Santosh Nigam

Phospholipase A(2)s and lipid peroxidation.

Lipid peroxidation of membrane phospholipids can proceed both enzymatically via the mammalian 15-lipoxygenase-1 or the NADPH-cytochrome P-450 reductase system and non-enzymatically. In some cells, such as reticulocytes, this process is biologically programmed, whereas in the majority of biological systems lipid peroxidation is a deleterious process that has to be repaired via a deacylation-reacylation cycle of phospholipid metabolism. Several reports in the literature pinpoint a stimulation by lipid peroxidation of the activity of secretory phospholipase A(2)s (mainly pancreatic and snake venom enzymes) which was originally interpreted as a repair function. However, recent experiments from our laboratory have demonstrated that in mixtures of lipoxygenated and native phospholipids the former are not preferably cleaved by either secretory or cytosolic phospholipase A(2)s. We propose that the platelet activating factor (PAF) acetylhydrolases of type II, which cleave preferentially peroxidised or lipoxygenated phospholipids, are competent for the phospholipid repair, irrespective of their role in PAF metabolism. A corresponding role of Ca(2+)-independent phospholipase A(2), which has been proposed to be involved in phospholipid remodelling in biomembranes, has not been addressed so far. Direct and indirect 15-lipoxygenation of phospholipids in biomembranes modulates cell signalling by several ways. The stimulation of phospholipase A(2)-mediated arachidonic acid release may constitute an alternative route of the arachidonic acid cascade. Thus, 15-lipoxygenase-mediated oxygenation of membrane phospholipids and its interaction with phospholipase A(2)s may play a crucial role in the pathogenesis of diseases, such as bronchial asthma and atherosclerosis.

Acetylation by Histone Acetyltransferase CREB-binding Protein/p300 of STAT6 Is Required for Transcriptional Activation of the 15-Lipoxygenase-1 Gene

Interleukin-4 (IL-4) induces expression of reticulocyte-type 15-lipoxygenase-1 (15-LOX-1) in various mammalian cells via the Janus kinase/signal transducer and activator of transcription 6 (STAT6) signaling system. We studied the mechanism of 15-LOX-1 induction in A549 lung epithelial cells and found that genistein, a potent tyrosine kinase inhibitor, prevented phopsphorylation of STAT6, its binding to the 15-LOX-1 promoter, and the expression of catalytically active enzyme. In contrast, cycloheximide did not prevent 15-LOX-1 induction. Surprisingly, we found that IL-4 up-regulated the histone acetyltransferase activity of CREB-binding protein (CBP)/p300, which is responsible for acetylation of nuclear histones and STAT6. The acetylation of both proteins appears to be essential for the IL-4-induced signal transduction cascade, because inhibition of CBP/p300 by the viral wild-type E1A oncoprotein abrogated acetylation of both histones and STAT6 and strongly suppressed transcriptional activation of the 15-LOX-1 gene. Moreover, we found that the inhibition by sodium butyrate of histone deacetylases, which apparently suppress 15-LOX-1 gene transcription, synergistically enhanced the IL-4-stimulated 15-LOX-1 expression. These data suggest that both phosphorylation and acetylation of STAT6 as well as acetylation of nuclear histones are involved in transcriptional activation of the 15-LOX-1 gene, although these reactions follow differential kinetics. STAT6 phosphorylation proceeds within the first hour of IL-4 stimulation. In contrast, CBP/p300-mediated acetylation requires 9–11 h, and similar kinetics were observed for the expression of the active enzyme. Thus, our results suggest that in the absence of IL-4, nuclear histones may be bound to regulatory elements of the 15-LOX-1 gene, preventing its transcription. IL-4 stimulation causes rapid phosphorylation of STAT6, but its binding to the promoter appears to be prevented by nonacetylated histones. After 9–11 h, when histones become acetylated, STAT6 binding sites may be demasked so that the phosphorylated and acetylated transcription factor can bind to activate gene transcription.

Increased endocannabinoid levels reduce the development of precancerous lesions in the mouse colon.

Colorectal cancer is an increasingly important cause of death in Western countries. Endocannabinoids inhibit colorectal carcinoma cell proliferation in vitro. In this paper, we investigated the involvement of endocannabinoids on the formation of aberrant crypt foci (ACF, earliest preneoplastic lesions) in the colon mouse in vivo. ACF were induced by azoxymethane (AOM); fatty acid amide hydrolase (FAAH) and cannabinoid receptor messenger ribonucleic acid (mRNA) levels were analyzed by the quantitative reverse transcription polymerase chain reaction (RT-PCR); endocannabinoid levels were measured by liquid chromatography–mass spectrometry; caspase-3 and caspase-9 expressions were measured by Western blot analysis. Colonic ACF formation after AOM administration was associated with increased levels of 2-arachidonoylglycerol (with no changes in FAAH and cannabinoid receptor mRNA levels) and reduction in cleaved caspase-3 and caspase-9 expression. The FAAH inhibitor N-arachidonoylserotonin increased colon endocannabinoid levels, reduced ACF formation, and partially normalized cleaved caspase-3 (but not caspase-9) expression. Notably, N-arachidonoylserotonin completely prevented the formation of ACF with four or more crypts, which have been show to be best correlated with final tumor incidence. The effect of N-arachidonoylserotonin on ACF formation was mimicked by the cannabinoid receptor agonist HU-210. No differences in ACF formation were observed between CB1 receptor-deficient and wild-type mice. It is concluded that pharmacological enhancement of endocannabinoid levels (through inhibition of endocannabinoid hydrolysis) reduces the development of precancerous lesions in the mouse colon. The protective effect appears to involve caspase-3 (but not caspase-9) activation.

Quantitative determination by HPLC of urinary 6β-hydroxycortisol, an indicator of enzyme induction by rifampicin and antiepileptic drugs

SummaryA method for determination of 6β-hydroxycortisol in urine by means of high performance liquid chromatography is described. After extraction of 10–30 ml aliquots of urine with ethylacetate, separation is accomplished on a silica gel column (30 cm, Lichrosorb Si 100) with a special two-phase four-component eluent of methylene chloride, n-hexane, ethanol and water. Complete separation of α- andβ-isomers requires 15 to 20 min. For routine determinations precolumn cleaning by backflush permits injections of samples at minimum time intervals. For quantitative determinations, each injection should contain at least 0.05–0.5 µg of 6β-hydroxycortisol, depending on the detector employed. The mean excretion rate in healthy male adults (26–40 years) was 273 µg/day (SD=74.5; n=12). In patients on long term mono-therapy with rifampicin, 6β-hydroxycortisol excretion had risen fourfold (1166 µg/d; SEM=248; n=7), paralleling the known enzyme-inducing effect of rifampicin. The relatively smaller increase to 498 µg/d observed in patients receiving triple therapy with rifampicin, isoniazid and ethambutol points to possible inhibition by isoniazid. The greatest stimulation of 6β-hydroxycortisol excretion (2352 µg/d) was found in patients receiving antiepileptic therapy (phenytoin and/or carbamazepine and other drugs). The HPLC technique for 6β-hydroxycortisol proved to be a tool routinely applicable to non-invasive evaluation of drug metabolizing enzyme activity in man.

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-κB

Candidiasis, in its mucocutaneous form as well as in an invasive form, is frequently associated with high morbidity. PGE2, 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 PGE2, 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, p38MAPK 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 p38MAPK and PKC pathways. No involvement of other stress kinases, e.g., c-Jun NH2-terminal kinase and extracellular-regulated kinases 1 and 2, was observed. Transient transfection of NF-κB promoter construct and dominant negative plasmid of IκBβ kinase showed that COX-2 transcription is mediated through p38MAPK and NF-κB pathways. That NF-κB up-regulates p38MAPK is novel and is in contradiction to earlier reports in which NF-κB was shown to inhibit p38MAPK. In conclusion, multiple converging signaling pathways, involving TLRs followed by PKC, p38MAPK, and/or NF-κB, are triggered by C. albicans in activation of COX-2 gene.

Candida albicans Modulates Host Defense by Biosynthesizing the Pro-Resolving Mediator Resolvin E1

Candida albicans is an opportunistic fungal pathogen of humans that resides commensally on epithelial surfaces, but can cause inflammation when pathogenic. Resolvins are a class of anti-inflammatory lipids derived from omega-3 polyunsaturated fatty acids (PUFA) that attenuate neutrophil migration during the resolution phase of inflammation. In this report we demonstrate that C. albicans biosynthesizes resolvins that are chemically identical to those produced by human cells. In contrast to the trans-cellular biosynthesis of human Resolvin E1 (RvE1), RvE1 biosynthesis in C. albicans occurs in the absence of other cellular partners. C. albicans biosynthesis of RvE1 is sensitive to lipoxygenase and cytochrome P450 monoxygenase inhibitors. We show that 10nM RvE1 reduces neutrophil chemotaxis in response to IL-8; 1nM RvE1 enhanced phagocytosis of Candida by human neutrophils, as well as intracellular ROS generation and killing, while having no direct affect on neutrophil motility. In a mouse model of systemic candidiasis, RvE1 stimulated clearance of the fungus from circulating blood. These results reveal an inter-species chemical signaling system that modulates host immune functions and may play a role in balancing host carriage of commensal and pathogenic C. albicans.

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.

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 γ 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 γ (PPARγ) nuclear receptors in macrophages and A549 cells. In this study we demonstrate that 15(S)-HETE binds to PPARγ nuclear receptors and induces apoptosis in A549 cells. Moreover, pretreatment of cells with nordihydroguaiaretic acid, a 15-lipoxygenase inhibitor, prevented PPARγ activation and apoptosis. The latter was accomplished by the interaction of the 15(S)-HETE/PPARγ 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 PPARγ 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-xL level. We therefore believe that in unstimulated cells Bcl-xL and Bax form a heterodimer, in which Bcl-xL dominates and prevents the induction of apoptosis, whereas in IL-4-stimulated cells the 15(S)-HETE/PPARγ complex down-regulates Bcl-xL, 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.

Biological dynamics and distribution of 3-hydroxy fatty acids in the yeast Dipodascopsis uninucleata as investigated by immunofluorescence microscopy. Evidence for a putative regulatory role in the sexual reproductive cycle

Dipodascopsis uninucleata has been recently shown to produce 3‐hydroxy polyenoic fatty acids from several exogenous polyenoic fatty acids. In order to examine whether endogenous 3‐hydroxy fatty acids (3‐OH‐FA) may be implicated in the developmental biology of this yeast, we mapped by immunofluorescence microscopy their occurrence in fixed cells with or without cell walls using an antibody raised against 3R‐hydroxy‐5Z,8Z,11Z,14Z‐eicosatetraenoic acid (3R‐HETE), the biotransformation product from arachidonic acid (AA). This antibody turned out to cross‐react with other 3‐OH‐FA. 3‐OH‐FA were detected in situ in gametangia, asci, as well as between released ascospores, and proved to be associated with the sexual reproductive stage of the life cycle of the yeast. Acetylsalicylic acid (1 mM), which is known to suppress the formation of 3‐OH‐FA from exogenous polyenoic fatty acids, inhibited the occurrence of immunoreactive material as well as the sexual phase of the life cycle suggesting a prominent regulatory role of 3‐OH‐FA for the latter.

Arachidonic acid stimulates cell growth and forms a novel oxygenated metabolite in Candida albicans

Infection of human tissues by Candida albicans has been reported to cause the release of arachidonic acid (AA), eicosanoids and other proinflammatory mediators from host cells. Therefore, we investigated the interaction of this pathogen with AA. AA stimulated cell growth at micromolar concentrations when used as a sole carbon source. Moreover, it selectively inhibited the antimycin A-resistant alternative oxidase. [1-(14)C]AA was completely metabolised by C. albicans. Only one-seventh of the radioactivity metabolised was found in CO(2), whereas two-thirds occurred in carbohydrates suggesting a predominant role of the glyoxalate shunt of citrate cycle. About 1% of radioactivity was found in polar lipids including eicosanoids. A novel AA metabolite, which revealed immunoreactivity with an antibody against 3(R)-hydroxy-oxylipins, was identified as 3, 18-dihydroxy-5,8,11,14-eicosatetraenoic acid. Using immunofluorescence microscopy, endogenous 3(R)-hydroxy-oxylipins were found in hyphae but not in yeast cells. Such compounds have recently been shown to be connected with the sexual stage of the life cycle of Dipodascopsis uninucleata. Together, we propose that infection-mediated release of AA from host cells may modulate cell growth, morphogenesis and invasiveness of C. albicans by several modes. A better understanding of its role is thus promising for novel approaches towards the treatment of human mycoses.