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Y Urade

Publications and source records attributed to Y Urade.

At least 37 records · Page 2Linked to original sources
PubMed2000

Structural basis of hematopoietic prostaglandin D synthase activity elucidated by site-directed mutagenesis.

Hematopoietic prostaglandin (PG) D synthase (PGDS) is the first identified vertebrate ortholog in the Sigma class of the glutathione S-transferase (GST) family and catalyzes both isomerization of PGH(2) to PGD(2) and conjugation of glutathione to 1-chloro-2, 4-dinitrobenzene. We introduced site-directed mutations of Tyr(8), Arg(14), Trp(104), Lys(112), Tyr(152), Cys(156), Lys(198), and Leu(199), which are presumed to participate in catalysis or PGH(2) substrate binding based on the crystallographic structure. Mutants were analyzed in terms of structure, GST and PGDS activities, and activation of the glutathione thiol group. Of all the mutants, only Y8F, W104I, K112E, and L199F showed minor but substantial differences in their far-UV circular dichroism spectra from the wild-type enzyme. Y8F, R14K/E, and W104I were completely inactive. C156L/Y selectively lost only PGDS activity. K112E reduced GST activity slightly and PGDS activity markedly, whereas K198E caused a selective decrease in PGDS activity and K(m) for glutathione and PGH(2) in the PGDS reaction. No significant changes were observed in the catalytic activities of Y152F and L199F, although their K(m) for glutathione was increased. Using 5,5'-dithiobis(2-nitrobenzoic acid) as an SH-selective agent, we found that only Y8F and R14E/K did not accelerate the reactivity of the glutathione thiol group under the low reactivity condition of pH 5.0. These results indicate that Lys(112), Cys(156), and Lys(198) are involved in the binding of PGH(2); Trp(104) is critical for structural integrity of the catalytic center for GST and PGDS activities; and Tyr(8) and Arg(14) are essential for activation of the thiol group of glutathione.

Amino Acids↗
PubMed2000

Prostaglandin D synthase gene is involved in the regulation of non-rapid eye movement sleep.

To examine the function of prostaglandin (PG) D synthase (PGDS) gene, as well as endogenously produced PGD(2) in sleep regulation in vivo, we generated transgenic (TG) mice that overexpress human PGDS gene to study their sleep behavior. Although no difference was observed in the sleep/wake patterns between wild-type and TG mice, a striking time-dependent increase in non-rapid eye movement (NREM), but not in rapid eye movement (REM), sleep was observed in two independent lines of TG mice after stimulation by tail clipping. Concomitantly, the spontaneous locomotor activity of TG animals was drastically decreased in response to the tail clip. Induction of NREM sleep in TG mice was positively correlated with the PGD(2) production in the brain. Sleep, locomotion, and PGD(2) content were essentially unchanged in wild-type mice after tail clipping. The results with TG mice demonstrate the involvement of the PGDS gene in the regulation of NREM sleep.

Animals↗
PubMed2000

Prostaglandin D2 as a mediator of allergic asthma.

Allergic asthma is caused by the aberrant expansion in the lung of T helper cells that produce type 2 (TH2) cytokines and is characterized by infiltration of eosinophils and bronchial hyperreactivity. This disease is often triggered by mast cells activated by immunoglobulin E (IgE)-mediated allergic challenge. Activated mast cells release various chemical mediators, including prostaglandin D2 (PGD2), whose role in allergic asthma has now been investigated by the generation of mice deficient in the PGD receptor (DP). Sensitization and aerosol challenge of the homozygous mutant (DP-/-) mice with ovalbumin (OVA) induced increases in the serum concentration of IgE similar to those in wild-type mice subjected to this model of asthma. However, the concentrations of TH2 cytokines and the extent of lymphocyte accumulation in the lung of OVA-challenged DP-/- mice were greatly reduced compared with those in wild-type animals. Moreover, DP-/- mice showed only marginal infiltration of eosinophils and failed to develop airway hyperreactivity. Thus, PGD2 functions as a mast cell-derived mediator to trigger asthmatic responses.

Allergens↗
PubMed2000

Prostaglandin D synthase: structure and function.

Prostaglandin (PG) D synthase catalyzes the isomerization of PGH2, a common precursor of various prostanoids, to produce PGD2 in the presence of sulfhydryl compounds. PGD2 induces sleep, regulates nociception, inhibits platelet aggregation, acts as an allergic mediator, and is further converted to 9 alpha, 11 beta-PGF2 or the J series of prostanoids, such as PGJ2, delta 12-PGJ2, and 15-deoxy-delta 12,14-PGJ2. We have purified two distinct types of PGD synthase; one is the lipocalin-type enzyme and the other is the hematopoietic enzyme. We isolated the cDNA and the gene for each enzyme and determined the tissue distribution profile and the cellular localization in several animal species. Lipocalin-type PGD synthase is localized in the central nervous system and male genital organs of various mammals and the human heart and is secreted into cerebrospinal fluid, seminal plasma, and plasma, respectively. The human enzyme was identified as beta-trace, which is a major protein in human cerebrospinal fluid. This enzyme is considered to be a dual-function protein; it acts as a PGD2-producing enzyme and also as a lipophilic ligand-binding protein, because the enzyme binds retinoids, thyroids, and bile pigments, with high affinities. Hematopoietic PGD synthase is widely distributed in the peripheral tissues and localized in the antigen-presenting cells, mast cells, and megakaryocytes. The hematopoietic enzyme is the first recognized vertebrate homolog of the sigma class of glutathione S-transferase. X-ray crystallographic analyses and generation of gene-knockout and transgenic mice for each enzyme have been performed.

Animals↗
PubMed2000

Identification of mu-class glutathione transferases M2-2 and M3-3 as cytosolic prostaglandin E synthases in the human brain.

Cytosolic prostaglandin (PG) E synthase was purified from human brain cortex. The N-terminal amino acid sequence, PMTLGYXNIRGL, was identical to that of the human mu-class glutathione transferase (GST) M2 subunit. Complementary DNAs for human GSTM2, GSTM3, and GSTM4 subunits were cloned, and recombinant proteins were expressed as homodimers in Escherichia coli. The recombinant GSTM2-2 and 3-3 catalyzed the conversion of PGH2 to PGE2 at the rates of 282 and 923 nmol/min/mg of protein, respectively, at the optimal pH of 8, whereas GSTM4-4 was inactive; although all three enzymes showed GST activity. The PGE synthase activity depended on thiols, such as glutathione, dithiothreitol, 2-mercaptoethanol, or L-cysteine. Michaelis-Menten constants and turnover numbers for PGH2 were 141 microM and 10.8 min(-1) for GSTM2-2 and 1.5 mM and 130 min(-1) for GSTM3-3, respectively. GSTM2-2 and 3-3 may play crucial roles in temperature regulation, nociception, and sleep-wake regulation by producing PGE2 in the brain.

Amino Acid Sequence↗
PubMed2000

Strong rebound of wakefulness follows prostaglandin D2- or adenosine A2a receptor agonist-induced sleep.

We studied the effect of sleep excess on the sleep-wakefulness pattern of rats. Subarachnoid infusion of prostaglandin D2 or the adenosine A2a receptor agonist CGS21680 effectively induced slow wave sleep (SWS) for the first 12 h of the night-time period, whereas they did not induce sleep during the following 24 h of infusion. An increase in the amount of wakefulness was seen during the last 12 h of prostaglandin D2 infusion. The amounts of wakefulness strongly increased during the following 36-h recovery period. Rebound wakefulness was extraordinarily strong after the cessation of CGS21680 infusion, reaching almost complete insomnia during the night-time. Treatment of animals with prostaglandin D2 overnight, following by treatment with CGS21680 on the next night, resulted in the strongest induction of wakefulness rebound. During the rebound period, the amount of wakefulness reached up to 50 min per hour in the daytime. Rebound of wakefulness depended on the amounts of preceding SWS induced by infusion of prostaglandin D2 for 6 or 12 h and of CGS21680 for 12 h. The larger the amount of SWS, the larger the amount of the following rebound of wakefulness. Rebounds of wakefulness occurred as a result of decrease in SWS amounts, whereas paradoxical sleep amounts did not change. Desensitization of adenosine A2a receptors and accumulation of prostaglandin E2 may be involved in the production of strong wakefulness rebound following relatively long treatments (more than 12 h) with prostaglandin D2 or CGS21680.

Adenosine↗
PubMed2000

Structure and chromosomal localization of human and mouse genes for hematopoietic prostaglandin D synthase. Conservation of the ancestral genomic structure of sigma-class glutathione S-transferase.

Hematopoietic prostaglandin D synthase (H-PGDS) is the key enzyme for the production of the D and J series of prostanoids, and the first recognized vertebrate homolog of sigma-class glutathione S-transferase (GST). We isolated the genes and cDNAs for human and mouse H-PGDSs. The human and mouse cDNAs contained a coding region corresponding to 199 amino-acid residues with calculated molecular masses of 23 343 and 23 226, respectively. Both H-PGDS proteins recombinantly expressed in Escherichia coli showed bifunctional activities for PGDS and GST, and had almost the same catalytic properties as the rat enzyme. Northern analyses demonstrated that the H-PGDS genes were expressed in a highly species-specific manner. Whereas the human gene was widely distributed, in contrast, the mouse gene was detected only in samples from oviduct and skin. By fluorescence in situ hybridization, the chromosomal localization of the human and mouse H-PGDS genes were mapped to 4q21-22 and 3D-E, respectively. The human and mouse H-PGDS genes spanned approximately 41 and 28 kb, respectively, and consisted of six exons divided by five introns. The exon/intron boundaries of both genes were completely identical to those of the sigma-class GST subfamily, although the amino-acid sequences of the latter were only 17.0-21.5% identical to those of either H-PGDS. These findings suggest that the H-PGDS genes evolved from the same ancestral gene as the members of the sigma-class GST family.

Amino Acid Sequence↗
PubMed2000

Dexamethasone induces lipocalin-type prostaglandin D synthase gene expression in mouse neuronal cells.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is responsible for the production of PGD(2), the main PG in the CNS. PGD(2) is an endogenous sleep inducer, and it is involved in the control of odor and pain responses and body temperature. In addition, PGD synthase transports lipophilic molecules in the subarachnoid space and CSF. By northern and western assays we show that the synthetic glucocorticoid dexamethasone, an inhibitor of PG production in most tissues, induces L-PGDS mRNA and protein in a dose- and time-dependent fashion in mouse neuronal GT1-7 cells. Accordingly, dexamethasone increases cellular L-PGDS enzymatic activity. Dexamethasone induced L-PGDS gene transcription in run-on assays and activated the mouse L-PGDS gene promoter in transiently transfected cells. It is interesting that the tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA), which induces the synthesis of PGs in many tissues, inhibited the increase in L-PGDS expression induced by dexamethasone. In contrast, neither dexamethasone nor TPA affected the expression of cyclooxygenases-1 and -2. Our data demonstrate that dexamethasone induces L-PGDS gene transcription in neuronal cells.

Animals↗
PubMed2000

Possible involvement of enhanced prostaglandin E2 production in the photosensitivity in xeroderma pigmentosum group A model mice.

Xeroderma pigmentosum group A (XPA) gene-deficient mice cannot repair UV-induced DNA damage and easily develop skin cancers by UV irradiation. Therefore, XPA-deficient mice are a useful model of human XP and represent a promising tool for photobiologic studies of the disorder. Exposure to ultraviolet (UV) B (280-320 nm) radiation greatly enhanced inflammation and immunosuppression in these mice. To investigate the molecular mechanisms of enhanced UV inflammation and immunosuppression, we determined the amount of prostaglandin (PG) E2, an inflammatory mediator and immunomodulator, and analysed the expression of cyclooxygenase (COX) mRNA in the ear skin of XPA-deficient mice after UV irradiation. In XPA-deficient mice, the amount of PGE2 significantly increased at 48 and 72 h after UVB irradiation to the level that was 8- and 16-fold higher than those in wild-type mice, respectively. The expression level of COX-2 mRNA increased in a time-dependent manner, although COX-1 mRNA was constantly expressed. Treatment with indomethacin, a potent inhibitor of PG biosynthesis, inhibited UV-induced ear swelling, abrogated local immunosuppression, and decreased the amount of PGE2 in the ear skin of XPA-deficient mice. These results indicate that the excess DNA photoproducts remaining in XPA-deficient cells after UV radiation may induce COX-2 expression. The induced production of PGE2 may be involved in the enhanced inflammation and immunosuppression caused by UV radiation in XPA-deficient mice and XP patients.

Animals↗
PubMed2000

Comparative study of the asparagine-linked sugar chains of human lipocalin-type prostaglandin D synthase purified from urine and amniotic fluid, and recombinantly expressed in Chinese hamster ovary cells.

Lipocalin-type prostaglandin D synthase (L-PGDS) is a highly glycosylated member of the lipocalin gene family and is secreted into various human body fluids. We comparatively analyzed the structures of asparagine-linked sugar chains of human L-PGDS produced by recombinant Chinese hamster ovary cells and naturally occurring human urine and amniotic fluid. After the sugar chains were liberated by hydrazinolysis followed by N-acetylation, they were derivatized with 2-aminobenzamide. All of the sugar chains of three L-PGDSs occur as biantennary complex-type sugar chains. Most of the sugar chains of three samples were fucosylated on the inner most N-acetylglucosamine residue. Although the sugar chains of the recombinant L-PGDS do not contain any bisecting N-acetylglucosamine residues, 58% and 34% of the fucosylated-sugar chains of amniotic fluid and urine L-PGDSs, respectively, contain bisecting N-acetylglucosamine residues. The sialic acid residues occur solely as Siaalpha2-->3Gal groups of the recombinant L-PGDS; the sialic acid residues of other L-PGDS occur as both Siaalpha2-->3Gal and Siaalpha2-->6Gal groups. Variations in L-PGDS glycosylation may prove useful as markers to further elucidate the role of L-PGDS glycoforms in different tissues.

Acetylglucosamine↗
PubMed2000

Immunocytochemical localization of lipocalin-type prostaglandin D synthase in the bull testis and epididymis and on ejaculated sperm.

Previously, we identified a 26-kDa fertility-associated protein in bull seminal plasma as lipocalin-type prostaglandin D synthase. The objective of the present study was to immunohistochemically localize this enzyme to the various cell types within the bull testis and seven subsegments of the epididymis, and on ejaculated sperm in order to gain further insight into its potential function in male reproduction. In the testis, immunoperoxidase staining was localized within the elongating spermatids and Sertoli cells of the seminiferous tubules, varying with the stage of the spermatogenic cycle. The highest level of staining occurred during stages III-VII. The cuboidal epithelial cells of the rete testis and efferent ducts were also immunoreactive. Expression of lipocalin-type prostaglandin D synthase was not uniform in the seven epididymal subsegments, suggesting a possible role in sperm maturation. In all epididymal regions, expression was limited to the epithelial principal cells; no immunoreactivity was apparent in other cell types. Lipocalin-type prostaglandin D synthase was strikingly localized in the caput epididymidis, while moderate to weak staining was observed in the remainder of the epididymis. Droplets of reaction product observed within the lumen increased progressively from the caput to cauda. Using fluorescence microscopy, we also localized lipocalin-type prostaglandin D synthase to the apical ridge of the acrosome on ejaculated sperm.

Animals↗
PubMed1999

Enhancement of lipocalin-type prostaglandin D synthase enzyme activity by guanidine hydrochloride.

The characterization of unfolding of mouse recombinant lipocalin-type prostaglandin D synthase (L-PGDS) by guanidine hydrochloride (GdnHCl) was carried out. In the presence of low concentrations of GdnHCl (up to 0.75 M), enhancement of the enzyme activity was observed. However, above a 1 M concentration of GdnHCl, the enzyme activity was reduced in a concentration-dependent manner. The maximum enzyme activity induced by GdnHCl was approximately 1. 5-fold compared with the activity under physiological conditions without GdnHCl. The ellipticity in circular dichroism (CD) spectrum of the L-PGDS at 218 nm, reflecting the beta-sheet content, was decreased by GdnHCl (up to 0.75 M), and the minimum ellipticity was observed at 0.5 M GdnHCl. The fluorescence quenching of the intrinsic tryptophan of L-PGDS due to the binding of bilirubin in the presence or absence of GdnHCl was measured. The K(d) values obtained in the presence and absence of 0.5 M GdnHCl were 447 and 115 nM, respectively, indicating lower affinity of the L-PGDS for bilirubin with GdnHCl than without it. Further, an NMR study revealed that the reorganization of hydrogen-bond network in the L-PGDS was observed in the presence of 0.5 M GdnHCl. These results, taken together, indicate that the enzyme activity of L-PGDS is enhanced by the conformational change, especially by the change in the secondary structure.

Animals↗
PubMed1999

Acute and transient increase of lipocalin-type prostaglandin D synthase (beta-trace) level in cerebrospinal fluid of patients with aneurysmal subarachnoid hemorrhage.

We measured the concentration of lipocalin-type prostaglandin D synthase (PGDS) in cerebrospinal fluid (CSF) and serum in patients 1, 3, 5, 7, 9, 11, 14 and 17 days after subarachnoid hemorrhage (SAH) due to ruptured cerebral aneurysms. The PGDS level in lumbar CSF increased about two-fold at day 3 (20.85 +/- 2.71 microg/ml, mean +/- SE) and at day 5 (25.24 +/- 3.76), as compared with the level at day 1 (11.25 +/- 1.07). The CSF level gradually decreased and returned to the day 1 level at day 17. The serum PGDS level was much lower than the CSF level (0.39 +/- 0.06 at day 1) and almost unchanged until day 17. The neuron-specific enolase level in CSF, as an index of brain damage, was maximum at day 1 (29.83 +/- 7.32 ng/ml) and decreased at day 3 and at day 5 (18.28 +/- 2.65 and 11.95 +/- 1.82, respectively). These results suggest that the transient and delayed increase in the PGDS level in CSF is due to its induction of PGDS in the arachnoid membrane after SAH.

Aged↗
PubMed1999

Binding of biliverdin, bilirubin, and thyroid hormones to lipocalin-type prostaglandin D synthase.

Lipocalin-type prostaglandin D synthase is a major protein of the cerebrospinal fluid and was originally known as beta-trace. We investigated the binding ability of prostaglandin D synthase toward bile pigments, thyroid hormones, steroid hormones, and fatty acids in this present study. We found that the recombinant enzyme binds bile pigments and thyroid hormones, resulting in quenching of the intrinsic tryptophan fluorescence, the appearance of induced circular dichroism of the lipophilic ligands, and a red shift of the absorption spectra of bilirubin and biliverdin. The binding of prostaglandin D synthase to lipophilic ligands was also demonstrated by the resonant mirror technique and surface plasmon resonance detection. The dissociation constants were calculated to be 33 nM, 37 nM, 660 nM, 820 nM, and 2.08 microM for biliverdin, bilirubin, L-thyroxine, 3,3',5'-triiodo-L-thyronine, and 3,3', 5-triiodo-L-thyronine, respectively. Biliverdin and bilirubin underwent a shift in their absorption peaks from 375 to 380 nm and from 439 to 446 nm, respectively, after binding to prostaglandin D synthase. Bilirubin bound to the enzyme showed a bisignate CD spectrum with a (-) Cotton effect at 422 nm and a (+) Cotton effect at 472 nm, indicating a right-handed chirality. The ligands also inhibited prostaglandin D synthase activity noncompetitively in a concentration-dependent manner, with IC50 values between 3.9 and 10. 9 microM. Epididymal retinoic acid-binding protein and beta-lactoglobulin, two other lipocalin proteins that bind retinoids such as prostaglandin D synthase, did not show any significant interaction with bile pigments or thyroid hormones. These results show that prostaglandin D synthase binds small lipophilic ligands with a specificity distinct from that of other lipocalins.

Animals↗
PubMed1999

Lack of tactile pain (allodynia) in lipocalin-type prostaglandin D synthase-deficient mice.

Prostaglandin (PG) D2 is the most abundant prostanoid produced in the central nervous system of mammals and has been implicated in the modulation of neural functions such as sleep induction, nociception, regulation of body temperature, and odor responses. We generated gene-knockout mice for lipocalin-type PGD2 synthase (L-PGDS) and found that the intrathecal administration of PGE2, an endogenous pain-producing substance, failed to elicit allodynia (touch-evoked pain), which is one typical phenomenon of neuropathic pain, whereas it evoked thermal hyperalgesia, in L-PGDS-/- mice. We also found that the allodynic response induced by the gamma-aminobutyric acid (GABA)A receptor antagonist bicuculline was selectively abolished in the L-PGDS-/- mice, among excitatory and inhibitory agents that induced allodynia in wild-type mice. Interestingly, simultaneous injection of a femtogram amount of PGD2 with PGE2 or bicuculline induced allodynia in L-PGDS-/- mice to the same extent as in wild-type mice. The PGE2- or bicuculline-evoked allodynia in wild-type and in PGD2-supplemented L-PGDS-/- mice was blocked by a PGD2 receptor antagonist given in a femtogram amount. These results reveal that endogenous PGD2 is essential for both PGE2- and bicuculline-induced allodynia.

Anesthesia↗
PubMed1999

Prostaglandin D2 and sleep regulation.

Prostaglandin (PG) D2 is recognized as the most potent endogenous sleep-promoting substance whose action mechanism is the best characterized among the various sleep-substances thus far reported. The PGD2 concentration in rat cerebrospinal fluid (CSF) shows a circadian change coupled to the sleep-wake cycle and elevates with an increase in sleep propensity during sleep deprivation. Lipocalin-type PGD synthase is dominantly produced in the arachnoid membrane and choroid plexus of the brain, and is secreted into the CSF to become beta-trace, a major protein component of the CSF. The PGD synthase as well as the PGD2 thus produced circulates in the ventricular system, subarachnoidal space, and extracellular space in the brain system. PGD2 then interacts with DP receptors in the chemosensory region of the ventro-medial surface of the rostral basal forebrain to initiate the signal to promote sleep probably via the activation of adenosine A2A receptive neurons. The activation of DP receptors in the PGD2-sensitive chemosensory region results in activation of a cluster of neurons within the ventrolateral preoptic area, which may promote sleep by inhibiting tuberomammillary nucleus, the source of the ascending histaminergic arousal system.

Animals↗