Nitric oxide synthase activity in tissues of the blowfly Chrysomya megacephala (Fabricius, 1794)

Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses, which include both cellular and Immoral components. Cellular responses are mediated by hemocytes, and Immoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In mammals, nitric oxide synthases (NOSs) are also present in the endothelium, the brain, the adrenal glands, and the platelets. Studies on the distribution of NO-producing systems in invertebrates have revealed functional similarities between NOS in this group and vertebrates. We attempted to localize NOS activity in tissues of naive (UIL), yeast-injected (YIL), and saline-injected (SIL) larvae of the blowfly Chrysomya megacephala, using the NADPH diaphorase technique. Our findings revealed similar levels of NOS activity in muscle, fat body, Malpighian tubule, gut, and brain, suggesting that NO synthesis may not be involved in the immune response of these larval systems. These results were compared to many studies that recorded the involvement of NO in various physiological functions of insects.

Structural bioinformatics study of EPSP synthase from Mycobacterium tuberculosis

The shikimate pathway is an attractive target for herbicides and antimicrobial agent development because it is essential in algae, higher plants, bacteria, and fungi, but absent from mammals. Homologues to enzymes in the shikimate pathway have been identified in the genome sequence of Mycobacterium tuberculosis. Among them, the EPSP synthase was proposed to be present by sequence homology. Accordingly, in order to pave the way for structural and functional efforts towards anti-mycobacterial agent development, here we describe the molecular modeling of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase isolated from M. tuberculosis that should provide a structural framework on which the design of specific inhibitors may be based on. Significant differences in the relative orientation of the domains in the two models result in open and closed conformations. The possible relevance of this structural transition in the ligand biding is discussed. (C) 2003 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray crystallographic analysis of chorismate synthase from Mycobacterium tuberculosis

The enzymes of the shikimate pathway are potential targets for the development of new therapies because they are essential for bacteria but absent from mammals. The last step in this pathway is performed by chorismate synthase (CS), which catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. Optimization of crystallization trials allowed the crystallization of homogeneous recombinant CS from Mycobacterium tuberculosis (MtCS). The crystals of MtCS belong to space group P6(4)22 (or P6(2)22) and diffract to 2.8 Angstrom resolution, with unit-cell parameters a = b = 129.7, c = 156.8 Angstrom. There are two molecules in the asymmetric unit. Molecular-replacement trials were not sucessful. Heavy-atom derivative screening is in progress.

Structure of chorismate synthase from Mycobacterium tuberculosis

In bacteria, fungi, plants, and apicomplexan parasites, the aromatics compounds, such as aromatics amino acids, are synthesized through seven enzymes from the shikimate pathway, which are absent in mammals. The absence of this pathway in mammals make them potential targets for development of new therapy against infectious diseases, such as tuberculosis, which is the world's second commonest cause of death from infectious disease. The last enzyme of shikimate pathway is the chorismate synthase (CS), which is responsible for conversion of the 5-enolpyruvylshikimate-3-phosphate to chorismate. Here, we report the crystallographic structure of CS from Mycobacterium tuberculosis (MtCS) at 2.65 angstrom resolution. The MtCS structure is similar to other CS structures, presenting beta-alpha-beta sandwich structural topology, in which each monomer of MtCS consists of a central helical core. The MtCS can be described as a tetramer formed by a dimer of dimers. However, analytical ultracentrifugation studies suggest the MtCS is a dimer with a more asymmetric shape than observed on the crystallographic dimer and the existence of a low equilibrium between dimer and tetramer. Our results suggest that the MtCS oligomerization is concentration dependent and some conformational changes must be involved on that event. (c) 2005 Elsevier B.V. All rights reserved.

Dynamics of glyphosate-induced conformational changes of Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19) determined by hydrogen-deuterium exchange and electrospray mass spectrometry

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the reaction between shikimate 3-phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate 3-phosphate, an intermediate in the shikimate pathway, which leads to the biosynthesis of aromatic amino acids. EPSPS exists in an open conformation in the absence of substrates and/or inhibitors and in a closed conformation when bound to the substrate and/or inhibitor. In the present report, the H/D exchange properties of EPSPS from Mycobacterium tuberculosis (Mt) were investigated for both enzyme conformations using ESI mass spectrometry and circular dichroism (CD). When the conformational changes identified by H/D exchanges were mapped on the 3-D structure, it was observed that the apoenzyme underwent extensive conformational changes due to glyphosate complexation, characterized by an increase in the content of alpha-helices from 40% to 57%, while the beta-sheet content decreased from 30% to 23%. These results indicate that the enzyme underwent a series of rearrangements of its secondary structure that were accompanied by a large decrease in solvent access to many different regions of the protein. This was attributed to the compaction of 71% of alpha-helices and 57% of beta-sheets as a consequence of glyphosate binding to the enzyme. Apparently, MtEPSPS undergoes a series of inhibitor-induced conformational changes, which seem to have caused synergistic effects in preventing solvent access to the core of molecule, especially in the cleft region. This may be part of the mechanism of inhibition of the enzyme, which is required to prevent the hydration of the substrate binding site and also to induce the cleft closure to avoid entrance of the substrates.

Understanding the Structure, Activity and Inhibition of Chorismate Synthase from Mycobacterium tuberculosis

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

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)

Molecular models of tryptophan synthase from Mycobacterium tuberculosis complexed with inhibitors

The development of new therapies against infectious diseases is vital in developing countries. Among infectious diseases, tuberculosis is considered the leading cause of death. A target for development of new drugs is the tryptophan pathway. The last enzyme of this pathway, tryptophan synthase (TRPS), is responsible for conversion of the indole 3-glycerol phosphate into indol and the condensation of this molecule with serine-producing tryptophan. The present work describes the molecular models of TRPS from Mycobacterium tuberculosis (MtTRPS) complexed with six inhibitors, the indole 3-propanol phosphate and five arylthioalkyl-phosphonated analogs of substrate of the a-subunit. The molecular models of MtTRPS present good stereochemistry, and the binding of the inhibitors is favorable. Thus, the generated models can be used in the design of more specific drugs against tuberculosis and other infectious diseases.

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.

Sources of errors in the quantitative analysis of food carotenoids by HPLC

Several factors render carotenoid determination inherently difficult. Thus, in spite of advances in analytical instrumentation, discrepancies in quantitative results on carotenoids can be encountered in the international literature. A good part of the errors comes from the pre-chromatographic steps such as sampling scheme that does not yield samples representative of the food lots under investigation; sample preparation which does not maintain representativity and guarantee homogeneity of the analytical sample; incomplete extraction; physical losses of carotenoids during the various steps, especially during partition or washing and by adsorption to glass walls of containers; isomerization and oxidation of carotenoids during analysis. on the otherhand, although currently considered the method of choice for carotenoids, high performance liquid chromatography (HPLC) is subject to various sources of errors, such as: incompatibility of the injection solvent and the mobile phase, resulting in distorted or split peaks; erroneous identification; unavailability, impurity and instability of carotenoid standards; quantification of highly overlapping peaks; low recovery from the HPLC column; errors in the preparation of standard solutions and in the calibration procedure; calculation errors. Illustrations of the possible errors in the quantification of carotenoids by HPLC are presented.

Updated Brazilian database on food carotenoids: Factors affecting carotenoid composition

This article updates the Brazilian database on food carotenoids. Emphasis is on carotenoids that have been demonstrated important to human health: alpha-carotene, beta-carotene, beta-cryptoxanthin, lycopene, lutein and zeaxanthin. The sampling and sample preparation strategies and the analytical methodology are presented. Possible sources of analytical errors, as well as the measures taken to avoid them, are discussed. Compositional variation due to such factors as variety/cultivar, stage of maturity, part of the plant utilized, climate or season and production technique are demonstrated. The effects of post-harvest handling, preparation, processing and storage of food on the carotenoid composition are also discussed. The importance of biodiversity is manifested by the variety of carotenoid sources and the higher levels of carotenoids in native, uncultivated or semi-cultivated fruits and vegetables in comparison to commercially produced crops. (C) 2008 Elsevier B.V. All rights reserved.

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)

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)