Síncope cardioinibitória 2b com pausa de 48 segundos em paciente com síndrome de down e defeito do septo av corrigido: descrição do caso e do plano terapêutico / Cardioinhibitory syncope 2b with a 48-second pause in a patient with down syndrome and corrected AV septal defect: case description and therapeutic plan

INTRODUÇÃO: Os quadros de Síncope habitualmente se iniciam na adolescência. Aproximadamente 20% da população experimenta o primeiro episódio de desmaio entre 10 e 20 anos. Apesar de extremamente angustiantes estes nem sempre são investigados. Apenas 25 a 50% dos pacientes são avaliados nos serviços de saúde. Entretanto alguns casos merecem tratamento pelo risco de eventos e necessitam de acompanhamento especializado, inclusive para monitorização da indicação de dispositivos de marcapasso (MP) em caso de refratariedade ao tratamento clínico. Isto ocorre em alguns caos de síncope de padrão neuromediado do tipo cardioinibitória com pausa (Síncope cardioinibitória 2b ao Teste de Inclinação) OBJETIVO: Descrever o caso de uma paciente púbere pós com Síndrome de Down e síncopes de repetição de início recente com teste de inclinação (TI) positivo (resposta 2b). DESCRIÇÃO DO CASO: Paciente 13 anos feminina, Síndrome de Down portadora de defeito do septo AV total em fase pós operatória de correção total com bom resultado cirúrgico. Iniciou concomitante a menarca quadro de sincope recorrente com pródromos (dor abdominal e palidez). Apresenta Holter sem alterações arrítmicas e ecocardiograma com FEVD no limite inferior da normalidade. Analise do ECG com sinais de bloqueio divisional ântero superior direito. Foi submetido a avaliação por TI. Realizou protocolo de inclinação passivo a 70 graus não sensibilizado. Após 5 minutos de repouso foi inclinada e se manteve estável por 7 minutos e no oitavo minuto de inclinação apresentou dor abdominal, seguida de sincope com resposta cardioinibitória 2b e pausa de 48 segundos (fig 1) apesar do retorno a posição de Trendelenburgo (-30 graus). Houve retorno dos batimentos cardíacos com recuperação imediata do nível de consciência. Iniciado tratamento clínico com orientação de aumento da ingesta hídrica, suspender fatores desencadeantes (ortostase prolongada, ambientes quentes por exemplo) e mantido a monitorização rigorosa com intuito de avaliar a refratariedade e a necessidade de indicação de MP. CONCLUSÃO: 1) Os quadros de síncope neuromediada são particularmente frequentes durante a adolescência, principalmente após o estirão do crescimento 2) As respostas cardioinibitórias podem se instalar subitamente e serem de grande repercussão. Apesar deste fato o tratamento clínico deve ser sempre priorizado; 3) O paciente deve ser monitorizado quanto as recorrências e refratariedade ao tratamento clínico para indicação precisa de MP em pacientes com síncope cardioinibitória 2b com grandes pausas refratária à medidas gerais.

Addition of chlorogenic acid and caffeine during the processing of cooled boar semen / Adição de ácido clorogênico e cafeína durante o processamento do sêmen suíno resfriado

A study was conducted to evaluate the effect of chlorogenic acid (ChA) added pre-cooling and its combination with caffeine added during warming on cooled-stored boar semen parameters. Ten ejaculates were diluted in commercial extender with or without 4.5mg/ml ChA and stored at 15°C. After 0, 24 and 72 hours of storage, aliquots of these doses were taken and incubated at 37°C in the presence or absence of 8.0mM caffeine. Semen quality was evaluated after 10 and 120 minutes of incubation. The ChA increased (P <0.01) the sperm motility, viability, acrosomal integrity and the percentage of spermatozoa with high mitochondrial activity (PMHA), however, decreased (P <0.01) the malondialdehyde (MDA) concentration. Caffeine increased (P<0.05) the sperm motility, viability, PMHA and the MDA concentration and reduced (P <0.05) the acrosome integrity. When associated (ChA+caffeine), there was an increase (P <0.05) in sperm motility and viability, PMHA and acrosome integrity. The addition of ChA to the dilution medium improves the quality of the swine inseminating doses. The addition of caffeine during re-warming is only recommended when the semen is stored for prolonged periods (72h), and the inseminating dose should be used immediately after its addition.(AU)

O objetivo deste estudo foi avaliar os efeitos da adição de ácido clorogênico (ChA) antes do resfriamento e sua combinação com cafeína adicionada durante o reaquecimento sobre a qualidade do sêmen suíno resfriado. Dez ejaculados foram diluídos em diluidor comercial com adição ou não de 4,5mg/mL de ChA e armazenados a 15°C. Após zero, 24 e 72 horas de armazenamento, 10mL foram retirados e incubados a 37°C na presença ou ausência de 8,0mM de cafeína. A qualidade seminal foi avaliada após 10 e 120 minutos de incubação. O ChA aumentou (P<0,01) a motilidade, a viabilidade, a integridade acrosomal e a porcentagem de espermatozoides com alta atividade mitocondrial (PMHA), entretanto diminuiu (P<0,01) a concentração de malondialdeído (MDA). A cafeína aumentou (P<0,05) a motilidade, a viabilidade, a PMHA e a concentração de MDA e reduziu a integridade acrossomal. Quando associados (ChA+cafeína), houve aumento (P<0,05) na motilidade, na PMHA, na viabilidade e na integridade acrossomal. Conclui-se que a adição de ChA ao meio de diluição melhora a qualidade das doses inseminantes de suínos. A adição de cafeína durante o reaquecimento só é recomendada ao sêmen adicionado de ChA quando esse for armazenado por períodos prolongados (72h), devendo a dose inseminante ser utilizada imediatamente após sua adição.(AU)

The multigene families of actinoporins (part II): Strategies for heterologous production in Escherichia coli.

The sea anemone venom contains pore-forming proteins (PFP) named actinoporins, due to their purification from organisms belonging to Actiniaria order and its ability to form pores in sphingomyelin-containing membranes. Actinoporins are generally basic, monomeric and single-domain small proteins (∼20 kDa) that are classified as α-type PFP since the pore formation in membranes occur through α-helical elements. Different actinoporin isoforms have been isolated from most of the anemones species, as was analyzed in the first part of this review. Several actinoporin full-length genes have been identified from genomic-DNA libraries or messenger RNA. Since the actinoporins lack carbohydrates and disulfide bridges, their expression in bacterial systems is suitable. The actinoporins heterologous expression in Escherichia coli simplifies their production, replaces the natural source reducing the ecological damage in anemone populations, and allows the production of site-specific mutants for the study of the structure-function relationship. In this second part of the review, the strategies for heterologous production of actinoporins in Escherichia coli are analyzed, as well as the different approaches used for their purification. The activity of the recombinant proteins with respect to the wild-type is also reviewed.

The multigene families of actinoporins (part I): Isoforms and genetic structure.

Actinoporins are basic pore-forming proteins produced by sea anemones, with molecular weight around 20 kDa showing high affinity for sphingomyelin-containing membranes. Most sea anemones produce more than one actinoporin isoform differing in isoelectric point, molecular weigth and cytolytic activity. Examples of sea anemones with actinoporin isoforms are: Actinia equina with at least five isoform genes; Actinia tenebrosa, three isoforms; Actinia fragacea, five isoforms; Actineria villosa, Phyllodiscus semoni, Stichodactyla helianthus and Oulactis orientalis, with two isoforms each one, and Heteractis crispa with twenty-four isoforms. Additionally, thirty-four different amino acid sequences were deduced from fifty-two nucleotide sequences of Heteractis magnifica toxins suggesting the presence of a large number of isoforms or allelic variants. Many amino acidic changes in the isoforms are located in important regions for pore formation. The genetic structure of actinoporins comprises a pre-propeptide and a mature toxin region; therefore, actinoporins could be synthetized in the Golgi apparatus as precursor forms. The subsequent maturation of the toxins involves a proteolytic processing during secretion. Here we hypothesize that sea anemones could have suffered duplication, conversion and mutation of genes that produced multigene families as an efficient response to evolutionary pressure, leading to successful strategies of predatory and defensive function.

Silagem de capim-elefante adicionada de casca de maracujá / Elephant grass silage added to passion fruit's peel

Avaliaram-se-se as características fermentativas e químico-bromatológicas da silagem de capim-elefante contendo diferentes níveis de inclusão do coproduto da extração da polpa do maracujá, a casca de maracujá (CM). Utilizaram-se níveis crescentes de adição desse coproduto (0,0%; 12,5%; 25,0%; 37,5% e 50,0%) em relação à matéria natural do capim-elefante durante a ensilagem. As silagens foram obtidas a partir do corte da forrageira aos 150 dias de idade. O material foi ensilado em silos laboratoriais e, após 180 dias, os silos foram abertos e coletaram-se amostras para a determinação dos teores de matéria seca (MS); carboidratos solúveis (CHOS); potencial hidrogeniônico (pH); nitrogênio amoniacal em porcentagem do nitrogênio total (NNH3/NT); extrato etéreo (EE); proteína bruta (PB); proteína insolúvel em detergente neutro em porcentagem do nitrogênio total (PIDN/NT); proteína insolúvel em detergente ácido em porcentagem do nitrogênio total (PIDA/NT); fibra em detergente neutro (FDN); fibra em detergente ácido (FDA); celulose (CEL); lignina (LIG); nitrogênio insolúvel em detergente neutro (NIDN); nitrogênio insolúvel em detergente ácido (NIDA) e digestibilidade in vitro da matéria seca (DIVMS). Para a avaliação do efeito dos tratamentos, utilizou-se delineamento experimental inteiramente ao acaso, com quatro repetições, executando-se o estudo de regressão para cada variável analisada (P<0,05). De acordo com as equações de regressão, para as características fermentativas, as silagens podem ser consideradas de muito boa qualidade. Quanto às frações fibrosas da silagem, a inclusão de níveis crescentes desse coproduto contribuiu para diminuí-las, o que resultou em maior DIVMS. Os parâmetros analisados permitem concluir que a inclusão da CM in natura durante a ensilagem do capim-elefante é recomendada em todos os níveis avaliados.

We evaluated the fermentative, chemical and qualitative characteristics of elephant grass silage containing different levels of inclusion of coproduct extraction of the passion fruit pulp in nature, passion fruit peel (CM). We used increasing levels of addition of this coproduct (0.0, 12.5, 25.0, 37.5 and 50.0%) in relation to natural field of elephant grass during ensiling. The silages were obtained from the cut forage at 150 days of age. The material was ensiled in laboratory silos, and after 180 days, the silos were opened and samples were collected for determination of dry matter (DM); soluble carbohydrates (CHOS); hydrogen potential (pH); ammonia nitrogen in percentage of total nitrogen (NNH3/NT); ether extract (EE); crude protein (CP); neutral detergent insoluble protein as a percentage of total nitrogen (NDIP/NT); acid detergent insoluble protein as a percentage of total nitrogen (PIDA/NT); neutral detergent fiber (NDF); acid detergent fiber (ADF); cellulose (CEL); lignin (LIG); neutral detergent insoluble nitrogen (NDIN); acid detergent insoluble nitrogen (ADIN) and in vitro digestibility of dry matter (IVDDM). To assess the effect of the treatments we used a completely randomized design with four replications, running the regression analysis for each variable analyzed (P<0.05). According to the regression equations for the fermentative characteristics, silage can be considered very good quality. As for fiber digestibility of silage, the increasing levels of this coproduct contributed to decrease it, which resulted in higher IVDDM. The parameters analyzed showed that the inclusion of CM in nature with elephant grass silage is recommended at all levels evaluated.

A route from darkness to light: emergence and evolution of luciferase activity in AMP-CoA-ligases inferred from a mealworm luciferase-like enzyme.

The origin of luciferases and of bioluminescence is enigmatic. In beetles, luciferases seem to have evolved from AMP-CoA-ligases. How the new oxygenase luminogenic function originated from AMP-ligases leading to luciferases is one of the most challenging mysteries of bioluminescence. Comparison of the cloned luciferase-like enzyme from the nonluminescent Zophobas morio mealworm and beetle luciferases showed that the oxygenase activity may have emerged as a stereoselective oxidative drift with d-luciferin, a substrate that cannot be easily thioesterified to CoA as in the case of the l-isomer. While the overall kcat displayed by beetle luciferases is orders of magnitude greater than that of the luciferase-like enzyme, the respective oxidation rates and quantum yields of bioluminescence are roughly similar, suggesting that the rate constant of the AMP-ligase activity exerted on the new d-luciferin substrate in beetle protoluciferases was the main enzymatic property that suffered optimization during the evolution of luciferases. The luciferase-like enzyme and luciferases boost the rate of luciferyl-adenylate chemiluminescent oxidation by factors of 10(6) and 10(7), respectively, as compared to the substrate spontaneous oxidation in buffer. A similar enhancement of luciferyl-adenylate chemiluminescence is provided by nucleophilic aprotic solvents, implying that the peptide bonds in the luciferin binding site of beetle luciferase could provide a similar catalytically favorable environment. These data suggest that the luciferase-like enzyme and other similar AMP-ligases are potential alternative oxygenases. Site-directed mutagenesis studies of the luciferase-like enzyme and the red light-producing luciferase of Phrixotrix hirtus railroadworm confirm here a critical role for T/S345 in luciferase function. Mutations such as I327T/S in the luciferase-like enzyme, which simultaneously increases luciferase activity and promotes blue shifts in the emission spectrum, could have been critical for evolving functional bioluminescence from red-emitting protoluciferases. Through the combination of I327T/S mutations and N-terminal fusion, the luminescence activity of this enzyme was increased to visible levels, with the development of a totally new orange-emitting luciferase. These results open the possibility of engineering luciferase activity in a set of AMP-CoA-ligases.

The Kazal-type inhibitors infestins 1 and 4 differ in specificity but are similar in three-dimensional structure.

Blood coagulation is an important process in haemostasis, and disorders of blood coagulation can lead to an increased risk of haemorrhage and thrombosis. Coagulation is highly conserved in mammals and has been comprehensively studied in humans in the investigation of bleeding or thrombotic diseases. Some substances can act as inhibitors of blood coagulation and may affect one or multiple enzymes throughout the process. A specific thrombin inhibitor called infestin has been isolated from the midgut of the haematophagous insect Triatoma infestans. Infestin is a member of the nonclassical Kazal-type serine protease inhibitors and is composed of four domains, all of which have a short central α-helix and a small antiparallel ß-sheet. Domains 1 and 4 of infestin (infestins 1 and 4) possess specific inhibitory activities. Infestin 1 inhibits thrombin, while infestin 4 is an inhibitor of factor XIIa, plasmin and factor Xa. Here, the structure determination and structural analysis of infestin 1 complexed with trypsin and of infestin 4 alone are reported. Through molecular modelling and docking, it is suggested that the protein-protein binding site is conserved in the infestin 1-thrombin complex compared with other Kazal-type inhibitors. Infestin 4 is able to bind factor XIIa, and the F9N and N11R mutants selected by phage display were shown to be more selective for factor XIIa in comparison to the wild type.

Avaliação da utilização de medicamentos na prática clínica em um hospital público / Assessment of drugs prescribed to medical in-patients in a public hospital

Através desta pesquisa, objetivou-se estudar o consumo dos medicamentos em pacientes hospitalizados na clínica médica de um hospital público na cidade de Campina Grande (PB). O estudo caracterizou-se como descritivo e exploratório, de caráter transversal, com abordagens quali-quantitativas, e foi constituído por uma amostra de 107 pacientes que iniciaram internação na clínica médica no período de agosto de 2007 a julho de 2008. Os 107 pacientes apresentavam 270 diagnósticos ativos, sendo as doenças do aparelho circulatório as de maior ocorrência. Dos pacientes, 65,4% eram idosos e 3,33% dos medicamentos prescritos foram considerados impróprios para eles. Os pacientes que apresentaram possíveis 107 RAMs totalizaram 43%, com média de 2,32 por paciente; as que afetaram o sistema gastrintestinal dos pacientes foram as identificadas com maior frequência. Houve 42 interações distintas, envolvendo 26 tipos de fármacos. Dessa forma, os resultados podem ser úteis no estímulo ao desenvolvimento de mecanismos de avaliação de processos que visem reduzir esses riscos, aumentando a chance de resultados terapêuticos positivos e benefícios para os pacientes.

We studied the consumption of drugs by in-patients in the medical ward of a public hospital in the city of Campina Grande, PB, Brazil. This paper describes a descriptive / exploratory cross-sectional quali-quantitative study of a sample of 107 patients who were admitted to the general medical ward, from August 2007 to July 2008. The 107 patients were diagnosed with 270 active complaints, mainly diseases of the circulatory system. Most of the patients (65.4%) were elderly and 3.33% of drugs prescribed for them are considered unfit for use in the elderly. Many patients (43%) presented 107 possible Adverse Drug Reactions, with an average of 2.32 per patient, those affecting the gastrointestinal system of the patients being identified most frequently. There were 42 different drug interactions, involving 26 types of drug. We hope these results may be useful in stimulating the development of means to assess drug treatment in hospital, so as to reduce these risks and increase the chance of positive outcomes and therapeutic benefits for the patients.

Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site.

The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases.

Conservação e fisiologia pós-colheita de folhas de Capuchinha (Tropaeolum majus L. ) / Conservation and postharvest physiology of leaves Capuchin (Tropaeolum majus L. )

Este trabalho teve como objetivo avaliar a vida útil pós-colheita de folhas de Capuchinha (Tropaeolum majus L.) embaladas com filme PVC de baixa densidade e proteínas nas concentrações de 1, 3 e 5 por cento. O experimento foi conduzido no Laboratório de Química e Bioquímica do Centro de Ciências Agrárias (CCA) da Universidade Federal da Paraíba (UFPB) Areia - PB. As folhas de Capuchinha colhidas no CCA foram levadas imediatamente ao Laboratório de Química e Bioquímica, para pré-seleção, desinfestação em água clorada contendo 100mg.L-1 de cloro ativo, e seca à temperatura ambiente. Após o controle fitossanitário, as folhas foram submetidas aos seguintes tratamentos: solução de proteína nas concentrações de 1, 3 e 5 por cento, filme PVC de baixa densidade e a testemunha sem recobrimento. As folhas foram armazenadas durante cinco dias em temperatura média de 26 - 29ºC e umidade relativa média de 59,5 - 71,5 por cento e outra parte foi armazenada em câmara fria com temperatura média de 12 ± 0,5 ºC e umidade relativa média de 95 ± 3 por cento. Durante o período de armazenamento as folhas foram avaliadas quanto à perda de massa fresca, determinação de ácido ascórbico, sólidos solúveis e acidez titulável. O armazenamento durante quatro dias sob refrigeração a temperatura média de 12 ± 0,5ºC associada ao recobrimento com filme PVC mostrou melhores resultados na conservação da vida útil pós-colheita das folhas de Capuchinha.

This study aimed to evaluate the shelf-life of Capuchin sheets (Tropaeolum majus L.) packed with plastic wrap and low density proteins at concentrations of 1, 3 and 5 percent. The experiment was conducted at the Laboratório de Química e Bioquímica and the Centro de Ciências Agrárias (CCA) of the Universidade Federal da Paraíba (UFPB) Areia - PB. Capuchin leaves harvested in the CCA were taken immediately to the Laboratório de Química e Bioquímica, for pre-screening, disinfection in chlorinated water containing 100mg.L-1 of active chlorine, and dried at room temperature. After spraying, the leaves were treated as follows: protein solution at concentrations of 1, 3 and 5 percent, low density plastic wrap and uncoated witness. The leaves were stored for five days in average temperature from 26 to 29ºC and relative humidity from 59.5 to 71.5 percent and another part was stored in cold with average temperature of 12 ± 0.5ºC and relative humidity of 95 ± 3 percent. During the storage period the leaves were evaluated for weight loss, determination of ascorbic acid, soluble solids and titratable acidity. Storage for four days under refrigeration at an average temperature of 12 ± 0.5ºC associated with PVC film coating showed better results in the conservation of shelf-life of Capuchin sheets.

Cloning, overexpression, purification and preliminary characterization of human septin 8.

Mammalian septins comprise a family of 14 genes that encode GTP-binding proteins involved in important cellular processes such as cytokinesis and exocytosis. Expression of three different constructs encoding human septin 8 were analyzed and the results show that SEPT8GC, a clone expressing the conserved domain plus C-terminal domain of human septin 8 yields the highest amount of recombinant protein. This protein was purified by affinity chromatography followed by a gel filtration chromatography. CD spectrum of SEPT8GC is characteristic of folded proteins and it presents a transition profile with a T (m) of 54 degrees C. Fluorescence emission spectra, analytic gel filtration and DLS reflect the sample oligomeric heterogeneity with the predominance of dimers in solution. Homology models indicate clearly that the preferred dimer interface is the one comprising the GTP binding site.

Crystallization, data collection and data processing of maltose-binding protein (MalE) from the phytopathogen Xanthomonas axonopodis pv. citri.

Maltose-binding protein is the periplasmic component of the ABC transporter responsible for the uptake of maltose/maltodextrins. The Xanthomonas axonopodis pv. citri maltose-binding protein MalE has been crystallized at 293 K using the hanging-drop vapour-diffusion method. The crystal belonged to the primitive hexagonal space group P6(1)22, with unit-cell parameters a = 123.59, b = 123.59, c = 304.20 A, and contained two molecules in the asymetric unit. It diffracted to 2.24 A resolution.

Crystallization, data collection and phasing of two digestive lysozymes from Musca domestica.

Lysozymes are mostly known for their defensive role against bacteria, but in several animals lysozymes have a digestive function. Here, the initial crystallographic characterization of two digestive lysozymes from Musca domestica are presented. The proteins were crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate or PEG/2-propanol as the precipitant. X-ray diffraction data were collected to a maximum resolution of 1.9 angstroms using synchrotron radiation. The lysozyme 1 and 2 crystals belong to the monoclinic space group P2(1) (unit-cell parameters a = 36.52, b = 79.44, c = 45.20 angstroms, beta = 102.97 degrees) and the orthorhombic space group P2(1)2(1)2 (unit-cell parameters a = 73.90, b = 96.40, c = 33.27 angstroms), respectively. The crystal structures were solved by molecular replacement and structure refinement is in progress.

Crystallization, data collection and phasing of the molybdate-binding protein of the phytopathogen Xanthomonas axonopodis pv. citri.

Xanthomonas axonopodis pv. citri ModA protein is the ABC periplasmic binding component responsible for the capture of molybdate. The protein was crystallized with sodium molybdate using the hanging-drop vapour-diffusion method in the presence of PEG or sulfate. X-ray diffraction data were collected to a maximum resolution of 1.7 A using synchrotron radiation. The crystal belongs to the orthorhombic space group C222(1), with unit-cell parameters a = 68.15, b = 172.14, c = 112.04 A. The crystal structure was solved by molecular-replacement methods and structure refinement is in progress.

Endosonographic characteristics of perigastrointestinal lymphnodes studied ex-corpore.

OBJECTIVES: Assessment of morphological characteristics of lymphnodes studied ex-corpore by echoendoscopy--shape, sharpness of limits, echogenicity and dimensions--to categorize them either as metastatic or non-metastatic. MATERIAL AND METHODS: Fifty-four lymphnodes were studied. They were identified and studied by echoendoscopy in fresh surgical specimens of oesophagus, stomach and rectum. Eight nodal characteristics were evaluated. The data obtained were studied by multivariate analysis using the logistical estimate method in two different statistical models. RESULTS: In model 1, logistical estimate demonstrated that well-defined limits (WDL) of the lymphnodes and the association of hypoechogenicity and round shape were the most significant variables suggesting the presence of invasion. In model 2, the most significant variables were WDL, round shape and association of round shape with WDL. The first model had a high sensitivity of 93.1% and specificity of 68% whereas the second model had a greater specificity of 84%, with a slight fall in sensitivity. CONCLUSION: These models might have an application to clinical practice particularly in the pre-operative assessment of patients with oesophageal carcinoma, gastric carcinoma or rectal carcinoma.

Crystallization, data collection and phasing of infestin 4, a factor XIIa inhibitor.

Infestin is a protein from Triatoma infestans (kissing bug) composed of seven Kazal-type domains that is further processed to yield several serine protease inhibitors with varying specificities. Infestins 3 and 4 are the last two domains of the infestin gene and are found in vivo in the insect's anterior midgut. The last domain, infestin 4, has been cloned, expressed and purified. Here, the crystallization of infestin 4 using the sitting-drop vapour-diffusion method with PEG 8000 as precipitant is described. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 25.89, b = 45.64, c = 57.41 A. X-ray diffraction data were collected to a maximum resolution of 1.8 A using a synchrotron-radiation source. Initial phases were calculated by molecular replacement using an edited rhodniin molecule as the search model. Structure refinement is in progress.

Crystallization, data collection and phasing of black-eyed pea trypsin/chymotrypsin inhibitor in complex with bovine beta-trypsin.

The black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) is a Bowman-Birk-type inhibitor from Vigna unguiculata seeds. A complex of BTCI with bovine beta-trypsin was crystallized by the hanging-drop vapour-diffusion method with ammonium sulfate as precipitant. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 59.3, b = 61.8, c = 80.0 A. Diffraction data were collected to 2.36 A resolution and were processed to give an overall R(sym) of 0.137. The Matthews coefficient for one complex per asymmetric unit is 2.2 A(3) Da(-1), with a corresponding solvent content of 43%. After molecular replacement and initial refinement, the model gives an R(cryst) of 0.361 and an R(free) of 0.432.

Efficacy and safety of omapatrilat with hydrochlorothiazide for the treatment of hypertension in subjects nonresponsive to hydrochlorothiazide alone.

This multicenter, double-blind study evaluated efficacy and safety of the vasopeptidase inhibitor omapatrilat, which simultaneously inhibits neutral endopeptidase and angiotensin converting enzyme, when given in conjunction with hydrochlorothiazide (HCTZ) to subjects nonresponsive to HCTZ alone. The study enrolled 657 subjects with mild to severe hypertension. After a 2-week placebo lead-in period and a 4-week HCTZ phase, 274 subjects were randomized to receive omapatrilat (10 or 20 mg, electively titrated to 20 or 40 mg, respectively, at week 4 if seated diastolic blood pressure [SeDBP] was > or =90 mm Hg) or matching placebo in addition to 25 mg of HCTZ as continuing therapy. The primary outcome measure was change in SeDBP from baseline to week 8. At week 8, placebo plus HCTZ-adjusted additional reductions in SeDBP in the omapatrilat 10/20 mg and 20/40 mg treatment groups (4 and 5 mm Hg, respectively) were significant (P < .001), as were changes in seated systolic blood pressure in both omapatrilat-treated groups (7 and 10 mm Hg, respectively; P < .001). Seated diastolic blood pressure was normalized (<90 mm Hg) in 38% of subjects in the placebo group compared to 59% and 64% of subjects in the omapatrilat groups (P < or = .008). Adverse events, serious adverse events, and discontinuations attributed to adverse events were infrequent. There were no clinically relevant changes in serum creatinine or potassium. Omapatrilat was effective and well tolerated when added to HCTZ in subjects whose blood pressure was not controlled with HCTZ alone.

Three-dimensional structure of 2-amino-3-ketobutyrate CoA ligase from Escherichia coli complexed with a PLP-substrate intermediate: inferred reaction mechanism.

2-Amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29) is a pyridoxal phosphate (PLP) dependent enzyme, which catalyzes the second reaction step on the main metabolic degradation pathway for threonine. It acts in concert with threonine dehydrogenase and converts 2-amino-3-ketobutyrate, the product of threonine dehydrogenation by the latter enzyme, with the participation of cofactor CoA, to glycine and acetyl-CoA. The enzyme has been well conserved during evolution, with 54% amino acid sequence identity between the Escherichia coli and human enzymes. We present the three-dimensional structure of E. coli KBL determined at 2.0 A resolution. KBL belongs to the alpha family of PLP-dependent enzymes, for which the prototypic member is aspartate aminotransferase. Its closest structural homologue is E. coli 8-amino-7-oxononanoate synthase. Like many other members of the alpha family, the functional form of KBL is a dimer, and one such dimer is found in the asymmetric unit in the crystal. There are two active sites per dimer, located at the dimer interface. Both monomers contribute side chains to each active/substrate binding site. Electron density maps indicated the presence in the crystal of the Schiff base intermediate of 2-amino-3-ketobutyrate and PLP, an external aldimine, which remained bound to KBL throughout the protein purification procedure. The observed interactions between the aldimine and the side chains in the substrate binding site explain the specificity for the substrate and provide the basis for a detailed proposal of the reaction mechanism of KBL. A putative binding site of the CoA cofactor was assigned, and implications for the cooperation with threonine dehydrogenase were considered.

Active site modulation in the N-acetylneuraminate lyase sub-family as revealed by the structure of the inhibitor-complexed Haemophilus influenzae enzyme.

The N-acetylneuraminate lyase (NAL) sub-family of (beta/alpha)(8) enzymes share a common catalytic step but catalyse reactions in different biological pathways. Known examples include NAL, dihydrodipicolinate synthetase (DHDPS), d-5-keto-4-deoxyglucarate dehydratase, 2-keto-3-deoxygluconate aldolase, trans-o-hydroxybenzylidenepyruvate hydrolase-aldolase and trans-2'-carboxybenzalpyruvate hydratase-aldolase. Little is known about the way in which the three-dimensional structure of the respective active sites are modulated across the sub-family to achieve cognate substrate recognition. We present here the structure of Haemophilus influenzae NAL determined by X-ray crystallography to a maximum resolution of 1.60 A, in native form and in complex with three substrate analogues (sialic acid alditol, 4-deoxy-sialic acid and 4-oxo-sialic acid). These structures reveal for the first time the mode of binding of the complete substrate in the NAL active site. On the basis of the above structures, that of substrate-complexed DHDPS and sequence comparison across the sub-family we are able to propose a unified model for active site modulation. The model is one of economy, allowing wherever appropriate the retention or relocation of residues associated with binding common substrate substituent groups. Our structures also suggest a role for the strictly conserved tyrosine residue found in all active sites of the sub-family, namely that it mediates proton abstraction by the alpha-keto acid carboxylate in a substrate-assisted catalytic reaction pathway.