Biomedical subjects

V Radhika

Publications and source records attributed to V Radhika.

8 recordsLinked to original sources

PubMed2006

Bioremediation of zinc using Desulfotomaculum nigrificans: bioprecipitation and characterization studies.

Desulfotomaculum nigrificans, a typical sulfate reducing bacterium (SRB) was successfully grown in the presence of 12-210 mg/L of zinc. Complete bioremoval of zinc was achieved in 2 days for 12 mg/L while the bioremoval efficiency was about 70% in 40 days in the presence of 210 mg/L initial concentration of zinc, attesting to the inhibition of bacterial cell growth at higher zinc concentrations. The bioremoval mechanism was predominantly governed by bioprecipitation with biosorption contributing to a minor extent. The amount of protein present in the extracellular secretions obtained by growth of SRB in modified Baars' medium devoid of iron was the highest followed by those obtained in the presence of zinc or iron, in that order. Bioremediation studies carried out using a specially designed set-up, facilitating the transfer of biogenically produced hydrogen sulfide gas to a separate precipitation assembly, confirmed that zinc could be successfully precipitated from its corresponding sulfate solution, varying in concentration from 10 to 20,000 mg/L. Detailed characterization of the various zinc sulfide precipitates by EDAX and X-ray diffraction analysis conformed to wurtzite structure. The isoelectric points of high purity zinc sulfide and that of chemically synthesized, biogenically produced and zinc sulfide precipitated using bacterially produced hydrogen sulfide gas (BPH-ZnS) were located at pH 3, 7.8, 2.8 and 8, respectively.

Adsorption↗

PubMed2005

Mitogenic signaling by lysophosphatidic acid (LPA) involves Galpha12.

Lysophosphatidic acid (LPA), a major G protein coupled receptor (GPCR)-activating ligand present in serum, elicits growth factor like responses by stimulating specific GPCRs coupled to heterotrimeric G proteins such as G(i), G(q), and G12/13. Previous studies have shown that the overexpression of wild-type Galpha12 (Galpha12WT) results in the oncogenic transformation of NIH3T3 cells (Galpha12WT-NIH3T3) in a serum-dependent manner. Based on the potent growth-stimulating activity of LPA and the presence of LPA and LPA-like molecules in the serum, we hypothesized that the serum-dependent neoplastic transformation of Galpha12WT-NIH3T3 cells was mediated by the stimulation of LPA-receptors (LPARs) by LPA in the serum. In the present study, using guanine nucleotide exchange assay and GST-TPR binding assay, we show that the treatment of Galpha12WT-NIH3T3 with 2 muM LPA leads to the activation of Galpha12. Stimulation of these cells with LPA promotes JNK-activation, a critical component of Galpha12-response and cell proliferation. We also show that LPA can substitute for serum in stimulating JNK-activity, DNA synthesis, and proliferation of Galpha12WT-NIH3T3 cells. LPA-mediated proliferative response in NIH3T3 cells involves Galpha12, but not the closely related Galpha13. Pretreatment of Galpha12WT-NIH3T3 cells with suramin (100 microM), a receptor-uncoupling agent, inhibited LPA-stimulated proliferation of these cells by 55% demonstrating the signal coupling between cell surface LPAR and Galpha12 in the neoplastic proliferation of NIH3T3 cells. As LPA and LPAR mediated mitogenic pathways have been shown to play a major role in tumor genesis and progression, a mechanistic understanding of the signal coupling between LPAR, Galpha12, and the downstream effectors is likely to unravel additional targets for novel cancer chemotherapies.

3T3 Cells↗

PubMed2005

Engineered Saccharomyces cerevisiae strain BioS-1, for the detection of water-borne toxic metal contaminants.

Saccharomyces cerevisiae responds to extracellular toxic stimuli by increasing intracellular cyclic AMP levels, leading to activation of a cAMP-dependent protein kinase, protein kinase A (PKA). Activated PKA phosphorylates downstream substrates, including specific DNA-binding proteins, to turn off the expression of most or all of the yeast genes. Such cAMP-PKA-mediated inhibition of gene expression in response to toxic stimuli appears to be unique to S. cerevisiae. For instance, in mammalian cells, the cAMP-PKA signaling pathway is rather responsive to growth factors and hormones in addition to being primarily involved in the activation of gene expression. Activation of gene expression by the cAMP-PKA pathway in mammalian cells is due mainly to the presence of cAMP-response elements (CREs) located in the promoters of many mammalian genes, and the expression of PKA-responsive stimulatory transcription factor CRE-binding protein, commonly referred as CREBP, which binds to the CREs. Thus, activation of the cAMP-PKA signaling pathway results in the phosphorylation of CREBP by PKA, and phosphorylated CREBP transactivates specific gene expression by interacting with the cognate CRE. Based on these findings, we sought to engineer a yeast-based biosensor, in which the stress-sensing cAMP-PKA pathway of yeast is coupled to the mammalian CREBP-CRE-stimulated gene expression pathway, which drives the expression of a reporter protein, such as green fluorescent protein (GFP). As a primary step toward the development of this biosensor, we engineered a yeast strain, BioS-1, by genetically altering YPH 501, a wild-type strain of S. cerevisiae, to express human CREBP and human CRE promoter-driven GFP. Exposure of BioS-1 to varying concentrations of As3+, Fe2+, Pb2+, and Cd2+ elicits concentration-dependent expression of the GFP reporter that can be easily monitored by the fluorescence emitted by GFP. The results also indicate that the engineered BioS-1 yeast cells can detect 2.5 ppm of these toxic metals and report it through the expression of GFP within 3 h. The results presented herein demonstrate that this engineered yeast strain can detect metal toxicants and can validate the use of this prototypic yeast strain to develop a biosensor that can be used to detect and monitor cytotoxic water-borne toxic heavy metals.

Biological Assay↗

PubMed2004

Galpha13 stimulates cell migration through cortactin-interacting protein Hax-1.

Galpha13, the alpha-subunit of the heterotrimeric G protein G13, has been shown to stimulate cell migration in addition to inducing oncogenic transformation. Cta, a Drosophila ortholog of G13, has been shown to be critical for cell migration leading to the ventral furrow formation in Drosophila embryos. Loss of Galpha13 has been shown to disrupt cell migration associated with angiogenesis in developing mouse embryos. Whereas these observations point to the vital role of G13-orthologs in regulating cell migration, widely across the species barrier, the mechanism by which Galpha13 couples to cytoskeleton and cell migration is largely unknown. Here we show that Galpha13 physically interacts with Hax-1, a cytoskeleton-associated, cortactin-interacting intracellular protein, and this interaction is required for Galpha13-stimulated cell migration. Hax-1 interaction is specific to Galpha13, and this interaction is more pronounced with the mutationally or functionally activated form of Galpha13 as compared with the wild-type Galpha13. Expression of Hax-1 reduces the formation of actin stress fibers and focal adhesion complexes in Galpha13-expressing NIH3T3 cells. Coexpression of Hax-1 also attenuates Galpha(13)-stimulated activity of Rho while potentiating Galpha13-stimulated activity of Rac. The presence of a quadnary complex consisting of Galpha13, Hax-1, Rac, and cortactin indicates the role of Hax-1 in tethering Galpha13 to the cytoskeletal component(s) involved in cell movement. Whereas the expression of Hax-1 potentiates Galpha13-mediated cell movement, silencing of endogenous Hax-1 with Hax-1-specific small interfering RNAs drastically reduces Galpha13-mediated cell migration. These findings, along with the observation that Hax-1 is overexpressed in metastatic tumors and tumor cell lines, suggest a novel role for the association of oncogenic Galpha13 and Hax-1 in tumor metastasis.

Actins↗

PubMed2001

Transforming G proteins.

Heterotrimeric guanine nucleotide binding proteins, commonly known as G proteins form a super-family of signal transduction proteins. They are peripherally associated with the plasma membrane and provide signal coupling to seven transmembrane surface receptors. G proteins are composed of monomers of alpha, beta, and gamma subunits. The beta- and gamma-subunits are tightly associated. The receptors activated by the appropriate "signal", interact catalytically with specific G-proteins to mediate guanine nucleotide exchange at the GDP/GTP binding site of the G-protein alpha-subunits, thus displacing the bound GDP for GTP. The GTP bound form of the g-protein alpha-subunit and in some cases the free betagamma-subunits initiate cellular response by altering the activity of specific effector molecules. Recent studies have indicated that the asyncronous activation of these proteins can lead to the oncogenic transformation of different cell types. The mechanism by which G-proteins regulate the various cell functions appear to involve a complex net-working between different signaling pathways. This review summarizes the signaling mechanisms involved in the regulation of cell proliferation by these transforming G proteins.

Cell Transformation, Neoplastic↗

PubMed2000

Actin polymerization in response to different chemoattractants is reduced in granulocytes from chronic myeloid leukemia patients.

Chronic myeloid leukemia (CML), a hematopoietic stem cell disorder, is characterized by the presence of Philadelphia chromosome (Ph1). Earlier studies have shown that various functions, such as chemotaxis, fluid phase pinocytosis, phagocytosis, and degranulation in response to chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP), were defective in polymorphonuclear leukocytes (PMNL) from CML patients. These functions depend on actin microfilaments (MF). Further studies showed that fMLP-induced actin polymerization was lower in CML PMNL. To see if this defect is specific to stimulation by fMLP alone or is a global phenomenon involving other chemoattractant receptors, chemotaxis and actin polymerization were studied in response to fMLP, an analog of fMLP, formyl-methionine-1 aminocyclooctane 1 carboxylic acid-phenyalanine-O-methionine (FACC8), platelet-activating factor (PAF), and leukotriene B4 (LTB4). These compounds bind to different chemoattractant receptors. Chemotaxis and actin polymerization in response to all four chemoattractants were significantly lower in CML PMNL compared with PMNL from normal subjects and were differentially affected for the different chemoattractants. These results suggest a global phenomenon involving all four chemoattractant-stimulated pathways. This lower amount of F-actin may be responsible for the defective chemotaxis seen in these cells.

Actin Cytoskeleton↗

PubMed1996

Granulocytes from chronic myeloid leukemia (CML) patients show differential response to different chemoattractants.

Binding of chemoattractant to polymorphonuclear leukocytes (PMNL) triggers a series of events like polymerization of actin and tubulin, orientation of cells, chemotaxis, increase in fluid pinocytosis and phagocytosis, and stimulation of microbicidal pathways which includes lysosomal degranulation and generation of reactive oxygen species. Earlier studies from our laboratory have shown that stimulation of chemotaxis, fluid pinocytosis, and actin polymerization of CML PMNL in response to a synthetic chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) is significantly lower than that in normal PMNL. It is not known whether this lower response of CML PMNL to fMLP is a global phenomenon involving different chemoattractant receptors or is restricted to the fMLP pathway. We have evaluated chemoattractant induced degranulation process in normal and CML PMNL to fMLP, platelet activating factor (PAF), leukotriene B4 (LTB4), and an analogue of fMLP viz formyl-methionine-1 aminocyclooctane 1 carboxylic acid-phenylalanine-O-methionine (FACC8) using release of lysozyme as a parameter. We find that after stimulation with fMLP and FACC8, the mean percent release of lysozyme was significantly lower in CML PMNL as compared to that in normal cells (P < 0.001). There was no significant difference between the two after stimulation with PAF and LTB4. The results indicate that the fMLP pathway is suppressed in CML granulocytes whereas PAF and LTB4 pathways appear unaltered in these cells. We therefore also studied the kinetics of peptide-receptor interaction with a labelled hexapeptide fNLPNTL which binds to the fMLP receptor. Our results show that the number of fMLP receptors/cell is significantly lower in CML PMNL (P < 0.05) than in normal PMNL, while their affinity constants and dissociation constants were comparable.

Chemotactic Factors↗

PubMedDate not supplied

Engineered Saccharomyces cerevisiae strain BioS-OS1/2, for the detection of oxidative stress.

One of the major stress factors during space and high-altitude flight is the oxidative damage caused by the release of reactive oxygen intermediates (ROIs) in human tissues. ROIs are released in response to several stress factors including radiation in space. Since ROIs contribute to several pathological conditions, there has been a great interest in developing a biosensor that can monitor the impact of ROIs on biological systems. Toward this goal, we sought to engineer a yeast stain that can monitor oxidative stress and be easily integrated into a biosensor platform. Saccharomyces cerevisiae respond to hyperoxidative stress by activating the expression of many proteins including the transcription factor, Yap1. Activated Yap1 primarily binds to the Yap-1 response elements in the promoters of genes that combat oxidative stress. Based on these observations, we genetically altered the Yap-1 pathway in the YCR094W BY4742 strain of S. cerevisiae by fusing the YREs in the promoter region of TRX2 gene to a cDNA-insert encoding green fluorescent protein (GFP). Exposure of this engineered yeast strain BioS-OS1 to varying levels of oxidative stress, as generated by different concentrations of H(2)O(2) or diamide, elicits robust expression of GFP that can be monitored by the fluorescence of GFP by as early as 1 h. BioS-OS1 can detect a H(2)O(2) concentration from 300 microM onward. We also show that the signaling strength of the strain can be increased by engineering multiple YREs in the upstream of the cDNA-insert encoding GFP. Thus, the results presented here demonstrate that the engineered BioS-OS yeast strain can detect ROI-generating oxidative stress and validate the use of this prototypic strain for the development of a biosensor to detect and monitor oxidative stress factors during space and high altitude flights.

Biosensing Techniques↗