Composição química, valores de energia metabolizável e aminoácidos digestíveis de subprodutos do arroz para frangos de corte

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

Sunflower meal in commercial layer diets formulated on total and digestible amino acids basis

An experiment was conduced to evaluate the inclusion of sunflower meal (SBM) in commercial layer diets formulated on total or digestible amino acids basis. One hundred forty-four 41-week-old Lohmann LSL layers were distributed in a completely randomized experimental design in a 2 x 4 factorial arrangement with three replications of six birds each. Treatments consisted of a combination of four SBM inclusion levels SBM(0%, 4%, 8%, and 12%) and feed formulation according two amino acid recommendations (total or digestible). The experimental period was divided into five periods of fourteen days. Performance parameters (egg production, feed intake, feed conversion, egg mass) were evaluated for each period. In the last two days of each period, three eggs per replication were collected to evaluate egg quality (Haugh units, specific gravity, egg weight, eggshell thickness, and eggshell percentage). Hens fed on total amino acid recommendation presented the highest values for egg weight. Diets formulated on digestible amino acids basis showed an improvement in eggshell percentage and egg specific gravity. SBM addition in commercial layer diets did not influence performance; however, increasing SBM dietary levels SBM improved eggshell quality.

Commercial laying hen diets formulated according to different recommendations of total and digestible amino acids

An experiment was conducted to evaluate different commercial laying hen diets formulated based on recommendations for total and digestible amino acids. One hundred and twenty Lohmann LSL commercial laying hens aged 25 weeks were distributed in a completely randomized experimental design involving five replications of six birds in four treatments. Diet formulation on a total amino acid basis followed the recommendations of NRC (1994) and Rostagno et al. (2000), whereas formulation on digestible amino acids basis was according to Rostagno et al. (2000) and Degussa (1997) recommendations. The experimental period was divided into five periods of fourteen days. Performance parameters (egg production, feed intake, feed conversion, egg mass) were evaluated for each period, and on the last two days of each period, three eggs per replication were collected to evaluate egg quality parameters (Haugh unit, egg specific gravity, egg weight, eggshell thickness and percentage). Means were compared by orthogonal contrasts. Results on feed intake, egg production, egg mass, feed conversion and egg specific gravity showed that total amino acid recommendations promoted better bird responses than digestible amino acid recommendations.

EBNA-1 gene sequences in Brazilian and American patients with Hodgkin's disease

We examined the types of Epstein-Barr virus-associated nuclear antigen-1 (EBNA-1) gene carboxy (C)-terminal mutations occurring in Hodgkin's disease (HD) and reactive tissues from two different geographic regions. Previously reported EBNA-1 C-terminal region amino acid sequence variants, based on the amino acid at codon 487, include Prototype (P)-ala, which is found in the B95.8-derived prototype virus, P-thr, Variant (V)-leu, V-val, and V-pro. Using polymerase chain reaction (PCR) to amplify portions of the EBNA-1 gene, followed by DNA sequencing, we found a single EBNA-1 gene sequence variant in each tissue, whether reactive or neoplastic and whether from Brazil or the United States. Variant EBNA-1 gene sequences were more common in both neoplastic and non-neoplastic tissues from different geographic areas than the so-called prototype sequence. In the 17 Brazilian HD cases, 4 cases had P-thr variants and 13 had V-leu variants. In the six reactive tissues from Brazil, one had a P-ala variant, two had P-thr variants, and three had V-leu variants. In the 12 American HD cases, 2 had P-ala variants, 6 had P-thr variants, and 4 had V-leu variants. The 11 American reactive tissues included 2 P ala variants, 5 P-thr variants, and 4 V-leu variants. In both countries, there were similar variant EBNA-1 sequences present in normal tissues and HD cases. Compared with the P ala and P-thr cases, the V-leu cases were more likely to have the 30-bp latent membrane protein 1 (LMP1) gene deletion (P = 0.0075). In addition, cases of HD with the V-leu were statistically associated with a substitution of asparagine for glutamine at codon 322 of the C-terminal portion of the LMP1 gene. Our results suggest that any variation in EBNA-1 gene sequence is caused by a polymorphism present in pre-existing viral strains in the underlying population, and not a mutation occurring during oncogenesis. (C) 1999 by the American Society of Hematology.

Single nucleotide polymorphisms in the bovine genome are associated with the number of oocytes collected during ovum pick up

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

Mutational analysis of Arabidopsis thaliana plant uncoupling mitochondrial protein

In this study, point mutations were introduced in plant uncoupling mitochondrial protein AtUCP1, a typical member of the plant uncoupling protein (UCP) gene subfamily, in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and in two more residues, Cys28 and His83, specific for plant UCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted proteoliposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to linoleic acid (LA) whereas His83 plays an important role in AtUCP1 transport activity. Residues Cys28, Lys147, and also Tyr269 are probably essential for correct protein function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to inhibitors (purine nucleotides). Interestingly, Cys28 substitution reduced ATP inhibitory effect on AtUCP1, while Tyr269Phe mutant exhibited 2.8-fold increase in sensitivity to ATP, in accordance with the reverse mutation Phe267Tyr of mammalian UCP1. (C) 2007 Elsevier B.V. All fights reserved.

Interaction of shikimic acid with shikimate kinase (Retracted Article. See vol 334, pg 967, 2005)

The crystal structure of shikimate kinase from Mycobacterium tuberculosis (MtSK) complexed with MgADP and shikimic acid (shikimate) has been determined at 2.3 Angstrom resolution, clearly revealing the amino acid residues involved in shikimate binding. In MtSK, the Glu61 strictly conserved in SK forms a hydrogen bond and salt-bridge with Arg58 and assists in positioning the guanidinium group of Arg58 for shikimate binding. The carboxyl group of shikimate interacts with Arg58, Gly81, and Arg136, and hydroxyl groups with Asp34 and Gly80. The crystal structure of MtSK-MgADP-shikimate will provide crucial information for elucidation of the mechanism of SK-catalyzed reaction and for the development of a new generation of drugs against tuberculosis. (C) 2004 Elsevier B.V. All rights reserved.

Structure of shikimate kinase from Mycobacterium tuberculosis reveals the binding of shikimic acid

Tuberculosis made a resurgence in the mid-1980s and now kills approximately 3 million people a year. The re-emergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons and the proliferation of multi-drug-resistant strains have created a need to develop new drugs. Shikimate kinase and other enzymes in the shikimate pathway are attractive targets for development of non-toxic antimicrobial agents, herbicides and anti-parasitic drugs, because the pathway is essential in these species whereas it is absent from mammals. The crystal structure of shikimate kinase from Mycobacterium tuberculosis (MtSK) complexed with MgADP and shikimic acid ( shikimate) has been determined at 2.3 Angstrom resolution, clearly revealing the amino-acid residues involved in shikimate binding. This is the first three-dimensional structure of shikimate kinase complexed with shikimate. In MtSK, the Glu61 residue that is strictly conserved in shikimate kinases forms a hydrogen bond and salt bridge with Arg58 and assists in positioning the guanidinium group of Arg58 for shikimate binding. The carboxyl group of shikimate interacts with Arg58, Gly81 and Arg136 and the hydroxyl groups interact with Asp34 and Gly80. The crystal structure of MtSK-MgADP-shikimate will provide crucial information for the elucidation of the mechanism of the shikimate kinase-catalyzed reaction and for the development of a new generation of drugs against tuberculosis.

Photoacoustic spectroscopy of aromatic amino acids in proteins

This paper concerns the use of photoacoustic spectroscopy (PAS) to study the presence of aromatic amino acid in proteins. We examined the aromatic amino acids in six proteins with well-known structures using absorption spectra of near ultraviolet PAS over the wavelength range 240-320 nm. The fundamental understanding of the physical and chemical properties that govern the absorption of light and a subsequent release of heat to generate a transient pressure wave was used to test the concept of monitoring aromatic amino acids with this method. Second derivative spectroscopy in the ultraviolet region of proteins was also used to study the regions surrounding the aromatics and the percentage area in each band was related in order to determine the contribution in function of the respective molar extinction coefficients for each residue. Further investigation was conducted into the interaction between sodium dodecyl sulphate (SDS) and bothropstoxin-I (BthTx-I), with the purpose of identifying the aromatics that participate in the interaction. The clear changes in the second derivative and curve-fitting procedures suggest that initial SDS binding to the tryptophan located in the dimer interface and above 10 SDS an increased intensity between 260 and 320 nm, demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interactions. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scattering is significant.

The activity of amino acids produced by Paenibacillus macerans and from commercial sources against the root-knot nematode Meloidogyne exigua

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular determinants of binding of a wasp toxin (PMTXs) and its analogs in the Na+ channels proteins

The structural specificity of alpha-PMTX, a novel peptide toxin derived from wasp venom has been studied on the neuromuscular synapse in the walking leg of the lobster. alpha-PMTX is known to induce repetitive action potentials in the presynaptic axon due to sodium channel inactivation. We synthesized 29 analogs of alpha-PMTX by substituting one or two amino acids and compared threshold concentrations of these mutant toxins for inducing repetitive action potentials. In 13 amino acid residues of alpha-PMTX, Arg-1, Lys-3 and Lys-12 regulate the toxic activity because substitution of these basic amino acid residues with other amino acid residues greatly changed the potency. Determining the structure-activity relationships of PMTXs will help clarifying the molecular mechanism of sodium channel inactivation. (C) 2000 Elsevier B.V. Ireland Ltd. All rights reserved.

Identification and characterization of novel mutations of the aspartoacylase gene in non-Jewish patients with Canavan disease

Canavan disease, an inherited leukodystrophy, is caused by mutations in the aspartoacylase (ASPA) gene. It is most common among children of Ashkenazi Jewish descent but has been diagnosed in many diverse ethnic groups. Two mutations comprise the majority of mutant alleles in Jewish patients, while mutations in the ASPA gene among non-Jewish patients are different and more diverse. In the present study, the ASPA gene was analysed in 22 unrelated non-Jewish patients with Canavan disease, and 24 different mutations were found. of these,14 are novel, including five missense mutations (E24G, D68A, D249V, C152W, H244R), two nonsense mutations (Q184X, E214X), three deletions (923delT, 33del13, 244delA), one insertion mutation (698insC), two sequence variations in one allele ([10T>G; 11insG]), an elimination of the stop codon (941A>G, TAG-->TGG, X314W), and one splice acceptor site mutation (IVS1 - 2A>T). The E24G mutation resulted in substitution of an invariable amino acid residue (Glu) in the first esterase catalytic domain consensus sequence. The IVS1 - 2A>T mutation caused the retention of 40 nucleotides of intron 1 upstream of exon 2. The results of transient expression of the mutant ASPA cDNA containing these mutations in COS-7 cells and assays for ASPA activity of patient fibroblasts indicated that these mutations were responsible for the enzyme deficiency. In addition, patients with the novel D249V mutation manifested clinically at birth and died early. Also, patients with certain other novel mutations, including C152W, E214X, X314W, and frameshift mutations in both alleles, developed clinical manifestations at an earlier age than in classical Canavan disease.

Kinetic and mechanistic characterization of the Sphingomyelinases D from Loxosceles intermedia spider venom

Envenomation by arachnids of the genus Loxosceles leads to local dermonecrosis and serious systemic toxicity mainly induced by sphingomyelinases D (SMase D). These enzymes catalyze the hydrolysis of sphingomyelin resulting in the formation of ceramide-phosphate and choline as well as the cleavage of lysophosphatidyl choline generating the lipid mediator lysophosphatidic acid. We have, previously, cloned and expressed two functional SMase D isoforms, named P1 and P2, from Loxosceles intertnedia venom and comparative protein sequence analysis revealed that they are highly homologous to SMase I from Loxosceles laeta which folds to form an (alpha/beta)(8) barrel. In order to further characterize these proteins, pH dependence kinetic experiments and chemical modification of the two active SMases D isoforms were performed. We show here that the amino acids involved in catalysis and in the metal ion binding sites are strictly conserved in the SMase D isoforms from L. intermedia. However, the kinetic studies indicate that SMase P1 hydrolyzes sphingomyelin less efficiently than P2, which can be attributed to a substitution at position 203 (Pro-Leu) and local amino acid substitutions in the hydrophobic channel that could probably play a role in the substrate recognition and binding. (c) 2005 Elsevier Ltd. All rights reserved.

Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP Two coding substitutions were found in comparison With the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429] PLAP, into wildtype Chinese hamster ovary cells. We determined the k(cat) and K-m, of the PLAP S, F. and D allozymes using the non,physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5'-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples. Hum Mutat 19:258-267, 2002. (C) 2002 Wiley-Liss, Inc.

Aspartic acid racemization dating of Holocene brachiopods and bivalves from the southern Brazilian shelf, South Atlantic

The extent of racemization of aspartic acid (Asp) has been used to estimate the ages of 9 shells of the epifaunal calcitic brachiopod Bouchardia rosea and 9 shells of the infaunal aragonitic bivalve Semele casali. Both taxa were collected concurrently from the same sites at depths of 10 m and 30 m off the coast of Brazil. Asp D/L values show an excellent correlation with radiocarbon age at both sites and for both taxa (r(Site)(2) (9) (B. rosea) = 0.97 r(Site)(2) (1) (B.) (rosea) = 0.997, r(Site)(2) (9) (S.) (casali) = 0.9998, r(2) (Site) (1) (S.casali) = 0.93). The Asp ratios plotted against reservoir-corrected AMS radiocarbon ages over the time span of multiple millennia can thus be used to develop reliable and precise geochronologies not only for aragonitic mollusks (widely used for dating previously), but also for calcitic brachiopods. At each collection site, Bouchardia specimens display consistently higher D/L values than specimens of Semele. Thermal differences between sites are also notable and in agreement with theoretical expectations, as extents of racemization for both taxa are greater at the warmer, shallower site than at the cooler, deeper one. In late Holocene marine settings, concurrent time series of aragonitic and calcitic shells can be assembled using Asp racemization dating, and parallel multi-centennial to multi-millennial records can be developed simultaneously for multiple biomineral systems. (c) 2006 University of Washington. All rights reserved.