Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus

Three Brazilian isolates of bovine viral diarrhea virus (BVDV), antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs). Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11), were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA) assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid) and 1:100 (hybridoma culture supernatant) in IFA and immunoperoxidase (IPX) staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes.

Systemic and regional hemodynamic effects of enalaprilat infusion in experimental normotensive sepsis

Angiotensin-converting enzyme inhibitors have been shown to improve splanchnic perfusion in distinct shock states. We hypothesized that enalaprilat potentiates the benefits of early fluid resuscitation in severe experimental sepsis, particularly in the splanchnic region. Anesthetized and mechanically ventilated mongrel dogs received an intravenous infusion of live Escherichia coli over a period of 30 min. Thereafter, two interventions were performed: fluid infusion (normal saline, 32 mL/kg over 30 min) and enalaprilat infusion (0.02 mg kg-1 min-1 for 60 min) in randomized groups. The following groups were studied: controls (fluid infusion, N = 4), E1 (enalaprilat infusion followed by fluid infusion, N = 5) and E2 (fluid infusion followed by enalaprilat infusion, N = 5). All animals were observed for a 120 min after bacterial infusion. Mean arterial pressure, cardiac output (CO), portal vein blood flow (PVBF), systemic and regional oxygen-derived variables, and lactate levels were measured. Rapid and progressive reductions in CO and PVBF were induced by the infusion of live bacteria, while minor changes were observed in mean arterial pressure. Systemic and regional territories showed a significant increase in oxygen extraction and lactate levels. Widening venous-arterial and portal-arterial pCO2 gradients were also detected. Fluid replacement promoted transient benefits in CO and PVBF. Enalaprilat after fluid resuscitation did not affect systemic or regional hemodynamic variables. We conclude that in this model of normotensive sepsis inhibition of angiotensin-converting enzyme did not interfere with the course of systemic or regional hemodynamic and oxygen-derived variables.

Recombinant E2 glycoprotein of bovine viral diarrhea virus induces a solid humoral neutralizing immune response but fails to confer total protection in cattle

Two recombinant baculoviruses were produced in order to obtain a bovine viral diarrhea virus (BVDV) immunogen: AcNPV/E2 expressing E2 glycoprotein, and AcNPV/E0E1E2 expressing the polyprotein region coding for the three structural proteins of BVDV (E0, E1, and E2). Mice were immunized with Sf9 cells infected with the recombinant baculoviruses in a water in oil formulation and the production of neutralizing antibodies was evaluated. Since E2 elicited higher neutralizing antibody titers than E0-E1-E2 polyprotein, it was selected to immunize cattle. Calves received two doses of recombinant E2 vaccine and were challenged with homologous BVDV 37 days later. The recombinant immunogen induced neutralizing titers which showed a mean value of 1.5 ± 0.27 on the day of challenge and reached a top value of 3.36 ± 0.36, 47 days later (84 days post-vaccination). On the other hand, sera from animals which received mock-infected Sf9 cells did not show neutralizing activity until 25 days post-challenge (62 days post-vaccination), suggesting that these antibodies were produced as a consequence of BVDV challenge. Even when no total protection was observed in cattle, in vitro viral neutralization assays revealed that the recombinant immunogen was able to induce neutralizing antibody synthesis against the homologous strain as well as against heterologous strains in a very efficient way.

Effects of a methanolic fraction of soybean seeds on the transcriptional activity of peroxisome proliferator-activated receptors (PPAR)

Since the anti-inflammatory, antidiabetic and hypolipidemic effects of soy isoflavones may be mediated by activation of peroxisome proliferator-activated receptors (PPAR), the present study investigated whether the methanolic fractions obtained from soybean seeds (E1) and soybean seed coats with hypocotyls (E2) could influence PPARα, PPARγ and PPARβ/δ transcriptional activity. The isoflavones from E1 and E2 were quantified by HPLC analysis. E1 and E2 were rich in isoflavones (daidzin, glycitin, genistin, malonyldaidzin, malonylglycitin, malonylgenistin, daidzein, glycitein, and genistein). Moreover, E1 and E2 showed no evidence of genetically modified material containing the gene CP4 EPSPS. To investigate PPAR transcriptional activity, human promonocytic U-937 cells were treated with E1 and E2 (200, 400, 800, and 1600 µg/mL), positive controls or vehicle. Data are reported as fold-activation of the luciferase reporter driven by the PPAR-responsive element. Dose-response analysis revealed that E1 and E2 induced the transcriptional activity of PPARα (P < 0.001), with activation comparable to that obtained with 0.1 mM bezafibrate (positive control) at 1600 µg/mL (4-fold) and 800 µg/mL (9-fold), respectively. In addition, dose-response analysis revealed that E1 and E2 activated PPARβ/δ (P < 0.05), and the activation at 800 µg/mL (4- and 9-fold, respectively) was comparable to that of 0.1 mM bezafibrate (positive control). However, no effect on PPARγ was observed. Activation of PPARα is consistent with the lipid-lowering activity of soy isoflavones in vivo, but further studies are needed to determine the physiological significance of PPARβ/δ activation.

Taurine inhibits serum deprivation-induced osteoblast apoptosis via the taurine transporter/ERK signaling pathway

Taurine has positive effects on bone metabolism. However, the effects of taurine on osteoblast apoptosis in vitro have not been reported. The aim of this study was to investigate the activity of taurine on apoptosis of mouse osteoblastic MC3T3-E1 cells. The data showed that 1, 5, 10, or 20 mM taurine resulted in 16.7, 34.2, 66.9, or 63.75% reduction of MC3T3-E1 cell apoptosis induced by the serum deprivation (serum-free α-MEM), respectively. Taurine (1, 5, or 10 mM) also reduced cytochrome c release and inhibited activation of caspase-3 and -9, which were measured using fluorogenic substrates for caspase-3/caspase-9, in serum-deprived MC3T3-E1 cells. Furthermore, taurine (10 mM) induced extracellular signal-regulated kinase (ERK) phosphorylation in MC3T3-E1 cells. Knockdown of the taurine transporter (TAUT) or treatment with the ERK-specific inhibitor PD98059 (10 μM) blocked the activation of ERK induced by taurine (10 mM) and abolished the anti-apoptotic effect of taurine (10 mM) in MC3T3-E1 cells. The present results demonstrate for the first time that taurine inhibits serum deprivation-induced osteoblast apoptosis via the TAUT/ERK signaling pathway.

Tetramethylpyrazine potentiates arsenic trioxide activity against HL-60 cell lines

The objective of this study was to evaluate the effects of tetramethylpyrazine (TMP) in combination with arsenic trioxide (As2O3) on the proliferation and differentiation of HL-60 cells. The HL-60 cells were treated with 300 µg/mL TMP, 0.5 µM As2O3, and 300 µg/mL TMP combined with 0.5 µM As2O3, respectively. The proliferative inhibition rates were determined with MTT. Differentiation was detected by the nitroblue tetrazolium (NBT) reduction test, Wright’s staining and the distribution of CD11b and CD14. Flow cytometry was used to analyze cell cycle distribution. RT-PCR and Western blot assays were employed to detect the expressions of c-myc, p27, CDK2, and cyclin E1. Combination treatment had synergistic effects on the proliferative inhibition rates. The rates were increased gradually after the combination treatment, much higher than those treated with the corresponding concentration of As2O3 alone. The cells exhibited characteristics of mature granulocytes and a higher NBT-reducing ability, being a 2.6-fold increase in the rate of NBT-positive ratio of HL-60 cells within the As2O3 treatment versus almost a 13-fold increase in the TMP + As2O3 group. Cells treated with both TMP and As2O3 expressed far more CD11b antigens, almost 2-fold compared with the control group. Small doses of TMP potentiate As2O3-induced differentiation of HL-60 cells, possibly by regulating the expression and activity of G0/G1 phase-arresting molecules. Combination treatment of TMP with As2O3 has significant synergistic effects on the proliferative inhibition of HL-60 cells.

Adenovirus-mediated siRNA targeting TNF-α and overexpression of bone morphogenetic protein-2 promotes early osteoblast differentiation on a cell model of Ti particle-induced inflammatory response in vitro

Wear particles are phagocytosed by macrophages and other inflammatory cells, resulting in cellular activation and release of proinflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty failure. During this pathological process, tumor necrosis factor-alpha (TNF-α) plays an important role in wear-particle-induced osteolysis. In this study, recombination adenovirus (Ad) vectors carrying both target genes [TNF-α small interfering RNA (TNF-α-siRNA) and bone morphogenetic protein 2 (BMP-2)] were synthesized and transfected into RAW264.7 macrophages and pro-osteoblastic MC3T3-E1 cells, respectively. The target gene BMP-2, expressed on pro-osteoblastic MC3T3-E1 cells and silenced by the TNF-α gene on cells, was treated with titanium (Ti) particles that were assessed by real-time PCR and Western blot. We showed that recombinant adenovirus (Ad-siTNFα-BMP-2) can induce osteoblast differentiation when treated with conditioned medium (CM) containing RAW264.7 macrophages challenged with a combination of Ti particles and Ad-siTNFα-BMP-2 (Ti-ad CM) assessed by alkaline phosphatase activity. The receptor activator of nuclear factor-κB ligand was downregulated in pro-osteoblastic MC3T3-E1 cells treated with Ti-ad CM in comparison with conditioned medium of RAW264.7 macrophages challenged with Ti particles (Ti CM). We suggest that Ad-siTNFα-BMP-2 induced osteoblast differentiation and inhibited osteoclastogenesis on a cell model of a Ti particle-induced inflammatory response, which may provide a novel approach for the treatment of periprosthetic osteolysis.

Modeling of allergen proteins found in sea food products

Shellfish are a source of food allergens, and their consumption is the cause of severe allergic reactions in humans. Tropomyosins, a family of muscle proteins, have been identified as the major allergens in shellfish and mollusks species. Nevertheless, few experimentally determined three-dimensional structures are available in the Protein Data Base (PDB). In this study, 3D models of several homologous of tropomyosins present in marine shellfish and mollusk species (Chaf 1, Met e1, Hom a1, Per v1, and Pen a1) were constructed, validated, and their immunoglobulin E binding epitopes were identified using bioinformatics tools. All protein models for these allergens consisted of long alpha-helices. Chaf 1, Met e1, and Hom a1 had six conserved regions with sequence similarities to known epitopes, whereas Per v1 and Pen a1 contained only one. Lipophilic potentials of identified epitopes revealed a high propensity of hydrophobic amino acids in the immunoglobulin E binding site. This information could be useful to design tropomyosin-specific immunotherapy for sea food allergies.

Avaliação da aplicação de fungicida às sementes de amendoim antes do envelhecimento acelerado

Os objetivos do trabalho foram os de avaliar o efeito da aplicação de fungicida às sementes de amendoim (Arachis hypogaea L.), que foram colhidas em distintos estádios de maturação e provenientes de plantas-mãe que foram ou não submetidas à calagem, nas condições de envelhecimento acelerado. Foram avaliados quatro lotes de sementes do cultivar Botucatu, provenientes das áreas que receberam (CC) ou não a aplicação de calcário (SC) e que foram colhidas aos 104 (E1) e 124 (E2) dias após a semeadura e denominados de lote 1 (E1/SC), 2 (E1/CC), 3 (E2/SC) e 4(E2/CC). De cada lote, foram retiradas duas amostras de sementes, uma original e outra tratada com fungicida thiram. Estas amostras foram submetidas às condições do teste de envelhecimento acelerado, e após o período de exposição, as sementes foram avaliadas na primeira e na segunda contagem do teste de germinação realizado com e sem secagem. Pelos resultados pode-se concluir que há maior incidência de patógenos nas sementes colhidas aos 104 DAS e provenientes de plantas-mãe submetidas à calagem; o tratamento fungicida favorece a germinação das sementes contaminadas por Aspergillus do grupo flavus e Aspergillus niger, até 24 horas após a exposição às condições de envelhecimento acelerado.

Efeito da irrigação, épocas de corte da forragem e doses de nitrogênio sobre o rendimento de sementes de milheto

O experimento foi conduzido na Estação Experimental Agronômica/UFRGS, (30º05'52"S; 51º40'08"W) com o objetivo de avaliar o efeito da irrigação (irrigado e não irrigado), de duas épocas de remoção da forragem (E1- remoção de aproximadamente 50% dos ápices dos meristemas apicais dos perfilhos primários e E2- remoção de 75%) e de quatro doses de nitrogênio (0, 50, 100 e 150kg.ha-1), sobre os componentes de rendimento e o rendimento de sementes de milheto (Pennisetum americanum (L.) Leeke). O delineamento experimental utilizado foi parcela sub-subdividida com quatro repetições. A semeadura foi realizada com máquina de plantio direto no dia 29/12/00. As doses de N foram parceladas em duas aplicações iguais, sendo a primeira em 16/01/01 e a segunda uma semana após os cortes em E1 e E2. Os cortes foram realizados a uma altura de 20cm (E1=09/02 e E2=19/02) e a colheita de sementes nos dias 30/04 e 01/05. A aplicação de doses de N teve influência sobre os componentes de rendimento, tais como, número de panículas.m-2, número de panículas com sementes.m-2, comprimento de panícula, peso de panícula e número de sementes.panícula-1. O componente panículas com sementes respondeu ao efeito das interações entre irrigação com doses de nitrogênio e épocas de corte com doses de nitrogênio. Maiores pesos de panícula e de mil sementes foram registrados no corte precoce (E1). O rendimento de sementes puras viáveis foi influenciado pelas épocas de corte e doses de nitrogênio, e no ponto de maior eficiência técnica do N (120kg.ha-1), foi de 769kg.ha-1. Os componentes do rendimento que mais se correlacionaram com o rendimento de sementes foram o número de sementes.panícula-1 e o peso de mil sementes. O rendimento de matéria seca total respondeu às doses de N, sendo expresso por uma regressão linear positiva. A irrigação não afetou a resposta das variáveis estudadas.

Qualidade fisiológica de sementes de soja cultivada em rotação com milheto

Para o sucesso do plantio direto há necessidade de que a cultura antecessora à principal seja boa produtora e mantenedora de cobertura vegetal. O milheto tem-se constituído em boa opção de cultivo no outono/inverno para rotação com soja, porém não há estudos avaliando o efeito do sistema na qualidade das sementes. O objetivo do trabalho foi estudar o efeito de três épocas de semeadura e cinco manejos com ceifas do milheto sobre a qualidade fisiológica de sementes de soja cultivada em sucessão, por plantio direto, na mesma área, por três ciclos de rotação (1999/2000; 2001/2002 e 2002/2003). As épocas de semeadura constituíram três experimentos (E1, E2 e E3), em delineamento experimental de blocos ao acaso com cinco tratamentos (M1 = ceifa a cada florescimento e retirada da massa vegetal; M2 = ceifa a cada florescimento e permanência da massa vegetal; M3 = ceifa no florescimento e retirada da massa vegetal; M4 = ceifa no florescimento e permanência da massa vegetal e M5 = sem ceifa até a produção de grãos, quando foi cortada a panícula, permanecendo o restante da massa vegetal), e quatro repetições. Os experimentos de milheto foram semeados em 1999: E1 = 05/03, E2 = 25/03 e E3 = 19/04; em 2001 = E1 = 17/04, E2 = 07/05 e E3 = 27/05, e em 2002: E1 = 25/04, E2 = 15/05 e E3. = 05/06. Em cada ciclo de rotação, a soja foi implantada nos três experimentos na mesma data após o manejo final do milheto com dessecação química; as colheitas foram realizadas na mesma data, também. As sementes de soja, cv. Embrapa-48, foram avaliadas quanto ao tamanho, massa de 100 sementes, teor de água, germinação e vigor. Sementes de soja com melhor qualidade fisiológica foram obtidas, nos três ciclos de sucessão, quando cultivada após a primeira época de semeadura do milheto, independentemente do manejo com ceifas do milheto, cujo efeito foi pouco evidente.

Efeito da irrigação, épocas de corte da forragem e doses de nitrogênio sobre a qualidade de sementes de milheto (Pennisetum americanum (L.) Leeke)

O trabalho foi conduzido com o objetivo de avaliar o efeito da irrigação, da épocas de remoção da forragem e de quatro doses de nitrogênio (N) (0, 50, 100 e 150 kg/ha) sobre a qualidade de sementes de milheto (Pennisetum americanum (L.) Leeke). Os cortes foram realizados a uma altura de 20 cm, aos 41 dias apos semeadura, na E1 e aos 51 na E2 . Foram determinados o peso de mil sementes (PMS), o teor de nitrogênio, a germinação e o vigor pelos testes de: condutividade elétrica, envelhecimento acelerado a 48 e 72 horas, primeira contagem de germinação e teste de emergência a campo. A remoção precoce da forragem (E1) afeta positivamente a qualidade das sementes de milheto. Aplicações de nitrogênio podem contribuir para a melhoria da qualidade fisiológica da semente de milheto e associadas à irrigação proporcionam sementes com maior capacidade de emergência.O conteúdo de nitrogênio das sementes não e afetado em função da aplicação de nitrogênio em cobertura.

Época de colheita e secagem na qualidade de sementes de pimenta Habanero Yellow

As pimentas constituem um dos principais produtos da olericultura brasileira. O interesse das indústrias por espécies picantes, como a pimenta Habanero, tem crescido a cada ano, sobretudo para a produção de molhos e preparados desidratados, o que aumentou a demanda no mercado por sementes de qualidade. O autor teve, através desta pesquisa, por objetivo avaliar os efeitos das épocas de colheita das sementes e da secagem na qualidade das sementes de pimenta Habanero Yellow. Foram utilizadas sementes extraídas de frutos em quatro épocas de colheita (E1 - 50, E2 - 60, E3 - 67 e E4 - 67 DAA e mantidos em repouso por 7 dias após a colheita). As sementes extraídas dos frutos em diferentes épocas de colheita foram submetidas a quatro métodos de secagem: secagem artificial, aos 45 °C, até 8% de teor de água; secagem artificial, aos 35 °C, até 20% de teor de água, seguida de secagem aos 45 °C, até 8% de teor de água; secagem artificial, aos 35 °C, até 8% de teor de água; e secagem natural à sombra até 8% de teor de água). A qualidade fisiológica das sementes antes da secagem foi avaliada por meio dos testes de germinação, emergência e envelhecimento acelerado, além da análise das isoenzimas esterase, superóxido dismutase, peroxidase, malato desidrogenase, álcool desidrogenase e da endo-β-mananase. Após a secagem, a qualidade fisiológica das sementes foi avaliada por meio dos testes de germinação, emergência e deterioração controlada. A qualidade fisiológica das sementes é máxima aos 67 DAA, quando os frutos estão completamente maduros. Maiores valores de germinação e vigor são observados em sementes secadas aos 35 ºC.