Desempenho de frangos de corte de 1 a 21 dias de idade alimentados com rações contendo extrato de leveduras e prebiótico e criados em diferentes temperaturas

The performance of broilers reared at different temperatures and fed diet with or without yeast extract and/or prebiotic in the starter phase was evaluated. One thousand, four hundred and forty 1-d male Cobb-500 (R) were reared at different climatic chambers. The diets with or without yeast extract or prebiotic were fed only in the starter phase (1 to 7 days), and from the 8(th) day on all broiler were fed the same commercial diet. It was used a complete randomized experimental design, as a 3 x 2 x 2 factorial arrangement, with 3 rearing temperatures (high 34 +/- 1 degrees C, control 32 +/- 2 degrees C and low 27 +/- 2 degrees C), 2 yeast extract level (with or without) and 2 prebiotic level (with or without). High and low temperatures decreased the performance of brids at 7 and 21 days old. The inclusion of prebiotic in the pre-starter phase increased weight gain of birds under low temperature at 21 days old and the viability until 21 days old. The inclusion of yeast extract increase feed conversion at 21 days old.

Morphological and quantitative aspects of nodule formation in hemolymph of the blowfly Chrysomya megacephala (Fabricius, 1794)

Insects manifest effective immune responses that include both cellular and humoral components. Morphological and quantitative aspects of cellular and Immoral cooperation during nodule formation in Chrysomya megacephala hemolymph against Saccharomyces cerevisae yeast cells were demonstrated for the first time. The analyses were performed in non-injected larvae (NIL), saline-injected larvae (SIL) and yeast-injected larvae (YIL). The hemolymph of injected groups was collected 0.5, 1, 2, 4, 12, 24, 36, or 48-h post-injection. Morphological aspects of YIL nodulation were investigated using transmission electron microscopy (TEM). Quantitative analyses consisted of total (THC) and differential hemocyte counts (DHC) in all the groups and total yeast count (TYC) in YIL, which were performed in an improved Neubauer chamber. Nodule formation was initiated at approximately 2-h post-injection. Twelve hours after the injection, TEM revealed the presence of an amorphous membrane, at the same time that circulating hemocyte number decreased significantly contrasting the increase of yeast number. Our results showed the ability of C megacephala hemolymph to perform humoral encapsulation when hemocyte population is insufficient to eliminate the microorganisms, warranting consideration in future investigations on the relative roles played by cellular and humoral elements of innate immunity of this calliphorid. (c) 2007 Elsevier B.V. All rights reserved.

Mutational analyses of human eIF5A-1-identification of amino acid residues critical for eIF5A activity and hypusine modification

The eukaryotic translation initiation factor 5A (eIF5A) is the only protein that contains hypusine [N-epsilon-(4-amino-2-hydroxybutyl)lysine], which is required for its activity. Hypusine is formed by post-translational modification of one specific lysine (Lys50 for human eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1, with a single amino acid substitution at the highly conserved residues or with N-terminal or C-terminal truncations, and tested mutant proteins in complementing the growth of a Saccharomyces cerevisiae eIF5A null strain. Growth-supporting activity was abolished in only a few mutant eIF5As (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A), with substitutions at or near the hypusine modification site or with truncation of 21 amino acids from either the N-terminus or C-terminus. The inactivity of the Lys50 substitution proteins is obviously due to lack of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A activity, possibly in its interaction with effector(s). By use of a UBHY-R strain harboring genetically engineered unstable eIF5A, we present evidence for the primary function of eIF5A in protein synthesis. When selected eIF5A mutant proteins were tested for their activity in protein synthesis, a close correlation was observed between their ability to enhance protein synthesis and growth, lending further support for a central role of eIF5A in translation.

Disulfide Biochemistry in 2-Cys Peroxiredoxin: Requirement of Glu50 and Arg146 for the Reduction of Yeast Tsa1 by Thioredoxin

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

Application of methylotrophic yeast Pichia pastoris in the field of food industry - A review

Since ancient times, the utilization of yeasts by the man has a great impact on the socio-economic development. After the advent of the technology of recombinant DNA, great advances have occurred due to the acquisition of strains of mutant yeasts in the field of applied research, and Saccharomyces cerevisiae has soon been outstanding as an interesting candidate for the expression of heterologous proteins of biotechnological interest. As the time goes by other alternative systems of expression have been shown because they have advantages over Saccharomyces cerevisiae. Among those new systems, Pichia pastoris is outstanding as methylotrophic yeast capable of growing in a culture medium containing methanol as the only source of carbon and energy. The induction of production of glycerol-3-phosphate dehydrogenase (GPD, NAD(+): oxido-redutase EC 1.1. 1.8) by Pichia pastoris was accomplished in the medium containing methanol. One of the most important key parameters in Pichia pastoris expression system is the methanol concentration. Bibliographic reviews on the Pichia pastoris production system have shown that the best culture conditions vary according to the strain used and/or kind of heterologous protein desired to be expressed. Therefore, we have sought to develop a system, involving expression of glycerol-3-phosphate dehydrogenase in the yeast Pichia pastoris, for generating sufficient quantities of the enzyme in order to asses its potential value for use in various food bioanalytical determination. Dehydrogenases have been widely used in the enzymatic assays of diverse composites of industrial interest, being enclosed among them glycerol and a number of important bioanalytical applications.

Persistence and Metabolism of Folpet in Grapes and Wine

The fate of folpet from the treatment on vine to the production of wine was studied. Sunlight degraded folpet to unknown products. Phthalimide was a minor metabolite formed on grapes from folpet. Folpet degraded in must, giving 80% phthalimide; the results obtained with model solutions showed that in must folpet can also give small amounts of phthalic acid. During wine-making folpet degraded completely, and at the end of fermentation phthalimide was only present in wine. This compound was stable in wine after several months. The presence of folpet in grapes inhibited the alcoholic fermentation of Saccharomyces cerevisiae and Kloeckera apiculata completely. Phthalimide, on the contrary, had no negative effect on the fermentative action of the two yeasts. GC and HPLC methods were developed to determine folpet and its metabolites.

During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1

In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (=GGS1=FDP1=BYP1=CIF1=GLC6=TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and tile wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of D-glucose and an equal concentration of radiolabelled L-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total B-glucose measured (intracellular + periplasmic/extracellular) and the total L-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mM-glucose to 0.5-2 mM in the wild-type strain, ± 10 mm in a hxk1Δ hxk2Δ glk1Δ and 2-3 mM in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict Properly up to 50-fold higher hexokinase activity.

Preparation of N2, N2,7-trimethylguanosine affinity columns

2,2,7-trimethylguanosine (TMG) binding proteins from human cells were purified through TMG-affinity columns. TMG synthesis was improved and the TMG obtained was shown to be similar to the TMG in the 5' cap of the UsnRNAs. The eluates obtained with TMG-affinity chromatographies were very different from those isolated with m7G-affinity columns, thus suggesting that specific TMG- binding proteins were obtained. The fraction may be enriched with factors associated with import and/or hypermethylation of UsnRNPs.

Arginine methylation and binding of Hrp1p to the efficiency element for mRNA 3'-end formation

Hrp1p is a heterogeneous ribonucleoprotein (hnRNP) from the yeast Saccharomyces cerevisiae that is involved in the cleavage and polyadenylation of the 3'-end of mRNAs and mRNA export. In addition, Hrp1p is one of several RNA-binding proteins that are posttranslationally modified by methylation at arginine residues. By using-functional recombinant Hrp1p, we have identified RNA sequences with specific high affinity binding sites. These sites correspond to the efficiency element for mRNA 3'-end formation, UAUAUA. To examine the effect of methylation on specific RNA binding, purified recombinant arginine methyltransferase (Hmt1p) was used to methylate Hrp1p. Methylated Hrp1p binds with the same affinity to UAUAUA-containing RNAs as unmethylated Hrp1p indicating that methylation does not affect specific RNA binding. However, RNA itself inhibits the methylation of Hrp1p and this inhibition is enhanced by RNAs that specifically bind Hrp1p. Taken together, these data support a model in which protein methylation occurs prior to protein-RNA binding in the nucleus.

Conserved sequences in the β subunit of archaeal and eukaryal translation initiation factor 2 (elF2), absent from elF5, mediate interaction with elF2γ

The eukaryotic translation initiation factor 2 (eIF2) binds the methionyl-initiator tRNA in a GTP-dependent mode. This complex associates with the 40 S ribosomal particle, which then, with the aid of other factors, binds to the 5' end of the mRNA and migrates to the first AUG codon, where eIF5 promotes GTP hydrolysis, followed by the formation of the 80 S ribosome. Here we provide a comparative sequence analysis of the β subunit of eIF2 and its archaeal counterpart (aIF2β). aIF2β differs from eIF2β in not possessing an N-terminal extension implicated in binding RNA, eIF5 and eIF2B. The remaining sequences are highly conserved, and are shared with eIF5. Previously isolated mutations in the yeast eIF2β, which allow initiation of translation at UUG codons due to the uncovering of an intrinsic GTPase activity in eIF2, involve residues that are conserved in aIF2β, but not in eIF5. We show that the sequence of eIF2B homologous to aIF2β is sufficient for binding eIF2γ, the only subunit with which it interacts, and comprises, at the most, 78 residues, eIF5 does not interact with eIF2γ, despite its similarity with eIF2β, probably because of a gap in homology in this region. These observations have implications for the evolution of the mechanism of translation initiation.

Genetic interactions of yeast eukaryotic translation initiation factor 5a (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIFSA domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIFSA may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.

Mapping eIF5A binding sites for Dys1 and Lia1: In vivo evidence for regulation of eIF5A hypusination

The evolutionarily conserved factor eIF5A is the only protein known to undergo hypusination, a unique posttranslational modification triggered by deoxyhypusine synthase (Dys1). Although eIF5A is essential for cell viability, the function of this putative translation initiation factor is still obscure. To identify eIF5A-binding proteins that could clarify its function, we screened a two-hybrid library and identified two eIF-5A partners in S. cerevisiae: Dys1 and the protein encoded by the gene YJR070C, named Lia1 (Ligand of eIF5A). The interactions were confirmed by GST pulldown. Mapping binding sites for these proteins revealed that both eIF5A domains can bind to Dys1, whereas the C-terminal domain is sufficient to bind Lia1. We demonstrate for the first time in vivo that the N-terminal α-helix of Dys1 can modulate enzyme activity by inhibiting eIF5A interaction. We suggest that this inhibition be abrogated in the cell when hypusinated and functional eIF5A is required. © 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Screening for yeast with antibacterial properties from an ethanol distillery

A general screening for the expression of antibacterial activity and non-flocculating type of yeast strains from must and fermented broth of alcohol distilleries was performed. From 60 strains only Saccharomyces sp. M26 presented a inhibitory halo in Lactobacillus fermentum culture and significant reduction in the culture turbidity (71%) and specific growth rate (56%) when compared to the control. Freezing did not affect the antibacterial activity of the Saccharomyces sp. M26 extract and heating at 90°C for 20 min completely destroyed this activity. It is expected the decrease of lactic acid bacteria growth in the S. cerevisiae alcoholic fermentation should allow for better control of these bacteria in the process. © 2003 Elsevier Ltd. All rights reserved.

COX24 codes for a mitochondrial protein required for processing of the COX1 transcript

In most strains of Saccharomyces cerevisiae the mitochondrial gene COX1, for subunit 1 of cytochrome oxidase, contains multiple exons and introns. Processing of COX1 primary transcript requires accessory proteins factors, some of which are encoded by nuclear genes and others by reading frames residing in some of the introns of the COX1 and COB genes. Here we show that the low molecular weight protein product of open reading frame YLR204W, for which we propose the name COX24, is also involved in processing of COX1 RNA intermediates. The growth defect of cox24 mutants is partially rescued in strains harboring mitochondrial DNA lacking introns. Northern blot analyses of mitochondrial transcripts indicate cox24 null mutants to be blocked in processing of introns aI2 and aI3. The dependence of intron aI3 excision on Cox24p is also supported by the growth properties of the cox24 mutant harboring mitochondrial DNA with different intron compositions. The intermediate phenotype of the cox24 mutant in the background of intronless mitochondrial DNA, however, suggests that in addition to its role in splicing of the COX1 pre-mRNA, Cox24p still has another function. Based on the analysis of a cox14-cox24 double mutant, we propose that the other function of Cox24p is related to translation of the COX1 mRNA. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.