The kinetic mechanism of human uridine phosphorylase 1: Towards the development of enzyme inhibitors for cancer chemotherapy

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Hydrogen/deuterium exchange mass spectrometry for characterizing phosphoenolpyruvate-induced structural transitions in Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.1)

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

Phosphate closes the solution structure of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Mycobacterium tuberculosis

The 5-enolpyruvylshikimate-3-phosphate synthase catalyses the sixth step of the shikimate pathway that is responsible for synthesizing aromatic compounds and is absent in mammals, which makes it a potential target for drugs development against microbial diseases. Here, we report the phosphate binding effects at the structure of the 5-enolpyruvyl shikimate-3-phosphate synthase from Mycobacterium tuberculosis. This enzyme is formed by two similar domains that close on each other induced by ligand binding, showing the occurrence of a large conformation change. We have monitored the phosphate binding effects using analytical ultracentrifugation, small angle X-ray scattering and, circular dichroism techniques. The low resolution results showed that the enzyme in the presence of phosphate clearly presented a more compact structure. Thermal-induced unfolding experiments followed by circular dichroism suggested that phosphate rigidified the enzyme. Summarizing, these data suggested that the phosphate itself is able to induce conformational change resulting in the closure movement in the M. tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase. (c) 2006 Elsevier B.V. All rights reserved.

The role of nitric oxide in regulation of the cardiovascular system in reptiles

The roles that nitric oxide (NO) plays in the cardiovascular system of reptiles are reviewed, with particular emphasis on its effects on central vascular blood flows in the systemic and pulmonary circulations. New data is presented that describes the effects on hemodynamic variables in varanid lizards of exogenously administered NO via the nitric oxide donor sodium nitroprusside (SNP) and, preliminary data on the effects of SNP inhibition of nitric oxide synthase (NOS) by L-nitroarginine methyl ester (L-NAME). Furthermore. on hemodynamic variables in the tegu lizard are presented. The findings are compared with previously published data from Our laboratory on three other species of reptiles: pythons (Skovgaard, N., Galli, G., Taylor, E.W., Conlon, J.M., Wang.. T., 2005. Hemodynamic effects of python neuropeptide gamma in the anesthetized python, Python regius. Regul. Pept. 18, 15-26), rattlesnakes (Galli, G., Skovgaard, N., Abe, A.S., Taylor, E.W., Wang, T., 2005. The role of nitric oxide in the regulation of the systemic and the pulmonary vasculature of the rattlesnake, Crotalus durissus terrificus. J. Comp. Physiol. 175B, 201-208) and turtles (Crossley, D.A., Wang, T., Altimiras, J., 2000. Role of nitric oxide in the systemic and pulmonary circulation of anesthetized turtles (Trachemys scripta). J. Exp. Zool. 286, 683-689). These five species of reptiles possess different combinations of division of the heart and structural complexity of the lungs. Comparison of their responses to NO donors and NOS inhibitors may reveal whether the potential contribution of NO to vascular tone correlates with pulmonary complexity and/or with blood pressure. All existing studies oil reptiles have clearly established a potential role for NO in regulating vascular tone in the systemic circulation and NO may be important for maintaining basal systemic vascular tone in varanid lizards, pythons and turtles, through a continuous release of NO. In contrast., the pulmonary circulation is less responsive to NO donors or NOS inhibitors, and it was only in pythons and varanid lizards that the lungs responded to SNP. Both species have a functionally separated heart, so it is possible that NO may exert a larger role in species with low pulmonary blood pressures, irrespective of lung complexity. (C) 2005 Elsevier B.V. All rights reserved.

Structural bioinformatics study of cyclin-dependent kinases complexed with inhibitors

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

Immunohistochemical study of the expression of fatty acid synthase and Ki-67 in salivary gland tumors

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

Flavonols from Pterogyne nitens and their evaluation as myeloperoxidase inhibitors

A myeloperoxidase inhibitory kaempferol derivative, namely pterogynoside (1), was isolated from fruits of Pterogyne nitens, along with six known flavonols, kaempferol, afzelin, kaempferitrin, quercetin, isoquercetrin and rutin. The structures of all compounds were elucidated primarily from 1D and 2D NMR spectroscopic analyses, as well as by high resolution mass spectrometry. All flavonols were screened to identify secondary metabolites as potential myeloperoxidase (MPO) inhibitors, and at concentrations of 0.50-50 nM, quercetin (5), isoquercitrin (6) and rutin (7) exhibited strong inhibitory effects with IC(50) values of 1.22 +/- 0.01, 3.75 +/- 0.02 and 3.60 +/- 0.02, respectively. The MPO activity detected for the new derivative 1 was markedly decreased (IC(50) 10.3 +/- 0.03) when compared with known flavonols 5-7, and interestingly increased when tested against ABTS scavenging activity. (C) 2008 Published by Elsevier Ltd.

cAMP signaling pathway controls glycogen metabolism in Neurospora crassa by regulating the glycogen synthase gene expression and phosphorylation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A systematic approach to identify STRE-binding proteins of the gsn glycogen synthase gene promoter in Neurospora crassa

The gene encoding glycogen synthase in Neurospora crassa (gsn) is transcriptionally down-regulated when mycelium is exposed to a heat shock from 30 to 45 degrees C. The gsn promoter has one stress response element (STRE) motif that is specifically bound by heat shock activated nuclear proteins. In this work, we used biochemical approaches together with mass spectrometric analysis to identify the proteins that bind to the STRE motif and could participate in the gsn transcription regulation during heat shock. Crude nuclear extract of heat-shocked mycelium was prepared and fractionated by affinity chromatography. The fractions exhibiting DNA-binding activity were identified by electrophoretic mobility shift assay (EMSA) using as probe a DNA fragment containing the STRE motif DNA-protein binding activity was confirmed by Southwestern analysis. The molecular mass (MM) of proteins was estimated by fractionating the crude nuclear extract by SDS-PAGE followed by EMSA analysis of the proteins corresponding to different MM intervals. Binding activity was detected at the 30-50 MM kDa interval. Fractionation of the crude nuclear proteins by IEF followed by EMSA analysis led to the identification of two active fractions belonging to the pIs intervals 3.54-4.08 and 6.77-7.31. The proteins comprising the MM and pI intervals previously identified were excised from a 2-DE gel, and subjected to mass spectrometric analysis (MALDI-TOF/TOF) after tryptic digestion. The proteins were identified by search against the MIPS and MIT N. crassa databases and five promising candidates were identified. Their structural characteristics and putative roles in the gsn transcription regulation are discussed.

Peptides based on CcdB protein as novel inhibitors of bacterial topoisomerases

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

Design and synthesis of peptides from bacterial ParE toxin as inhibitors of topoisomerases

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

Ambient pH Controls Glycogen Levels by Regulating Glycogen Synthase Gene Expression in Neurospora crassa. New Insights into the pH Signaling Pathway

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