Doryctopambolus nunes & Zaldívar-Riverón (Braconidae), a new Neotropical doryctine wasp genus with propodeal spines

The new Neotropical doryctine genus Doryctopambolus gen. n. is erected to contain D. pilcomayensis (van Achterberg & Braet, 2004), comb. n., which was previously placed within Pambolus (Pambolinae), as well as three new species, D. clebschi sp. n., D. dominicanus sp. n. and D. sarochensis sp. n. Membersof this new genus are mainly characterised by the presence of at least one pair of conspicuous propodeal apico-lateral projections, which are similar to those present in all members of Pambolinae and in species of three Australasian doryctine genera. We generated DNA barcoding sequences for the three newly described species. We discuss the morphological similarity between species of the Australasian Echinodoryctes Belokobylskij, Iqbal & Austin and Doryctopambolus. A key for the described species of Doryctopambolus is provided.Fil: Nunes, Juliano Fiorelini. Universidade Federal de Minas Gerais; BrasilFil: Zaldívar Riverón, Alejandro. Universidad Nacional Autónoma de México; MéxicoFil: de Castro, Clóvis Sormus. Universidade Federal do São Carlos; BrasilFil: Marsh, Paul M.. No especifica;Fil: Penteado Dias, Angélica Maria. Universidade Federal do São Carlos; BrasilFil: Briceño, Rosa. Universidad Centroccidental Lissandro Alvarado; VenezuelaFil: Martinez, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”; Argentin

The role of Tyr(605) and Ala(607) of thimet oligopeptidase and Tyr(606) and Gly(608) of neurolysin in substrate hydrolysis and inhibitor binding

The physicochemical properties of TOP (thimet oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series AbzGFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X = Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr(605) and Ala(607) in TOP and at Tyr(606) and Gly(608) in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. the kinetic parameters for the hydrolysis of substrates with systematic variations at position P-1 showed that Tyr(605) and Tyr(606) of TOP and NEL respectively, played a role in subsite S-1. Ala(607) of TOP and Gly(608) of NEL contributed to the flexibility of the loops formed by residues 600-612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599-611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P-1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Ty,105 was an important anchor for its interaction with wild-type TOP. the hydroxy groups of Tyr 605 and Tyr.. did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH-k(cat)/K-m dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis.Universidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilInst Butantan, Lab Especial Toxinol Aplicada, CAT, CEPID, BR-05467010 São Paulo, BrazilUniv São Paulo, Inst Ciencias Biomed, Dept Biol Celular & Desenvolvimento, Programa Biol Celular, BR-05508900 São Paulo, BrazilUniv São Paulo, Lab Neurociencias, BR-03071000 São Paulo, BrazilUniv Mogi das Cruzes, CIIB, BR-08780911 Mogi Das Cruzes, SP, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

A structure-based site-directed mutagenesis study on the neurolysin (EC 3.4.24.16) and thimet oligopeptidase (EC 3.4.24.15) catalysis

Neurolysin (EP24.16) and thimet oligopeptidase (EP24.15) are closely related metalloendopeptidases. Site-directed mutagenesis of Tyr(613) (EP24.16) or Tyr(612) (EP24.15) to either Phe or Ala promoted a strong reduction of k(cat)/K-M for both enzymes. These data suggest the importance of both hydroxyl group and aromatic ring at this specific position during substrate hydrolysis by these peptidases. Furthermore, the EP24.15 A607G mutant showed a k(cat)/K-M of 2x10(5) M-1 s(-1) for the Abz-GFSIFRQ-EDDnp substrate, similar to that of EP24.16 (k(cat)/K-M = 3x10(5) M-1 s(-1)) which contains Gly at the corresponding position; the wild type EP24.15 has a k(cat)/K-M of 2.5x10(4) M-1 s(-1) for this substrate. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.Univ Mogi das Cruzes, CIIB, BR-08780911 Mogi Das Cruzes, SP, BrazilInst Butantan, CAT, Ctr Toxicol Aplicada, BR-05467010 São Paulo, BrazilUniv São Paulo, Inst Ciencias Biomed, Program Biol Celular, Dept Histol & Embriol, BR-05508900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

Characterization of thrombin inhibitory mechanism of rAaTI, a Kazal-type inhibitor from Aedes aegypti with anticoagulant activity

Saliva of blood-sucking arthropods contains a complex mixture of anti-haemostatic, anti-inflammatory and immune-modulator compounds. Among anti-haemostatic factors, there are anticoagulants, vasodilators and platelet aggregation inhibitors. Previous analyses of the sialotranscriptome of Aedes aegypti showed the potential presence of a Kazal-type serine protease inhibitor in the female salivary glands, carcass and also in the whole male, which inhibitor we named AaTI (A. aegypti thrombin inhibitor). Recently, we expressed and characterized rAaTI as a trypsin inhibitor, and its anticoagulant activity [1]. in this work we characterized the thrombin inhibition mechanism of rAaTI. Recombinant AaTI was able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. in contrast, AaTI Delta (rAaTI truncated form) and C-terminal AaTI acidic tail prolong only thrombin time. in the competition assay, rAaTI, AaTI Delta or C-terminal AaTI acidic tail thrombin interactions seem to be affected by heparin but not by hirudin, suggesting that rAaTI binds to thrombin exosite 2. Finally, the thrombin inhibition assay of rAaTI showed an uncompetitive inhibition mechanism. in conclusion, rAaTI can probably inhibit thrombin by interacting with thrombin exosite 2, and the interaction is not mediated by the AaTI C-terminal region, since the truncated AaTI Delta form also prolongs thrombin time. (C) 2010 Elsevier Masson SAS. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilInst Butantan, Lab Fisiopatol, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilFAPESP: 05/03514-9FAPESP: 07/56614-6CNPq: 470070/2004-8CNPq: 575829/2008-7Web of Scienc

Results of nutritional intervention in children and adolescents with cystic fibrosis

Objective: few studies have verified longitudinally the evolution of the nutritional status of patients with cystic fibrosis. The objective of this study is to follow the evolution of the nutritional status, body composition and energy consumption, macronutrients and micronutrients ingested by children and adolescents by means of nutritional interventions at the Clinic of Cystic Fibrosis/Pediatric Pneumology of the Department of Pediatrics of Universidade Federal de São Paulo (UNIFESP). Methods: 18 patients were involved in this study, thirteen males and five females with ages ranging from 0.3 to 18.4 years. We performed three evaluations: evaluation 1 (M1- prenutritional intervention), M2 after 6 months, and M3 after 12 months. In these three instances we verified: the z score for weight/age, weight/height and height/age and the calculation of a 3-day diet record. We verified the body composition (anthropometry) in M1 and M3. The nutritional interventions were hypercaloric, hyperproteic, with adequate amount of ingested macronutrients and micronutrients.Results: we observed an increase in the z score for height/age (M1=-1.07; M2=-0.69; M3=-0.50) and fat-free mass after the nutritional interventions, without improvement in the z score for weight/height and fat mass. We verified an increase in the energy intake during M2 (139%) and M3 (132%) compared to M1 (106%). Remarkable increase in the intake of protein, calcium, iron and vitamin C by the patients was found. The occurrence of anemia was found in 44% (8/18) of the patients.Conclusion: the improvement of the z score in height/age and fat-free mass was probably due to the increase in energy consumption after the nutritional intervention. A significant improvement in the z score for weight/height and fat mass was not found, probably due to a gain in height and fat-free mass.Objetivo: poucos estudos têm verificado longitudinalmente a evolução do estado nutricional de pacientes com fibrose cística. O objetivo deste estudo foi acompanhar a evolução do estado nutricional, composição corporal e consumo de energia, macro e micronutrientes ingeridos por crianças e adolescentes, mediante intervenção nutricional, no Ambulatório de Fibrose Cística/Pneumologia Pediátrica, do Departamento de Pediatria da UNIFESP.Métodos: a casuística constituiu-se de 18 pacientes, sendo 13 do sexo masculino e 5 do feminino, faixa etária de 0,3 a 18,4 anos. Realizaram-se 3 avaliações: no momento 1 (M1: pré-intervenção nutricional), no M2, após 6 meses, e no M3, após 12 meses. Foram analisados nos 3 momentos: o escore Z de peso/idade (P/I), peso/estatura (P/E) e estatura/idade (E/I) e o cálculo do registro alimentar de 3 dias. No M1 e M3, verificou-se a composição corporal (antropometria). A conduta nutricional foi hipercalórica, hiperprotéica e houve adequação de macro e micronutrientes.Resultados: observou-se aumento significante do escore Z de E/I (M1=-1,07; M2=-0,69; M3=-0,50) e de massa magra após a intervenção nutricional, sem melhora no escore Z de P/E e massa gorda. Verificou-se aumento no consumo energético nos M2 (139%) e M3 (132%) em relação ao M1 (106%). Houve considerável aumento no consumo de proteína, cálcio, ferro e vitamina C pelos pacientes. A presença de anemia ocorreu em 44,4% (8/18) dos pacientes.Conclusão: o aumento no escore Z de E/I e massa magra ocorreu devido a adequação no consumo de energia, após a intervenção nutricional. Não houve melhora significante no escore Z de P/E e massa gorda, em função do ganho de estatura e de massa magra.UNIFESP-EPM Dep. de PediatriaHospital São Paulo Ambulatório de Fibrose CísticaUNISA Faculdade de MedicinaUNISA Dep. de PediatriaUNIFESP, EPM, Dep. de PediatriaHospital São Paulo Ambulatório de Fibrose CísticaSciEL

Antioxidant dietary deficiency induces caspase activation in chick skeletal muscle cells

Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambdaexc = 320 nm and lambdaem = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BioquímicaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BiofísicaUniversidade de São Paulo Faculdade de Zootecnia e Engenharia de Alimentos Departamento de CiênciasUNIFESP, EPM, Depto. de BioquímicaUNIFESP, EPM, Depto. de BiofísicaSciEL

Genotyping And Descriptive Proteomics Of A Potential Zoonotic Canine Strain Of Giardia Duodenalis, Infective To Mice

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The zoonotic potential of giardiasis, as proposed by WHO since the late 70's, has been largely confirmed in this century. The genetic assemblages A and B of Giardia duodenalis are frequently isolated from human and canine hosts. Most of the assemblage A strains are not infective to adult mice, which can limit the range of studies regarding to biology of G. duodenalis, including virulence factors and the interaction with host immune system. This study aimed to determine the infectivity in mice of an assemblage A Giardia duodenalis strain (BHFC1) isolated from a dog and to classify the strain in sub-assemblages (Al, All, AIII) through the phylogenetic analysis of beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. In addition, the proteomic profile of soluble and insoluble protein fractions of trophozoites was analyzed by 2D-electrophoresis. Accordingly, trophozoites of BHFC1 were highly infective to Swiss mice. The phylogenetic analysis of tpi and gdh revealed that BHFC1 clustered to sub-assemblage Al. The proteomic map of soluble and insoluble protein fractions led to the identification of 187 proteins of G. duodenalis, 27 of them corresponding to hypothetical proteins. Considering both soluble and soluble fractions, the vast majority of the identified proteins (n = 82) were classified as metabolic proteins, mainly associated with carbon and lipid metabolism, including 53 proteins with catalytic activity. Some of the identified proteins correspond to antigens while others can be correlated with virulence. Besides a significant complementation to the proteomic data of G. duodenalis, these data provide an important source of information for future studies on various aspects of the biology of this parasite, such as virulence factors and host and pathogen interactions.1110CNPq (Brazilian National Council for Scientific and Technological Development)FAPEMIG (State Funding Agency of Minas Gerais (FAPEMIG))INCTV (National Institute of Science and Technology in Vaccines)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Branched-chain amino acids do not improve muscle recovery from resistance exercise in untrained young adults.

The purpose of this study was to investigate the effects of BCAA supplementation on muscle recovery from resistance exercise (RE) in untrained young adults. Twenty-four young adults (24.0 ± 4.3 years old) were assigned to 1 of 2 groups (n = 12 per group): a placebo-supplement group or a BCAA-supplement group. The groups were supplemented for a period of 5 days. On day 1 and 3, both groups underwent a RE session involving two lower body exercises (hack squat and leg press) and then were evaluated for muscle recovery on the 3 subsequent moments after the RE session [30 min (day 3), 24 h (day 4), and 48 h (day 5)]. The following indicators of muscle recovery were assessed: number of repetitions, rating of perceived exertion in the last RE session, muscle soreness and countermovement jump (CMJ) during recovery period (30 min, 24 h, and 48 h after RE session). Number of repetitions remained unchanged over time (time, P > 0.05), while the rating of perceived exertion increased (time, P  0.05). Muscle soreness increased (time, P  0.05). The results indicate that BCAA supplementation does not improve muscle recovery from RE in untrained young adults