A Ca2+/H+ antiport system driven by the tonoplast pyrophosphate-dependent proton pump from maize roots.

Calcium uptake by tonoplast enriched membrane vesicles from maize (Zea mays L. cv. LG 11) primary roots was studied. A pH gradient, measured by the fluorescence quenching of quinacrine, was generated across sealed vesicles driven by the pyrophosphate-dependent proton pump. The fluorescence quenching was strongly inhibited by Ca2+; moreover, when increasing Ca2+ concentrations were added to vesicles at steady-state, a concomitant decrease in the proton gradient was observed. Ca2+ uptake using Ca-45(2+) was linear from 10 min when oxalate (10 mM) was present, while Ca2+ uptake was completely inhibited with proton ionophores (FCCP and monensin), indicating a Ca2+/H+ antiport. Membranes were further fractionated using a linear sucrose density gradient (10-45%) and were identified with marker enzymes. Ca2+ uptake co-migrated with the tonoplast pyrophosphate-dependent proton pumping, pyrophosphatase and ATPase activities: the Ca2+/H+ antiport is consequently located at the tonoplast.

Tonoplast localization of a calmodulin-stimulated Ca2+-pump from maize roots.

The subcellular localization of a calmodulin-stimulated calcium (Ca2+)-ATPase activity from maize roots (Zea mays L., cv LG 11) was studied. For this purpose, an efficient procedure was developed to prepare sealed plasma membrane vesicles allowing the measurement of proton and Ca2+ transport activities. Two-day-old root membranes were fractionated by sucrose and dextran density gradient centrifugation. Marker enzymes were used to study the distribution of the different membranes in the gradients and a filtration technique was developed to measure Ca-45(2+) transport in sealed vesicles. Most of the ATP-dependent Ca2+ transport activity was associated with the ER. However, a small part of this activity was associated with the tonoplast (corresponding to the activity of the H+/Ca2+ antiport) and the plasma membrane. When the Ca2+ transport was measured in the presence of exogenous calmodulin (1 muM), a 3-5-fold increase of uptake was measured. The calmodulin-stimulated activity was associated with the tonoplast vesicles only. This activity was insensitive to monensin, a proton ionophore, ruling out a direct effect of calmodulin on the H+/Ca2+ antiport. In conclusion, four different Ca2+ transporters are present in young maize root cells. A Ca2+/H+ antiport system is present on the tonoplast, whereas, the plasma membrane and the ER possess each a calmodulinin-sensitive Ca2+-ATPase. Finally, a calmodulin-stimulated Ca2+-ATPase is associated with the tonoplast.

Chemical Biology Screen for Prostate Cancer Therapeutics

Prostate cancer initially responds to hormone-based therapeutics such as anti-androgen treatment or chemotherapeutics but eventually becomes resistant. Novel treatment options are therefore urgently needed. This thesis study applied a high-throughput screen of 4910 known drugs and drug-like small molecules to identify compounds that selectively inhibit growth of prostate cancer cells. In addition, the mechanisms underlying the cellular sensitivity to potent cancer selective compounds were addressed. Surprisingly, many of the compounds currently used in the clinics or studied in clinical trials were not cancer-selective. Only four drugs, aldehyde dehydrogenase inhibitor disulfiram (Antabus), antibiotic ionophore monensin, histone deacetylase inhibitor tricostatin A and fungicide thiram inhibited prostate cancer cell growth at nanomolar concentrations without major effects on non-malignant prostate epithelial cells. Disulfiram, monensin and a structurally similar compound to monensin, salinomycin, induced oxidative stress and inhibited aldehyde dehydrogenase activity. Moreover, monensin and salinomycin reduced androgen receptor signalling and steroidogenesis, enforced cell differentiation and reduced the overall levels of cancer stem cells. Taken together, novel and potentially prostate cancer-selective therapeutic agents were identified in this study, including the description of a multitude of intoxicating mechanisms such as those relating to oxidative stress. The results provide novel insights into prostate cancer biology and exemplify useful means of considering novel approaches to cancer treatment.

Leptospira interrogans activation of peripheral blood monocyte glycolipoprotein demonstrated in whole blood by the release of IL-6

Glycolipoprotein (GLP) from pathogenic serovars of Leptospira has been implicated in the pathogenesis of leptospirosis by its presence in tissues of experimental animals with leptospirosis, the inhibition of the Na,K-ATPase pump activity, and induced production of cytokines. The aims of the present study were to investigate the induction of IL-6 by GLP in peripheral blood mononuclear cells (PBMC) and to demonstrate monocyte stimulation at the cellular level in whole blood from healthy volunteers. PBMC were stimulated with increasing concentrations (5 to 2500 ng/ml) of GLP extracted from the pathogenic L. interrogans serovar Copenhageni, lipopolysaccharide (positive control) or medium (negative control), and supernatants were collected after 6, 20/24, and 48 h, and kept at -80ºC until use. Whole blood was diluted 1:1 in RPMI medium and cultivated for 6 h, with medium, GLP and lipopolysaccharide as described above. Monensin was added after the first hour of culture. Supernatant cytokine levels from PBMC were measured by ELISA and intracellular IL-6 was detected in monocytes in whole blood cultures by flow-cytometry. Monocytes were identified in whole blood on the basis of forward versus side scatter parameters and positive reactions with CD45 and CD14 antibodies. GLP ( > or = 50 ng/ml)-induced IL-6 levels in supernatants were detected after 6-h incubation, reaching a peak after 20/24 h. The percentage of monocytes staining for IL-6 increased with increasing GLP concentration. Thus, our findings show a GLP-induced cellular activation by demonstrating the ability of GLP to induce IL-6 and the occurrence of monocyte activation in whole blood at the cellular level.

FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely. E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Produção de metano entérico em pastagens tropicais

Na presente revisão, buscou-se apresentar os principais impactos ambientais causados pela pecuária, sobretudo, em relação às emissões de gases efeito estufa (GEE). Além disso, buscou-se apresentar possíveis formas de mitigar essas externalidades. A criação de bovinos, no Brasil, acontece de forma extensiva, muitas vezes em áreas com pastagem degradada e, portanto, de baixa produtividade. Isso possibilita à atividade uma oportunidade de redução do impacto causado ao meio ambiente, uma vez que ações tomadas, no sentido de melhorar o rendimento animal, devem resultar em um menor consumo de recursos naturais (terra e água) e maior eficiência do sistema digestivo animal. Os principais problemas apontados pelos pesquisadores, no que tange à pecuária extensiva, são o metano emitido pela fermentação entérica dos ruminantes, o óxido nitroso emitido pelos dejetos dos animais em pastejo e o dióxido de carbono trocado pelo solo e vegetação. Muitos fatores influenciam a produção de CH4 entérico dos ruminantes, inclusive o tipo de carboidrato fermentado, o sistema digestivo do animal, a quantidade e o tipo de alimentos consumidos. Diante do exposto, pesquisadores têm desenvolvido tecnologias para reduzir a emissão de metano, através da melhoria das práticas de manejo alimentar, manipulação ruminal, por meio de suplementação com monensina, lipídios, ácidos orgânicos e compostos de plantas. Outras estratégias de redução de metano que foram investigadas são: defaunação e vacinas, que buscam inibir micro-organismos metanogênicos e a metanogênese. Assim, a busca por sistemas de produção eficientes tem sido uma das perspectivas da pecuária mundial para reduzir a emissão de poluentes e intensificar a produção animal.

Fermentação ruminal e produção de metano em bovinos alimentados com feno de capim-tifton 85 e concentrado com aditivos

Objetivou-se avaliar o efeito do uso de monensina, complexo de leveduras, ácidos graxos poliinsaturados e aminoácidos no consumo de matéria seca e nutrientes, na estimativa da digestibilidade ruminal, nos parâmetros de fermentação ruminal (pH, concentração de nitrogênio amoniacal e de ácidos graxos de cadeia curta), na população de protozoários e na produção de metano. Foram utilizados seis bovinos e com peso corporal de 530 ± 15 kg, recebendo complexo de leveduras, ácidos graxos poliinsaturados e aminoácidos (5 g/dia); monensina (5 g/dia); caulim (5 g/dia), usado como controle adicionado à dieta composta de feno de capim-tifton 85 (Cynodon spp.); e concentrado, na relação 80:20. O delineamento experimental adotado para análise do consumo e da digestibilidade foi o de blocos completos casualizados e, para análise dos parâmetros ruminais e da produção de metano, o de parcelas subdivididas. O consumo foi influenciado pelo uso de monensina na dieta, mas não diferiu entre os aditivos. As digestibilidades da matéria seca e dos nutrientes não foram influenciadas pelo fornecimento dos aditivos. A relação acetato:propionato nos animais alimentados com a dieta com monensina foi menor que naqueles que receberam o complexo de leveduras e ácidos graxos poliinsaturados e aminoácidos, diminuindo a perda de energia na forma de metano. O pH e a concentração de nitrogênio amoniacal foram adequados para o crescimento bacteriano. A concentração de metano não é alterada pelo uso dos aditivos testados.

Molecular Identification and Characterization of Ileal and Cecal Fungus Communities in Broilers Given Probiotics, Specific Essential Oil Blends, and Under Mixed Eimeria Infection

Broiler digestive tract fungal communities have gained far less scrutiny than that given corresponding bacterial communities. Attention given poultry-associated fungi have focused primarily on feed-associated toxin-producers, yeast, and yeast products. The current project focused on the use of pyrosequencing and denaturing gradient gel electrophoresis (DGGE) to identify and monitor broiler digestive fungal communities. Eight different treatments were included. Four controls were an Uninfected-Unmedicated Control, an Unmedicated-Infected Control, the antibiotic bacitracin methylene disalicylate plus the ionophore monensin as Positive Control, and the ionophore monensin alone as a Negative Control. Four treatments were two probiotics (BC-30 and Calsporin) and two specific essential oil blends (Crina Poultry Plus and Crina Poultry AF). All chickens except the Unmedicated-Uninfected Control were given, at 15 days of age, a standard oral Eimeria inoculum of sporulated oocysts. Ileal and cecal digesta were collected at pre-Eimeria infection at 14 days of age and at 7 days post-Eimeria infection at 22 days of age. Extracted cecal DNA was analyzed by pyrosequencing to examine the impact of diet supplements and Eimeria infection on individual constituents in the fungal community, while DGGE was used to compare more qualitative changes in ileal and cecal communities. Pyrosequencing identified three phyla, seven classes, eight orders, 13 families, 17 genera, and 23 fungal species. Ileal and cecal DGGE patterns showed fungal communities were clustered mainly into pre- and post-infection patterns. Post-infection Unmedicated-Uninfected patterns were clustered with pre-infection groups demonstrating a strong effect of Eimeria infection on digestive fungal populations. These combined techniques offered added versatility towards unraveling the effects of enteropathogen infection and performance enhancing feed additives on broiler digestive microflora.

Efeito da monensina sódica sobre a produção e composição do leite, a produção de mozzarela e o escore de condição corporal de búfalas Murrah

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Características da carcaça e qualidade da carne de ovinos terminados em pastagens cultivadas

The objective of this study was to evaluate the quality of housing and the physical and chemical characteristics of meat from sheep raised on pasture Brachiaria brizantha and Panicum maximum. The experiment was conducted in the physical area of the Study Group on Forage (GEFOR), located in the Academic Unit Specialized in Agricultural Sciences - Federal University of Rio Grande do Norte - UFRN in Macaíba, RN, Brazil. We used 32 lambs SPRD, obtained from herds in the state, with liveweight (LW) of 24.5 kg were assigned randomly to four treatments consisting of tropical grasses, two cultivars of Brachiaria brizantha, Marandu and Piatã, and two of Panicum maximum, Aruana and Massai. The experimental area was 2.88 ha, divided into 4 paddocks of 0.72 ha, where each picket consisted of a farm and was divided into six plots of 0.12 ha, where the animals remained under rotational grazing. The period of adaptation to the pickets was seven days. At the beginning of the experiment the animals were weighed, identified with plastic earrings and necklaces colored according to the treatment, and treated against. The lambs were loose in the paddock at 8 am and collected at 16 hours, which returned to collective pens. During the time of grazing animals had free access to mineral supplement with monensin Ovinofós ® and water. Before entering the paddocks of pasture were sampled to characterize the chemical composition. Every seven days occurred at weighing, with fasting, to monitor the weight development. Cultivars Marandu, Aruana, Piatã and Massai were grazed for 133, 129, 143 and 142 days, respectively, until the lambs reach slaughter weight. Arriving at 32 kg lambs were evaluated subjectively for body condition score by, passed through fasting period, diet and water for 16 hours were slaughtered. Measurements were made in the inner and outer casings in addition to subjective evaluations regarding muscling, finish and quantity of pelvic-renal fat, then each was divided longitudinally into two half-carcases and cuts were made in the commercial left half, and after heavy calculated their income. Between the 12th and 13th thoracic vertebrae, was performed a cut to expose the cross section of the Longissimus dorsi, which was drawn on the rib eye area (REA) in transparent film. Fat thickness and extent of AOL GR were determined using a caliper. A tissue composition was determined by dissection of the legs. Analyzes were performed physical (color, cooking loss and shear force) and chemical composition of meat (moisture, ash, protein and lipids) in Longissimus dorsi muscle. Grazing tropical grass Brachiaria brizantha cvs. Marandu and Piatã and Panicum maximum cvs. Aruana and Massai can be used for lambs SRPD in the rainy season, because not alter the physico-chemical and chemical composition of meat

Avaliação da monensina sódica em vacas leiteiras

Utilizaram-se quinze vacas da raça Holandesa, submetidas a três tratamentos: T1-controle, T2-monensina sódica 300 mg/animal/dia, T3-monensina sódica 225 mg/animal/dia. A produção de leite, consumo de ração e o peso dos animais foram mensurados, juntamente com a avaliação da condição corporal Após o período experimental de 112 dias, evidenciou-se aumento na produção de leite de 13,5% e 10% (T2) em relação a TI e T3 respectivamente. Houve redução de consumo de ração de 4,5% (T2) e 3% (T3) em relação ao controle.

Performance, carcass characteristics and gain cost of feedlot cattle fed a high level of concentrate and different feed additives

The objective of this study was to evaluate the effects of feeding cattle with isoprotein and isoenergetic diets, with and without the addition of polyclonal antibody preparation (PAP), yeasts (YST) or monensin sodium (MON) on performance, carcass characteristics and gain cost in feedlot. Ninety-five 20-month old bullocks (323.3±21.8 kg) were distributed in 25 pens. The completely randomized experimental design had a 2 × 2 + 1 factorial arrangement and the treatments were replicated 5 times. There was no effect of MON for DMI throughout the feedlot period; however, MON reduced the dry matter intake (DMI) in g/kg of BW in the first 28 days when compared with the other treatments. The gain cost decreased with MON addition in relation to the other treatments. Inclusion of YST decreased average daily gain (ADG), final body weight, hot carcass weight, carcass weight, gain to feed ratio and DMI in g/kg body weight, worsening feed conversion and increasing the gain cost in the feeding periods. Inclusion of PAP increased ADG and decreased the gain cost, besides improving feed conversion. For MON and PAP, a difference was found for kidney-pelvic fat and kidney-pelvic fat per 100 kg of hot carcass weight. For MON and YST, there was a difference in ADG, feed conversion, gain cost and carcass yield and kidney-pelvic fat per 100 kg of hot carcass. Treatment YST worsened performance in relation to the non-supplemented treatments. Feeding PAP to animals did not influence performance and carcass characteristics of bullocks in feedlot negatively. Thus, PAP shows potential to substitute MON in cattle feeding using isoprotein and isoenergetic diets.

Parâmetros de fermentação ruminal em bovinos alimentados com grãos de milho ou sorgo de alta umidade ensilados

Os parâmetros de fermentação ruminal de dietas contendo silagem de sorgo úmido em substituição à de milho úmido foram estudados em 12 fêmeas bovinas, com peso médio de 584 kg. O delineamento foi inteiramente casualizado com três tratamentos: substituição do milho úmido pelo sorgo úmido ensilado, nos níveis de 0, 50 e 100%. As dietas continham grão úmido de milho ou de sorgo ensilados, soja extrusada, uréia, feno de aveia (Avena sativa sp.), suplemento mineral e monensina. Adicionalmente, foi avaliada a degradabilidade in situ da matéria seca e da fibra em detergente neutro do feno de aveia. Não houve diferença sobre produção total de ácidos graxos voláteis (AGVs) no rúmen, porcentagem molar dos ácidos acético, propiônico e butírico, relação acético/propiônico, pH ruminal, concentração de N-NH3 no rúmen, fluxo e volume de líquidos do rúmen, nos diferentes tratamentos. A degradabilidade da matéria seca e da fibra em detergente neutro do feno não apresentou diferenças. Não se constatou melhora nos parâmetros de fermentação ruminal com a associação dos grãos.

Effects of feeding a multivalent polyclonal antibody preparation on feedlot performance, carcass characteristics, rumenitis, and blood gas profile in Bos indicus biotype yearling bulls

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of adding polyclonal antibody preparations on ruminal fermentation patterns and digestibility of cows fed different energy sources

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)