Identification of organoselenium compounds that possess chemopreventive properties in human prostate cancer LNCaP cells

The process of cancer development consists of three sequential stages termed initiation, promotion, and progression. Oxidative stress damages DNA and introduces mutations into oncogenes or tumor suppressor genes, thus contributing to cancer development. Cancer chemoprevention is defined to prevent or delay the development of cancer by the use of natural or synthetic substances. In the present study, we synthesized a series of organoselenium compounds and evaluated their possible chemopreventive properties in human prostate cancer LNCaP cells. Among 42 organoselenium compounds tested, two compounds, 3-selena-1-dethiacephem 13 and 3-selena-1-dethiacephem 14 strongly activated the Nrf2/ARE (antioxidant response element) signaling and thus markedly increased expression of heme oxygenase-1 (HO-1), a phase II antioxidant enzyme. Translocation of Nrf2 to the nucleus preceded HO-1 protein induction by two compounds. The intracellular ROS level was strongly reduced immediately after treatment with these compounds, showing that they are potent antioxidants. Finally, both compounds inhibited cell growth via cell cycle arrest. Our findings suggest that compounds 13 and 14 could not only attenuate oxidative stress through Nrf2/ARE activation and direct ROS scavenging but also inhibit cell growth. Thus, these compounds possess the potential as pharmacological agents for chemoprevention of human prostate cancer.     

Biosynthesis of dihydrochalcomycin: Characterization of a deoxyallosyltransferase (gerGTI)

Through an inactivation experiment followed by complementation, the gerGTII gene was previously characterized as a chalcosyltransferase gene involved in the biosynthesis of dihydochalcomycin. The glycosyltransferase gerGTI was identified as a deoxyallosyltransferase required for the glycosylation of D-mycinose sugar. This 6-deoxyhexose sugar was converted to mycinose, via bis-O-methylation, following attachment to the polyketide lactone during dihydrochalcomycin biosynthesis. Gene sequence alignment of gerGTI to several glycosyltransferases revealed a consensus sequence motif that appears to be characteristic of the enzymes in this sub-group of the glycosyltransferase family. To characterize its putative function, genetic disruption of gerGTI in the wild-type strain Streptomyces sp. KCTC 0041BP and in the gerGTII-deleted mutant (S. sp. ΔgerGTII), as well as complementation of gerGTII in S. sp. ΔgerGTII-GTI, were carried out, and the products were analyzed by LC/MS. S. sp. ΔgerGTII-GTI mutant produced dihydrochalconolide macrolide. S. sp. ΔgerGTI and S. sp. ΔgerGTII-GTI complementation of gerGTII yielded dihydrochalconolide without the mycinose sugar. The intermediate shows that gerGTI encodes a deoxyallosyltransferase that acts after gerGTII.     

Analysis of Loss of heterozygosity and immunohistochemistry in BRCA1 gene in sporadic breast cancers

BRCA1 is a tumour suppressor gene (TSG), which predisposes cancer to both breast and ovary. The primary objective of the present study is to ascertain the involvement of BRCA1 gene in the pathogenesis of sporadic breast cancer women in Chennai (South India) by analysing its protein expression by immunohistochemistry (IHC) and loss of heterozygosity (LOH) for confirmation of the involvement of TSG in the study population. We found down regulation of BRCA1 protein (54%) in IHC and it was correlated with the clinicopathological parameters of the patients. We found near significant correlation (P < 0.063) between BRCA1 protein expression and clinicopathological parameters. We found 30% LOH in our study and it was also correlated with the clinicopathological parameters. No correlation was found between LOH and clinicopathological parameters. Though we found no correlation, the results revealed in this study support the involvement of BRCA1 TSG in the pathogenesis of sporadic breast cancer women in Chennai (South India).     

Calcium store contents control the expression of TRPC1, TRPC3 and TRPV6 proteins in LNCaP prostate cancer cell line

The mammalian homologues of the Drosophila transient receptor potential (TRP) represent a superfamily of ion channels involved in Ca(2+) homeostasis. Several members of this family are activated either by a depletion of the internal stores of Ca(2+) or by stimulation of G protein-coupled receptors. In androgen responsive prostate cancer cell line LNCaP, TRPC1, TRPC4 and/or TRPV6 have been reported to function as store-operated channels (SOCs) while TRPC3 might be involved in the response to agonist stimulation, possibly through the induction of diacylglycerol production by phospholipase C. However, the control of expression of these TRP proteins is largely unknown. In the present study, we have investigated if the expression of the TRP proteins possibly involved in the capacitative influx of calcium is influenced by the contents of Ca(2+) in the endoplasmic reticulum. Using real-time PCR and Western blot techniques, we show that the expression of TRPC1, TRPC3 and TRPV6 proteins increases after a prolonged (24-48 h) depletion of the stores with thapsigargin. The upregulation of TRPC1 and TRPC3 depends on the store contents level and involves the activation of the Ca(2+)/calmodulin/calcineurin/NFAT pathway. Functionally, cells overexpressing TRPC1, TRPC3 and TRPV6 channels after a prolonged depletion of the stores showed an increased [Ca(2+)](i) response to alpha-adrenergic stimulation. However, the store-operated entry of calcium was unchanged. The isolated overexpression of TRPV6 (without overexpression of TRPC1 and TRPC3) did not produce this increased response to agonists, therefore suggesting that TRPC1 and/or TRPC3 proteins are responsible for the response to alpha-adrenergic stimulation but that TRPC1, TPRC3 and TRPV6 proteins, expressed alone or concomitantly, are not sufficient for SOC formation.     

Identification of mirtrons in rice using MirtronPred: a tool for predicting plant mirtrons

Studies from flies and insects have reported the existence of a special class of miRNA, called mirtrons that are produced from spliced-out introns in a DROSHA-independent manner. The spliced-out lariat is debranched and refolded into a stem-loop structure resembling the pre-miRNA, which can then be processed by DICER into mature ~21 nt species. The mirtrons have not been reported from plants. In this study, we present MirtronPred, a web based server to predict mirtrons from intronic sequences. We have used the server to predict 70 mirtrons in rice introns that were put through a stringent selection filter to shortlist 16 best sequences. The prediction accuracy was subsequently validated by northern analysis and RT-PCR of a predicted Os-mirtron-109. The target sequences for this mirtron were also found in the rice degradome database. The possible role of the mirtron in rice regulon is discussed. The MirtronPred web server is available at http://bioinfo.icgeb.res.in/mirtronPred.     

Metabolic Modeling of Dynamic Brain 13C NMR Multiplet Data: Concepts and Simulations with a Two-Compartment Neuronal-Glial Model

Metabolic modeling of dynamic ¹³C labeling curves during infusion of ¹³C-labeled substrates allows quantitative measurements of metabolic rates in vivo. However metabolic modeling studies performed in the brain to date have only modeled time courses of total isotopic enrichment at individual carbon positions (positional enrichments), not taking advantage of the additional dynamic ¹³C isotopomer information available from fine-structure multiplets in ¹³C spectra. Here we introduce a new ¹³C metabolic modeling approach using the concept of bonded cumulative isotopomers, or bonded cumomers. The direct relationship between bonded cumomers and ¹³C multiplets enables fitting of the dynamic multiplet data. The potential of this new approach is demonstrated using Monte-Carlo simulations with a brain two-compartment neuronal-glial model. The precision of positional and cumomer approaches are compared for two different metabolic models (with and without glutamine dilution) and for different infusion protocols ([1,6-¹³C₂]glucose, [1,2-¹³C₂]acetate, and double infusion [1,6-¹³C₂]glucose?+?[1,2-¹³C₂]acetate). In all cases, the bonded cumomer approach gives better precision than the positional approach. In addition, of the three different infusion protocols considered here, the double infusion protocol combined with dynamic bonded cumomer modeling appears the most robust for precise determination of all fluxes in the model. The concepts and simulations introduced in the present study set the foundation for taking full advantage of the available dynamic ¹³C multiplet data in metabolic modeling.     

Identification and characterization of a novel Ribose 5-phosphate isomerase B from Leishmania donovani

Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Due to the emergence of resistance to the available antileishmanial drugs there is an immediate need to identify molecular targets on which to base future treatment strategies. Ribose 5-phosphate isomerase (Rpi; EC 5.3.1.6) is a key enzyme of the pentose phosphate pathway (PPP) which catalyses the reversible aldose-ketose isomerization between Ribose 5-phosphate (R5P) and Ribulose 5-phosphate (Ru5P). It exists in two isoforms A and B. These two are completely unrelated enzymes catalyzing the same reaction. Analysis of the Leishmania infantum genome revealed that though the RpiB gene is present, RpiA homologs are completely absent. An absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for the chemotherapy of Leishmaniasis. In this paper, we report for the first time the presence of B isoform of the Rpi enzyme in Leishmania donovani (LdRpiB) by cloning and molecular characterization of the enzyme. An amplified L. donovani RpiB gene is 519 bp and encodes for a putative 172 amino acid protein with a molecular mass of ～19 kDa. An ～19 kDa protein with poly-His tag at the C-terminal end was obtained by heterologous expression of LdRpiB in Escherichia coli. The recombinant form of RpiB was obtained in soluble and active form. The LdRpiB exists as a dimer of dimers i.e. the tetramer form. The polyclonal antibody against Trypanosoma cruzi RpiB could detect a band of ～19 kDa with the purified recombinant RpiB as well as native RpiB from the L. donovani promastigotes. Recombinant RpiB obeys the classical Michaelis-Menten kinetics utilizing R5P as the substrate with a K(m) value of 2.4±0.6 mM and K(cat) value of 30±5.2 s(-1). Our study confirms the presence of Ribose 5-phosphate isomerase B in L. donovani and provides functional characterization of RpiB for further validating it as a potential drug target.     

Synthesis of graft copolymer (CgOH-g-AGA): Physicochemical properties, characterization and application

A graft copolymer of κ-carrageenan and 2-acrylamidoglycolic acid (CgOH-g-AGA) was synthesized via free radical polymerization initiated by potassium peroxymonosulphate/malonic acid redox pair. For affording maximum percentage of grafting, optimum conditions were determined by varying the concentrations of κ-carrageenan, 2-acrylamidoglycolic acid, potassium peroxymonosulphate, malonic acid, hydrogen ion, time and temperature. The swelling, metal ion uptake and flocculation studies were investigated with water, three metals (Ni(2+), Pb(2+) and Zn(2+)) solutions, coal (coking and non-coking) suspensions, respectively. Both, polymer backbone and its corresponding graft copolymer samples were characterized by Fourier transform infrared spectroscopy and thermogravimetric analysis.     

Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications

Clostridia are anaerobic Firmicutes producing a large array of metabolites by utilizing simple and complex carbohydrates, such as cellulose, as well as CO2/H2 or CO. Their exceptional substrate diversity is enhanced by their ability to produce a broad spectrum of chemicals that can be used as precursors to or directly as biofuels and industrial chemicals. Genetic and genomic tools are under intense development, and recent efforts to metabolically engineer clostridia demonstrate their potential for biofuel and biorefinery applications. Pathway engineering to combine established substrate-utilization programs, such as for cellulose, CO2/H2 or CO, with desirable metabolic programs could lead to modular design of strains suitable for many applications. Engineering complex phenotypes--aerotolerance, abolished sporulation, and tolerance to toxic chemicals--could lead to superior bioprocessing strains.