Quantitative structure-activity studies on a series of migrastatin analogs as inhibitors of cancer cell metastasis

Migrastatin, a macrolide natural product, and its structurally related analogs are potent inhibitors of cancer cell metastasis, invasion and migration. In the present work, a specialized fragment-based method was employed to develop QSAR models for a series of migrastatin and isomigrastatin analogs. Significant correlation coefficients were obtained (best model, q(2) = 0.76 and r(2) = 0.91) indicating that the QSAR models possess high internal consistency. The best model was then used to predict the potency of an external test set, and the predicted values were in good agreement with the experimental results (R(2) (pred) = 0.85). The final model and the corresponding contribution maps, combined with molecular modeling studies, provided important insights into the key structural features for the anticancer activity of this family of synthetic compounds based on natural products.

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier Inc. All rights reserved.

Normalization procedure for relaxation studies in NMR quantum information processing

NMR quantum information processing studies rely on the reconstruction of the density matrix representing the so-called pseudo-pure states (PPS). An initially pure part of a PPS state undergoes unitary and non-unitary (relaxation) transformations during a computation process, causing a ""loss of purity"" until the equilibrium is reached. Besides, upon relaxation, the nuclear polarization varies in time, a fact which must be taken into account when comparing density matrices at different instants. Attempting to use time-fixed normalization procedures when relaxation is present, leads to various anomalies on matrices populations. On this paper we propose a method which takes into account the time-dependence of the normalization factor. From a generic form for the deviation density matrix an expression for the relaxing initial pure state is deduced. The method is exemplified with an experiment of relaxation of the concurrence of a pseudo-entangled state, which exhibits the phenomenon of sudden death, and the relaxation of the Wigner function of a pseudo-cat state.

P-Glycoprotein Expression, Tumor Weight, Age, and Relapse in Patients with Stage I and II Favorable-Histology Wilms` Tumor

Fifteen percent of patients with Wilms`` tumor (WT) experience relapse. It has been suggested that weight and age may affect the chances of relapse. Few studies have investigated the role, if any, between P-glycoprotein (P-gp) and relapse. The authors assessed the prognostic value of tumor weight and age at diagnosis and asked whether some other potential biological markers, specifically P-gp protein expression, had a prognostic value in favorable-histology WT. No association between age and relapse could be found. Patients with tumor weight >= a parts per thousand yen550 g were 6 times more likely to relapse, whereas P-gp expression was positive in 18/40 (45%%) of the patients, of which 10/12 (83.3%%) relapsed and 8/28 (28.6%%) did not. Further studies are necessary to elucidate whether or not P-gp is related to relapse in patients with histologically favorable Wilms`` tumor. If confirmed, the protein may be used in the future as a target for new drugs and treatments for this group of patients.

Bcl-2 expression and apoptosis induction in human HL60 leukaemic cells treated with a novel organotellurium(IV) compound RT-04

Organotellurium(]V) compounds have been reported to have multiple biological activities including cysteine protease-inhibitory activity, mainly cathepsin B. As cathepsin B is a highly predictive indicator for prognosis and diagnosis of cancer, a possible antitumor potential for these new compounds is expected. In this work, it was investigated the effectiveness of organotellurium(IV) RT-04 to produce lethal effects in the human promyelocytic leukaemia cell line HL60. Using the MTT tetrazolium reduction test, and trypan blue exclusion assay, the IC50 for the compound after 24 h incubation was 6.8 and 0.35 mu M, respectively. Moreover, the compound was found to trigger apoptosis in HL60 cells, inducing DNA fragmentation and caspase-3, -6, and -9 activations. The apoptsosis-induced by RT-04 is probably related to the diminished Bcl-2 expression, observed by RT-PCR, in HL60-treated cells. In vivo studies demonstrated that the RT-04 treatment (2.76 mg/kg given for three consecutive days) produces no significant toxic effects for bone marrow and spleen CFU-GM. However, higher doses (5.0 and 10 mg/kg) produced a dose-dependent reduction in the number of CFU-GM of RT-04-treated mice. These results suggest that RT-04 is able to induce apoptosis in HL60 cells by Bcl-2 expression down-modulation. Further studies are necessary to better clarify the effects of this compound on bone marrow normal cells. (C) 2008 Elsevier Ltd. All rights reserved.

Directed Differentiation of Neural Progenitors into Neurons Is Accompanied by Altered Expression of P2X Purinergic Receptors

Neural differentiation has been extensively studied in vitro in a model termed neurospheres, which consists of aggregates of neural progenitor cells. Previous studies suggest that they have a great potential for the treatment of neurological disorders. One of the major challenges for scientists is to control cell fate and develop ideal culture conditions for neurosphere expansion in vitro, without altering their features. Similar to human neural progenitors, rat neurospheres cultured in the absence of epidermal and fibroblast growth factors for a short period increased the levels of beta-3 tubulin and decreased the expression of glial fibrillary acidic protein and nestin, compared to neurospheres cultured in the presence of these factors. In this work, we show that rat neurospheres cultured in suspension under mitogen-free condition presented significant higher expression of P2X2 and P2X6 receptor subunits, when compared to cells cultured in the presence of growth factors, suggesting a direct relationship between P2X2/6 receptor expression and induction of neuronal differentiation in mitogen-free cultured rat neurospheres.

alpha-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization

Xylella fastidiosa is a xylem-restricted plant pathogen that causes a range of diseases in several and important crops. Through comparative genomic sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. The experimental determination of the primary sequence of some markedly expressed proteins for X fastidiosa and the comparison with the nucleic acids sequence of genome identified one of them as being SCJ21.16 (XFa0032) gene product. The comparative analysis of this protein against SWISSPROT database, in special, resulted in similarity with a-hydroxynitrile lyase enzyme (HNL) from Arabidopsis thaliana, causing interest for being one of the most abundant proteins both in the whole cell extract as well as in the extracellular protein fraction. It is known that HNL enzyme are involved in a process termed ""cyanogenesis"", which catalyzes the dissociation of alpha-hydroxinitrile into carbonyle and HCN when plant tissue is damaged. Although the complete genome sequences of X.fastidiosa are available and the cyanogenesis process is well known, the biological role of this protein in this organism is not yet functionally characterized. In this study we presented the cloning, expression, characterization of recombinant HNL from X fastidiosa, and its probable function in the cellular metabolism. The successful cloning and heterologous expression in Escherichia coli resulted in a satisfactory amount of the recombinant HNL expressed in a soluble, and active form giving convenient access to pure enzyme for biochemical and structural studies. Finally, our results confirmed that the product of the gene XFa0032 can be positively assigned as FAD-independent HNLs. (C) 2009 Elsevier Ltd. All rights reserved.

Changes in Apoptotic Gene Expression Induced by DNA Cross-Linkers

The Millard Research Laboratory is interested in the cytotoxic mechanisms of the bifunctional alkylators diepoxybutane (DEB), epichlorohydrin (ECH), and (1-chloroethenyl) oxirane (COX). Studies performed in the laboratory examine the dual nature of these DNA cross-linking compounds that can act as carcinogens or anti-cancer agents. The mechanisms through which these compounds induce cell death are explored in this study. Cells either undergo cell death due to necrosis or apoptosis. HL-60 cells were treated with varying concentrations of DEB, ECH, or COX. A caspase 3/7 assay was used to test for induction of apoptosis in the treated cells at varying incubation times. It was concluded that DEB induces apoptosis in HL-60 cells treated with 100 μM for 24 hours. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was then used to explore the changes in gene expression of various genes involved in apoptosis signaling. The results were inconclusive as to specific genes involved in DEB induced apoptosis, but the data does suggest that apoptosis is induced by a mitochondrial-mediated apoptosis signaling pathway.

Functional characterization by genetic complementation of aroB-Encoded dehydroquinate synthase from Mycobactetium tuberculosis H37Rv and its heterologous expression and purification

The recent recrudescence of Mycobacterium tuberculosis infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in M. tuberculosis, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The aroB-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the aroB gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The Escherichia coli AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.

Xylella fastidiosa: An in vivo system to study possible survival strategies within citrus xylem vessels based on global gene expression analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Whole-genome expression profiling of Xylella fastidiosa in response to growth on glucose

Xylella fastidiosa is the etiologic agent of diseases in a wide range of economically important crops including citrus variegated chlorosis, a major threat to the Brazilian citrus industry. The genomes of several strains of this phytopathogen have been completely sequenced enabling large-scale functional studies. In this work we used whole-genome DNA microarrays to investigate the transcription profile of X. fastidiosa grown in defined media with different glucose concentrations. Our analysis revealed that while transcripts related to fastidian gum production were unaffected, colicin-V-like and fimbria precursors were induced in high glucose medium. Based on these results, we suggest a model for colicin-defense mechanism in X. fastidiosa.

Analysis of gene expression profiles under water stress in tolerant and sensitive sugarcane plants

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

Gene Expression Profiles Stratified according to Type 1 Diabetes Mellitus Susceptibility Regions

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Pharmacological Evaluation and Preliminary Pharmacokinetics Studies of a New Diclofenac Prodrug without Gastric Ulceration Effect

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