The Trypanosoma cruzi proteins TcCox10 and TcCox15 catalyze the formation of heme A in the yeast Saccharomyces cerevisiae

Trypanosoma cruzi, the etiologic agent for Chagas` disease, has requirements for several cofactors, one of which is heme. Because this organism is unable to synthesize heme, which serves as a prosthetic group for several heme proteins (including the respiratory chain complexes), it therefore must be acquired from the environment. Considering this deficiency, it is an open question as to how heme A, the essential cofactor for eukaryotic CcO enzymes, is acquired by this parasite. In the present work, we provide evidence for the presence and functionality of genes coding for heme O and heme A synthases, which catalyze the synthesis of heme O and its conversion into heme A, respectively. The functions of these T. cruzi proteins were evaluated using yeast complementation assays, and the mRNA levels of their respective genes were analyzed at the different T. cruzi life stages. It was observed that the amount of mRNA coding for these proteins changes during the parasite life cycle, suggesting that this variation could reflect different respiratory requirements in the different parasite life stages.

Biochemical characterization of serine acetyltransferase and cysteine desulfhydrase from Leishmania major

Cysteine metabolism exhibits atypical features in Leishmania parasites. The nucleotide sequence annotated as LmjF32.2640 encodes a cysteine desulfhydrase, which specifically catalyzes the breakdown of cysteine into pyruvate, NH(3) and H(2)S. Like in other pathogens, this capacity might be associated with regulatory mechanisms to control the intracellular level of cysteine, a highly toxic albeit essential amino acid, in addition to generate pyruvate for energy production. Besides, our results provide the first insight into the biochemical properties of Leishmania major serine acetyltransferase (SAT), which is likely involved in the two routes for de novo synthesis of cysteine in this pathogen. When compared with other members of SAT family, the N-terminal region of L. major homologue is uniquely extended, and seems to be essential for proper protein folding. Furthermore, unlike plant and bacterial enzymes, the carboxy-terminal-C(10) sequence stretch of L major SAT appears not to be implicated in forming a tight bi-enzyme complex with cysteine synthase. (C) 2010 Elsevier B.V. All rights reserved.

Trypanosome Prereplication Machinery Contains a Single Functional Orc1/Cdc6 Protein, Which Is Typical of Archaea

In unicellular eukaryotes, such as Saccharomyces cerevisiae, and in multicellular organisms, the replication origin is recognized by the heterohexamer origin recognition complex (ORC) containing six proteins, Orc1 to Orc6, while in members of the domain Archaea, the replication origin is recognized by just one protein, Orc1/Cdc6; the sequence of Orc1/Cdc6 is highly related to those of Orc1 and Cdc6. Similar to Archaea, trypanosomatid genomes contain only one gene encoding a protein named Orc1. Since trypanosome Orc1 is also homologous to Cdc6, in this study we named the Orc1 protein from trypanosomes Orc1/Cdc6. Here we show that the recombinant Orc1/Cdc6 from Trypanosoma cruzi (TcOrc1/Cdc6) and from Trypanosoma brucei (TbOrc1/Cdc6) present ATPase activity, typical of prereplication machinery components. Also, TcOrc1/Cdc6 and TbOrc1/Cdc6 replaced yeast Cdc6 but not Orc1 in a phenotypic complementation assay. The induction of Orc1/Cdc6 silencing by RNA interference in T. brucei resulted in enucleated cells, strongly suggesting the involvement of Orc1/Cdc6 in DNA replication. Orc1/Cdc6 is expressed during the entire cell cycle in the nuclei of trypanosomes, remaining associated with chromatin in all stages of the cell cycle. These results allowed us to conclude that Orc1/Cdc6 is indeed a member of the trypanosome prereplication machinery and point out that trypanosomes carry a prereplication machinery that is less complex than other eukaryotes and closer to archaea.

Use of L-Proline and ATP Production by Trypanosoma cruzi Metacyclic Forms as Requirements for Host Cell Invasion

The process of host cell invasion by Trypanosoma cruzi depends on parasite energy. What source of energy is used for that event is not known. To address this and other questions related to T. cruzi energy requirements and cell invasion, we analyzed metacyclic trypomastigote forms of the phylogenetically distant CL and G strains. For both strains, the nutritional stress experienced by cells starved for 24, 36, or 48 h in phosphate-buffered saline reduced the ATP content and the ability of the parasite to invade HeLa cells proportionally to the starvation time. Inhibition of ATP production by treating parasites with rotenone plus antimycin A also diminished the infectivity. Nutrient depletion did not alter the expression of gp82, the surface molecule that mediates CL strain internalization, but increased the expression of gp90, the negative regulator of cell invasion, in the G strain. When L-proline was given to metacyclic forms starved for 36 h, the ATP levels were restored to those of nonstarved controls for both strains. Glucose had no such effect, although this carbohydrate and L-proline were transported in similar fashions. Recovery of infectivity promoted by L-proline treatment of starved parasites was restricted to the CL strain. The profile of restoration of ATP content and gp82-mediated invasion capacity by L-proline treatment of starved Y-strain parasites was similar to that of the CL strain, whereas the Dm28 and Dm30 strains, whose infectivity is downregulated by gp90, behaved like the G strain. L-Proline was also found to increase the ability of the CL strain to traverse a gastric mucin layer, a property important for the establishment of T. cruzi infection by the oral route. Efficient translocation of parasites through gastric mucin toward the target epithelial cells in the stomach mucosa is an essential requirement for subsequent cell invasion. By relying on these closely associated ATP-driven processes, the metacyclic trypomastigotes effectively accomplish their internalization.

Effect of 3,4-ethylenedioxy-extension of thiophene core on the DNA/RNA binding properties and biological activity of bisbenzimidazole amidines

Novel bisbenzimidazoles (4-6), characterized by 3,4-ethylenedioxy-extension of thiophene core, revealed pronounced affinity and strong thermal stabilization effect toward ds-DNA. They interact within ds-DNA grooves as dimmers or even oligomers and agglomerate along ds-RNA. Compounds 4-6 have shown moderate to strong antiproliferative effect toward panel of eight carcinoma cell lines. Compound 5 displayed the best inhibitory potential and in equitoxic concentration (IC(50) = 1 x 10 (6) M) induced accumulation of cells in G2/M phase after 48 h of incubation. Fluorescence microscopy showed that 5 entered into live HeLa cells within 30 min, but did not accumulate in nuclei even after 2.5 h. Compound 5 inhibited the growth of Trypanosome cruzi epimastigotes (IC(50) = 4.3 x 10 (6) M). (C) 2009 Elsevier Ltd. All rights reserved.

L-Proline uptake in Crithidia deanei is influenced by its endosymbiont bacterium

Crithidia deanei, a monoxenic trypanosomatid, presents an endosymbiotic bacterium in its cytoplasm. Both the protozoan and the bacterium maintain intensive metabolic exchange, resulting in an interesting model to study the coevolution of metabolisms. The relevance of L-proline for the growth of C. deanei and its transport into these cells was studied. Both the endosymbiont-containing (wild) and the endosymbiont-free protozoa (aposymbiont or cured) strains, when grown in medium supplemented with L-proline, reached higher cell densities than those grown in unsupplemented media. We biochemically characterized the uptake of L-proline in both the wild (K(m)=0.153 +/- 0.022 mM, V(max)=0.239 +/- 0.011 nmol min(-1) per 4 x 10(7) cells) and the aposymbiont strains (K(m)=0.177 +/- 0.049 mM, V(max)=0.132 +/- 0.012 nmol min(-1) per 4 x 10(7) cells). These data suggest a single type of proline transporter whose activity is upregulated by the presence of the symbiotic bacterium. Proline transport was further characterized and was found to be insensitive to the extracellular concentration of Na(+), but sensitive to K(+) and pH. The abolition of proline uptake by respiratory chain inhibitors and valinomycin indicates that the proline transport in C. deanei is dependent on the plasma membrane K(+) gradient.

Glucose uptake in the mammalian stages of Trypanosoma cruzi

Trypanosoma cruzi, the agent of Chagas` disease, alternates between different morphogenetic stages that face distinct physiological conditions in their invertebrate and vertebrate hosts, likely in the availability of glucose. While the glucose transport is well characterized in epimastigotes of T cruzi, nothing is known about how the mammalian stages acquire this molecule. Herein glucose transport activity and expression were analyzed in the three developmental stages present in the vertebrate cycle of T cruzi. The infective trypomastigotes showed the highest transport activity (V(max) = 5.34 +/- 0.54 nmol/min per mg of protein: K(m) = 0.38 +/- 0.01 mM) when compared to intracellular epimastigotes (V(max) = 2.18 +/- 0.20 nmol/min per mg of protein; K(m) = 0.39 +/- 0.01 mM). Under the conditions employed no transport activity could be detected in amastigotes. The gene of the glucose transporter is expressed at the mRNA level in trypomastigotes and in intracellular epimastigotes but not in amastigotes, as revealed by real-time PCR. In both trypomastigotes and intracellular epimastigotes protein expression could be detected by Western blot with an antibody raised against the glucose transporter correlating well with the transport activity measured experimentally. Interestingly, anti-glucose transporter antibodies showed a strong reactivity with glycosome and reservosome organelles. A comparison between proline and glucose transport among the intracellular differentiation forms is presented. The data suggest that the regulation of glucose transporter reflects different energy and carbon requirements along the intracellular life cycle of T cruzi. (C) 2009 Elsevier B.V. All rights reserved.

Eficiência de nematoides entomopatogênicos e inseticidas químicos contra Sphenophorus levis e Leucothyreus sp. em cana-de-açúcar

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

Disponibilidade biológica do fósforo de diferentes fontes para eqüinos em crescimento

Objetivou-se neste estudo, determinar a disponibilidade biológica do P de diferentes fontes, para eqüinos em fase de crescimento. Utilizaram-se dezesseis eqüinos machos em fase de crescimento, submetidos à aplicação de quatro fontes fosfatadas -- fosfato de rocha de Tapira (TAP), fosfato de rocha de Patos de Minas (PAT), fosfato bicálcico (BIC) e farinha de osso (FOS) --, adicionadas à dieta basal em quantidades suficientes para fornecer 22 g de P/animal/dia. No 16º dia, foram-lhes injetados 30 MBq de 32P/animal, e coletaram-se amostras de sangue, fezes e urina, durante sete dias. Foram determinadas as atividades específicas no plasma, fezes e urina e calculou-se a perda endógena fecal e a absorção real de P. Os valores obtidos quanto ao P consumido, P excretado, P no plasma e P retido não apresentaram diferenças estatísticas (P>0,05). Os valores de absorção real do P do TAP, PAT, BIC e da FOS foram, respectivamente, 25,23%, 33,97%, 31,71% e 29,36%. Não houve diferenças estatísticas (P>0,05) entre as fontes estudadas. em relação ao BIC, as rochas fosfáticas apresentaram altos valores de disponibilidade biológica.

Perda endógena e absorção real de fósforo em dietas para eqüinos em crescimento

O trabalho teve por objetivo avaliar os efeitos de diferentes níveis de P na dieta de eqüinos em crescimento sobre sua perda endógena fecal, e verificar qual seja a exigência mínima diária desse elemento na sua alimentação. Foram utilizados 16 eqüinos machos em crescimento, recebendo dieta basal sem suplementação de P, e dieta basal suplementada com fosfato bicálcico, para fornecer 15, 20 e 25 g P/animal/dia. No 16º dia experimental, foram injetados 30 MBq de 32P/animal e coletaram-se amostras de sangue, fezes e urina, durante sete dias. Foram determinadas as atividades específicas do P no plasma, nas fezes e na urina, e calculou-se a perda endógena fecal e a absorção real de P. A perda endógena fecal e a absorção real de P não foram afetadas (P>0,05) pelos tratamentos, e foram estimados, em média, 10,34 mg P/kg PV/dia e 47,07%, respectivamente, o que indica que, nas condições experimentais, animais com idade média de 19 meses necessitam de 21,96 mg de P/kg PV/dia para manter o balanço da perda metabólica fecal, e a quantidade diária de P de 14,75 g.

Exigência mínina de fósforo em novilhos da raça Nelore

A determinação da absorção real de fósforo (P) em bovinos deve levar em consideração a fração endógena mínina do mineral, que se perde nas fezes. de modo geral esses cálculos são feitos utilizando-se tabelas cujos valores foram obtidos em outros países, com outras raças de animais e em condições bem diferentes das condições brasileiras. O trabalho teve como objetivo determinar a perda endógena de P nas fezes e estimar a exigência mínima de P em novilhos da raça Nelore (Bos indicus). Foram utilizados 18 novilhos castrados, com peso médio de 190,82±27,53 kg e idade aproximada de 12 meses, divididos em três grupos de seis animais, e mantidos em gaiolas metabólicas individuais. Os animais receberam dieta básica constituída de feno de Brachiaria decumbens e uma mistura de concentrados durante os 30 dias de período experimental. Os tratamentos consistiram de diferentes quantidades de fosfato bicálcico em níveis de 0,12, 0,24 e 0,36% de P, com base na dieta total. Foram aplicadas injeções de 32P nos animais para a determinação da perda endógena fecal de P. A perda endógena mínima fecal de P para uma ingestão zero do mineral, calculada por interpolação, foi de 5,72 mg/kg de peso vivo e para um balanço zero, o requerimento mínimo foi de 8,84 mg/kg de peso vivo por dia.

Efeito da seleção quanto ao peso corporal nas características reprodutivas em linhagens de frangos de corte

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Resídue de polpas de frutas desidratadas na alimentação de leitões em fase de creche

It was evaluated chemically and biologically the powdered fruits pulp residue, used in human food industry. In the digestibility study it was used 12 commercial hybrids barrows piglets, with initial weight 12.2 ± 1,6 kg, allotted in individual cages. The treatments were a basal and a test diet. In the test diet the fruits pulp residue replaced 30% of the basal dry matter. The following values were obtained: dry matter 89,54%, starch 71,1%, glucose 5.4%, fructose 2,2%, crude protein 5,33%, gross energy 3771 kcal/kg, apparent digestible dry matter 96,01%, digestible energy 3448 kcal/kg, metabolizable energy 3389 kcal/kg. By bromatologic results and metabolism study, the powdered fruits pulp residue was characterized as an alternative to be evaluated in piglet diets. In the performance assay 90 piglets with initial weight of 6,60 ± 0,76 kg were allotted in a randomized block design, with six replications and three animals per experimental unit. The treatments were levels of powdered fruits pulp residue (0. 25, 50, 75 and 100%) replacing the corn of the control diet. The studied phases were initial-1 (14 days), initial-2 (21 days) and total period. On the performance there was no difference between the studied inclusion levels. For meal diets, the fruits pulp residue can replace the corn.

Setores exportador e importador da India

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