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Biomedical subjects

Santosh Nigam

Publications and source records attributed to Santosh Nigam.

17 recordsLinked to original sources
PubMed2006

Phospholipase A2 and phospholipase B activities in fungi.

As saprophytes or disease causing microorganisms, fungi acquire nutrients from dead organic material or living host organisms. Lipids as structural components of cell membranes and storage compartments play an important role as energy-rich food source. In recent years, it also has become clear that lipids have a wide range of bioactive properties including signal transduction and cell to cell communication. Thus, it is not surprising that fungi possess a broad range of hydrolytic enzymes that attack neutral lipids and phospholipids. Especially during infection of a mammalian host, phospholipase A(2) (PLA(2)) enzymes released by fungi could play important roles not only for nutrient acquisition and tissue invasion, but for intricate modulation of the host's immune response. Sequencing of fungal genomes has revealed a wide range of genes encoding PLA(2) activities in fungi. We are just beginning to become aware of the significance these enzymes could have for the fungal cells and their interaction with the host.

Animals↗
PubMed2006

Inactivation of the phospholipase B gene PLB5 in wild-type Candida albicans reduces cell-associated phospholipase A2 activity and attenuates virulence.

Phospholipases are critical for modification and redistribution of lipid substrates, membrane remodeling and microbial virulence. Among the many different classes of phospholipases, fungal phospholipase B (Plb) proteins show the broadest range of substrate specificity and hydrolytic activity, hydrolyzing acyl ester bonds in phospholipids and lysophospholipids and further catalyzing lysophospholipase-transacylase reactions. The genome of the opportunistic fungal pathogen Candida albicans encodes a PLB multigene family with five putative members; we present the first characterization of this group of potential virulence determinants. CaPLB5, the third member of this multigene family characterized herein is a putative secretory protein with a predicted GPI-anchor attachment site. Real-time RT-PCR gene expression analysis of CaPLB5 and the additional CaPLB gene family members revealed that filamentous growth and physiologically relevant environmental conditions are associated with increased PLB gene activity. The phenotypes expressed by null mutant and revertant strains of CaPLB5 indicate that this lipid hydrolase plays an important role for cell-associated phospholipase A(2) activity and in vivo organ colonization.

Amino Acid Sequence↗
PubMed2006

Oxylipin-coated hat-shaped ascospores of Ascoidea corymbosa.

We previously implicated 3-hydroxy oxylipins and ascospore structure in ascospore release from enclosed asci. Using confocal laser scanning microscopy on cells stained with fluorescein-coupled, 3-hydroxy oxylipin-specific antibodies, we found that oxylipins are specifically associated with ascospores and not the vegetative cells or ascus wall of Ascoidea corymbosa. Using gas chromatography--mass spectrometry the oxylipin 3-hydroxy 17:0 could be identified. Here, we visualize for the first time the forced release of oxylipin-coated, hat-shaped ascospores from terminally torn asci, probably through turgor pressure. We suggest that oxylipin-coated, razor-sharp, hat-shaped ascospore brims may play a role in rupturing the ascus to affect release.

Cell Wall↗
PubMed2005

Mapping the distribution of 3-hydroxy oxylipins in the ascomycetous yeast Saturnispora saitoi.

The distribution of 3-hydroxy oxylipins in Saturnispora saitoi was mapped using immunofluorescence microscopy. Fluorescence was observed on aggregating ascospores, indicating the presence of 3-hydroxy oxylipins on the surface or between ascospores. The oxylipin was identified as 3-hydroxy 9:1 using gas chromatography mass spectrometry. Furthermore, ultrastructural studies using scanning and transmission electron microscopy on ascospores revealed a clear equatorial ledge surrounding oval-shaped ascospores.

Fatty Acids↗
PubMed2005

Hepoxilin A3 synthase.

Hepoxilins constitute a group of 12S-hydroperoxyeicosatetraenoic acid (12S-HpETE)-derived epoxy-hydroxy fatty acids that have been detected in various cell types and tissues. Although hepoxilin A3 (HXA3) exhibits a myriad of biological activities, its biosynthetic mechanism was not investigated in detail. Here we review the isolation, cloning, and characterization of a leukocyte-type 12S-lipoxygenase (12S-LOX) from rat insulinoma cells RINm5F, which exhibits an intrinsic hepoxilin A3 synthase activity. Confirmation for this observation was achieved by coimmunoprecipitation of HXA3 synthase activity with an anti-leukocyte 12S-LOX antibody, preparation of recombinant rat 12S-LOX enzyme from RINm5F cells, and assay of HXA3 synthase activity therein. Site-directed mutagenesis studies performed on rat 12S-LOX showed that 12-lipoxygenating enzyme species exhibit a strong HXA3 synthase activity that is impaired when the positional specificity of arachidonic acid is altered in favor of 15-lipoxygenation. Inasmuch as cellular glutathione peroxidases (cGPx and PHGPx) and HXA3 synthase compete for the same substrate 12S-HpETE, it can be proposed that the overall activity of glutathione peroxidases, representing the overall peroxide tone, finely tunes the rate of HXA3 formation.

8,11,14-Eicosatrienoic Acid↗
PubMed2005

Oxygenation by COX-2 (cyclo-oxygenase-2) of 3-HETE (3-hydroxyeicosatetraenoic acid), a fungal mimetic of arachidonic acid, produces a cascade of novel bioactive 3-hydroxyeicosanoids.

Cyclo-oxygenases-1/2 (COX-1/2) catalyse the oxygenation of AA (arachidonic acid) and related polyunsaturated fatty acids to endoperoxide precursors of prostanoids. COX-1 is referred to as a constitutive enzyme involved in haemostasis, whereas COX-2 is an inducible enzyme expressed in inflammatory diseases and cancer. The fungus Dipodascopsis uninucleata has been shown by us to convert exogenous AA into 3(R)-HETE [3(R)-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid]. 3R-HETE is stereochemically identical with AA, except that a hydroxy group is attached at its C-3 position. Molecular modelling studies with 3-HETE and COX-1/2 revealed a similar enzyme-substrate structure as reported for AA and COX-1/2. Here, we report that 3-HETE is an appropriate substrate for COX-1 and -2, albeit with a lower activity of oxygenation than AA. Oxygenation of 3-HETE by COX-2 produced a novel cascade of 3-hydroxyeicosanoids, as identified with EI (electron impact)-GC-MS, LC-MS-ES (electrospray) and LC-MS-API (atmospheric pressure ionization) methods. Evidence for in vitro production of 3-hydroxy-PGE2 (3-hydroxy-prostaglandin E2) was obtained upon infection of HeLa cells with Candida albicans at an MOI (multiplicity of infection) of 100. Analogous to interaction of AA and aspirin-treated COX-2, 3-HETE was transformed by acetylated COX-2 to 3,15-di-HETE (3,15-dihydroxy-HETE), whereby C-15 showed the (R)-stereochemistry. 3-Hydroxy-PGs are potent biologically active compounds. Thus 3-hydroxy-PGE2 induced interleukin-6 gene expression via the EP3 receptor (PGE2 receptor 3) in A549 cells, and raised cAMP levels via the EP4 receptor in Jurkat cells. Moreover, 3R,15S-di-HETE triggered the opening of the K+ channel in HTM (human trabecular meshwork) cells, as measured by the patch-clamp technique. Since many fatty acid disorders are associated with an 'escape' of 3-hydroxy fatty acids from the b-oxidation cycle, the production of 3-hydroxyeicosanoids may be critical in modulation of effects of endogenously produced eicosanoids.

Adenocarcinoma↗
PubMed2005

Ascospore release from bottle-shaped asci in Dipodascus albidus.

Yeasts utilize different mechanisms to release ascospores of different lengths from bottle-shaped asci. Using electron microscopy, confocal laser scanning microscopy, gas chromatography-mass spectrometry and digital live imaging, the individual release of oval ascospores from tight-fitting narrow bottle-necks, is reported in the yeast Dipodascus albidus. These ascospores are surrounded by compressible, oxylipin-coated sheaths enabling ascospores to slide past each other when forced by turgor pressure and by possible sheath contractions towards the narrowing ascus-neck. In this paper, the release mechanisms of ascospores of various lengths from bottle-shaped asci and produced by different yeasts are compared. We suggest that different release mechanisms, utilizing compressible sheaths or geared-alignment, have possibly evolved to compensate for variation in ascospore length. Alternatively, sheaths and ridges might be two evolutionary solutions to the same biomechanical problem, i.e. to release ascospores irrespective of length from bottle-shaped asci.

Gas Chromatography-Mass Spectrometry↗
PubMed2005

The presence of 3-hydroxy oxylipins on the ascospore surfaces of some species representing Saccharomycopsis Schiönning.

Through gas chromatography - mass spectrometry, the presence of oxylipins, mainly 3-hydroxy 9:1 and 3-hydroxy 10:1, was detected in Saccharomycopsis fermentans, Saccharomycopsis javanensis, and Saccharomycopsis vini. The distribution of these compounds was mapped using immunofluorescence microscopy, and they were found to be closely associated with the surfaces of aggregating ascospores.

Fatty Acids, Unsaturated↗
PubMed2004

The rat leukocyte-type 12-lipoxygenase exhibits an intrinsic hepoxilin A3 synthase activity.

Hepoxilins are biologically relevant eicosanoids formed via the 12-lipoxygenase pathway of the arachidonic acid cascade. Although these eicosanoids exhibit a myriad of biological activities, their biosynthetic mechanism has not been investigated in detail. We examined the arachidonic acid metabolism of RINm5F rat insulinoma cells and found that they constitutively express a leukocyte-type 12S-lipoxygenase. Moreover, we observed that RINm5F cells exhibit an active hepoxilin A(3) synthase that converts exogenous 12S-HpETE (12S-5Z,8-Z,10E,14Z-12-hydro(pero)xy-eicosa-5,8,10,14-tetraenoic acid) or arachidonic acid predominantly to hepoxilin A(3). 12S-lipoxygenase and hepoxilin A(3) synthase activities were co-localized in the cytosol; immunoprecipitation with an anti-12S-lipoxygenase antibody co-precipitated the two catalytic activities. These data suggested that hepoxilin A(3) synthase activity may be considered an intrinsic catalytic property of the leukocyte-type 12S-lipoxygenase. To test this hypothesis we cloned the leukocyte-type 12S-LOX from RINm5F cells, expressed it in Pichia pastoris, and found that the recombinant enzyme exhibited both 12S-lipoxygenase and hepoxilin A(3) synthase activities. The recombinant human platelet-type 12S-lipoxygenase and the porcine leukocyte-type 12S-lipoxygenase also exhibited hepoxilin A(3) synthase activity. In contrast, the native rabbit reticulocyte-type 15S-lipoxygenase did not convert 12S-HpETE to hepoxilin isomers. These data suggest that the positional specificity of lipoxygenases may be crucial for this catalytic function. This hypothesis was confirmed by site-directed mutagenesis studies that altered the positional specificity of the rat leukocyte-type 12S- and the rabbit reticulocyte-type 15-lipoxygenase. In summary, it may be concluded that naturally occurring 12S-lipoxygenases exhibit an intrinsic hepoxilin A(3) synthase activity that is minimal in lipoxygenase isoforms with different positional specificity.

Amino Acid Sequence↗
PubMed2004

Report on the discovery of a novel 3-hydroxy oxylipin cascade in the yeast Saccharomycopsis synnaedendra.

A novel cascade of 3-hydroxy fatty acids was discovered in the yeast Saccharomycopsis synnaedendra. The cascade, probably derived from incomplete beta-oxidation, comprises both even and uneven carbon numbered as well as saturated and unsaturated 3-hydroxy oxylipins. This yeast may now be used as model to further study the metabolism of these compounds as well as their biotechnological production.

Ascomycota↗
PubMed2003

Candida albicans induces selectively transcriptional activation of cyclooxygenase-2 in HeLa cells: pivotal roles of Toll-like receptors, p38 mitogen-activated protein kinase, and NF-kappa B.

Candidiasis, in its mucocutaneous form as well as in an invasive form, is frequently associated with high morbidity. PGE(2), which is generated by enzymatic activity of cyclooxygenases (COXs) 1 and 2, has been shown to trigger morphogenesis in Candida albicans. In the present study, we investigated whether C. albicans altered COX-2 expression in HeLa cells. RT-PCR and Western blot analyses revealed a time-dependent biphasic behavior of COX-2 mRNA expression and COX-2 protein level. COX-1 protein remained unaffected. Neutralization with Abs against Toll-like receptors (TLR) 2 and 4 inhibited the Candida-induced production of PGE(2), suggesting a vital role for TLRs in the recognition and signaling in mammalian cells upon infection with C. albicans. Transient transfections with COX-2 promoter-luciferase construct and various inhibitors of mitogen-activated protein kinases (MAPK), such as protein kinase C (PKC) inhibitor GF203190X, p38(MAPK) inhibitor SB203109, and extracellular-regulated kinases 1 and 2 inhibitor PD98509 showed that C. albicans up-regulates selectively COX-2, but not COX-1, through p38(MAPK) and PKC pathways. No involvement of other stress kinases, e.g., c-Jun NH(2)-terminal kinase and extracellular-regulated kinases 1 and 2, was observed. Transient transfection of NF-kappaB promoter construct and dominant negative plasmid of IkappaBbeta kinase showed that COX-2 transcription is mediated through p38(MAPK) and NF-kappaB pathways. That NF-kappaB up-regulates p38(MAPK) is novel and is in contradiction to earlier reports in which NF-kappaB was shown to inhibit p38(MAPK). In conclusion, multiple converging signaling pathways, involving TLRs followed by PKC, p38(MAPK), and/or NF-kappaB, are triggered by C. albicans in activation of COX-2 gene.

Candida albicans↗
PubMed2003

Biosynthesis of hepoxilins: evidence for the presence of a hepoxilin synthase activity in rat insulinoma cells.

The 12(S)-lipoxygenase (12-LOX) pathway of arachidonic acid (AA) metabolism after dioxygenation to 12(S)-hydroperoxy-eicosatetraenoic acid is bifurcated in a reduction route to formation of 12(S)-hydroxy-eicosatetraenoic acid (12-HpETE) and an isomerization route to formation of hepoxilins. Interestingly, we found that the rat insulinoma RINm5F cells, which are devoid of cytoplasmic glutathione peroxidase (cGPx)/phospholipid hydroperoxide glutathione peroxidase (PHGPx), produce solely hepoxilin A(3) (HXA(3)). Since HXA(3) synthesis was abolished in heat-denatured or cGPx- or PHGPx-transfected cells, it was tempting to speculate that a HXA(3) synthase activity regulated by cGPx/PHGPx is present. To confirm this assumption we incubated AA with HeLa cells overexpressing the rat leukocyte-type 12-LOX. Neither HXA(3) nor 12(S)-HETE were detected due to abundance of cGPx/PHGPx. But, pretreatment of transfected cells with diethyl maleate, an inhibitor of glutathione and PHGPx, restored HXA(3) synthase and 12-LOX activities. Thus, we conclude, that cells containing rat leukocyte-type 12-LOX also possess an intrinsic HXA(3) synthase activity, which is activated by inhibition of cGPx/PHGPx. In normal cells HXA(3) is down-regulated by cGPx/PHGPx, but, it is persistently activated in oxidatively stressed cells deficient in cGPx/PHGPx, such as RINm5F.

8,11,14-Eicosatrienoic Acid↗
PubMed2003

IL-4 induces apoptosis in A549 lung adenocarcinoma cells: evidence for the pivotal role of 15-hydroxyeicosatetraenoic acid binding to activated peroxisome proliferator-activated receptor gamma transcription factor.

The proinflammatory cytokine IL-4 is secreted in large amounts during allergic inflammatory response in asthma and plays a pivotal role in the airway inflammation. IL-4 has been shown to up-regulate 15-lipoxygenase and produce 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) in A549 cells via the Janus kinase/STAT6 pathway under coactivation of CREB binding protein/p300. IL-4 has also been shown to up-regulate peroxisome proliferator-activated receptor gamma (PPARgamma) nuclear receptors in macrophages and A549 cells. In this study we demonstrate that 15(S)-HETE binds to PPARgamma nuclear receptors and induces apoptosis in A549 cells. Moreover, pretreatment of cells with nordihydroguaiaretic acid, a 15-lipoxygenase inhibitor, prevented PPARgamma activation and apoptosis. The latter was accomplished by the interaction of the 15(S)-HETE/PPARgamma complex with the adapter protein Fas-associating protein with death domain and caspase-8, as shown by transfection of Fas-associating protein with death domain dominant negative vector and cleavage of caspase 8 to active subunits p41/42 and p18. Whereas IL-4 and PPARgamma ligands failed to induce cleavage of Bid and release of cytochrome c from mitochondria, they caused translocation of the proapoptotic protein Bax from cytoplasm to mitochondria with a concomitant decrease in the Bcl-x(L) level. We therefore believe that in unstimulated cells Bcl-x(L) and Bax form a heterodimer, in which Bcl-x(L) dominates and prevents the induction of apoptosis, whereas in IL-4-stimulated cells the 15(S)-HETE/PPARgamma complex down-regulates Bcl-x(L), and the resulting overweight of Bax commits the cell to apoptosis via caspase-3. However, this pathway does not rule out the direct caspase-8-mediated activation of caspase-3. In conclusion, IL-4-induced apoptosis may contribute to severe loss of alveolar structures and infiltration of eosinophils, mononuclear phagocytes, etc., into the lung tissue of chronic asthma patients.

Adaptor Proteins, Signal Transducing↗
PubMed2003

The distribution of 3-hydroxy oxylipins in fungi.

One of the best-kept secrets by fungi especially yeast is the function of the different shapes and surface structures of their vegetative and sexual cells. They definitely do not produce these shapes (e.g. round, elongated, kidney, needle, hat, saturnoid, etc.) and surfaces (e.g. smooth, rough, hairy, warty, etc.) for our curiosity or to be classified, but surely produce these for their own benefit. This mini-review will show that a large variety of 3-hydroxy oxylipins are widely distributed in the fungal domain and closely associated with these surface ornamentations. In concert with nano-scale surface structures, they probably play a role in cell aggregation as well as spore release from sexual structures such as asci.

Fluorescent Antibody Technique↗
PubMed2002

A simple method for the preparation of (5Z,8Z,11Z,14Z)-16-hydroxyeicosa-5,8,11,14-tetraenoic acid enantiomers and the corresponding 14,15-dehydro analogues: role of the 16-hydroxy group for the lipoxygenase reaction.

(5Z,8Z,11Z,13E)-15-Hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) is not well oxygenated by arachidonate 15-lipoxygenases because of two structural reasons: (i) it contains a hydrophilic OH-group in close proximity to its methyl end and (ii) it lacks the bisallylic methylene at C(13). We synthesized racemic (5Z,8Z,11Z,14Z)-16-hydroxy-5,8,11,14-eicosatetraenoic acid (16-HETE) which still contains the bisallylic C(13), separated the enantiomers reaching an optical purity of >99% and tested them as substrates for 5- and 15-lipoxygenases. Our synthetic pathway, which is based on stereospecific hydrogenation of a polyacetylenic precursor, yielded substantial amounts (30%) of 14,15-dehydro-16-HETE in addition to 16-HETE. When 16-HETE was tested as lipoxygenase substrate, we found that it is well oxygenated by the soybean 15-lipoxygenase and by the recombinant human 5-lipoxygenase. Analysis of the reaction products suggested an arachidonic acid-like alignment at the active site of the two enzymes. In contrast, the product pattern of 16-HETE methyl ester oxygenation by the soybean lipoxygenase (5-lipoxygenation) may be explained by an inverse head to tail substrate orientation.

Chromatography, High Pressure Liquid↗