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Y Urade

Publications and source records attributed to Y Urade.

At least 55 records · Page 3Linked to original sources
PubMed1999

Selective activation of excitation-contraction coupling pathways by ET(A) and ET(B) receptors in guinea-pig tracheal smooth muscle.

1. Signalling events responsible for endothelin(A) (ET(A)) and ET(B) receptor-induced contraction were examined in epithelium-denuded guinea-pig tracheal smooth muscle strips. Selective stimulation of each subtype was achieved by a combination of ET-1 (100 nM) and ET(A) and ET(B) receptor-selective antagonists, BQ-123 (10 microM) and BQ-788 (3 microM), respectively. 2. Both ET(A) and ET(B) receptors induced long-lasting contraction that was totally dependent on Ca2+ influx. Stimulation of ET(A) receptor induced both transient and sustained (Ca2+)i increases whereas that of ET(B) receptor induced only a sustained increase. Suppression of the transient (Ca2+)i increase by U73122 (3 microM) did not affect the ET(A)-induced sustained (Ca2+)i increase and tension development. Stimulation of ET(A) receptor, but not ET(B), induced phosphoinositide breakdown and protein kinase C (PKC). The activated PKC contributed to the contraction by increasing the Ca2+ sensitivity of the contractile apparatus. 3. Thus, ET(A) receptor is coupled both with phospholipase C/Ca2+/PKC signalling and Ca2+ influx pathways whereas ET(B) receptor was coupled only with the latter. 4. Stimulation of ET(B) receptor, but not ET(A), caused membrane depolarization measured with a fluorescent indicator, bis-(1,3 dibutylbarbituric acid)-trimethine oxonol. Both nifedipine (1 microM) and verapamil (10 microM) abolished ET(B)-induced Ca2+ influx and contraction, while they barely affected ET(A)-induced responses. 5. Therefore, the Ca2+ influx pathways activated by each subtype appeared to be completely different; ET(A) and ET(B) receptors opens voltage-independent Ca2+ channels and L-type voltage-dependent Ca2+ channels, respectively.

Animals↗
PubMed1999

An apoptotic depletion of oligodendrocytes in the twitcher, a murine model of globoid cell leukodystrophy.

Morphological alterations of oligodendrocytes (OLs) leading to their depletion were studied in the genetic demyelinating mutant, twitcher, a murine model of globoid cell leukodystrophy (GLD). With pi-glutathione-S-transferase immunostaining, OLs with multiple varicose processes were recognized in the early stages and adjacent areas of demyelination and then the OLs cytoplasm as well as the processes became shrunken with progression of the disease. These shrunken OLs were labeled by the TUNEL method, indicative of apoptotic cell death. The ultrastructural features of apoptotic cells were noted in these OLs and DNA laddering was noted in the twitcher brain in advanced stages. This is the first report describing the gradual depletion of OLs by apoptosis in genetic demyelination.

Animals↗
PubMed1998

Plasmodium falciparum produces prostaglandins that are pyrogenic, somnogenic, and immunosuppressive substances in humans.

Plasmodium falciparum causes the most severe form of human malaria, which kills approximately 1.5-2.7 million people every year, but the molecular mechanisms underlying the clinical symptoms and the host-parasite interaction remain unclear. We show here that P. falciparum produces prostaglandins (PGs) D2, E2, and F2alpha. After incubation with 1 mM arachidonic acid (AA), cell homogenates of P. falciparum produced PGs as determined by enzyme immunoassay and gas chromatography-selected ion monitoring. PG production in the parasite homogenate was not affected by the nonsteroidal antiinflammatory drugs aspirin and indomethacin, and was partially heat resistant, whereas PG biosynthesis by mammalian cyclooxygenase was completely inhibited by these chemicals and by heat treatment. Addition of AA to the parasite cell culture markedly increased an ability of the parasite cell homogenate to produce PGs and of parasitized red blood cells to accumulate PGs in the culture medium. PGD2 and PGE2 accumulated in the culture medium at the stages of trophozoites and schizonts more actively than at the ring stage. These findings are the first evidence of the direct involvement of a malaria parasite in the generation of substances that are pyrogenic and injurious to the host defenses. We will discuss a possible contribution of the parasite-produced PGs to pathogenesis and host-parasite interaction of P. falciparum.

Animals↗
PubMed1998

Activation of ventrolateral preoptic neurons by the somnogen prostaglandin D2.

Prostaglandin D2 (PGD2) is an extensively studied sleep-promoting substance, but the neuroanatomical basis of PGD2-induced sleep is only partially understood. To determine potential regions involved in this response, we used Fos immunohistochemistry to identify neurons activated by infusion of PGD2 into the subarachnoid space below the rostral basal forebrain. PGD2 increased nonrapid eye movement sleep and induced striking expression of Fos in the ventrolateral preoptic area (VLPO), a cluster of neurons that may promote sleep by inhibiting the tuberomammillary nucleus, the source of the ascending histaminergic arousal system. Fos expression in the VLPO was positively correlated with the preceding amount of sleep and negatively correlated with Fos expression in the tuberomammillary nucleus. PGD2 also increased Fos immunoreactivity in the basal leptomeninges and several regions implicated in autonomic regulation. These observations suggest that PGD2 may induce sleep via leptomeningeal PGD2 receptors with subsequent activation of the VLPO.

Animals↗
PubMed1998

Sodium dodecyl sulfate-capillary gel electrophoretic analysis of molecular mass microheterogeneity of beta-trace protein in cerebrospinal fluid from patients with central nervous system diseases.

Molecular mass (M(r)) microheterogeneity of beta-trace protein (beta TP) in cerebrospinal fluid (CSF) from patients with various neurological disorders was analyzed by sodium dodecyl sulfate capillary gel electrophoresis. Under the conditions employed, beta TP with a M(r) distribution of 23,000-30,000 was roughly separated into two subfractions containing the major peaks with M(r) of 26,000 and 28,500, respectively. The peak area ratios of the two subfractions of the electropherograms varied among the samples examined, and elevation in the total beta TP level in the CSF from patients with organic diseases in the central nervous system (CNS) was often accompanied by changes in the ratios of the subfractions. The quantitative changes in the subfraction level in CSF beta TP are considered to reflect the pathological alterations in the CNS.

Adult↗
PubMed1998

Identification of a thyroid hormone response element in the promoter region of the rat lipocalin-type prostaglandin D synthase (beta-trace) gene.

We have previously reported that mRNA levels for the rat lipocalin-type prostaglandin (PG) D synthase/beta-trace (PGDS) gene, the enzyme responsible for the production of PGD2 in the central nervous system, are regulated by thyroid hormone in vivo. In this study, we describe the identification of a thyroid hormone (T3) response element (T3RE) in the 5'-flanking region of the rat PGDS gene. By radioimmunoprecipitation of genomic fragments using thyroid hormone receptor (TR) protein and specific anti-TR antibodies, gel-shift, foot-printing, mutational analysis, and transactivation assays we have identified a spaced four imperfect direct repeat (DR4) element, GGTTCACTTCAGGGTA (positions -586/-571), which functions as a T3RE when fused to a heterologous promoter. Our results suggest that thyroid hormone regulates the expression of the rat lipocalin-type PGDS gene through this element. Remarkably, the element identified also confers regulation by retinoic acid. Giving the important roles proposed for the PGDS enzyme and its product, PGD2, the major PG in the mammalian brain, the altered expression of the PGDS gene may contribute to the deleterious effects of hypothyroidism in the central nervous system.

5' Untranslated Regions↗
PubMed1998

Dominant expression of rat prostanoid DP receptor mRNA in leptomeninges, inner segments of photoreceptor cells, iris epithelium, and ciliary processes.

Prostaglandin (PG) D2 is one of the major prostanoids in the mammalian brain and eye tissues. Its function is mediated by the prostanoid DP receptor, which is specific for PGD2 among the various prostanoids. In this study, we cloned the full-length cDNA for the rat DP receptor and used it for detection of DP receptor mRNA in various rat tissues. Northern blotting and RT-PCR analyses revealed that this DP receptor was expressed most intensely in the eye tissues, moderately in the leptomeninges and oviduct, and weakly in the epididymis. The tissue distribution profile of the mRNA for the rat DP receptor is overlapped with those of hematopoietic and lipocalin-type PGD synthases. Among rat eye tissues, the expression was the highest in the iris. In situ hybridization and in situ RT-PCR revealed DP receptor mRNA to be localized in the epithelium of the iris and ciliary body and in photoreceptor cells of the retina, suggesting the involvement of the receptor in the physiological regulation of intraocular pressure and the vision process. In the brain, DP receptor mRNA was dominantly expressed in the leptomeninges and was not detected in the brain parenchyma including the ventral rostral forebrain, the surface area of which is reportedly involved in sleep induction by PGD2.

Amino Acid Sequence↗
PubMed1998

Lipocalin-type prostaglandin D synthase in human male reproductive organs and seminal plasma.

Prostaglandin D synthase (PGDS) activity was detected in human seminal plasma (0.05-1.83 nmol/min per milligram protein). The enzyme was purified from human seminal plasma by immunoaffinity chromatography and found to be 27 kDa in size and N-glycosylated, similar to PGDS in the cerebrospinal fluid. The N-terminal amino acid sequence of 16 residues of the seminal enzyme, APEAQVSVQPNFQQDK, was identical to that of the cerebrospinal fluid PGDS. Although PGDS activity and the content determined by the immunoassay each highly varied in the seminal plasma, the concentration was significantly (p < 0.001) lower in the oligozoospermic group (2.47 +/- 0.51 microg/ml) than in the normozoospermic group (9.75 +/- 1.49 microg/ml). Prostaglandin (PG) D2 was detected in the seminal plasma (5.00 +/- 0.65 ng/ml) with a positive correlation to the PGDS concentration (p < 0.05). PGD2 was converted to the J series of PGs in the seminal plasma with a half-life of 6.5 h. Northern blot analysis revealed that mRNA for PGDS was expressed in the testis, prostate, and epididymis. Through immunohistochemistry, PGDS was localized in Leydig cells of the testis and in epithelial cells of the prostate and ductus epididymidis.

Adult↗
PubMed1998

Identification of a fertility-associated protein in bull seminal plasma as lipocalin-type prostaglandin D synthase.

The objective of this study was to characterize a 26-kDa seminal plasma protein previously shown to be prevalent in bulls of high fertility. Spots of this protein, excised and electroeluted from two-dimensional SDS-PAGE gels, were used for N-terminal amino acid sequencing and for preparation of antiserum in rabbits. The N-terminal amino acid sequence (ALQPNFEEDKFLGRWFTSGL) was 75% identical and 100% homologous to lipocalin-type prostaglandin (PG) D synthase isolated from human cerebrospinal fluid (CSF). Western blots of purified 26-kDa protein cross-reacted with polyclonal antibodies against lipocalin-type PGD synthase isolated from rat brain and human CSF. Immunoreactive bands at 26 kDa appeared in Western blots of seminal plasma and cauda epididymal fluid (CEF). A 29-kDa band appeared in blots of rete testis fluid (RTF). PGD synthase activity was detected in seminal plasma, CEF, and RTF. The cDNA for bovine lipocalin-type PGD synthase, isolated by reverse transcription-polymerase chain reaction, contained a coding region of 573 base pairs corresponding to 191 amino acids. The amino acid sequence was 63-80% identical to that of the enzyme of other mammals. These results establish that the 26-kDa fertility-associated protein in bull seminal plasma is lipocalin-type PGD synthase. Although we do not yet know the role of lipocalin-type PGD synthase in the male genital tract, we speculate that this protein may play an important role in both the development and the maturation of sperm.

Amino Acid Sequence↗
PubMed1998

Localization of lipocalin-type prostaglandin D synthase (beta-trace) in iris, ciliary body, and eye fluids.

PURPOSE: Prostaglandin (PG) D synthase is present in neural tissues and cerebrospinal fluid (beta-trace). This enzyme belongs to the lipocalin family which consists of transporter proteins for lipophilic substances in the extracellular space. PGD synthase is found in retinal pigment epithelium, from where it is secreted into the interphotoreceptor matrix. The authors have undertaken the localization of this unique enzyme within the tissues and spaces of the anterior segment of the eye. METHODS: Iris, ciliary body, lens, and aqueous and vitreous humors were collected from adult rats and mice. PGD synthase activity was determined, and the protein was quantified by Western blot analysis and localized immunohistochemically. Finally, in situ hybridization was performed to localize PGD synthase mRNA. RESULTS: PGD synthase was most abundant in the aqueous and vitreous humors. It was less abundant in tissue cytosolic fractions; these fractions had almost 10-fold as much as their corresponding membrane-bound fractions. Lens tissue had the lowest amount observed. PGD synthase was localized to the epithelial cells of the iris and the ciliary body and to the adjacent extracellular chambers, but PGD synthase mRNA was found only within the epithelial cells. Several glycosylated forms of PGD synthase were also detected. CONCLUSIONS: PGD synthase was synthesized within the epithelial cells of the iris and the ciliary body and was then secreted into the aqueous and vitreous humors, where it accumulated as an active enzyme.

Animals↗
PubMed1998

[Gene engineering studies on sleep].

Recent advancements in molecular engineering techniques have enabled us to study sleep of animals lacking or overproducing any protein molecule we are interested in. Abnormalities in sleep have already been observe in knockout mice lacking the gene for prion protein, 55 kD-receptor of tumor necrosis factor, etc. We also started a project to examine the effect of the loss or the overproduction of prostaglandin D synthase in sleep. The enzyme is responsible for biosynthesis of prostaglandin D2, the most potent endogenous substance to promote sleep reported so far. We generated both knockout and transgenic mice of prostaglandin D synthase. We are currently analysing the sleep of these mutant mice.

Animals↗
PubMed1997

Expression of lipocalin-type prostaglandin D synthase (beta-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 beta-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 microg/ml, n = 7) than in the coronary artery (0.545 +/- 0.034 microg/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 microg/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.

Angina Pectoris↗
PubMed1997

Prostaglandin D synthase in human megakaryoblastic cells.

The cytosol fraction of human platelets did not convert prostaglandin (PG) H2 to PGD2. However, a homogenate of human megakaryoblastic CMK cells (precursor cells of platelets) produced PGD2 from PGH2. The PGD synthase activity was localized in the cytosol of CMK cells, and absolutely required glutathione. The catalytic properties and Western and Northern blottings indicated that the enzyme was PGD synthase of the hematopoietic type rather than the lipocalin type. When CMK cells were differentiated to megakaryocytes with phorbol ester along with induction of cyclooxygenase-1, the PGD synthase activity increased about 2-fold for 2 days and then decreased. In another human megakaryoblastic cell line, Dami, the PGD synthase increased about 10-fold by the addition of phorbol ester. Thus, the PGD synthase, which was undetectable in platelets, appeared during differentiation of megakaryoblasts to megakaryocytes.

Chromatography, Affinity↗
PubMed1997

Cloning and crystal structure of hematopoietic prostaglandin D synthase.

Hematopoietic prostaglandin (PG) D synthase is the key enzyme for production of the D and J series of prostanoids in the immune system and mast cells. We isolated a cDNA for the rat enzyme, crystallized the recombinant enzyme, and determined the three-dimensional structure of the enzyme complexed with glutathione at 2.3 A resolution. The enzyme is the first member of the sigma class glutathione S-transferase (GST) from vertebrates and possesses a prominent cleft as the active site, which is never seen among other members of the GST family. The unique 3-D architecture of the cleft leads to the putative substrate binding mode and its catalytic mechanism, responsible for the specific isomerization from PGH2 to PGD2.

Animals↗