Avaliação do perfil metabólico de vacas da raça Holandesa durante o período de transição

O período de transição é um momento de grande desafio para vacas de aptidão leiteira, uma vez que, a maioria dos problemas metabólicos ocorre nesta fase podendo prejudicar toda a expectativa de produção durante a lactação, resultando em impacto econômico significativo para fazendas de produção de leite. Este trabalho teve como objetivo avaliar o perfil metabólico de vacas da raça Holandesa durante o período de transição. Doze vacas Holandesas foram avaliadas, três semanas pré-parto até três semanas pós-parto, em sistema free-stall, localizado em Inhaúma, Minas Gerais, no período de outubro a dezembro de 2012. Avaliou-se o perfil metabólico através da concentração sérica de ácidos graxos não esterificados (AGNE), beta hidroxibutirato (BHBA), colesterol (COLES), proteína total (PT), albumina (ALB), cálcio, fósforo, magnésio bem como a atividade sérica das enzimas aspartato transaminase (AST) e lactato desidrogenase (LDH). As concentrações séricas de AGNE e BHBA foram diferentes entre o pré-parto e pós-parto (p<0,05). Observou-se diminuição na concentração de COLES com a aproximação do parto com posterior aumento (p<0,05). As concentrações séricas dos minerais, PT e ALB não apresentaram diferenças (p>0,05) no período avaliado. A atividade enzimática de AST e LDH foram maiores no período pós-parto (p<0,05). A avaliação do perfil metabólico é uma importante ferramenta de monitoramento e, na situação estudada, demonstrou alterações do perfil energético das vacas entre os períodos pré e pós-parto, relacionadas provavelmente a diminuição da ingestão de alimentos. A luz dos resultados do perfil metabólico, o rebanho avaliado possui pequeno risco para a ocorrência de enfermidades no pós-parto relacionadas ao período de transição

Eletroforese de isoenzimas de plântulas na identificação de espécies de Brachiaria

Lotes de sementes de braquiária comercializados no Brasil vem apresentando contaminações com sementes de outras espécies pertencentes ao mesmo gênero. Deste modo, uma das espécies de braquiária atuaria como planta infestante da outra no agroecossistema e a erradicação da espécie infestante seria dificultada pela agressividade característica do gênero e pela falta de seletividade dos herbicidas disponíveis no mercado. Esses fatores ressaltam a importância da comercialização e utilização de lotes de sementes isento de sementes de outras espécies e a utilização de metodologias precisas de identificação das principais espécies de braquiária no controle de qualidade das empresas produtoras de sementes. Neste trabalho, buscou-se avaliar o potencial discriminante da técnica de eletroforese, utilizando quatro sistemas enzimáticos presentes em plântulas de quatro espécies do gênero Brachiaria, quer sejam B. brizantha cv. Marandu, B. decumbens cv. Basilisk, B. humidicola cv. comercial e B. plantaginea. Foram realizadas análises de eletroforese de isoenzimas testando-se 50 indivíduos de cada espécie por tratamento, utilizando-se coleóptilos de plântulas obtidas a partir de sementes germinadas a 30°C, no escuro. Para a eletroforese foi utilizado como meio suporte géis de poliacrilamida, nas concentrações de 7,0 e 7,5%. As isoenzimas Glutamato desidrogenase e Glucose-6-fosfato desidrogenase, embora eficientes na diferenciação entre B. plantaginea e B. humidicola e entre as sementes dessas espécies e as de B. brizantha ou B. decumbens, não se mostraram capazes de diferenciar as sementes destas duas últimas espécies. Entretanto, as izoenzimas α- e β-esterase possibilitaram uma nítida diferenciação das quatro espécies de Brachiaria estudadas.

Glyphosate: influência na bioatividade do solo e ação de minhocas sobre sua dissipação em terra agrícola

Com a difusão do uso de agrotóxicos, torna-se necessário aprofundar os conhecimentos sobre seu comportamento no ambiente, visando diminuir os riscos à biota e a possível contaminação de água, solo e alimentos. A influência da presença de minhocas da espécie Eisenia foetida sobre a dissipação do herbicida glyphosate, a bioacumulação do herbicida nestes vermes e a influência do herbicida sobre a bioatividade da microbiota endógena foram avaliadas em amostra de terra agrícola tratada com solução aquosa de 14C-glyphosate. Os estudos foram conduzidos em sistemas mantidos por dois ou quatro meses em amostras de terra tratadas com três diferentes concentrações de 14C-glyphosate, contendo ou não minhocas. Após esses períodos, amostras de terra e de minhocas foram submetidas à extração e combustão, para quantificação da radioatividade por espectrometria de cintilação em líquido (ECL). Paralelamente, a atividade da microbiota foi avaliada em relação à atividade da enzima desidrogenase. Os resultados mostraram que a presença de minhocas não alterou a dissipação de glyphosate na terra independentemente do período de contato, embora elas tenham bioacumulado resíduos do herbicida, numa relação diretamente proporcional ao tempo de contato com a terra tratada. O período de quatro meses favoreceu a formação de 14C-resíduos não-extraíveis ou ligados. Já a bioatividade não sofreu qualquer alteração, nem pela presença de minhocas nem dos tratamentos ou do tempo de exposição.

Evolução de CO2 e atividades enzimáticas em amostras de solo tratado com herbicidas

Os efeitos dos herbicidas bentazon, metolachlor, trifluralin, imazethapyr, imazethapyr+lactofen, haloxyfop-methyl, glyphosate e chlorimuron-ethyl, testados em duas concentrações (duas e dez vezes a dose média recomendada por hectare), sobre a atividade microbiana foram estudados em amostras de solo que nunca haviam recebido tratamento com pesticidas. Como bioindicadores, utilizou-se a respiração microbiana, quantificando a emissão de CO2 aos 2, 4, 8, 12, 16, 20 e 24 dias após incubação, a atividade da enzima desidrogenase e a hidrólise de diacetato de fluoresceína (FDA), aos 8 e 28 dias. Bentazon e a mistura de imazethapyr+lactofen na maior concentração e o haloxyfop-methyl nas duas concentrações apresentaram efeitos inibitórios na respiração edáfica, embora diferentes em época e duração do efeito. Nenhum dos tratamentos herbicidas afetou a hidrólise da FDA. A atividade da desidrogenase foi inibida, o que foi verificado em análise realizada aos oito dias,nas amostras de solo com alta concentração de bentazon e imazethapyr; no entanto, foi estimulada nos tratamentos com baixa concentração de metolachlor e imazethapyr e na maior concentração de glyphosate. A respiração basal e a atividade da desidrogenase mostraram maior sensibilidade na detecção de efeitos dos herbicidas sobre a microbiota do solo que as determinações da hidrólise de FDA. Apenas foi encontrada correlação significativa entre a atividade da desidrogenase e a respiração basal aos oito dias de incubação. Os resultados destacam a importância da consideração de múltiplos indicadores na avaliação dos efeitos de herbicidas na microbiota do solo.

Caracterização genética de populações naturais de araticunzeiro (Annona crassiflora Mart. - Annonaceae) no Estado de Goiás

No intuito de avaliar a magnitude e a distribuição da variabilidade genética existente em populações naturais de araticunzeiro, seis populações oriundas de duas regiões do Estado de Goiás foram amostradas (30 indivíduos cada) e analisadas para quatro sistemas enzimáticos: 6-Fosfogluconato Desidrogenase (6PGD), Fosfoglucomutase (PGM), Malato Desidrogenase (MDH) e Leucina Aminopeptidase (LAP). Dois locos diméricos foram encontrados para a enzima 6PGD, e um loco monomérico para os demais sistemas. Somente a enzima MDH apresentou monomorfismo em todas as populações, sendo que o número médio de alelos por loco polimórfico foi igual a dois. A heterozigosidade total média foi de 0,357, denotando a existência de elevada variabilidade genética na região para esta espécie. Cerca de 19% da variação genética total foram devidos a diferenças interpopulacionais, indicando uma elevada divergência genética entre as populações. A análise de variância das freqüências alélicas para os locos polimórficos no conjunto de populações evidenciou um elevado grau de estruturação genética em nível populacional, tendo-se observado coeficientes significativos para o parentesco entre indivíduos dentro de populações e para a endogamia total em nível individual. A avaliação da divergência genética entre as populações sugeriu a existência de um efeito da distribuição espacial sobre a magnitude da similaridade entre as mesmas. Os resultados sugerem que a espécie se reproduz preferencialmente por alogamia, em conformidade com achados de outros autores.

Superoxide: a two-edged sword

Superoxide (O2-) is the compound obtained when oxygen is reduced by one electron. For a molecule with an unpaired electron, O2- is surprisingly inert, its chief reaction being a dismutation in which it reacts with itself to form H2O2 and oxygen. The involvement of O2- in biological systems was first revealed by the discovery in 1969 of superoxide dismutase, an enzyme that catalyzes the dismutation of O2-. Since then it has been found that biological systems produce a bewildering variety of reactive oxidants, all but a few arising ultimately from O2-. These oxidants include O2- itself, H2O2 and alkyl peroxides, hydroxyl radical and other reactive oxidizing radicals, oxidized halogens and halamines, singlet oxygen, and peroxynitrite. These various oxidants are able to damage molecules in their environment, and are therefore very dangerous. They are thought to participate in the pathogenesis of a number of common diseases, including among others malignancy, by their ability to mutate the genome, and atherosclerosis, by their capacity for oxidizing lipoproteins. Their properties are put to good use, however, in host defense, where they serve as microbicidal and parasiticidal agents, and in biological signalling, where their liberation in small quantities results in redox-mediated changes in the functions of enzymes and other proteins

Horizontal projections of area 17 in Cebus monkeys: metric features, and modular and laminar distribution

Metric features and modular and laminar distributions of intrinsic projections of area 17 were studied in Cebus apella. Anterogradely and retrogradely labeled cell appendages were obtained using both saturated pellets and iontophoretic injections of biocytin into the operculum. Laminar and modular distributions of the labeled processes were analyzed using Nissl counterstaining, and/or cytochrome oxidase and/or NADPH-diaphorase histochemistry. We distinguished three labeled cell types: pyramidal, star pyramidal and stellate cells located in supragranular cortical layers (principally in layers IIIa, IIIb a, IIIb ß and IIIc). Three distinct axon terminal morphologies were found, i.e., Ia, Ib and II located in granular and supragranular layers. Both complete and partial segregation of group I axon terminals relative to the limits of the blobs of V1 were found. The results are compatible with recent evidence of incomplete segregation of visual information flow in V1 of Old and New World primates

Erythrocyte glucose-6-phosphate dehydrogenase activity assay and affinity for its substrate under "physiological" conditions

Glucose-6-phosphate dehydrogenase (G6PD) activity and the affinity for its substrate glucose-6-phosphate were investigated under conditions similar to the physiological environment in terms of ionic strength (I: 0.188), cation concentration, pH 7.34, and temperature (37oC). A 12.4, 10.4 and 21.4% decrease was observed in G6PD B, G6PD A+ and G6PD A- activities, respectively. A Km increase of 95.1, 94.4 and 95.4% was observed in G6PD B, G6PD A+ and G6PD A-, respectively, leading to a marked decrease in affinity. In conclusion, the observation of the reduced activity and affinity for its natural substrate reflects the actual pentose pathway rate. It also suggests a much lower NADPH generation, which is crucial mostly in G6PD-deficient individuals, whose NADPH availability is poor.

Effect of the injection of an extract of Ascaris suum on macrophage activation during the early phase of Mycobacterium bovis BCG infection in C57Bl/6 mice

Injection of an Ascaris suum extract (Asc) affects both the humoral and cellular immune responses to unrelated antigens when it is co-administered with these antigens. In the present study we evaluated the effect of Asc on macrophage activation in the early phase of Mycobacterium bovis BCG (Pasteur strain TMCC 1173) infection in C57Bl/6 mice. C57Bl/6 mice were injected intraperitoneally (ip) with 0.1 mg BCG (BCG group) or BCG plus 1 mg Asc (BCG + Asc group). The peritoneal exudates were obtained at 2, 7 and 14 days after infection. The numbers of IFN-g-secreting cells were assessed by the ELISPOT assay. Nitric oxide (NO) production was measured by the Griess method and by the evaluation of NADPH diaphorase activity in the peritoneal exudates. The administration of Asc extract increased NADPH diaphorase activity (2 days: control = 0, BCG = 7%, BCG + Asc = 13%, and Asc = 4%; 7 days: control = 4, BCG = 13%, BCG + Asc = 21%, and Asc = 4.5%) and TNF-a levels (mean ± SD; 2 days: control = 0, BCG = 169 ± 13, BCG + Asc = 202 ± 37, and Asc = 0; 7 days: control = 0, BCG = 545 ± 15.5, BCG + Asc = 2206 ± 160.6, and Asc = 126 ± 26; 14 days: control = 10 ± 1.45, BCG = 9 ± 1.15, BCG + Asc = 126 ± 18, and Asc = 880 ± 47.67 pg/ml) in the early phase of BCG infection. Low levels of NO production were detected at 2 and 7 days after BCG infection, increasing at 14 days (mean ± SD; 2 days: control = 0, BCG = 3.7 ± 1.59, BCG + Asc = 0.82 ± 0.005, Asc = 0.48 ± 0.33; 7 days: control = 0, BCG = 2.78 ± 1.54, BCG + Asc = 3.07 ± 1.05, Asc = 0; 14 days: control = 0, BCG = 9.05 ± 0.53, BCG + Asc = 9.61 ± 0.81, Asc = 10.5 ± 0.2 (2 x 106) cells/ml). Furthermore, we also observed that Asc co-injection induced a decrease of BCG-colony-forming units (CFU) in the spleens of BCG-infected mice during the first week of infection (mean ± SD; 2 days: BCG = 1.13 ± 0.07 and BCG + Asc = 0.798 ± 0.305; 7 days: BCG = 1.375 ± 0.194 and BCG + Asc = 0.548 ± 0.0226; 14 days: BCG = 0.473 ± 0.184 and BCG + Asc = 0.675 ± 0.065 (x 102) CFU). The present data suggest that Asc induces the enhancement of the immune response in the early phase of BCG infection.

Inhibition of rat microsomal lipid peroxidation by the oral administration of D002

The effect of D002, a defined mixture of higher primary alcohols purified from bee wax, on in vivo and in vitro lipid peroxidation was studied. The extent of lipid peroxidation was measured on the basis of the levels of thiobarbituric acid reactive substances (TBARS). When D002 (5-100 mg/kg body weight) was administered orally to rats for two weeks, a partial inhibition of the in vitro enzymatic and non-enzymatic lipid peroxidation was observed in liver and brain microsomes. Maximal protection (46%) occurred at a dose of 25 mg/kg. D002 behaved differently depending on both the presence of NADPH and the integrity of liver microsomes, which suggests that under conditions where microsomal metabolism was favored the protective effect of D002 was increased. D002 (25 mg/kg) also completely inhibited carbon tetrachloride- and toluene-induced in vivo lipid peroxidation in liver and brain. Also, D002 significantly lowered in a dose-dependent manner the basal level of TBARS in liver (19-40%) and brain (28-44%) microsomes. We conclude that the oral administration of D002 (5, 25 and 100 mg/kg) for two weeks protected rat liver and brain microsomes against microsomal lipid peroxidation in vitro and in vivo. Thus, D002 could be useful as a dietary natural antioxidant supplement. More studies are required before these data can be extrapolated to the recommendation for the use of D002 as a dietary antioxidant supplement for humans.

Glutamine and glutamate as vital metabolites

Glucose is widely accepted as the primary nutrient for the maintenance and promotion of cell function. This metabolite leads to production of ATP, NADPH and precursors for the synthesis of macromolecules such as nucleic acids and phospholipids. We propose that, in addition to glucose, the 5-carbon amino acids glutamine and glutamate should be considered to be equally important for maintenance and promotion of cell function. The functions of glutamine/glutamate are many, i.e., they are substrates for protein synthesis, anabolic precursors for muscle growth, they regulate acid-base balance in the kidney, they are substrates for ureagenesis in the liver and for hepatic and renal gluconeogenesis, they act as an oxidative fuel for the intestine and cells of the immune system, provide inter-organ nitrogen transport, and act as precursors of neurotransmitter synthesis, of nucleotide and nucleic acid synthesis and of glutathione production. Many of these functions are interrelated with glucose metabolism. The specialized aspects of glutamine/glutamate metabolism of different glutamine-utilizing cells are discussed in the context of glucose requirements and cell function.

A method for multiple sequential analyses of macrophage functions using a small single cell sample

Microbial pathogens such as bacillus Calmette-Guérin (BCG) induce the activation of macrophages. Activated macrophages can be characterized by the increased production of reactive oxygen and nitrogen metabolites, generated via NADPH oxidase and inducible nitric oxide synthase, respectively, and by the increased expression of major histocompatibility complex class II molecules (MHC II). Multiple microassays have been developed to measure these parameters. Usually each assay requires 2-5 x 10(5) cells per well. In some experimental conditions the number of cells is the limiting factor for the phenotypic characterization of macrophages. Here we describe a method whereby this limitation can be circumvented. Using a single 96-well microassay and a very small number of peritoneal cells obtained from C3H/HePas mice, containing as little as <=2 x 10(5) macrophages per well, we determined sequentially the oxidative burst (H2O2), nitric oxide production and MHC II (IAk) expression of BCG-activated macrophages. More specifically, with 100 µl of cell suspension it was possible to quantify H2O2 release and nitric oxide production after 1 and 48 h, respectively, and IAk expression after 48 h of cell culture. In addition, this microassay is easy to perform, highly reproducible and more economical.

Arachidonic acid triggers an oxidative burst in leukocytes

The change in cellular reducing potential, most likely reflecting an oxidative burst, was investigated in arachidonic acid- (AA) stimulated leukocytes. The cells studied included the human leukemia cell lines HL-60 (undifferentiated and differentiated into macrophage-like and polymorphonuclear-like cells), Jurkat and Raji, and thymocytes and macrophages from rat primary cultures. The oxidative burst was assessed by nitroblue tetrazolium reduction. AA increased the oxidative burst until an optimum AA concentration was reached and the burst decreased thereafter. In the leukemia cell lines, optimum concentration ranged from 200 to 400 µM (up to 16-fold), whereas in rat cells it varied from 10 to 20 µM. Initial rates of superoxide generation were high, decreasing steadily and ceasing about 2 h post-treatment. The continuous presence of AA was not needed to stimulate superoxide generation. It seems that the NADPH oxidase system participates in AA-stimulated superoxide production in these cells since the oxidative burst was stimulated by NADPH and inhibited by N-ethylmaleimide, diphenyleneiodonium and superoxide dismutase. Some of the effects of AA on the oxidative burst may be due to its detergent action. There apparently was no contribution of other superoxide-generating systems such as xanthine-xanthine oxidase, cytochromes P-450 and mitochondrial electron transport chain, as assessed by the use of inhibitors. Eicosanoids and nitric oxide also do not seem to interfere with the AA-stimulated oxidative burst since there was no systematic effect of cyclooxygenase, lipoxygenase or nitric oxide synthase inhibitors, but lipid peroxides may play a role, as indicated by the inhibition of nitroblue tetrazolium reduction promoted by tocopherol.

The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease

Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by a defective oxidative burst of phagocytes and subsequent impairment of their microbicidal activity. Mutations in one of the NADPH-oxidase components affect gene expression or function of this system, leading to the phenotype of CGD. Defects in gp91-phox lead to X-linked CGD, responsible for approximately 70% of CGD cases. Investigation of the highly heterogeneous genotype of CGD patients includes mutation analysis, Northern blot or Western blot assays according to the particular case. The aim of the present study was to use reverse transcription (RT)-PCR for the analysis of molecular defects responsible for X-linked CGD in eight Brazilian patients and to assess its potential for broader application to molecular screening in CGD. Total RNA was prepared from Epstein B virus-transformed B-lymphocytes and reverse transcribed using random hexamers. The resulting cDNA was PCR-amplified by specific and overlapping pairs of primers designed to amplify three regions of the gp91-phox gene: exons 1-5, 3-9, and 7-13. This strategy detected defective gp91-phox expression in seven patients. The RT-PCR results matched clinical history, biochemical data (nitroblue tetrazolium or superoxide release assay) and available mutation analysis in four cases. In three additional cases, RT-PCR results matched clinical history and biochemical data. In another case, RT-PCR was normal despite a clinical history compatible with CGD and defective respiratory burst. We conclude that this new application of RT-PCR analysis - a simple, economical and rapid method - was appropriate for screening molecular defects in 7 of 8 X-linked CGD patients.

Expression of neuronal nitric oxide synthase in the developing superficial layers of the rat superior colliculus

We investigated the level of expression of neuronal nitric oxide synthase (nNOS) in the retinorecipient layers of the rat superior colliculus during early postnatal development. Male and female Lister rats ranging in age between the day of birth (P0) and the fourth postnatal week were used in the present study. Two biochemical methods were used, i.e., in vitro measurement of NOS specific activity by the conversion of [³H]-arginine to [³H]-citrulline, and analysis of Western blotting immunoreactive bands from superior colliculus homogenates. As revealed by Western blotting, very weak immunoreactive bands were observed as early as P0-2, and their intensity increased progressively at least until P21. The analysis of specific activity of NOS showed similar results. There was a progressive increase in enzymatic activity until near the end of the second postnatal week, and a nonsignificant tendency to an increase until the end of the third week was also observed. Thus, these results indicated an increase in the amount of nNOS during the first weeks after birth. Our results confirm and extend previous reports using histochemistry for NADPH-diaphorase and immunocytochemistry for nNOS, which showed a progressive increase in the number of stained cells in the superficial layers during the first two postnatal weeks, reaching an adult pattern at the end of the third week. Furthermore, our results suggested that nNOS is present in an active form in the rat superior colliculus during the period of refinement of the retinocollicular pathway.