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991.
Uyama T Kitagawa H Tanaka J Tamura J Ogawa T Sugahara K 《The Journal of biological chemistry》2003,278(5):3072-3078
We identified a novel human chondroitin N-acetylgalactosaminyltransferase, designated chondroitin GalNAcT-2 after a BLAST analysis of the GenBank(TM) data base using the sequence of a previously described human chondroitin N-acetylgalactosaminyltransferase (chondroitin GalNAcT-1) as a probe. The new cDNA sequence contained an open reading frame encoding a protein of 542 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 60% identity to that of human chondroitin GalNAcT-1. Like chondroitin GalNAcT-1, the expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which not only transferred beta1,4-N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to a polymer chondroitin representing growing chondroitin chains (beta-GalNAc transferase II activity) but also to GlcUA beta 1-3Gal beta 1-O-C(2)H(4)NHCbz, a synthetic substrate for beta-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein-linkage region of chondroitin sulfate. In contrast, the tetrasaccharide serine (GlcUA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser) derived from the linkage region, which is an inert acceptor substrate for chondroitin GalNAcT-1, served as an acceptor substrate. The coding region of this enzyme was divided into seven discrete exons, which is similar to the genomic organization of the chondroitin GalNAcT-1 gene, and was localized to chromosome 10q11.22. Northern blot analysis revealed that the chondroitin GalNAcT-2 gene exhibited a ubiquitous but differing expression in human tissues, and the expression pattern differed from that of chondroitin GalNAcT-1. Thus, we demonstrated redundancy in the chondroitin GalNAc transferases involved in the biosynthetic initiation and elongation of chondroitin sulfate, which is important for understanding the biosynthetic mechanisms leading to the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate and heparin/heparan sulfate chains. 相似文献
992.
Kim BT Kitagawa H Tanaka J Tamura J Sugahara K 《The Journal of biological chemistry》2003,278(43):41618-41623
Heparan, the common unsulfated precursor of heparan sulfate (HS) and heparin, is synthesized on the glycosaminoglycan-protein linkage region tetrasaccharide GlcUA-Gal-Gal-Xyl attached to the respective core proteins presumably by HS co-polymerases encoded by EXT1 and EXT2, the genetic defects of which result in hereditary multiple exostoses in humans. Although both EXT1 and EXT2 exhibit GlcNAc transferase and GlcUA transferase activities required for the HS synthesis, no HS chain polymerization has been demonstrated in vitro using recombinant enzymes. Here we report in vitro HS polymerization. Recombinant soluble enzymes expressed by co-transfection of EXT1 and EXT2 synthesized heparan polymers with average molecular weights greater than 1.7 x 105 using UDP-[3H]GlcNAc and UDP-GlcUA as donors on the recombinant glypican-1 core protein and also on the synthetic linkage region analog GlcUA-Gal-O-C2H4NH-benzyloxycarbonyl. Moreover, in our in vitro polymerization system, a part time proteoglycan, alpha-thrombomodulin, that is normally modified with chondroitin sulfate served as a polymerization primer for heparan chain. In contrast, no polymerization was achieved with a mixture of individually expressed EXT1 and EXT2 or with acceptor substrates such as N-acetylheparosan oligosaccharides or the linkage region tetrasaccharide-Ser, which are devoid of a hydrophobic aglycon, suggesting the critical requirement of core protein moieties in addition to the interaction between EXT1 and EXT2 for HS polymerization. 相似文献
993.
Kobayashi H Suzuki M Tanaka Y Kanayama N Terao T 《The Journal of biological chemistry》2003,278(10):7790-7799
Bikunin is a Kunitz-type protease inhibitor, acting at the level of tumor invasion and metastasis. The goal of this study was to investigate the effect of bikunin-dependent signal transduction involved in the expression of a plasminogen activator (PA) system and invasion. We report here the following. 1) The human ovarian cancer cell line HRA produced secreted and cell-associated urokinase-type PA (uPA) and PA inhibitor type 1 (PAI-1). The plasma membrane of the cells showed enzymatically active uPA even in the presence of high level of PAI-1, as measured by zymography, Western blot, chromogenic assay, enzyme-linked immunosorbent assay, and Northern blot. 2) HRA cells leading to invasion are induced through up-regulation of uPA expression. 3) HRA cells specifically released transforming growth factor-beta type 1 (TGF-beta1) participating in an autocrine/paracrine regulation of cell invasion. 4) Elimination of endogenous TGF-beta1 could induce change in uPA/PAI-1 expression, which could in turn modify the invasive behavior of the cells. 5) The constitutive expression of TGF-beta1 as well as up-regulation of the PA system observed in HRA cells was inhibited by preinoculation of the cells with bikunin or calcium channel blocker SK&F 96365 but not with nifedipine or verapamil, with an IC(50) of approximately 100 nm for bikunin or approximately 30 microm for SK&F 96365, respectively, as measured by enzyme-linked immunosorbent assay. Bikunin showed no additive effect on SK&F 96365-mediated suppression of TGF-beta1 expression. 6) The ability of TGF-beta1 to elevate free intracellular Ca(2+), followed by activation of Src and ERK, was reduced by preincubation of the cells with bikunin. In conclusion, bikunin could inhibit the constitutive expression of TGF-beta1 and TGF-beta1-mediated, Src- and ERK-dependent, PA system signaling cascade, at least in part, through inhibition of a non-voltage-sensitive calcium channel. 相似文献
994.
995.
Medroxyprogesterone acetate (MPA) is a drug commonly used in endocrine therapy for advanced breast cancer, although it is known to cause thrombosis as a serious side effect. Recently, we found that cytochrome P450 3A4 (CYP3A4) mainly catalyzed the metabolism of MPA via CYP in human liver microsomes. However, the metabolic products of MPA in humans and rats have not been elucidated. In addition, it is not clear whether thrombosis could be induced by MPA itself or by its metabolites. In this study, we determined the overall metabolism of MPA as the disappearance of the parent drug from an incubation mixture, and identified the enzymes catalyzing the metabolism of MPA via CYP in rats. Moreover, the effects of CYP-modulators on MPA-induced hypercoagulation in vivo were examined. Intrinsic clearance of MPA in rat liver microsomes was increased by treatment with CYP3A-inducers. The intrinsic clearance of MPA in liver microsomes of rats treated with various CYP-inducers showed a significant correlation with CYP3A activity, but not CYP1A activity, CYP2B activity or CYP2C contents. Among the eight recombinant rat CYPs studied, CYP3A1, CYP3A2 and CYP2A2 catalyzed the metabolism of MPA. However, since CYP3A2 and CYP2A2 are male-specific isoforms, CYP3A1 appears to be mainly involved in the metabolism of MPA in liver microsomes of female rats. In an in vivo study, pretreatment of female rats with SKF525A, an inhibitor of CYPs including CYP3A1, significantly (p < 0.05) enhanced MPA-induced hypercoagulation, whereas pretreatment with phenobarbital, an inducer of CYPs including CYP3A1, reduced it. These findings suggest that CYP-catalyzed metabolism of MPA is mainly catalyzed by CYP3A1 and that MPA-induced hypercoagulation is predominantly caused by MPA itself in female rats. 相似文献
996.
The interaction of cell surface hormone receptors with heterotrimeric G proteins is crucial for hormonal actions. The domains of the receptor, which interact with and activate G protein, have been extensively studied. However, precise molecular mechanisms underlying regulation of the receptor-induced G protein activation are still poorly understood. Prostaglandin E(2) (PGE(2)) receptors comprise of four subtypes, EP1, EP2, EP3 and EP4. Among them, EP2 and EP4 couple to Gs and EP3 to Gi. To assess the functional domains essential for Gs activation in prostanoid receptors, EP2, EP3beta and each intracellular loop- (IC-) interchanged EP2/EP3 chimeras were tested for agonist binding and functional responses. In EP2 receptor, substitution of IC1 or IC3 resulted in loss of binding activity, while substitution of IC2, N- (IC2N) or C-terminal half region of IC2 (IC2C) had no effects on the binding activity. Wild-type EP2 and IC2C-substituted EP2 showed agonist-induced Gs activity, but IC2- and IC2N-substituted EP2 failed to elicit Gs activity upon agonist stimulation. On the other hand, in EP3 receptor substitution of IC1 resulted in loss of PGE(2) binding, while substitution of IC2, IC3, IC2N or IC2C had no effects on binding activity. Wild-type EP3beta, IC3- or IC2C-substituted EP3 failed to show Gs activity upon agonist stimulation, but IC2- or IC2N-substituted EP3 chimera showed agonist-dependent Gs activity. These results indicated that the second intracellular loop of the EP2 plays an essential role in activation of Gs. 相似文献
997.
The present study was designed to investigate the antipsychotic-like effects of selective group II metabotropic glutamate receptor (mGluR) agonists, 5-[2-[4-(6-fluoro-1H-indole-3-yl) piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (MGS0008) and (1R, 2S, 5S, 6S)-2-amino-6-fluoro-4-oxobicyclo[3.1.0]hexane-2,6-dicarboxylic acid monohydrate (MGS0028) on conditioned avoidance responses in rats. MGS0008 (1, 3 and 10 mg/kg, p.o.) and MGS0028 (0.3, 1 and 3 mg/kg, p.o.) significantly and reduced conditioned avoidance responses in a dose-dependent fashion. Similar effects were seen with LY418426 (0.3, 1 and 3 mg/kg, p.o.), but not with LY354740 (3, 10 and 30 mg/kg, p.o.), both of which are selective agonists for group II mGluR. Since this effect is seen with a wide range of antipsychotics, such as haloperidol and clozapine [Life Sciences 71 (2002) 947], group II mGluR agonists deserve further attention for possible antipsychotic activity. 相似文献
998.
999.
It is thought that sodium dodecyl sulfate (SDS), an anionic detergent, binds to hydrophobic moieties of peptide to destroy the conformational structure of protein. Because of this property, it is involved in many biochemical procedures such as separations of protein and proteolytic digestion. In the course of our study on a solid-phase protein assay, we found that SDS acts as an effective reagent for protein blotting onto a hydrophobic membrane of polyvinylidene difluoride with a manifold dot-blot apparatus. At least 0.1% SDS in an acid-ethanol blotting solution, while reducing the bias of pronounced interferers for protein assay to protein-membrane interaction, quantitatively retains protein on the membrane. Presumably, protein denatures by SDS to become an unfolded state and adsorbs into the membrane by hydrophobic interaction, even in the presence of excess SDS. Therefore, bolts stained with a pyrogallol red-molybdate complex (Pyromolex) reagent unreactive to the membrane allowed a precise protein determination without significant interference of materials, especially detergents in the sample solution. The filtration-blotting with SDS would be a crucial procedure for quantitative analyses such as immunoblotting in detergent-containing samples, together with the solid-phase protein assay with limited sample volumes, such as 20 microL or less. 相似文献
1000.
True hermaphroditism with 46,X,+22p/46,XY and gonadal mosaicism detected by fluorescence in situ hybridization 总被引:1,自引:0,他引:1
A Japanese girl was diagnosed as true hermaphroditism with 46,X,+mar/46,XY and the marker chromosome was determined on the short arm of chromosome 22 without alpha-satellite by fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) methods. At birth, she showed intersexual external genitalia, urethral-vaginal fistula and right inguinal hernia. The right gonad was revealed as an ovotestis, and the left was as an undifferentiated testis. The gonadal mosaicism was demonstrated directly in gonadal tissue by interphase FISH. 相似文献