Preparation and evaluation of atrazine immunoconjugate

In order to study the affinity reaction between the anti-atrazine antibody and atrazine, an enzyme was incorporated, as a marker, to an atrazine carboxylic derivative. The hapten and conjugate were synthesized and characterized by MS, IR and NMR. The interaction between monoclonal antibodies and hapten-HRP conjugate was investigated by enzyme linked immunosorbent assay (ELISA).

Estudo do comportamento eletroquímico da enzima peroxidase na presença de peróxido de hidrogênio e ácido 5-aminossalicílico

O comportamento eletroquímico da enzima peroxidase (HRP) foi estudado utilizando o peróxido de hidrogênio como substrato enzimático e o ácido 5-aminossalicílico (5-ASA) como mediador de elétrons sobre eletrodo de grafite. Diversos parâmetros foram otimizados, tais como, o potencial aplicado à técnica amperométrica fixado em -0,125V, a solução tampão fosfato-citrato 0,1 mol L-1 pH 5,0 como eletrólito suporte e a proporção entre o 5-ASA e H2O2 em 1:7, entre outros. Foi observada a catálise da reação de oxidação do peróxido de hidrogênio na presença da enzima HRP e do mediador 5-ASA. O produto dessa oxidação foi reduzido na superfície do eletrodo, evidenciando um significativo aumento na intensidade da corrente catódica.

Biossíntese e recentes avanços na produção de celulose bacteriana

O presente trabalho discute os recentes avanços na biossíntese e na produção de celulose bacteriana (CB) pela gram-negativa, aeróbia e aceto-ácida Gluconacetobacter. xylinus. A CB se difere de seu par vegetal, principalmente devido ao seu caráter de fibras nanométricas, contra o caráter micrométrico da vegetal, são extruídas através da parede celular de G. xylinus, com isso sua estrutura macroscópica é mecanicamente e fisicamente mais resistente, abrindo grandes oportunidades de aplicações tecnológicas e biológicas, muito além das obtidas pela celulose vegetal. O desafio atual está no aumento da produção de CB, que se debruça num maior entendimento de sua biossíntese para que seja possível uma posterior manipulação genético-bioquímica oriundas do recente avanço na biologia molecular e nos bioprocessos. São relacionados trabalhos utilizando a CB como base para produção de compósitos como também o que a está sendo feito de mais atual com este material biológico.

Characterization of Xanthomonas axonopodis pv. phaseoli isolates

A simple, quick and easy protocol was standardized for extraction of total DNA of the bacteria Xanthomonas axonopodis pv. phaseoli. The DNA obtained by this method had high quality and the quantity was enough for the Random Amplified Polymorphic DNA (RAPD) reactions with random primers, and Polymerase Chain Reaction (PCR) with primers of the hypersensitivity and pathogenicity gene (hrp). The DNA obtained was free of contamination by proteins or carbohydrates. The ratio 260nm/380nm of the DNA extracted ranged from 1.7 to 1.8. The hrp gene cluster is required by bacterial plant pathogen to produce symptoms on susceptible hosts and hypersensitive reaction on resistant hosts. This gene has been found in different bacteria as well as in Xanthomonas campestris pv. vesicatoria (9). The primers RST21 and RST22 (9) were used to amplify the hrp gene of nine different isolates of Xanthomonas axonopodis pv. phaseoli from Botucatu, São Paulo State, Brazil, and one isolate, "Davis". PCR amplified products were obtained in all isolates pathogenic to beans.

Desenvolvimento do feijoeiro sob o uso de biofertilizante e adubação mineral

A produção intensiva de alimentos exige manejo adequado do solo para garantir a produtividade e a sustentabilidade ambiental. Uma das alternativas é a utilização de resíduos orgânicos no desenvolvimento das culturas, diminuindo a dependência de adubos minerais. Com o objetivo de avaliar o desenvolvimento da cultura de feijão (Phaseolus vulgaris L.), utilizando biofertilizante e adubação mineral, conduziu-se o experimento com seis tratamentos dispostos ao acaso, em esquema fatorial, em quatro blocos, com parcelas de 8,0 x 5,0 m. Os tratamentos sob solo cultivado com a cultura de feijão caracterizaram-se como: com e sem biofertilizante (CB e SB, respectivamente) e para a adubação mineral foram utilizadas a dose recomendada no plantio, ½ dose de adubação e sem adubação mineral (AM, 1/2AM, SAM). Adotaram-se práticas culturais convencionais para o preparo inicial do solo, e em seguida foi efetuada a aplicação de biofertilizante de origem bovina na dosagem de 100 m³ ha-1, com antecedência de três meses da semeadura. Foram avaliados os parâmetros massa da matéria seca acumulada na parte aérea da planta, área foliar e produtividade da cultura. Os resultados mostraram semelhanças entre as características analisadas, obtendo-se melhor desenvolvimento à cultura que recebeu biofertilizante.

Seedlings of Acrocomia aculeata in diferent substrates and protected environments

The seedling production stage is the key to achieve uniformity in tree breeding stage. This study evaluated "bocaiúva" (Acrocomia aculeata) seedling formation, with pre-germinated seeds in different substrates and protected environments, in the University of Mato Grosso do Sul State, Aquidauana, MS. As substrates, we used 100% cattle manure (M), 100% cassava branches (CB), 100% vermiculite (V), 50% cattle manure + 50% cassava branches, 50 % cattle manure + 50% vermiculite, 50% cassava branches + 50% vermiculite and ⅓ cattle manure + ⅓ cassava branches + ⅓ vermiculite. These substrates were tested in a greenhouse covered with 150 µm low density polyethylene (LDPE) film under thermo-reflective screen with 50% shading under film; black screen with 50% shading on the sides; black monofilament screen with 50% shading set on roof and sides; and aluminized thermo- reflective screen with 50% shading set on roof and sides. The completely randomized experimental design with 5 replications of 5 plants each was adopted. Initially, data were submitted to analysis of substrate individual variance in each growing environment, then performing the waste mean square evaluation and their environment joint analysis for comparison. The best growing environment is the thermo-reflective screen compared to LDPE greenhouse and black screen set. All substrates containing manure are recommended for bocaiúva seedlings formation. The pure cassava branch is not indicated for seedling, even using chemical fertilizer.

Avaliação nutricional pré-operatória dos pacientes com megaesôfago não-avançado

OBJETIVO: Avaliar o estado nutricional de pacientes com megaesôfago não-avançado no pré-operatório. MÉTODO: Foram selecionados dez pacientes adultos (seis do sexo masculino, quatro do feminino) provenientes dos Ambulatórios de Cirurgia do Hospital Regional do Vale do Paraíba-SAB e do Hospital Universitário de Taubaté. O diagnóstico do megaesôfago foi realizado por meio de um protocolo com informações referentes à origem epidemiológica, quadro clínico, sorologia para doença de Chagas, esofagrafia, endoscopia digestiva alta e eletromanometria. A avaliação do estado nutricional foi feita com dados antropométricos (Peso, IMC, CB, PCT e CMB) e bioquímicos (albumina, transferrina e linfocitometria). RESULTADOS: O peso foi o parâmetro mais comprometido, sendo a porcentagem de perda de peso considerada grave em oito pacientes. Somente um paciente classificou-se como percentil 5. Não houve alteração bioquímica significante. CONCLUSÃO: A maioria dos pacientes é eutrófica, sendo um paciente considerado desnutrido.

Organophosphorus and carbamates residues in milk and feedstuff supplied to dairy cattle

Considering acute and chronic toxicity effects on human and animal health caused by pesticide residues in food, this study aimed to analyze organophosphorate (OP) and carbamate (CB) in feedstuff and water destined for dairy cattle, as well as in the milk produced by these animals, through gas chromatography (GC). In the Agreste region of Pernambuco, Brazil, 30 raw milk samples and all components of the animals' diet were collected from several farms. Out of the 30 milk of milk analyzed, six (20%) were contaminated with OP, five (16.7%) with CB, and one sample with both pesticides. From 48 analyzed feed samples, 15 (31.25%) were contaminated with residues of OP, six (12.50%) with CB, and one sample was contaminated with both pesticides. Out of 16 water samples analyzed, six (37.50%) were contaminated with OP residues, but non with CB. In four dairy farms the pesticides detected in milk were compatible with the active principles found in water and/or foodstuff, suggesting them to be the source of contamination.

Efeitos de herbicidas nos teores de macronutrientes e nas características tecnológicas da cana-de-açúcar (Saccharum spp.). I - Misturas de herbicidas em pós-emergência

A presente pesquisa foi conduzida em um Latossol Roxo, com 3,9% de matéria orgânica, na Usina São Carlos, município de Jaboticabal - SP com o objetivo de verificar o comportamento das principais misturas de herbicidas aplicadas em pós-emergência e suas possíveis interferências nos teores de macronutrientes, e no desenvolvimento da variedade CB 41-14. Os tratamentos utilizados com as respectivas doses do ingrediente ativo em kg/ha foram: ametryne + 2,4 - D (formulação comercial) a 1,47 - 2,03, diuron + 2,4 -D a 1,60 + 2,16; ametryne + 2,4-D a 1,60 + 2,16; alachlor + 2,4-D a 1,09 + 1,80; asulam + ioxynil - 2,4-D a 2,80 + 1,75; MCPA + 2,4-D a 0,83 + 0,83; oxadiazon + 2,4 -D a 0,50 + 2,16; ametryne + simazine + 2,4-D a 1,25 + 1,25 + 2,16; ametryne + secbumetone + 2,4-D a 2,00 + 2,00 + 2,16; diuron + hexazinone + surfatante a 0,80 + 0,45 + 0,5% e MCPA + 2,3,6-TBA + pendimethalin + surfatante a 1,50 + 0,48 + 0,66 + 0,5%. O controle das plantas daninhas foi avaliado através de contagens por espécie botânica e por avaliações visuais . Sobre a cultura os efeitos foram constatados pela contagem da brotação inicial, avaliações visuais dos efeitos fitotóxicos, análise dos teores de macronutrientes aos 5 e 8 meses, medidas do comprimento dos colmos, análise dos teores de fibra, pol, brix, % de cana e pureza, assim como valores de pol/ha, e peso de colmos por hectare. Não houve interferência das diferentes misturas de herbicidas na brotação inicial, no comprimento médio dos colmos, e nos teores de fibra, pol, brix e pureza por ocasião da colheita. Em relação às principais plantas daninhas presentes na área, que eram capim-colchão (Digitaria sanguinalis (L). Scop.) capim-coloniâo (Panicum maximum Jacq), beldroega (Portulacca oleracea L.) e caruru (Amaranthus spp), as melhores porcentagens de controle foram obtidas com as misturas de ametryne + 2,4-D, MCPA + 2,4-D, oxadiazon + 2,4-D, ametryne + secbumetone, diuron + hexazinone e MCPA + 2,3,6-TBA + pendimethalin. Aos cinco meses o teor de P foi diminuido pela mistura de ametryne + secbumetone + 2,4-D, e o teor de Ca decresceu pelas misturas de diuron + 2,4-D, ametryne + 2,4-D, alachlor + 2,4-D, asulam + ioxynil + 2,4-D, MCPA + 2,4-D, oxadiazon + 2,4-D, ametryne + simazine + 2,4-D e ametryne + secbumetone + 2,4-D. Já aos oito meses não houve diferença estatística para qualquer dos nutrientes estudados. Os teores de pol/ha e produção de colmos/ha não foram significativamente afetados por qualquer dos tratamentos quando comparados com a testemunha com capina. Todavia, todos os tratamentos apresentaram produção inferior a esta, sugerindo certa precaução com as aplicações pós-emergentes em canade-açúcar.

Ultrastructural and biochemical detection of biotin and biotinylated polypeptides in Schistosoma mansoni

Biotinylation is proposed for the identification of surface proteins in Schistosoma mansoni using the streptavidin-HRP conjugate for the detection of labeled polypeptides. However, control samples also showed several endogenous biotinylated polypeptides. In an attempt to determine the possibility of nonspecific binding between the streptavidin-HRP conjugate and polypeptides from S. mansoni, the conjugate was blocked with biotinamidecaproate-N-hydroxysuccinimide ester (BcapNHS) before biotin-streptavidin blotting. No bands were detected on the nitrocellulose sheet, demonstrating the specific recognition of biotin by the streptavidin present in the conjugate. Whole cercariae and cercarial bodies and tails showed several endogenous biotinylated polypeptides. The biotin concentration was 13 µg/190,000 cercariae. Adult worms presented less endogenous biotinylated polypeptides than cercariae. These results may be due to changes in the environment from aerobic to anaerobic conditions when cercarial bodies (schistosomula) are transformed into adult worms and a decrease in CO2 production may occur. Cercariae, cercarial bodies and adult male worms were examined by transmission electron microscopy employing an avidin-colloidal gold conjugate for the detection of endogenous biotin. Gold particles were distributed mainly on the muscle fibers, but dispersed granules were observed in the tegument, mitochondria and cytosol. The discovery of endogenous biotin in S. mansoni should be investigated in order to clarify the function of this vitamin in the parasite

Distribution of calcium-binding proteins in the chick visual system

The calcium-binding proteins calbindin (CB), calretinin (CR), and parvalbumin (PV) have been extensively studied over the last decade since they appear to be important as buffers of intracellular calcium. In the present study we investigated the distribution of these proteins in the chick visual system by means of conventional immunocytochemistry. The results indicated that CB, CR, and PV are widely distributed in retinorecipient areas of the chick brain. In some regions, all three calcium-binding proteins were present at different intensities and often in different neurons such as in the dorsolateral thalamic complex. In other areas, such as the nucleus geniculatus lateralis ventralis, only CB and CR were detected, whereas PV was absent. These results show that these three calcium-binding proteins are differentially distributed in the visual system of the chick, with varying degrees of co-localization

Colocalization of coilin and nucleolar proteins in Cajal body-like structures of micronucleated PtK2 cells

Cajal bodies (CB) are ubiquitous nuclear structures involved in the biogenesis of small nuclear ribonucleoproteins and show narrow association with the nucleolus. To identify possible relationships between CB and the nucleolus, the localization of coilin, a marker of CB, and of a set of nucleolar proteins was investigated in cultured PtK2 cells undergoing micronucleation. Nocodazol-induced micronucleated cells were examined by double indirect immunofluorescence with antibodies against coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM/Scl, and To/Th. Cells were imaged on a BioRad 1024-UV confocal system attached to a Zeiss Axiovert 100 microscope. Since PtK2 cells possess only one nucleolus organizer region, micronucleated cells presented only one or two micronuclei containing nucleolus. By confocal microscopy we showed that in most micronuclei lacking a typical nucleolus a variable number of round structures were stained by antibodies against fibrillarin, NOR-90/hUBF protein, and coilin. These bodies were regarded as CB-like structures and were not stained by anti-PM/Scl and anti-To/Th antibodies. Anti-RNA polymerase I antibodies also reacted with CB-like structures in some micronuclei lacking nucleolus. The demonstration that a set of proteins involved in RNA/RNP biogenesis, namely coilin, fibrillarin, NOR-90/hUBF, and RNA polymerase I gather in CB-like structures present in nucleoli-devoid micronuclei may contribute to shed some light into the understanding of CB function.

Density, proportion, and dendritic coverage of retinal ganglion cells of the common marmoset (Callithrix jacchus jacchus)

We performed a quantitative analysis of M and P cell mosaics of the common-marmoset retina. Ganglion cells were labeled retrogradely from optic nerve deposits of Biocytin. The labeling was visualized using horseradish peroxidase (HRP) histochemistry and 3-3'diaminobenzidine as chromogen. M and P cells were morphologically similar to those found in Old- and New-World primates. Measurements were performed on well-stained cells from 4 retinas of different animals. We analyzed separate mosaics for inner and outer M and P cells at increasing distances from the fovea (2.5-9 mm of eccentricity) to estimate cell density, proportion, and dendritic coverage. M cell density decreased towards the retinal periphery in all quadrants. M cell density was higher in the nasal quadrant than in other retinal regions at similar eccentricities, reaching about 740 cells/mm² at 2.5 mm of temporal eccentricity, and representing 8-14% of all ganglion cells. P cell density increased from peripheral to more central regions, reaching about 5540 cells/mm² at 2.5 mm of temporal eccentricity. P cells represented a smaller proportion of all ganglion cells in the nasal quadrant than in other quadrants, and their numbers increased towards central retinal regions. The M cell coverage factor ranged from 5 to 12 and the P cell coverage factor ranged from 1 to 3 in the nasal quadrant and from 5 to 12 in the other quadrants. These results show that central and peripheral retinal regions differ in terms of cell class proportions and dendritic coverage, and their properties do not result from simply scaling down cell density. Therefore, differences in functional properties between central and peripheral vision should take these distinct regional retinal characteristics into account.

Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca²+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca²+]i oscillations similar to those evoked by sperm (95 vs 100%; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 µg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6%, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83%, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7% ethanol (62 vs 62%, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62%, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca²+]i oscillations, completion of meiosis and parthenogenetic development.

Water extracts of cabbage and kale inhibit ex vivo H2O2-induced DNA damage but not rat hepatocarcinogenesis

The chemopreventive potential of water extracts of the Brassica vegetables cabbage and kale was evaluated by administering their aqueous extracts in drinking water ad libitum to Wistar rats submitted to Ito’s hepatocarcinogenesis model (CB group and K group, respectively - 14 rats per group). Animals submitted to this same model and treated with water were used as controls (W group - 15 rats). Treatment with the vegetable extracts did not inhibit (P > 0.05) placental glutathione S-transferase-positive preneoplastic lesions (PNL). The number of apoptotic bodies did not differ (P > 0.05) among the experimental groups. Ex vivo hydrogen peroxide treatment of rat livers resulted in lower (P < 0.05) DNA strand breakage in cabbage- (107.6 ± 7.8 µm) and kale- (110.8 ± 10.0 µm) treated animals compared with control (120.9 ± 12.7 µm), as evaluated by the single cell gel (comet) assay. Treatment with cabbage (2 ± 0.3 µg/g) or kale (4 ± 0.2 µg/g) resulted in increased (P < 0.05) hepatic lutein concentration compared with control (0.5 ± 0.07 µg/g). Despite the absence of inhibitory effects of cabbage and kale aqueous extracts on PNL, these Brassica vegetables presented protection against DNA damage, an effect possibly related to increased hepatic lutein concentrations. However, it must be pointed out that the cause-effect relationship between lutein levels and protection is hypothetical and remains to be demonstrated.