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Papers by deepa j
Background: Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of inn... more Background: Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometrybased proteomics methods.
Proteome Science, 2007
Background: Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of inn... more Background: Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometrybased proteomics methods.
PLoS Pathogens, 2009
SM1 is a twelve-amino-acid peptide that binds tightly to the Anopheles salivary gland and inhibit... more SM1 is a twelve-amino-acid peptide that binds tightly to the Anopheles salivary gland and inhibits its invasion by Plasmodium sporozoites. By use of UV-crosslinking experiments between the peptide and its salivary gland target protein, we have identified the Anopheles salivary protein, saglin, as the receptor for SM1. Furthermore, by use of an anti-SM1 antibody, we have determined that the peptide is a mimotope of the Plasmodium sporozoite Thrombospondin Related Anonymous Protein (TRAP). TRAP binds to saglin with high specificity. Point mutations in TRAP's binding domain A abrogate binding, and binding is competed for by the SM1 peptide. Importantly, in vivo down-regulation of saglin expression results in strong inhibition of salivary gland invasion. Together, the results suggest that saglin/TRAP interaction is crucial for salivary gland invasion by Plasmodium sporozoites.
Molecular & Cellular Proteomics, 2010
Microbiology, 1997
We have purified proline permease to homogeneity from Candida albicans using an L-proline-linked ... more We have purified proline permease to homogeneity from Candida albicans using an L-proline-linked agarose matrix as an affinity column. The eluted protein produced two bands of 64 and 67 kDa by SDS-PAGE, whereas it produced a single band of 67 kDa by native PAGE and Western blotting. The apparent Km for L-proline binding to the purified protein was 153 pM. The purified permease was reconstituted into proteoliposomes and its functionality was tested by imposing a valinomycin-induced membrane potential. The main features of L-proline transport in reconstituted systems, viz. specificity and sensitivity to N-ethylmaleimide, were very similar to those of intact cells. The antifungal cispentacin, which enters C. albicans cells via an inducible proline permease, competitively inhibited the L-praline binding and translocation in reconstituted proteoliposomes. However, the uptake of Lproline in proteoliposomes reconstituted with the purified protein displayed monophasic kinetics with an apparent Km of 40 pM.
Infection and Immunity, 2005
A-deficient mice compared to that in wild-type C57BL/6 mice when they were challenged with Plasmo... more A-deficient mice compared to that in wild-type C57BL/6 mice when they were challenged with Plasmodium berghei sporozoites. These data demonstrate that the extracellular region of TRAP interacts with fetuin-A on hepatocyte membranes and that this interaction enhances the parasite's ability to invade hepatocytes.
Free Radical Biology and Medicine, 2002
We previously used mouse macrophage-like RAW264.7 cells as an experimental system for the study o... more We previously used mouse macrophage-like RAW264.7 cells as an experimental system for the study of nitric oxide (NO)-associated genotoxicity under physiologically relevant conditions, and characterized genotoxic effects in the NO-producing cells. Here we report experiments utilizing a co-culture system enabling parallel studies of cytotoxic and genotoxic responses in co-cultured target cells as well as in macrophages stimulated to produce NO. We found that co-cultivation with macrophages stimulated to produce NO for 38 -42 h resulted in significant increases in mutant fraction in the endogenous genes of target human TK6 and hamster CHO-AA8 cells and in the macrophages themselves, accompanied by a substantial decrease in cell viability. Addition of N-methyl-L-arginine, an NO synthase inhibitor, abrogated much of the cytotoxicity and genotoxicity in both target and macrophages cells, verifying the role of NO in the induction of these responses. We also showed that NO-associated genotoxic response in macrophages could be influenced by culture medium. Collectively, these results support the hypothesis that NO production by activated macrophages may contribute to genotoxic risks associated with chronic inflammation.
Background: Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of inn... more Background: Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometrybased proteomics methods.
Proteome Science, 2007
Background: Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of inn... more Background: Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometrybased proteomics methods.
PLoS Pathogens, 2009
SM1 is a twelve-amino-acid peptide that binds tightly to the Anopheles salivary gland and inhibit... more SM1 is a twelve-amino-acid peptide that binds tightly to the Anopheles salivary gland and inhibits its invasion by Plasmodium sporozoites. By use of UV-crosslinking experiments between the peptide and its salivary gland target protein, we have identified the Anopheles salivary protein, saglin, as the receptor for SM1. Furthermore, by use of an anti-SM1 antibody, we have determined that the peptide is a mimotope of the Plasmodium sporozoite Thrombospondin Related Anonymous Protein (TRAP). TRAP binds to saglin with high specificity. Point mutations in TRAP's binding domain A abrogate binding, and binding is competed for by the SM1 peptide. Importantly, in vivo down-regulation of saglin expression results in strong inhibition of salivary gland invasion. Together, the results suggest that saglin/TRAP interaction is crucial for salivary gland invasion by Plasmodium sporozoites.
Molecular & Cellular Proteomics, 2010
Microbiology, 1997
We have purified proline permease to homogeneity from Candida albicans using an L-proline-linked ... more We have purified proline permease to homogeneity from Candida albicans using an L-proline-linked agarose matrix as an affinity column. The eluted protein produced two bands of 64 and 67 kDa by SDS-PAGE, whereas it produced a single band of 67 kDa by native PAGE and Western blotting. The apparent Km for L-proline binding to the purified protein was 153 pM. The purified permease was reconstituted into proteoliposomes and its functionality was tested by imposing a valinomycin-induced membrane potential. The main features of L-proline transport in reconstituted systems, viz. specificity and sensitivity to N-ethylmaleimide, were very similar to those of intact cells. The antifungal cispentacin, which enters C. albicans cells via an inducible proline permease, competitively inhibited the L-praline binding and translocation in reconstituted proteoliposomes. However, the uptake of Lproline in proteoliposomes reconstituted with the purified protein displayed monophasic kinetics with an apparent Km of 40 pM.
Infection and Immunity, 2005
A-deficient mice compared to that in wild-type C57BL/6 mice when they were challenged with Plasmo... more A-deficient mice compared to that in wild-type C57BL/6 mice when they were challenged with Plasmodium berghei sporozoites. These data demonstrate that the extracellular region of TRAP interacts with fetuin-A on hepatocyte membranes and that this interaction enhances the parasite's ability to invade hepatocytes.
Free Radical Biology and Medicine, 2002
We previously used mouse macrophage-like RAW264.7 cells as an experimental system for the study o... more We previously used mouse macrophage-like RAW264.7 cells as an experimental system for the study of nitric oxide (NO)-associated genotoxicity under physiologically relevant conditions, and characterized genotoxic effects in the NO-producing cells. Here we report experiments utilizing a co-culture system enabling parallel studies of cytotoxic and genotoxic responses in co-cultured target cells as well as in macrophages stimulated to produce NO. We found that co-cultivation with macrophages stimulated to produce NO for 38 -42 h resulted in significant increases in mutant fraction in the endogenous genes of target human TK6 and hamster CHO-AA8 cells and in the macrophages themselves, accompanied by a substantial decrease in cell viability. Addition of N-methyl-L-arginine, an NO synthase inhibitor, abrogated much of the cytotoxicity and genotoxicity in both target and macrophages cells, verifying the role of NO in the induction of these responses. We also showed that NO-associated genotoxic response in macrophages could be influenced by culture medium. Collectively, these results support the hypothesis that NO production by activated macrophages may contribute to genotoxic risks associated with chronic inflammation.