de la electroforesis, un anexo indicando el modo de preparación de soluciones de electroforesis, unidades y fórmulas básicas a ser aplicadas y algunas importantes medidas de bioseguridad que deben considerarse durante la manipulación de los reactivos. Es importante señalar que la electroforesis en asociación con otras técnicas tales como PCR, e hibridación, brinda una gran ayuda en el campo de la salud. Gracias a esta técnica es posible visualizar las bandas de ARN o ADN de patógenos, detectar cambios o mutaciones a nivel genético, visualizar una nueva proteína, entre otros

A lethal neurotoxic protein from Indian king cobra (Ophiophagus hannah) venom.

A lethal neurotoxin protein (Toxin CM36) was isolated and purified from the Indian King Cobra (Ophiophagus hannah) venom by CM-Sephadex ion exchange chromatography and HPLC. The purified toxin had a SDS-molecular weight of 15 +/- 0.5 kD. The UV absorption spectra of Toxin CM36 showed a peak at 280 nm and an Emax at 343.8 nm, when excited at 280 nm fluorescence. Toxin CM36 had an LD50 of 3.5 microg/20 g (i.v.) in male albino mice. It exhibited neurotoxicity and produced irreversible blockade of isolated chick biventer cervicis and rat phrenic nerve diaphragm. The neurotoxicity was found to be Ca2+ dependent. Toxin CM36 had no significant effect on isolated guineapig heart and auricle. It also had no effect on blood pressure of cat and rat but produced respiratory apnoea in rat and guineapig. Toxin CM36 lacked phospholipase activity.

Valoracion nutricional de 10 variedades de Quinua Chenopodium quinoa Willd del altiplano boliviano / Nutricional valuation of 10 varieties of Quinua Chenopodium quinoa Willd of the Bolivian plateau

El presente trabajo de investigación se desarrollo con el propósito de dar a conocer y difundir las características y propiedades nutritivas de 10 variedades de quinua (chenopodiurn qtunoa) propias de nuestra región (altiplano boliviano). La valoración comprendió el estudio de la naturaleza bioquímica el producto mediante evaluaciones de las propiedades químicas y nutritivas. Los resultados de este estudio muestran que estas, son mayores en rendimientos de proteínas (14 -18 porciento) en referencia a los reportados en la bibliografía (6) (13 porciento) como promedio. Se destaca que la Variedad REAL BLANCA presenta un 17 porciento y AJARA 18 porciento en proteínas, asimismo presentan los mas altos rendimientos respecto a la composición de sus macro nutrientes y micro nutrientes como Vitaminas como la tiamina 0.56mg/ lOOg en la REAL BLANCA y Oligoelementos como el Calcio, Magnesio, Manganeso en la variedad AJARA

Síntesis in vitro de la proteína de la envoltura del virus peruano de la fiebre amarilla / In vitro synthesis of the envelope protein of the peruvian yellow fever virus

Objetivo: Sintetizar la proteína recombinante de la envoltura (Er) del virus peruano de la Fiebre Amarilla (FA) utilizando técnicas moleculares. Materiales y métodos: El gen de la proteína de interés fue amplificado por transcripción reversa - reacción en cadena e la polimerasa (RT-PCR) y clonado en un vector plasmídico para ser analizado mediante secuenciamiento de ADN. El inserto de ADN fue subclonado en un vector de expresión para ser traducido a proteína. Se purificó la proteína mediante cromatografía de afinidad bajo condiciones denaturantes, siendo visualizada por electroforésis en SDS-PAGE.Resultados: Se sintetizó y purificó la pr E del virus de la FA. Presentó un peso de && kDa, y luego de tres horas de inducción a partir de 1x10 células (OD=0,5) se obtuvo 10mg/mL de la proteína a miniescala. Conclusión: El análisis de la secuencia de aminoácidos demostró que Er podría ser un buen candidato a ser evaluado serológicamente y luego usado como herramienta de diagnóstico específico para la FA

Isolation and characterization of a reserve protein from the seeds of Opuntia ficus-indica (Cactaceae)

We describe here the isolation and characterization of a major albumin from the seeds of Opuntia ficus-indica (Cactaceae). This protein has a molecular mass of 6.5 kDa and was isolated by a combination of gel filtration chromatography and reverse-phase HPLC. The amino acid composition of this protein was determined and it was shown to have similarities with the amino acid composition of several proteins from the 2S albumin storage protein family. The N-terminal amino acid sequence of this protein is Asp-Pro-Tyr-Trp-Glu-Gln-Arg