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Biomedical subjects

M Negishi

Publications and source records attributed to M Negishi.

At least 37 records · Page 2Linked to original sources
PubMed2001

Crystal structure-based studies of cytosolic sulfotransferase.

Sulfation is a widely observed biological reaction conserved from bacterium to human that plays a key role in various biological processes such as growth, development, and defense against adversities. Deficiencies due to the lack of the ubiquitous sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) are lethal in humans. A large group of enzymes called sulfotransferases catalyze the transfer reaction of sulfuryl group of PAPS to the acceptor group of numerous biochemical and xenochemical substrates. Four X-ray crystal structures of sulfotransferases have now been determined: cytosolic estrogen, hydroxysteroid, aryl sulfotransferases, and a sulfotransferase domain of the Golgi-membrane heparan sulfate N-deacetylase/N-sulfotransferase 1. These have revealed the conserved core structure of the PAPS binding site, a common reaction mechanism, and some information concerning the substrate specificity. These crystal structures introduce a new era of the study of the sulfotransferases.

Animals↗
PubMed2001

Promoter CpG methylation of Hox-a10 and Hox-a11 in mouse uterus not altered upon neonatal diethylstilbestrol exposure.

Mouse abdominal B-like Hoxa genes are expressed and functionally required in the developing reproductive tracts. Mice lacking either Hoxa-10 or Hoxa-11, two of the AbdB Hoxa genes, exhibit abnormal uterine development similar to that induced by in utero diethylstilbestrol (DES) exposure. Indeed, uterine Hoxa-10 and Hoxa-11 expression is potently repressed by perinatal DES exposure, providing a potential molecular mechanism for DES-induced reproductive tract malformations. We have shown previously that DES can permanently alter uterine lactoferrin gene expression through modulation of the lactoferrin promoter methylation pattern. Here we ask whether a similar mechanism also functions to deregulate uterine Hoxa-10 or Hoxa-11 expression during neonatal DES exposure. We mapped the Hoxa-10 promoter by cloning a 1.485 kb DNA fragment 5' of the Hoxa-10 exon1a. A 5' rapid amplification of cDNA ends (RACE) experiment revealed a transcription start site for the a10-1 transcript. Functional analysis of the proximal 200-bp sequences demonstrated significant promoter activity, confirming the location of the Hoxa-10 promoter. Moreover, methylation assays performed on eight CpGs in Hoxa-10 and 19 CpGs in Hoxa-11 proximal promoters demonstrated that all these CpGs were highly unmethylated in both control and DES-dosed mice from postnatal day 5 to day 30. Significant methylation around Hoxa-10 and Hoxa-11 promoters was only observed in DES-induced uterine carcinomas in 18-mo-old mice. Our results suggest that DES-induced downregulations of Hoxa-10 or Hoxa-11 gene expression are not associated with methylation changes in their proximal promoters and that gene imprinting by developmental DES exposure may be a gene-specific phenomenon.

Animals↗
PubMed2001

Percutaneous transvenous embolisation through the occluded sinus for transverse-sigmoid dural arteriovenous fistulas with sinus occlusion.

We report six cases of transverse-sigmoid dural arteriovenous fistulae (TS DAVF) treated with percutaneous transvenous embolisation through the occluded sinus. All patients had sinus occlusive lesions: an isolated sinus in five cases and a distal occlusion of the affected sinus in one. Leptomeningeal retrograde venous drainage via the vein of Labbé or the sylvian vein was observed in all patients with an isolated sinus. In five patients a microcatheter was easily passed through the occluded sinus. In four of them, a complete angiographic cure was achieved by packing the sinus with coils. However, in one, sinus packing was ineffective and surgical excision of the affected sinus was necessary. The microcatheter could not be passed through the occluded sinus in one case, and direct packing of the isolated sinus was later required. In all cases, complete cure was achieved without complications. This safe, not very invasive and highly effective treatment for TS DAVF with sinus occlusion is thus worth trying when the occluded segment is relatively short.

Aged↗
PubMed2001

Prostaglandin EP3 receptor protein in serotonin and catecholamine cell groups: a double immunofluorescence study in the rat brain.

Prostaglandin E(2) exerts diverse physiological actions in the central nervous system with unknown mechanisms. We have reported the immunohistochemical localization of the EP3 receptor, one of the prostaglandin E receptor subtypes, in various brain regions including many monoaminergic nuclei. In the present study, a double immunofluorescence technique with an antibody to EP3 receptor and antibodies to markers for monoamine neurons was employed to examine the expression of the receptor in serotonin and catecholamine neurons, and to reveal the distribution of the receptor-expressing monoamine neurons in the rat brain. Almost all serotonergic cells in the medulla oblongata (B1-B4) exhibited EP3 receptor-like immunoreactivity, whereas mesencephalic and pontine serotonergic cell groups (B5-B9) contained relatively small populations of EP3 receptor-immunoreactive cells. In the catecholaminergic cell groups, many of the noradrenergic A7 cells in the subcoeruleus nucleus showed immunoreactivity for the receptor. The locus coeruleus exhibited EP3 receptor-like immunoreactivity densely in the neuropil and occasionally in neuronal cell bodies, all of which were immunopositive for dopamine beta-hydroxylase, as observed by confocal laser microscopy. Many of the other noradrenergic and adrenergic cell groups contained small populations of EP3 receptor-like immunoreactive cells. In contrast, no EP3 receptor-like immunoreactivity was detected in the noradrenergic A2 and A4, the adrenergic C2, and all the dopaminergic cell groups. The expression of EP3 receptor by most of the serotonergic, noradrenergic and adrenergic cell groups suggests that prostaglandin E(2) modulates many physiological processes mediated by widely distributed monoaminergic projections through activation of the EP3 receptor on the monoaminergic neurons; for instance, it may modulate nociceptive and autonomic processes by affecting the descending serotonergic pathway from the raphe magnus nucleus to the spinal cord.

Animals↗
PubMed2001

Potentiation of PGE(2)-mediated cAMP production during neuronal differentiation of human neuroblastoma SK-N-BE(2)C cells.

The prostaglandin-evoked cAMP production was studied in human neuroblastoma SK-N-BE(2)C cells during neuronal differentiation induced by all-trans retinoic acid. The incubation with 5 microM all-trans retinoic acid for 4-6 days promoted neurite outgrowth of cells. After differentiation, prostaglandin E(2) (PGE(2))-induced cAMP production was dramatically increased, whereas forskolin- and AlF-induced cAMP productions were not changed. The increase reached maximum after 4-days of incubation with all-trans retinoic acid. The differentiation caused an increase in the maximal response and a decrease in the half-maximal effective concentration of the PGE(2)-induced cAMP production. In addition, the binding of [(3)H]PGE(2) to membrane receptors was enhanced in differentiated cells. However, the order of potency of the various prostaglandins (PGE(1) = PGE(2) > PGD(2) = PGF(2alpha) = PGI(2)) in cAMP production did not change during the differentiation, suggesting that mainly E-prostanoid (EP) receptors were involved. Butaprost, an EP(2) receptor specific agonist, increased the cAMP level in a concentration dependent manner and had a similar potentiating effect on cAMP production as PGE(2) upon differentiation. Northern blot analysis using the human cDNA probes shows that the EP(2) mRNA level was about seven times higher in differentiated cells, while the dopamine beta-hydroxylase (DBH) mRNA completely disappeared. Our results, thus, suggest that elevated gene expression of the prostanoid EP(2) receptor results in an increase in the PGE(2)-evoked cAMP production in SK-N-BE(2)C cells during neuronal differentiation.

Alprostadil↗
PubMed2001

The phenobarbital response enhancer module in the human bilirubin UDP-glucuronosyltransferase UGT1A1 gene and regulation by the nuclear receptor CAR.

The UDP-glucuronosyltransferase, UGT1A1, is the critical enzyme responsible for detoxification of the potentially neurotoxic bilirubin by conjugating it with glucuronic acid. For decades, phenobarbital (PB) treatment for hyperbilirubinemia has been known to increase expression of the UGT1A1 gene in liver. We have now delineated the PB response activity to a 290-bp distal enhancer sequence (-3483/-3194) of the UGT1A1 gene. The enhancer contains 3 putative nuclear receptor motifs, and it was activated by the nuclear orphan receptor, human constitutive active receptor (hCAR), in cotransfected HepG2 cells. Bacterially expressed hCAR, acting as a heterodimer with in vitro-translated retinoid X receptor (RXRalpha), only bound to 1 of the 3 NR motifs, named gtNR1 in a gel-shift assay. Consistently, mutations of the gtNR1 site significantly decreased the activation by hCAR of the 290-bp DNA in transfection assays. Moreover, the 290-bp DNA was effectively activated in mouse primary hepatocytes in response to PB, offering an excellent clinical test for the examination of the responsiveness of the UGT1A1 to PB in the human population, particularly individuals with hyperbilirubinemia.

Base Sequence↗
PubMed2001

Preliminary report: effect of adrenal androgen and estrogen on bone maturation and bone mineral density.

To clarify the independent physiological roles of adrenal androgen and estrogen on bone growth, we compared the lumbar spine bone mineral density (BMD) in prepubertal girls with virilizing congenital adrenal hyperplasia (CAH) (n = 17) and girls with central precocious puberty (CPP) (n = 18). When BMD was analyzed according to chronologic age, no significant differences were found between CPP and CAH patients. However, when adjusted to bone age, BMD was statistically higher in CAH than in CPP subjects. This finding suggests that adrenal androgen, as well as estrogen, plays an important role in increasing BMD. Adrenal androgen may act on bone not only as androgen, but as estrogen after having been metabolized into an aromatized bone-active compound in peripheral tissues, such as bone and fat. Therefore, adrenal androgen may have a more important role in increasing BMD than previously realized.

Absorptiometry, Photon↗
PubMed2001

Release behavior of ketoprofen from poly(acryloyl-L-proline methyl ester) gels having different crosslinked networks.

In order to clarify the relationship between the crosslinked structure of thermo-responsive polymer and drug release profile, polymer gels based on acryloyl-L-proline methyl ester (A-ProOMe) were synthesized in a mixture of water and acetone by the following two methods: a simultaneously occurring process of radiation-induced polymerization and crosslinking without a crosslinker (self-bridging method), and radiation-induced polymerization in the presence of the crosslinker tetradecaethylene glycol dimethacrylate (crosslinker method). The pronounced gap in thermo-response between two A-ProOMe gels, with an apparent degree of crosslinking of 11 for 1-propanol, shows a different shrinking pattern in the initial stage of time. The gels, which were obtained with the self-bridging method and the crosslinker method, were kept constant at a swelling ratio of 17 in water at 0 degree C for all systems. However, those values fell to 0.5 and 4, respectively, at 10 min after the temperature was increased to 37 degrees C. The release mechanism of ketoprofen from two gel devices showed an anomalous (non-Fickian) transport, in which the release of ketoprofen with a low water-solubility could be directly related to the rapid release of water accompanying a gel shrinkage.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2001

[Two cases of typhoid fever with reduced susceptibility to fluoroquinolones].

Two separate febrile Indian patients who reside in Japan and had recently returned from their country were diagnosed as suffering from typhoid fever. Fluoroquinolone therapy was clinically ineffective and the addition of a third-generation cephalosporin was required in each case. Each strain of Salmonella Typhi was resistant to nalidixic acid in vitro and also showed higher minimal inhibitory concentration to other quinolones than usual susceptible strains. Similar cases of typhoid fever responding poorly to quinolone treatment have been observed in the Indian subcontinent, south-east Asia and central Asia since the early 1990s, and potential spread by travelers into Japan is of serious concern. Although quinolones still remain the drugs of choice for treatment of typhoid fever, physicians should be aware of the possibility and implications of clinical treatment failure.

Adult↗
PubMed2001

Nuclear receptor CAR as a regulatory factor for the sexually dimorphic induction of CYB2B1 gene by phenobarbital in rat livers.

The nuclear receptor constitutive active receptor (CAR) translocates into liver nuclei after phenobarbital (PB) treatment, and activates the conserved enhancer called the PB-response element module (PBREM) found in CYP2B genes. We have examined whether CAR regulates the dimorphic induction by PB of the CYP2B1 gene in Wistar Kyoto (WKY) rats. Northern blot analysis showed that PB induced CYP2B1 mRNA in male WKY rats but not female rats. An in situ injected PBREM-luciferase reporter gene was activated by PB only in the male livers. Western blot analysis revealed extremely low levels of CAR in the cytosols of female livers compared with male counterparts. CAR was accumulated in the liver nucleus of male rats in response to PB treatment, whereas the receptor was barely detectable in the liver nuclei of PB-induced females. These sexually dimorphic responses of PBREM and CAR to PB treatment were not observed with Fisher 344 rats, in which CYP2B1 mRNA was induced in both sexes. Thus, these results indicate that CAR is a regulatory factor that leads to the sexual dimorphic induction of CYP2B1 gene in WKY rats.

Amino Acid Sequence↗
PubMed2001

Endoplasmic reticulum stress-induced formation of transcription factor complex ERSF including NF-Y (CBF) and activating transcription factors 6alpha and 6beta that activates the mammalian unfolded protein response.

The levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) are controlled by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis-acting ER stress response element (ERSE), the consensus sequence of which is CCAAT-N(9)-CCACG. We recently proposed that ER stress response factor (ERSF) binding to ERSE is a heterologous protein complex consisting of the constitutive component NF-Y (CBF) binding to CCAAT and an inducible component binding to CCACG and identified the basic leucine zipper-type transcription factors ATF6alpha and ATF6beta as inducible components of ERSF. ATF6alpha and ATF6beta produced by ER stress-induced proteolysis bind to CCACG only when CCAAT is bound to NF-Y, a heterotrimer consisting of NF-YA, NF-YB, and NF-YC. Interestingly, the NF-Y and ATF6 binding sites must be separated by a spacer of 9 bp. We describe here the basis for this strict requirement by demonstrating that both ATF6alpha and ATF6beta physically interact with NF-Y trimer via direct binding to the NF-YC subunit. ATF6alpha and ATF6beta bind to the ERSE as a homo- or heterodimer. Furthermore, we showed that ERSF including NF-Y and ATF6alpha and/or beta and capable of binding to ERSE is indeed formed when the cellular UPR is activated. We concluded that ATF6 homo- or heterodimers recognize and bind directly to both the DNA and adjacent protein NF-Y and that this complex formation process is essential for transcriptional induction of ER chaperones.

Activating Transcription Factor 6↗
PubMed2001

The peptide near the C terminus regulates receptor CAR nuclear translocation induced by xenochemicals in mouse liver.

In response to phenobarbital (PB) and other PB-type inducers, the nuclear receptor CAR translocates to the mouse liver nucleus (T. Kawamoto et al., Mol. Cell. Biol. 19:6318-6322, 1999). To define the translocation mechanism, fluorescent protein-tagged human CAR (hCAR) was expressed in the mouse livers using the in situ DNA injection and gene delivery systems. As in the wild-type hCAR, the truncated receptor lacking the C-terminal 10 residues (i.e., AF2 domain) translocated to the nucleus, indicating that the PB-inducible translocation is AF2 independent. Deletion of the 30 C-terminal residues abolished the receptor translocation, and subsequent site-directed mutagenesis delineated the PB-inducible translocation activity of the receptor to the peptide L313GLL316AEL319. Ala mutations of Leu313, Leu316, or Leu319 abrogated the translocation of CAR in the livers, while those of Leu312 or Leu315 did not affect the nuclear translocation. The leucine-rich peptide dictates the nuclear translocation of hCAR in response to various PB-type inducers and appears to be conserved in the mouse and rat receptors.

Amino Acid Sequence↗
PubMed2001

Phenobarbital response elements of cytochrome P450 genes and nuclear receptors.

Phenobarbital (PB) response elements are composed of various nuclear receptor (NR)-binding sites. A 51-bp distal element PB-responsive enhancer module (PBREM) conserved in the PB-inducible CYP2B genes contains two NR-binding direct repeat (DR)-4 motifs. Responding to PB exposure in liver, the NR constitutive active receptor (CAR) translocates to the nucleus, forms a dimer with the retinoid X receptor (RXR), and activates PBREM via binding to DR-4 motifs. For CYP3A genes, a common NR site [DR-3 or everted repeat (ER)-6] is present in proximal promoter regions. In addition, the distal element called the xenobiotic responsive module (XREM) is found in human CYP3A4 genes, which contain both DR-3 and ER-6 motifs. Pregnane X receptor (PXR) could bind to all of these sites and, upon PB induction, a PXR:RXR heterodimer could transactivate XREM. These response elements and NRs are functionally versatile, and capable of responding to distinct but overlapping groups of xenochemicals.

Animals↗
PubMed2001

Upregulatory expression of furin and transforming growth factor-beta by fluid shear stress in vascular endothelial cells.

Furin, a yeast Kex2-family endoprotease, converts many vasoregulatory propeptides, including pro-transforming growth factor (TGF)-beta to their mature forms. We examined whether furin expression is regulated by shear stress in vivo and in vitro. When an arteriovenous shunt was placed between the carotid artery and external jugular vein in rabbits, furin and TGF-beta were highly expressed in shear stress-loaded endothelial cells. Exposure of bovine aortic endothelial cells in culture to shear stress induced furin and TGF-beta expression in a similar manner. Molecular analysis of furin expression in bovine aortic endothelial cells revealed that shear stress increases the furin gene expression at transcriptional levels. Furthermore, TGF-beta itself increased the furin mRNA levels. Shear-mediated furin expression was partly mediated by TGF-beta because shear-induced furin mRNA levels were considerably decreased by overexpression of the truncated form of the TGF-beta type II receptor. Likewise, blockade of furin activity by a furin inhibitor significantly decreased the endothelial production of mature TGF-beta. Taken together, the results indicate that furin expression is induced and maintained by a coordination of shear stress and TGF-beta. Increased furin expression may facilitate the formation of mature TGF-beta, resulting in the enhanced effects of TGF-beta on endothelial cells and vascular smooth muscle cells in the vasculature.

Animals↗
PubMed2001

Mechanism of induction of cytochrome p450 enzymes by the proestrogenic endocrine disruptor pesticide-methoxychlor: interactions of methoxychlor metabolites with the constitutive androstane receptor system.

Methoxychlor, a structural analog of the DDT pesticide, was previously shown to induce rat hepatic CYP2B and -3A mRNAs and the corresponding proteins [J Biochem Mol Toxicol 1998;12:315-323], Additionally, methoxychlor was found to activate the constitutive androstane receptor (CAR) system and induce CYP2B6 (J Biol Chem 1999;274:6043-6046), suggesting a mechanism for methoxychlor-mediated cytochrome P450 (P450) 2B induction. However, it has not been established whether CAR activation and P450 induction was due to methoxychlor per se and/or due to its metabolites. Also, a possible link between the estrogenic potency of methoxychlor metabolites and CAR activation or P450 induction was not investigated. The current study explores the ability of methoxychlor and its metabolites to activate CAR and whether their potency of CAR activation correlates with their respective estrogenicity. Methoxychlor and its metabolites [mono-OH-M [1,1,1-trichloro-2 (4-hydroxyphenyl)-2'-(4-methoxyphenyl)ethane]; bis-OH-M [1,1,1-trichloro-2,2'-bis(4-hydroxyphenyl)ethane]; ring-OH-M [1,1,1-trichloro-2(4-methoxyphenyl)-2'-(3-hydroxy-4-methoxyphenyl)ethane]; and tris-OH-M [1,1,1-trichloro-2(4-hydroxyphenyl)-2'-(3,4-dihydroxyphenyl)ethane]] were found to be potent activators of CAR. Dose response curves indicated that tris-OH-M is a more potent CAR activator than methoxychlor, mono-OH-M, and bis-OH-M. Since tris-OH-M is a much weaker estrogen receptor-alpha agonist than mono-OH-M and bis-OH-M, it seems that estrogenicity is not a significant factor in CAR activation. These findings indicate that alteration of methoxychlor-benzene rings, i.e., generation of phenolic constituents, does not appreciably alter CAR activation and suggest that a common structural motif in the methoxychlor class of compounds controls CAR activation. Studies are needed to identify the structural motif necessary for CAR activation and CYP2B induction.

Androstanes↗
PubMed2000

Rnd1, a novel rho family GTPase, induces the formation of neuritic processes in PC12 cells.

Rho family GTPases have been shown to be involved in the regulation of neuronal cell morphology, including neurite extension and retraction. Rho activation leads to neurite retraction and cell rounding, whereas Rac and Cdc42 are implicated in the promotion of filopodia and lamellipodia formation in growth cones and, therefore, in neurite extension. In this study, we examined the morphological role of Rnd1, a new member of Rho family GTPases, in PC12 cells, and found that expression of Rnd1 by itself caused the formation of many neuritic processes from the cell body with disruption of the cortical actin filaments, the processes having microtubules but few filamentous actin and neurofilaments. Treatment with cytochalasin D, an inhibitor of actin polymerization, could mimic the effects of expression of Rnd1, in that this inhibitor disrupted the cortical actin filaments and induced the formation of many thin processes containing microtubules. The process formation induced by Rnd1 was inhibited by dominant negative Rac1. These results suggest that Rnd1 induces the Rac-dependent neuritic process formation in part by disruption of the cortical actin filaments.

Animals↗
PubMed2000

Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I.

Human beta1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis. GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Gal beta 1-3Gal beta 1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. We have now determined the crystal structure of GlcAT-1 at 2.3 A in the presence of the donor substrate product UDP, the catalytic Mn(2+) ion, and the acceptor substrate analog Gal beta 1-3Gal beta 1-4Xyl. The enzyme is a alpha/beta protein with two subdomains that constitute the donor and acceptor substrate binding site. The active site residues lie in a cleft extending across both subdomains in which the trisaccharide molecule is oriented perpendicular to the UDP. Residues Glu(227), Asp(252), and Glu(281) dictate the binding orientation of the terminal Gal-2 moiety. Residue Glu(281) is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor. The conserved DXD motif (Asp(194), Asp(195), Asp(196)) has direct interaction with the ribose of the UDP molecule as well as with the Mn(2+) ion. The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.

Amino Acid Sequence↗