Crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of ADP-glucose pyrophosphorylase from potato tuber

ADP-glucose pyrophosphorylase is the key regulatory enzyme in the biosynthesis of starch in plants and glycogen in bacteria. The enzyme from potato tuber is comprised of a regulatory subunit and a catalytic subunit and is present as a heterotetramer (alpha(2)beta(2)) the catalytic subunit from potato tuber (50 kDa) was crystallized in four different forms, two of which are suitable for structural studies. A tetragonal crystal form obtained in the presence of the substrate analog Cr-ATP diffracted to 2.2 Angstrom and belongs to space group P4(1) (or its enantiomorph), with unit-cell parameters a = b = 110.57, c = 190.14 Angstrom. A second crystal form obtained diffracted to 2.8 Angstrom and belongs to space group PZ, with unit-eel parameters a = 80.06, b = 138.84, c = 92.20 Angstrom, beta = 112.40 degrees. As this protein displays no significant homology to any currently known protein structure, a search for heavy-atom derivatives has been initiated.

Molecular adaptability of nucleoside diphosphate kinase b from trypanosomatid parasites: stability, oligomerization and structural determinants of nucleotide binding

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

Structural bioinformatics study of cyclin-dependent kinases complexed with inhibitors

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

Efeito analgésico de longa duração da dipirona sobre a hiperalgesia persistente induzida pela constrição do nervo ciático em ratos: participação do óxido nítrico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Solução cardioplégica polarizante: estado da arte

A exegese do termo cardioplegia remete aos significados de lesão, golpe, ataque ou ferimento, bem diferente, portanto, do sentido em que o termo é empregado na maior parte dos centros de cirurgia cardíaca do Brasil e do mundo, ou seja, como correspondendo à proteção miocárdica. Daí a melhor denominação de solução cardioplégica, para caracterizar as soluções empregadas com finalidade de promover a parada cardíaca controlada do coração. A parada cardíaca induzida por solução cardioplégica pode acontecer por hiperpolarização, despolarização ou com bloqueadores da bomba de cálcio. No presente trabalho, discorreremos sobre os principais agentes que promovem a parada cardíaca por hiperpolarização da membrana miocárdica. Com a solução hiperpolarizante, o coração pára no período diastólico, havendo uma redução ainda maior no seu gasto energético, o que propicia melhores condições ao coração quando este reinicia sua contração ao final do procedimento cirúrgico.

Consumo de frutas, verduras e legumes associado à Síndrome Metabólica e seus componentes em amostra populacional adulta

A síndrome metabólica (SM) aumenta o risco de eventos cardiovasculares e o consumo adequado de frutas, verduras e legumes (FVL) está relacionado a sua prevenção. Objetivo: Identificar o consumo de FVL e sua relação com a SM e seus componentes em amostra populacional adulta. Estudo prospectivo de corte transversal com 636 indivíduos adultos, no período de 2004 a 2008. Foram realizadas avaliações antropométrica, clínica, dietética e bioquímica com todos participantes. A SM foi classificada pelo NCEP ATP-III, modificada para a glicose >100mg/dl. A regressão logística foi utilizada para verificar a razão de chance entre o consumo de FVL com a SM e seus componentes, e o nível de significância adotado foi de 5%. O consumo de frutas adequado se mostrou protetor para obesidade abdominal (OR: 0,77; IC: 0,38-0,93), hipertrigliceridemia (OR: 0,76; IC: 0,35-0,96) e presença de SM (OR: 0,78; IC: 0,39-0,96), e o consumo adequado de FVL apresentou efeito protetor para a presença de SM (OR: 0,79; IC: 0,41-0,95). A análise isolada do consumo adequado de verduras e legumes não mostrou efeitos de proteção/risco para a presença de SM e de seus componentes. O consumo adequado de FVL apresentou efeito protetor para a presença de SM e o consumo adequado de frutas exerceu efeito protetor tanto para a presença de SM e como de seus componentes.

Efeitos da correção da acidose metabólica com bicarbonato de sódio sobre o catabolismo protéico na insuficiência renal crônica

A desnutrição protéico-energética constitui problema comum aos pacientes com insuficiência renal crônica, influenciando diretamente na sua morbi-mortalidade. A acidose metabólica tem papel no catabolismo protéico, ativando a via proteolítica proteasoma-ubiquitina, dependente de adenosina trifosfato, e conjuntamente com glicocorticóides induz uma maior atividade na desidrogenase que degrada os aminoácidos de cadeia ramificada. Esta revisão teve como objetivo descrever o mecanismo pelo qual a acidose metabólica nos pacientes com insuficiência renal crônica promove o catabolismo protéico, favorecendo assim a desnutrição, bem como avaliar os efeitos do uso de bicarbonato de sódio na correção da acidose e conseqüentemente redução do catabolismo protéico. Pesquisas mostram melhora da acidose pelo uso de bicarbonato de sódio e conseqüente redução do catabolismo protéico na insuficiência renal crônica, podendo ser esta uma conduta promissora na atenuação da desnutrição nestes pacientes.

Parâmetros bioquímicos de equinos submetidos à simulação de prova de enduro recebendo dietas com adição de óleo de soja

Com o objetivo de verificar o efeito da adição de níveis crescentes de óleo no concentrado sobre a atividade plasmática das enzimas creatina quinase (CK), aspartato aminotransferase (AST) e lactato desidrogenase (LDH) como indicativo de metabolismo energético, foram fornecidas dietas experimentais compostas de cinco níveis de óleo (controle, 6, 12, 18 e 24% do concentrado). Utilizaram-se 20 equinos da raça Árabe, peso médio de 400 kg, submetidos a prova de enduro de 80 km em esteira rolante. O enduro foi dividido em quatro anéis de 20 km, com duração média de 1 hora e dez minutos. A adição de óleo e a distância percorrida tiveram efeitos sobre as variáveis AST, CK e LDH, que apresentaram as respectivas expressões: AST (7,045-0,2292x+0,007991x2+0,008517z- 0,0003282xz), CK (8,06-,07020x+0,05546x2-0,001262x3+0,01204z+0,0006207xz) e LDH (6,624-0,3522x+0,03448x2-0,0008382x3+0,02401z-0,0007489xz) . O óleo é uma importante e bem aproveitada fonte de energia para equinos em exercício, pois sua adição na dieta de animais submetidos a prova de enduro promoveu alteração metabólica que favorece a produção de energia. O metabolismo animal poupou suas reservas energéticas oriundas da glicose, favorecendo a utilização do óleo. A menor atividade plasmática das enzimas AST, CK e LDH com a adição de óleo nas dietas indica direcionamento do metabolismo energético para a β-oxidação. Como apresentam várias isoenzimas, as enzimas estudadas atuam amplamente no metabolismo energético, favorecendo a constante reposição de ATP ao longo do exercício.

beta(2)-Agonists and cAMP inhibit protein degradation in isolated chick (Gallus domesticus) skeletal muscle

1. The role of beta(2)-agonist and of cAMP in chick skeletal muscle proteolytic pathways and protein synthesis was investigated using an in vitro preparation that maintains tissue glycogen stores and metabolic activity for several hours.2. In extensor digitorum longus (EDL) muscle total proteolysis decreased by 15 to 20% in the presence of equimolar concentrations of epinephrine, clenbuterol, a selective beta(2)-agonist, or dibutyryl-cAMP. Rates of protein synthesis were not altered by clenbuterol or dibutyryl-cAMP.3. The decrease in the rate of total protein degradation induced by 10(-5) M clenbuterol was paralleled by a 44% reduction in Ca2+-dependent proteolysis, which was prevented by 10(-5) M ICI 118.551, a selective beta(2)-antagonist.4. No change was observed in the activity of the lysosomal, ATP-dependent, and ATP-independent proteolytic systems. Ca2+-dependent proteolytic activity was also reduced by 58% in the presence of 10(-4) M dibutyryl-cAMP or isobutylmethylxanthine.5. The data suggest that catecholamines exert an inhibitory control of Ca2+-dependent proteolysis in chick skeletal muscle, probably mediated by beta(2)-adrenoceptors, with the participation of a cAMP-dependent pathway.

Shikimate kinase: A potential target for development of novel antitubercular agents

Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. However, no new classes of drugs for TB have been developed in the past 30 years. Therefore there is an urgent need to develop faster acting and effective new antitubercular agents, preferably belonging to new structural classes, to better combat TB, including MDR-TB, to shorten the duration of current treatment to improve patient compliance, and to provide effective treatment of latent tuberculosis infection. The enzymes in the shikimate pathway are potential targets for development of a new generation of antitubercular drugs. The shikimate pathway has been shown by disruption of aroK gene to be essential for the Mycobacterium tuberculosis. The shikimate kinase (SK) catalyses the phosphorylation of the 3-hydroxyl group of shikimic acid (shikimate) using ATP as a co-substrate. SK belongs to family of nucleoside monophosphate (NMP) kinases. The enzyme is an alpha/beta protein consisting of a central sheet of five parallel beta-strands flanked by alpha-helices. The shikimate kinases are composed of three domains: Core domain, Lid domain and Shikimate-binding domain. The Lid and Shikimate-binding domains are responsible for large conformational changes during catalysis. More recently, the precise interactions between SK and substrate have been elucidated, showing the binding of shikimate with three charged residues conserved among the SK sequences. The elucidation of interactions between MtSK and their substrates is crucial for the development of a new generation of drugs against tuberculosis through rational drug design.

Molecular models of cyclin-dependent kinase 1 complexed with inhibitors

Roscovitine and flavopiridol have been shown to potently inhibit cyclin-dependent kinase 1 and 2 (CDK1 and 2). The structures of CDK2 complexed with roscovitine and deschoroflavopiridol have been reported, however no crystallographic structure is available for complexes of CDK1 with inhibitors. The present work describes two molecular models for the binary complexes CDK1:roscovitine and CDK1:flavopiridol. These structural models indicate that both inhibitors strongly bind to the ATP-binding pocket of CDKI and structural comparison of the CDK complexes correlates the structures with differences in inhibition of these CDKs by flavopiridol and roscovitine. This article explains the structural basis for the observed differences in activity of these inhibitors. (C) 2004 Elsevier B.V. All rights reserved.

A 4.2 kDa synthetic peptide as a potential probe to evaluate the antibacterial activity of coumarin drugs

The coumarin antibiotics are potent inhibitors of DNA replication whose target is the enzyme DNA gyrase, an ATP-dependent bacterial type II topoisomerase. The coumarin drugs inhibit gyrase action by competitive binding to the ATP-binding site of DNA gyrase B protein. The production of new biologically active products has stimulated additional studies on coumarin-gyrase interactions. In this regard, a 4.2 kDa peptide mimic of DNA gyrase B protein from Escherichia coli has been designed and synthesized. The peptide sequence includes the natural fragment 131-146 (coumarin resistance-determining region) and a segment containing the gyrase-DNA interaction region (positions 753-770). The peptide mimic binds to novobiocin (K-a = 1.4 +/- 0.3 x 10(5) m(-1)), plasmid (K-a = 1.6 +/- 0.5 x 10(6) m(-1)) and ATP (K-a = 1.9 f 0.4 x 10(3) m(-1)), results previously found with the intact B protein. on the other hand, the binding to novobiocin was reduced when a mutation of Arg-136 to Leu-136 was introduced, a change previously found in the DNA gyrase B protein from several coumarin-resistant clinical isolates of Escherichia coLi. In contrast, the binding to plasmid and to ATP was not altered. These results suggest that synthetic peptides designed in a similar way to that described here could be used as mimics of DNA gyrase in studies which seek a better understanding of the ATP, as well as coumarin, binding to the gyrase and also the mechanism of action of this class of antibacterial drugs. Copyright (C) 2004 European Peptide Society and John Wiley Sons, Ltd.

EFFECT OF EXERCISE DURING PREGNANCY AND DAM AGE ON MATERNAL BLOOD-CHEMISTRY AND FETAL GROWTH

In order to determine the effect of maternal exercise on maternal nutritional status and fetal growth, young (Y = 45-50 days old) Wistar rats were divided into 4 groups of 5 to 8 animals: control pregnant (CP), control non-pregnant (CNP), exercise-trained (swimming 1 h/day, 5 days/week, for 19 days) pregnant (TP) and exercise-trained non-pregnant (TNP). Four equivalent groups of adult rats (A - 90-100 days old) were also formed. Serum glucose, total protein, albumin, hematocrit and liver glycogen were determined in female rats and pups. There were no statistical differences in serum glucose, total protein and albumin levels, litter size ot birth weight among exercise-trained animals, controls and their respective pups. Hematocrit was significantly lower in pups of exercise-trained young rats than in all other groups (YCP = 38.6 +/- 3.0; YTP = 32.6 +/- 2.1; ACP = 39.0 +/- 2.5; ATP = 39.2 +/- 2.9%). Liver glycogen levels were lower in pregnant than in non-pregnant rats but similar in exercise-trained and control rats of the same age and physiological status (YCNP = 4.1 +/- 0.2; YCP = 2.7 +/- 0.9; YTNP = 4.9 +/- 0.8; YTP = 2.7 +/-0.4; ACNP = 6.1 +/- 0.6; ACP = 3.1 +/- 0.8; ATNP = 6.6 +/- 0.8; ATP = 2.2 +/- 0.9 mg/100 mg). We conclude that pups of adult female rats are spared from the effects of this kind of exercise training during pregnancy. on the other hand, it appears that maternal adaptations to exercise training in young rats are able to preserve only some aspects of pup metabolism.

OSSEOUS PLATE ALKALINE-PHOSPHATASE IS ANCHORED BY GPI

Alkaline phosphatase activity was released up to 100% from the membrane by using 0.1 U of phosphatidylinositol-specific phospholipase C from B. thuringiensis. The Mr of solubilized enzyme was 145,000 by Sephacryl S-300 gel filtration and 66,000 by SDS-PAGE, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyze p-nitrophenyl phosphate (PNPP) (264.3 mu mol min(-1) mg(-1)), ATP (42.0 mu mol min(-1) mg(-1)) and pyrophosphate (28.4 mu mol min(-1) mg(-1)). The hydrolysis of ATP and PNPP by solubilized enzyme exhibited ''Michaelian'' kinetics with K-0.5 = 70 and 979 mu M, respectively. For pyrophosphate, K-0.5 was 128 mu M and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K-d = 1.5 mM) but zinc ions were powerful non-competitive inhibitors (K-d = 6.2 mu M) of solubilized enzyme. Treatment of solubilized alkaline phosphatase with Chellex 100 reduced the original PNPPase activity to 5%. Cobalt (K-0.5 = 10.1 mu M), magnesium (K-0.5 = 29.5 mu M) and manganese ions (K-0.5 = 5 mu M) restored the activity of the apoenzyme with positive cooperativity, suggesting that phosphatidylinositol-specific phospholipase C-solubilized alkaline phosphatase is a metalloenzyme. The stimulation of the apoenzyme by calcium ions (K-0.5 = 653 mu M) was lower than that observed for the other ions (26%) and exhibited site-site interactions (n = 0.7). Zinc ions had no effect on the apoenzyme of the solubilized enzyme.