Expression of lipocalin-type prostaglandin D synthase (β-trace) in human heart and its accumulation in the coronary circulation of angina patients

Lipocalin-type prostaglandin D synthase (L-PGDS) is localized in the central nervous system and male genital organs of various mammals and is secreted as β-trace into the closed compartment of these tissues separated from the systemic circulation. In this study, we found that the mRNA for the human enzyme was expressed most intensely in the heart among various tissues examined. In human autopsy specimens, the enzyme was localized immunocytochemically in myocardial cells, atrial endocardial cells, and a synthetic phenotype of smooth muscle cells in the arteriosclerotic intima, and accumulated in the atherosclerotic plaque of coronary arteries with severe stenosis. In patients with stable angina (75–99% stenosis), the plasma level of L-PGDS was significantly (P < 0.05) higher in the great cardiac vein (0.694 ± 0.054 μg/ml, n = 7) than in the coronary artery (0.545 ± 0.034 μg/ml), as determined by a sandwich enzyme immunoassay. However, the veno-arterial difference in the plasma L-PGDS concentration was not observed in normal subjects without stenosis. After a percutaneous transluminal coronary angioplasty was performed to compress the stenotic atherosclerotic plaques, the L-PGDS concentration in the cardiac vein decreased significantly (P < 0.05) to 0.610 ± 0.051 μg/ml at 20 min and reached the arterial level within 1 h. These findings suggest that L-PGDS is present in both endocardium and myocardium of normal subjects and the stenotic site of patients with stable angina and is secreted into the coronary circulation.

Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect.

Evidence from epidemiological studies, clinical trials, and animal experiments indicates that inhibitors of prostaglandin synthesis lower the risk of colon cancer. We tested the hypothesis that abnormal expression of prostaglandin H synthase 2 (PHS-2), which can be induced by oncogenes and tumor promoters, occurs during colon carcinogenesis by examining its level in colon tumors. Human colon cancers were found to have an increased expression of PHS-2 mRNA compared with normal colon specimens from the same patient (n = 5). In situ hybridization showed that the neoplastic colonocytes had increased expression of PHS-2 (n = 4). Additionally, five colon cancer cell lines were shown to express high levels of PHS-2 mRNA even in the absence of a known inducer of PHS-2. To study the basis for this increased gene expression, we transfected a colon cancer cell line, HCT-116, with a reporter gene containing 2.0 kb of the 5' regulatory sequence of the PHS-2 gene. Constitutive transcription of the reporter gene was observed, whereas normal control cell lines transcribed the reporter only in response to an exogenous agonist. We conclude that PHS-2 is transcribed abnormally in human colon cancers and that this may be one mechanism by which prostaglandins or related compounds that support carcinogenesis are generated.

Interleukin 4 suppresses c-kit ligand-induced expression of cytosolic phospholipase A2 and prostaglandin endoperoxide synthase 2 and their roles in separate pathways of eicosanoid synthesis in mouse bone marrow-derived mast cells.

Mouse bone marrow-derived mast cells (BMMCs) developed with interleukin 3 (IL-3) can be stimulated by c-kit ligand (KL) and accessory cytokines over a period of hours for direct delayed prostaglandin (PG) generation or over a period of days to prime for augmented IgE-dependent PG and leukotriene (LT) production, as previously reported. We now report that IL-4 is counterregulatory for each of these distinct KL-dependent responses. BMMCs cultured for 4 days with KL + IL-3 or with KL + IL-10 produced 5- to 7-fold more PGD2 and approximately 2-fold more LTC4 in response to IgE-dependent activation than BMMCs maintained in IL-3 alone. IL-4 inhibited the priming for increased IgE-dependent PGD2 and LTC4 production to the level obtained by activation of BMMCs maintained in IL-3 alone with an IC50 of approximately 0.2 ng/ml. IL-4 inhibited the KL-induced increase in expression of cytosolic phospholipase A2 (cPLA2) but had no effect on the incremental expression of PG endoperoxide synthase 1 (PGHS-1) and hematopoietic PGD2 synthase or on the continued baseline expression of 5-lipoxygenase, 5-lipoxygenase activating protein, and LTC4 synthase. BMMCs stimulated by KL + IL-10 for 10 h exhibited a delayed phase of PGD2 generation, which was dependent on de novo induction of PGHS-2. IL-4 inhibited the induction of PGHS-2 expression and the accompanying cytokine-initiated delayed PGD2 generation with an IC50 of approximately 6 ng/ml. IL-4 had no effect on the expression of PGHS-2 and the production of PGD2 elicited by addition of IL-1 beta to the combination of KL + IL-10. IL-4 had no effect on the immediate phase of eicosanoid synthesis elicited by KL alone or by IgE and antigen in BMMCs maintained in IL-3. Thus, the counterregulatory action of IL-4 on eicosanoid generation is highly selective for the induced incremental expression of cPLA2 and the de novo expression of PGHS-2, thereby attenuating time-dependent cytokine-regulated responses to stimulation via Fc epsilon receptor I and stimulation via c-kit, respectively.

Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis

Peroxynitrite activates the cyclooxygenase activities of constitutive and inducible prostaglandin endoperoxide synthases by serving as a substrate for the enzymes’ peroxidase activities. Activation of purified enzyme is induced by direct addition of peroxynitrite or by in situ generation of peroxynitrite from NO coupling to superoxide anion. Cu,Zn-superoxide dismutase completely inhibits cyclooxygenase activation in systems where peroxynitrite is generated in situ from superoxide. In the murine macrophage cell line RAW264.7, the lipophilic superoxide dismutase-mimetic agents, Cu(II) (3,5-diisopropylsalicylic acid)2, and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin dose-dependently decrease the synthesis of prostaglandins without affecting the levels of NO synthase or prostaglandin endoperoxide synthase or by inhibiting the release of arachidonic acid. These findings support the hypothesis that peroxynitrite is an important modulator of cyclooxygenase activity in inflammatory cells and establish that superoxide anion serves as a biochemical link between NO and prostaglandin biosynthesis.

The effects of aging on gene expression in the hypothalamus and cortex of mice

A better understanding of the molecular effects of aging in the brain may help to reveal important aspects of organismal aging, as well as processes that lead to age-related brain dysfunction. In this study, we have examined differences in gene expression in the hypothalamus and cortex of young and aged mice by using high-density oligonucleotide arrays. A number of key genes involved in neuronal structure and signaling are differentially expressed in both the aged hypothalamus and cortex, including synaptotagmin I, cAMP-dependent protein kinase C β, apolipoprotein E, protein phosphatase 2A, and prostaglandin D. Misregulation of these proteins may contribute to age-related memory deficits and neurodegenerative diseases. In addition, many proteases that play essential roles in regulating neuropeptide metabolism, amyloid precursor protein processing, and neuronal apoptosis are up-regulated in the aged brain and likely contribute significantly to brain aging. Finally, a subset of these genes whose expression is affected by aging are oppositely affected by exposure of mice to an enriched environment, suggesting that these genes may play important roles in learning and memory.

Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-d-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol

In Escherichia coli, 1-deoxy-d-xylulose (or its 5-phosphate, DXP) is the biosynthetic precursor to isopentenyl diphosphate [Broers, S. T. J. (1994) Dissertation (Eidgenössische Technische Hochschule, Zürich)], thiamin, and pyridoxol [Himmeldirk, K., Kennedy, I. A., Hill, R. E., Sayer, B. G. & Spenser, I. D. (1996) Chem. Commun. 1187–1188]. Here we show that an open reading frame at 9 min on the chromosomal map of E. coli encodes an enzyme (deoxyxylulose-5-phosphate synthase, DXP synthase) that catalyzes a thiamin diphosphate-dependent acyloin condensation reaction between C atoms 2 and 3 of pyruvate and glyceraldehyde 3-phosphate to yield DXP. We have cloned and overexpressed the gene (dxs), and the enzyme was purified 17-fold to a specific activity of 0.85 unit/mg of protein. The reaction catalyzed by DXP synthase yielded exclusively DXP, which was characterized by 1H and 31P NMR spectroscopy. Although DXP synthase of E. coli shows sequence similarity to both transketolases and the E1 subunit of pyruvate dehydrogenase, it is a member of a distinct protein family, and putative DXP synthase sequences appear to be widespread in bacteria and plant chloroplasts.

Cloning and characterization of human inducible nitric oxide synthase splice variants: A domain, encoded by exons 8 and 9, is critical for dimerization

The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alternative splicing, including deletion of exons 8 and 9 that encode amino acids 242–335 of the oxygenase domain. In this study, iNOS8−9− and full-length iNOS (iNOSFL) were cloned from bronchial epithelial cells. Expression of iNOS8−9− in 293 cell line resulted in generation of iNOS8−9− mRNA and protein but did not lead to NO production. In contrast to iNOSFL, iNOS8−9− did not form dimers. Similar to iNOSFL, iNOS8−9− exhibited NADPH-diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-binding domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOSFL was used to construct 12 mutants, each with deletion of eight residues in the region encoded by exons 8 and 9. In addition, two “control” iNOS deletion mutants were synthesized, lacking either residues 45–52 of the oxygenase domain or residues 1131–1138 of the reductase domain. Whereas both control deletion mutants generated NO and formed dimers, none of the 12 other mutants formed dimers or generated NO. The region encoded by exons 8 and 9 is critical for iNOS dimer formation and NO production but not for reductase activity. This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synthesis.

Nitric oxide in the contractile action of bradykinin, oxytocin, and prostaglandin F2 α in the estrogenized rat uterus

Experiments were performed on uteri from estrogen-primed female rats. Bradykinin (BK) (10−8 M) significantly augmented biosynthesis of prostaglandin F2 α (PGF2α) and prostaglandin E2 (PGE2), and this synthesis was completely blocked by NG-monomethyl l-arginine (NMMA) (300 μM), a competitive inhibitor of nitric oxide synthase (NOS). Blockade of prostaglandin synthesis by indomethacin caused rapid dissipation of isometric developed tension (IDT) induced by BK. Blockade of NOS with NMMA had similar but less marked effects. Combining the two inhibitors produced an even more rapid decay in IDT, suggesting that BK-induced NO release maintains IDT by release of prostanoids. The decline of frequency of contraction (FC) was not significantly altered by either indomethacin or NMMA but was markedly accelerated by combination of the inhibitors, which suggests that PGs maintain FC and therefore FC decline is accelerated only when PG production is blocked completely by combination of the two inhibitors of PG synthesis. The increase in IDT induced by oxytocin was unaltered by indomethacin, NMMA or their combination indicating that neither NO nor PGs are involved in the contractions induced by oxytocin. However, the decline in FC with time was significantly reduced by the inhibitor of NOS, NMMA, suggesting that FC decay following oxytocin is caused by NO released by the contractile process. In the case of PGF2α, NMMA resulted in increased initial IDT and FC. The decline in FC was rapid and dramatically inhibited by NMMA. Receptor-mediated contraction by BK, oxytocin, and PGF2α is modulated by NO that maintains IDT by releasing PGs but reduces IDT and FC via cyclic GMP.

Structure of the thrombin complex with triabin, a lipocalin-like exosite-binding inhibitor derived from a triatomine bug

Triabin, a 142-residue protein from the saliva of the blood-sucking triatomine bug Triatoma pallidipennis, is a potent and selective thrombin inhibitor. Its stoichiometric complex with bovine α-thrombin was crystallized, and its crystal structure was solved by Patterson search methods and refined at 2.6-Å resolution to an R value of 0.184. The analysis revealed that triabin is a compact one-domain molecule essentially consisting of an eight-stranded β-barrel. The eight strands A to H are arranged in the order A-C-B-D-E-F-G-H, with the first four strands exhibiting a hitherto unobserved up-up-down-down topology. Except for the B-C inversion, the triabin fold exhibits the regular up-and-down topology of lipocalins. In contrast to the typical ligand-binding lipocalins, however, the triabin barrel encloses a hydrophobic core intersected by a unique salt-bridge cluster. Triabin interacts with thrombin exclusively via its fibrinogen-recognition exosite. Surprisingly, most of the interface interactions are hydrophobic. A prominent exception represents thrombin’s Arg-77A side chain, which extends into a hydrophobic triabin pocket forming partially buried salt bridges with Glu-128 and Asp-135 of the inhibitor. The fully accessible active site of thrombin in this complex is in agreement with its retained hydrolytic activity toward small chromogenic substrates. Impairment of thrombin’s fibrinogen converting activity or of its thrombomodulin-mediated protein C activation capacity upon triabin binding is explained by usage of overlapping interaction sites of fibrinogen, thrombomodulin, and triabin on thrombin. These data demonstrate that triabin inhibits thrombin via a novel and unique mechanism that might be of interest in the context of potential therapeutic applications.

The mycosubtilin synthetase of Bacillus subtilis ATCC6633: A multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase

Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic. Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a β-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Gln-Pro-Ser-Asn, with the second, third, and sixth position present in the D-configuration. The gene cluster from B. subtilis ATCC6633 specifying the biosynthesis of mycosubtilin was identified. The putative operon spans 38 kb and consists of four ORFs, designated fenF, mycA, mycB, and mycC, with strong homologies to the family of peptide synthetases. Biochemical characterization showed that MycB specifically adenylates tyrosine, as expected for mycosubtilin synthetase, and insertional mutagenesis of the operon resulted in a mycosubtilin-negative phenotype. The mycosubtilin synthetase reveals features unique for peptide synthetases as well as for fatty acid synthases: (i) The mycosubtilin synthase subunit A (MycA) combines functional domains derived from peptide synthetases, amino transferases, and fatty acid synthases. MycA represents the first example of a natural hybrid between these enzyme families. (ii) The organization of the synthetase subunits deviates from that commonly found in peptide synthetases. On the basis of the described characteristics of the mycosubtilin synthetase, we present a model for the biosynthesis of iturin lipopeptide antibiotics. Comparison of the sequences flanking the mycosubtilin operon of B. subtilis ATCC6633, with the complete genome sequence of B. subtilis strain 168 indicates that the fengycin and mycosubtilin lipopeptide synthetase operons are exchanged between the two B. subtilis strains.

Down-regulation of the expression of endothelial NO synthase is likely to contribute to glucocorticoid-mediated hypertension

Hypertension is a side effect of systemically administered glucocorticoids, but the underlying molecular mechanism remains poorly understood. Ingestion of dexamethasone by rats telemetrically instrumented increased blood pressure progressively over 7 days. Plasma concentrations of Na+ and K+ and urinary Na+ and K+ excretion remained constant, excluding a mineralocorticoid-mediated mechanism. Plasma NO2−/NO3− (the oxidation products of NO) decreased to 40%, and the expression of endothelial NO synthase (NOS III) was found down-regulated in the aorta and several other tissues of glucocorticoid-treated rats. The vasodilator response of resistance arterioles was tested by intravital microscopy in the mouse dorsal skinfold chamber model. Dexamethasone treatment significantly attenuated the relaxation to the endothelium-dependent vasodilator acetylcholine, but not to the endothelium-independent vasodilator S-nitroso-N-acetyl-d,l-penicillamine. Incubation of human umbilical vein endothelial cells, EA.hy 926 cells, or bovine aortic endothelial cells with several glucocorticoids reduced NOS III mRNA and protein expression to 60–70% of control, an effect that was prevented by the glucocorticoid receptor antagonist mifepristone. Glucocorticoids decreased NOS III mRNA stability and reduced the activity of the human NOS III promoter (3.5 kilobases) to ≈70% by decreasing the binding activity of the essential transcription factor GATA. The expressional down-regulation of endothelial NOS III may contribute to the hypertension caused by glucocorticoids.

Identification of nitric oxide synthase as a protective locus against tuberculosis

Mutagenesis of the host immune system has helped identify response pathways necessary to combat tuberculosis. Several such pathways may function as activators of a common protective gene: inducible nitric oxide synthase (NOS2). Here we provide direct evidence for this gene controlling primary Mycobacterium tuberculosis infection using mice homozygous for a disrupted NOS2 allele. NOS2−/− mice proved highly susceptible, resembling wild-type littermates immunosuppressed by high-dose glucocorticoids, and allowed Mycobacterium tuberculosis to replicate faster in the lungs than reported for other gene-deficient hosts. Susceptibility appeared to be independent of the only known naturally inherited antimicrobial locus, NRAMP1. Progression of chronic tuberculosis in wild-type mice was accelerated by specifically inhibiting NOS2 via administration of N6-(1-iminoethyl)-l-lysine. Together these findings identify NOS2 as a critical host gene for tuberculostasis.

Direct measurement of nitric oxide generation from nitric oxide synthase

Although nitric oxide synthase (NOS) is widely considered as the major source of NO in biological cells and tissues, direct evidence demonstrating NO formation from the purified enzyme has been lacking. It was recently reported that NOS does not synthesize NO, but rather generates nitroxyl anion (NO−) that is subsequently converted to NO by superoxide dismutase (SOD). To determine if NOS synthesizes NO, electron paramagnetic resonance (EPR) spectroscopy was applied to directly measure NO formation from purified neuronal NOS. In the presence of the NO trap Fe2+-N-methyl-d-glucamine dithiocarbamate, NO gives rise to characteristic EPR signals with g = 2.04 and aN = 12.7 G, whereas NO− is undetectable. In the presence of l-arginine (l-Arg) and cofactors, NOS generated prominent NO signals. This NO generation did not require SOD, and it was blocked by the specific NOS inhibitor N-nitro-l-arginine methyl ester. Isotope-labeling experiments with l-[15N]Arg further demonstrated that NOS-catalyzed NO arose from the guanidino nitrogen of l-Arg. Measurement of the time course of NO formation demonstrated that it paralleled that of l-citrulline. The conditions used in the prior study were shown to result in potent superoxide generation, and this may explain the failure to measure NO formation in the absence of SOD. These experiments provide unequivocal evidence that NOS does directly synthesize NO from l-Arg.

No ·NO from NO synthase

The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H4Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of l-arginine (Arg) associated with stoichiometric consumption of dioxygen (O2) and 1.5 mol of NADPH to form l-citrulline (Cit) and nitric oxide (·NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we therefore sought to directly quantify ·NO formation from purified NOS. However, by two independent methods, NOS did not yield detectable ·NO unless superoxide dismutase (SOD; EC 1.15.1.1) was present. In the presence of H4Bip, internal ·NO standards were only partially recovered and the dismutation of superoxide (O2⨪), which otherwise scavenges ·NO to yield ONOO−, was a plausible mechanism of action of SOD. Under these conditions, a reaction between NADPH and ONOO− resulted in considerable overestimation of enzymatic NADPH consumption. SOD lowered the NADPH:Cit stoichiometry to 0.8–1.1, suggesting either that additional reducing equivalents besides NADPH are required to explain Arg oxidation to ·NO or that ·NO was not primarily formed. The latter was supported by an additional set of experiments in the absence of H4Bip. Here, recovery of internal ·NO standards was unaffected. Thus, a second activity of SOD, the conversion of nitroxyl (NO−) to ·NO, was a more likely mechanism of action of SOD. Detection of NOS-derived nitrous oxide (N2O) and hydroxylamine (NH2OH), which cannot arise from ·NO decomposition, was consistent with formation of an ·NO precursor molecule such as NO−. When, in the presence of SOD, glutathione was added, S-nitrosoglutathione was detected. Our results indicate that ·NO is not the primary reaction product of NOS-catalyzed Arg turnover and an alternative reaction mechanism and stoichiometry have to be taken into account.

Dynamic regulation of neuronal NO synthase transcription by calcium influx through a CREB family transcription factor-dependent mechanism

Neuronal nitric oxide (NO) synthase (nNOS) is dynamically regulated in response to a variety of physiologic and pathologic stimuli. Although the dynamic regulation of nNOS is well established, the molecular mechanisms by which such diverse stimuli regulate nNOS expression have not yet been identified. We describe experiments demonstrating that Ca2+ entry through voltage-sensitive Ca2+ channels regulates nNOS expression through alternate promoter usage in cortical neurons and that nNOS exon 2 contains the regulatory sequences that respond to Ca2+. Deletion and mutational analysis of the nNOS exon 2 promoter reveals two critical cAMP/Ca2+ response elements (CREs) that are immediately upstream of the transcription start site. CREB binds to the CREs within the nNOS gene. Mutation of the nNOS CREs as well as blockade of CREB function results in a dramatic loss of nNOS transcription. These findings suggest that nNOS is a Ca2+-regulated gene through the interactions of CREB on the CREs within the nNOS exon 2 promoter and that these interactions are likely to be centrally involved in the regulation of nNOS in response to neuronal injury and activity-dependent plasticity.