Effects of 60Co gamma radiation on crotamine

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide (pI 10.3) from Crotalus durissus terrificus venom composed of 42 amino acid residues. It induces skeletal muscle spasms leading to a spastic paralysis of hind limbs in mice. The objective of the present study was to carry out a biochemical study and a toxic activity assay on native and irradiated crotamine. Crotamine was purified from C.d. terrificus venom by Sephadex G-100 gel filtration followed by ion-exchange chromatography, and irradiated at 2 mg/ml in 0.15 M NaCl with 2.0 kGy gamma radiation emitted by a 60Co source. The native and irradiated toxins were evaluated in terms of structure and toxic activity (LD50). Irradiation did not change the protein concentration, the electrophoretic profile or the primary structure of the protein although differences were shown by spectroscopic techniques. Gamma radiation reduced crotamine toxicity by 48.3%, but did not eliminate it.

Study of the proteins in the defatted flour and protein concentrate of baru nuts (Dipteryx alata Vog)

O baru (Dipteryx alata Vog.) é uma leguminosa abundante no Cerrado brasileiro, cuja castanha pode ser explorada através do uso sustentável para o aproveitamento das frações proteicas e lipídicas. Este trabalho teve como objetivo estudar as proteínas desta castanha, presentes na farinha desengordurada e no concentrado proteico, quanto as suas propriedades funcionais, ao perfil das frações proteicas e à digestibilidade in vitro. A farinha desengordurada com hexano foi submetida à extração no pH de maior solubilidade das proteínas, obtendo-se o concentrado proteico. O perfil eletroforético das frações proteicas foi avaliado em gel de SDS-PAGE. As propriedades funcionais indicaram a possibilidade de emprego em diversos alimentos, assim como a soja, conferindo capacidade de absorção de água, capacidade de absorção de óleo, propriedades emulsificantes e espumabilidade. As globulinas, seguidas das albuminas, são as frações majoritárias da farinha e do concentrado proteico, respectivamente. A digestibilidade foi superior no concentrado em relação à farinha desengordurada.

Quadro seroproteico como auxílio diagnóstico na anemia hemolítica imunomediada em cães

No presente protocolo experimental, determinaram-se os proteinogramas séricos, por intermédio da eletroforese em gel de poliacrilamida contendo duodecil sulfato de sódio (SDS-PAGE), de 120 cães com raças e idades variadas e atendidos junto ao Hospital Veterinário Governador Laudo Natel da FCAV/Unesp, com o objetivo principal de comparar diferentes frações seroproteicas em estados anêmicos regenerativos, arregenerativos, imunomediados primários e secundários. Os referidos animais foram distribuídos em cinco grupos experimentais: grupo 1: 20 cães de controle; grupo 2: 28 cães com anemia regenerativa não imune; grupo 3: 27 cães com anemia arregenerativa não imune; grupo 4: 10 cães com anemia hemolítica imunomediada primária; grupo 5: 35 cães com anemia hemolítica imunomediada secundária. A técnica SDS-PAGE permitiu o fracionamento de 24 proteínas, cujos pesos moleculares (PM) variaram de 18.000 a 165.000 daltons (Da). Os cães com AHIM primária e secundária apresentaram 24 frações proteicas em seus traçados eletroforéticos, enquanto que cães de controle (1) e portadores de anemia regenerativa (2) e arregenerativa (3) de natureza não imune apresentaram 23 frações de proteínas, cuja proteína de peso molecular 68.000Da não foi encontrada. Dessa forma, 23 frações proteicas foram detectadas e revelaram-se comuns aos proteinogramas dos cães de controle e daqueles dos quatro grupos experimentais. Destas, identificaram-se nominalmente 11 frações proteicas, e as demais foram estudadas com base nos seus respectivos pesos moleculares. em relação aos cães de controle, os anêmicos (grupos 2, 3, 4 e 5) apresentaram maiores concentrações de transferrina sérica e entre estes os animais portadores da AHIM primária. Todos os cães anêmicos apresentaram teores séricos de haptoglobina e fosforilase significativamente maiores que os controles, enquanto que a concentração sérica de ceruloplasmina foi significativamente maior nestes. Tais achados analisados em conjunto agregam informações adicionais úteis à elucidação das AHIMs em cães.

Detecção e conscientização de portadores de hemoglobinopatias nas regiões de São José do Rio Preto e Presidente Prudente, SP (Brasil)

Foram analisadas 7.657 amostras de sangue provenientes de 48 cidades das regiões de São José do Rio Preto e de Presidente Prudente, com o objetivo de detectar e conscientizar os portadores de hemoglobinas anormais. As análises efetuadas mostraram que 3,47% tinham hemoglobinas anormais, sendo 2,26% portadores de variantes moleculares (Hbs, AS, AC, SS, SC, AJ, AB2) e 1,21% de talasse-mias (alfa e beta). Os resultados obtidos evidenciaram que estudos semelhantes, além de propiciar o melhor conhecimento das causas genéticas, bioquímicas e hematológicas dessas alterações hereditárias, oferecem também a oportunidade de estimar a importância que essas patologias representam para a saúde pública do nosso país. A ação preventiva foi estabelecida por meio de reuniões de esclarecimentos médicos e biológicos aos portadores de hemoglobinas anormais.

Tipos de miosinas de linhagens de frangos de corte criados em sistemas de confinamento e semiconfinamento

O objetivo neste trabalho foi avaliar o peso vivo, o peso de pernas, os aspectos morfológicos das fibras musculares do músculo flexor longo do hálux e o perfil eletroforético das miosinas de cadeia pesada de quatro linhagens de frangos de corte criados nos sistemas de confinamento e semiconfinamento. Foram utilizados 1.440 pintos distribuídos em delineamento inteiramente casualisado em esquema fatorial 4 × 2, composto de quatro linhagens (Ross 305, Máster Gris, Label Rouge e Vermelhão Pesado) e dois sistemas de criação (confinamento e semiconfinamento), cada combinação avaliada com quatro repetições. Aos 28 e 84 dias de idade, foram abatidas quatro aves por tratamento, totalizando 64 aves. A eletroforese identificou a presença das três isoformas de miosinas, tipo MyHC-I, MyHC-IIa e MyHC-IIb, no músculo flexor longo do hálux dos frangos de corte. Com aumento da idade, a isoforma de miosina MyHC-II aumenta, enquanto a MyHC-I diminui. Somente aos 84 dias de idade, a expressão das isoformas de miosina do tipo MyHC-II foram influenciadas pela linhagem, confirmando o reflexo da seleção na linhagem Ross no músculo mais glicolítico.A linhagem Ross apresenta maior peso vivo, peso de perna, peso e área do músculo flexor longo do hálux em comparação às linhagens tipo caipira.

Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique

Background: Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. Objective: the objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. Methods: CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. Results: Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. Conclusion: the microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations

Investigation of the systems silica and silica containing chromium in alcohol medium

The purpose of this work is to obtain micrometer sized spherical particles of silica and silica-chromium from sodium silicate. Spherical particles were prepared by sol-gel method from hydrolysis to polycondensation of aqueous sodium silicate in alcohol medium. Chromium was added to the system for some samples. Compositions and morphologies were achieved by changing the precipitation agent. X-ray diffractometry, electrophoretic mobility, infrared spectroscopy and scanning electron microscopies were carried out on these particles to identify phases, determine particle mobility, morphology, particle sizes, shapes and order at short distance. Non-crystalline silica particles with spherical shapes and micrometric size were obtained. The surface potentials of the silica particles differed from that of the silica-chromium particles. (C) 1999 Elsevier B.V. B.V. All rights reserved.

TEMPERATURE-SENSITIVE FLOCCULATION DURING GROWTH OF SACCHAROMYCES CEREVISIAE

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Allergenic extracts from Metarhizium anisopliae: Obtainment and characterization

Metarhizium anisopliae is used as a biopesticide for insects that damage agricultural plantations like sugar cane and forage plants. In a previous study the sensitization to this fungus of asthmatic patients coming from sugar cane areas was showed. The aims of this work were: to compare crude extracts obtained with Tris-HCl and Coca liquid from several growth phases of M. anisopliae concerning the total content of proteins and their electrophoretic analysis profile; to evaluate in vivo allergic sensitization in Balb/c mice and allergic patients from a sugar cane area, and to characterize the allergenic fractions in the sera of patients positive for the prick test by means of Western-blotting. The extract obtained with Coca liquid on the 16(th) day was the one that presented the greatest number of proteic fractions, including all those present in the other extracts. Twelve fractions were verified in this extract with approximate molecular weights from 94 to 14 kDa. The allergenicity of the extract obtained on the 16(th) day was proven by the production of IgE antibodies in Balb/c mice, with titres of 200. Prick tests carried out with the extract of the 16(th) day in 79 atopic individuals (from sugar cane area), 35 atopic individuals (from urban area) and 11 non- atopic individuals showed respective positivity of 29%, 9% and 0%. The allergenic characterization in vitro was performed by means of Western blotting, and the fractions that reacted with the positive individuals' sera were those of approximate molecular weights of 67 kDa (95%); 20 kDa (55%); 94 kDa (36%); 34 and 36 kDa (23%); 43 and 48 kDa (14%): 16 kDa (9%) and 54kDa (5%). It was concluded that the crude allergenic extract, obtained with Coca liquid from the 16(th) day growth of Metarhizium anisopliae, contains allergenic fractions and can be used in diagnostic screening tests.

Relação Cálcio/Fósforo Disponível em Rações para Tilápia do Nilo (Oreochromis niloticus)

The objective of this work was to evaluate the effect of Ca: available P (P-a) ratio in the diets on performance and bone mineral depositions of juvenile Nile tilapia (2.5 +/- 0.5g). Seven isoproteic and isoenergetic diets (28% CP and 3100 Kcal of DE/Kg of diet), with similar crude fiber and sulfur aminoacids content, were used. The diets had 0.50 and 0.25; 0.50 and 0.50; 0.50 and 0.75; 0.80 and 0.40; 0.80 and 0.80; and 0.80 and 1.20% of Ca and P-a which corresponded to Ca/P-a ratios of 2:1;1:1 and 1:1.5, respectively. An additional control diet, with low levels of Ca and Pa (0.02 and 0.12%, respectively) was used. A completely randomized design with a 2'3 factorial arrangement (Ca level and Ca:P-a ratio) on treatments, plus the additional control diet and three replications was used. Five fishes per aquaria were randomly allotted to 21 fiberglass aquaria (80L). The temperature of the water was maintained close to 26degreesC, and the oxygen, pH and ammonia levels of the water were monitored. The results showed that it was necessary a minimum dietary level of 0.25% of P-a for a satisfactory bone mineral depositions, and that the best performance results were achieved using diets with Ca: P-a ratios between 1:1 and 1:1.5.

Genomic organization of the Neurospora crassa gsn gene: possible involvement of the STRE and HSE elements in the modulation of transcription during heat shock

Glycogen synthase, an enzyme involved in glycogen biosynthesis, is regulated by phosphorylation and by the allosteric ligand glucose-6-phosphate (G6P). In addition, enzyme levels can be regulated by changes in gene expression. We recently cloned a cDNA for glycogen synthase (gsn) from Neurospora crassa, and showed that gsn transcription decreased when cells were exposed to heat shock (shifted from 30degreesC to 45degreesC). In order to understand the mechanisms that control gsn expression, we isolated the gene, including its 5' and 3' flanking regions, from the genome of N. crassa. An ORF of approximately 2.4 kb was identified, which is interrupted by four small introns (II-V). Intron I (482 bp) is located in the 5'UTR region. Three putative Transcription Initiation Sites (TISs) were mapped, one of which lies downstream of a canonical TATA-box sequence (5'-TGTATAAA-3'). Analysis of the 5'-flanking region revealed the presence of putative transcription factor-binding sites, including Heat Shock Elements (HSEs) and STress Responsive Elements (STREs). The possible involvement of these motifs in the negative regulation of gsn transcription was investigated using Electrophoretic Mobility Shift Assays (EMSA) with nuclear extracts of N. crassa mycelium obtained before and after heat shock, and DNA fragments encompassing HSE and STRE elements from the 5'-flanking region. While elements within the promoter region are involved in transcription under heat shock, elements in the 5'UTR intron may participate in transcription during vegetative growth. The results thus suggest that N. crassa possesses trans-acting elements that interact with the 5'-flanking region to regulate gsn transcription during heat shock and vegetative growth.

The venom gland of queens of Apis mellifera (Hymenoptera, Apidae): morphology and secretory cycle

The venom gland of queens of Apis mellifera was examined through light and transmission electron microscopy and subjected to electrophoretic analyses. Virgin queens exhibited prismatic secretory cells containing large amounts of rough endoplasmic reticulum with dilated cisternae, open secretory spaces, numerous vacuoles and granules scattered in the cytoplasm, and spherical nuclei with numerous nucleoli. The secretion produced was non-refringent under polarized light and the electrophoretic analysis of glandular extracts revealed five main protein bands. In mated queens, the venom gland exhibited a high degree of degeneration. Its secretion was refringent under polarized light and one of the main bands was absent in the electrophoretic pattern obtained. The morphological aspects observed are in agreement with the function of this gland in queens, given that virgin queens use venom in battles for the dominance of the colony, a situation that occurs as soon as they emerge, while fertilized queens rarely use venom. (c) 2006 Elsevier Ltd. All rights reserved.

Identification, content, and distribution of type VI collagen in bovine tendons

Tendon composition changes according to differentiation, mechanical load, and aging. In this study, we attempted to identify, localize, and quantify type VI collagen in bovine tendons. Type VI collagen was identified by the electrophoretic behavior of the alpha chains and Western blotting, and by rotary shadowing. Type VI collagen was extracted from powdered tendon with three sequential 24-h extractions with 4 M guanidine-HCl. The amount of type VI collagen was determined by enzyme-linked immunosorbent assay for purely tensional areas and for the compressive fibrocartilage regions of the deep flexor tendon of the digits, for the corresponding fetal and calf tendons, and for the extensor digital tendon. The distal fibrocartilaginous region of the adult tendon was richer in type VI collagen than the tensional area, reaching as much as 3.3 mg/g (0.33%) of the wet weight. Calf tendons showed an accumulation of type VI at the fibrocartilage site. Immunocytochemistry demonstrated that type VI collagen was evenly distributed in the tensional areas of tendons but was highly concentrated around the fibrochondrocytes in the fibrocartilages. The results demonstrate that tendons are variable with regard to the presence and distribution of type VI collagen. The early accumulation of type VI collagen in the region of calf tendon that will become fibrocartilage in the adult suggests that it is a good marker of fibrocartilage differentiation. Furthermore, the distribution of type VI collagen in tendon fibrocartilage indicates that it organizes the pericellular environment and may represent a survival factor for these cells.

Preparation of ceramic membranes from surface modified tin oxide nanoparticles

The preparation of crack-free SnO2 supported membranes requires the development of new strategies of synthesis capable to allow controlled changes of surface chemistry and to improve the processability of supported layers. In this way, the controlled modification of the SnO2 nanoparticle surface by adding capping molecules like Tiron(R) ((OH)(2)C6H2(SO3Na)(2)) during the sol-gel process was studied, aiming to obtain high performance membranes. Colloidal suspensions were prepared by hydrolyzing SnCl4.5H(2)O aqueous solution with NH4OH in presence of Tiron(R). The effect of the amount of Tiro(R) (from I to 20 wt.%) on the structural features of nanoparticles, powder redispersability and particle-solution interface properties was investigated by X-ray powder diffraction (XRPD), extended X-ray absorption fine structure (EXAFS), quasi-elastic light scattering and electrophoretic mobility measurements. XRPD and EXAFS results showed that the addition of Tiron(R) up to 20 wt.% to colloidal suspensions does not affect the crystallite size of SnO2 primary particles, determined around 2-3 nm. This value is comparable to the hydrodynamic size measured after redispersion of powder prepared with amount of Tiro(R) higher than 7.5 wt.%, indicating the absence of condensation reactions between primary particles after the initial precipitation step. As a consequence the powder with amount of Tiron(R) > 7.5 wt.%, can be fully redispersed in aqueous solution at pH greater than or equal to I I until a nanoparticle concentration of 6 vol.%. The electrophoresis measurements showed a decrease of the isoelectric point by increasing the amount of grafted Tiron(R) at the SnO2 nanoparticle surface, resulting in negatively charged particle-solution interface in all the studied pH range (2-11). These features govern the gelation process favoring the preparation of crack-free SnO2 supported membranes. The control exercised by Tiron(R) modifying agent in the aggregation process allows the fine-tuning of the porosity, from 0.124 to 0.065 cm(3) g(-1), and mean pore size, from 6.4 to 1.9 nm, as the amount of grafted molecules increases from 0 to 10 wt.%. In consequence, the membrane cut-off determined by filtration of polyethylene glycol standard solutions can be screened from 1500 to 3500 g mol(-1). (C) 2002 Elsevier B.V. B.V. All rights reserved.

Determination of olive oil acidity by CE

In this work, a CE method for the determination of olive oil acidity was proposed. The method was based on an ethanolic extraction (at 60 degrees C) of the oil long-chain free fatty acids (LC-FFAs) components followed by CE determination in pH 6.86 phosphate buffer at 15 mmol/L concentration containing 4 mmol/L sodium dodecylbenzenesulfonate (SDBS), 10 mmol/L polyoxyethylene 23 lauryl ether (Brij 35((R))), 2% v/v 1-octanol and 45% v/v ACN under indirect UV detection at 224 nm. Although this electrolyte promoted baseline separation of myristic acid (C14:0) (internal standard (IS)) and olive oil major components (palmitic acid (C16:0), oleic acid (C18:1c) and linoleic acid (C18:2cc)) in less than 8 min, after a few injections, the electropherogram profiles were severely altered (peak broadening, migration time shifts, etc.) and the current increased substantially. An adsorption study was conducted revealing that the dissolution of the capillary external polyimide coating during the electrophoretic run caused the detrimental effect. After removal of the capillary tip coating, ten consecutive injections could be performed without any disturbances and this simple procedure was, therefore, implemented during quantitative purposes. The reliability of the proposed method was further investigated by the determination of acidity of an extra virgin olive oil sample in comparison to the established methodology (AOCS method Ca 5a40, alkaline volumetric titration (AVT)). No statistical differences were found within 95% confidence level. A % acidity of 0.39 +/- 0.02 was found for the olive oil sample under consideration.