Biomedical subjects

Smita Misra

Publications and source records attributed to Smita Misra.

9 recordsLinked to original sources

PubMed2006

Human SLUG does not directly bind to CtBP1.

SLUG is a transcriptional repressor protein implicated to have major role in the oncogenesis and metastasis of human breast cells. We previously have shown by chromatin immunoprecipitation assay that human SLUG (hSLUG) is co-localized with the co-repressor protein CtBP1 as bound to the BRCA2 gene silencer [M.K. Tripathi, S. Misra, S.V. Khedkar, N. Hamilton, C. Irvin-Wilson,, C. Sharan, L. Sealy, G. Chaudhuri, J. Biol. Chem. 280 (2005) 17163-17171]. hSLUG was predicted to be binding directly to CtBP1 because of an apparent presence of CtBP1 binding site in its amino acid sequences. Here, we provide evidence through yeast two-hybrid and in vitro co-immunoprecipitation analyses that hSLUG does not directly interacts with hCtBP1. This observation will help in the study of the mode of action of hSLUG in human cells.

Alcohol Oxidoreductases↗

PubMed2006

Rural communities' preferences for arsenic mitigation options in Bangladesh.

In the context of arsenic contamination of groundwater in Bangladesh, this paper analyses rural people's preferences for arsenic-free drinking water options. A particular focus is on rural households' willingness to pay for piped water supply which can provide a sustainable solution to the arsenic problem, and how the preference for piped water supply compares with that for various other household/community-based arsenic mitigation technologies. The analysis is based on data collected in a survey of over 2700 households in rural Bangladesh. Six arsenic mitigation technologies were selected for the study: three-kolshi (pitcher) method, activated alumina method (household-based and community-based), dugwell, pond sand filter and deep tubewell (handpump). The survey results indicate that, after taking into consideration the initial and recurring costs, convenience, associated risks and the advantages and disadvantages of each selected technology, the preference of the rural people is overwhelmingly in favor of deep tubewells, followed by the three-kolshi method. The analysis reveals a strong demand for piped water in both arsenic-affected and arsenic-free rural areas, and scope of adequate cost recovery. Between piped water and other arsenic mitigation technologies, the preference of the rural people is found to be predominantly in favor of the former.

Arsenic↗

PubMed2005

Down-regulation of 7SL RNA expression and impairment of vesicular protein transport pathways by Leishmania infection of macrophages.

The parasitic protozoan Leishmania specifically manipulates the expression of host macrophage genes during initial interactions, as revealed by mRNA differential display reverse transcription-PCR and cDNA microarray analyses. The genes that are down-regulated in mouse (J774G8) or human (U937) macrophages upon exposure to Leishmania include small RNA transcripts from the short interspersed element sequences. Among the short interspersed element RNAs that are down-regulated is 7SL RNA, which is the RNA component of the signal recognition particle. Because the microbicidal functions of macrophages profoundly count on vesicular protein transport processes, down-regulation of 7SL RNA may be significant in the establishment of infection by Leishmania in macrophage phagolysosomes. To evaluate whether down-regulation of 7SL RNA results in inhibition of signal recognition particle-mediated vesicular protein transport processes, we have tested and found that the targeting of proteins to the endoplasmic reticulum and plasma membrane and the secretion of proteins by macrophages are compromised in Leishmania-infected J774G8 and U937 cells. Knocking down 7SL RNA using small interfering RNA mimicked the effect of exposure of macrophages to Leishmania. The overexpression of 7SL RNA in J774G8 or U937 cells made these cells resistant to Leishmania infection, suggesting the possible biological significance of down-regulation of 7SL RNA synthesis in the establishment of infection by Leishmania. We conclude that Leishmania down-regulates 7SL RNA in macrophages to manipulate the targeting of many proteins that use the vesicular transport pathway and thus favors its successful establishment of infection in macrophages.

Animals↗

PubMed2005

Negative regulation of the expressions of cytokeratins 8 and 19 by SLUG repressor protein in human breast cells.

Invasiveness of tumor cells is often determined by the profile of their expressed genes. To determine the gene expression differences between an invasive and a non-invasive human breast tumor cells, we selected BT-549 (invasive) and MDA-MB-468 (non-invasive) cells, and compared their transcriptomes by cDNA microarray analysis. Among the significant differences in gene expressions, notable are the up-regulation of cytokeratins 8 and 19, and down-regulation of metallothioneins 1G and IL in MDA-MB-468 cells. Since MDA-MB-468 cells do not express SLUG, a member of a small family of E2-box-binding zinc finger silencer proteins, we studied whether the cytokeratin gene overexpressions in these cells are due to the absence of SLUG. Inducible expression of SLUG in MDA-MB-468 cells inhibited the expressions of the cytokeratin 8 and 19 but not others as was revealed by microarray analysis. Similarly, siRNA knock down of SLUG in BT-549 cells increased the expressions of those cytokeratin mRNAs. SLUG levels in the cell regulated the function of cytokeratins 8 and 19 gene promoters. We conclude that the expressions of cytokeratins and metallothioneins may be associated with the differential invasive behaviors of these breast tumor cells and SLUG may have regulatory roles in this process.

Breast Neoplasms↗

PubMed2005

Regulation of BRCA2 gene expression by the SLUG repressor protein in human breast cells.

The expression of the breast cancer susceptibility protein BRCA2 is highly regulated in human breast, ovary, and pancreatic cells. BRCA2 is not expressed in the non-dividing cells, and expression is cell cycle stage-dependent and is elevated in the sporadic cancer cells. Mutational analysis of the upstream sequence of the human BRCA2 gene revealed an E2-box-containing silencer at the -701 to -921 position. The E2-box is essential for the cell-cycle stage-dependent activity of the silencer. We affinity-purified a 29-kDa silencer-binding protein (SBP) from the nuclear extracts of human breast cells BT-549 and MDA-MB-231. We explored whether the E2-box-binding repressor protein SLUG, which is of similar molecular size, is involved in the silencing process. Supershift assay with the purified SBP and anti-SLUG antibody revealed the identity of the SBP as SLUG. We found that silencer is inactive in the human breast cancer cells such as MDA-MB-468 and MCF-7 that do not express SLUG, further suggesting the involvement of SLUG in the BRCA2 gene silencing. Inducible expression of human SLUG in the dividing MDA-MB-468 cells reduced BRCA2 RNA levels with the activation of the silencer. Furthermore, small interfering RNA-mediated knockdown of SLUG mRNA in the BT-549 cells caused inhibition of the silencer function. Chromatin immunoprecipitation assays suggested that SLUG mediates its action by recruiting C-terminal-binding protein-1 (CtBP-1) and histone deacetylase-1 (HDAC-1) at the silencer E2-box. The general HDAC inhibitor, trichostatin A, inhibited the SLUG-mediated regulation of the silencer function. It thus appears that SLUG is a negative regulator for BRCA2 gene expression.

Acetylation↗

PubMed2005

Value of arsenic-free drinking water to rural households in Bangladesh.

Using contingent valuation survey data for about 2700 households in rural Bangladesh, and applying a multinomial logit model, the paper estimates the value of arsenic-free drinking water to the rural people. The estimates indicate that the rural people in arsenic-affected areas of Bangladesh place a low value on arsenic-free drinking water. It is about 10-14 percent of the amount they are willing to pay for piped water and only about 0.2-0.3 percent of the average household income. The implication of the result is that robust but costly arsenic reduction technologies such as activated alumina technology may find little social acceptance, unless heavily subsidized.

Aluminum Oxide↗

PubMed2005

A type II ribonuclease H from Leishmania mitochondria: an enzyme essential for the growth of the parasite.

Replication of kDNA in the mitochondrion of the kinetoplastid protozoan is an essential process. One of the proteins that may be required for the kDNA replication is the ribonuclease H (RNase H; EC 3.1.26.4). We have identified four distinct ribonuclease H genes in Leishmania, one type I (LRNase HI) and three type II (LRNase HIIA, LRNase HIIB and LRNase HIIC). We detail here molecular characterization of LRNase HIIC. The coding sequence of LRNase HIIC is 1425 bp in length encoding a 474-amino acid protein with a calculated molecular mass of approximately 53 kDa. While LRNase HIIC shares several conserved domains with mitochondrial RNase H from other organisms, it has three extra patches of amino acid sequences unique to this enzyme. Functional identity of this protein as an RNase H was verified by genetic complementation in RNase H-deficient Escherichia coli. The precursor protein may be enzymatically inactive as it failed to complement the E. coli mutant. The mitochondrial localization signal in LRNase HIIC is within the first 40 amino acid residues at the N-terminus. In vitro import of the protein by the mitochondrial vesicles showed that the precursor protein is processed to a 49-kDa protein. Antisense ablation of LRNase HIIC gene expression is lethal to the parasite cells both in vitro and in vivo. This study not only reveals the significance of the LRNase HIIC in the kinetoplast biology but also identifies a potential molecular target for antileishmanial chemotherapy.

Amino Acid Sequence↗

PubMed2005

Expression, purification, and characterization of Leishmania donovani trypanothione reductase in Escherichia coli.

Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in all the trypanosomatids including Leishmania. The unique presence of this enzyme in trypanosomatids and absence in mammalian host make this enzyme an attractive target for the development of the antileishmanials. Complete open reading frame encoding trypanothione reductase from Leishmania donovani (Dd8 strain, causative agent of Indian visceral leishmaniasis) was cloned, sequenced, and expressed in Escherichia coli strain BL21 (DE3) as glutathione S-transferase fusion protein. The conditions were developed for overexpression of fusion protein in soluble form and purification of the recombinant protein to homogeneity. The recombinant LdTR was 54.68 kDa in size, dimeric in nature, and reduces oxidized trypanothione to reduced form. The kinetic parameters for trypanothione disulfide are K(m), 50 microM; k(cat), 18,181 min(-1); and k(cat)/K(m), 6.06x10(6) M(-1) s(-1). The yield of recombinant LdTR was approximately 16 mg/L bacterial culture and accounted for 6% of the total soluble proteins. The expressed protein was inhibited by known TR inhibitors as well as by SbIII, the known antileishmanial compound. This is the first report of large-scale production of any leishmanial TR in E. coli.

Amino Acid Sequence↗

PubMed2005

Molecular characterization of a human BRCA2 homolog in Leishmania donovani.

The breast cancer susceptibility protein BRCA2 is implicated in the DNA double-strand break (DSB) repair pathway through its association with Rad51. It is almost a ubiquitous eukaryotic protein; homologs of the BRCA2 gene (BRH2) have been identified in many mammals, as well as nonmammals. As a part of our quest to understand the DNA damage, repair, and recombination process in the parasitic protozoan, Leishmania sp., we have cloned and characterized a BRCA2 homolog from Leishmania sp. (LBRH2). LBRH2 is coded by a single-copy gene (ORF = 3,498 bp) located at the 700-kb chromosome 16. The transcripts in both the promastigotes and the amastigotes are approximately 3.9 kilonucleotides (knt) in size, corresponding to a protein with a calculated molecular mass of 128 kDa. The primary transcript of the gene is alternatively trans-spliced to produce 3 distinct mRNAs with altered folded structures at their 5' ends. This study will contribute toward the understanding of a potential RAD51-mediated DNA recombination/repair pathway in Leishmania sp.

Alternative Splicing↗