Biomedical subjects

K Uchida

Publications and source records attributed to K Uchida.

At least 199 records · Page 11Linked to original sources

PubMed2000

Poly(ADP-ribose) polymerase localizes to the centrosomes and chromosomes.

Poly(ADP-ribose) polymerase (PARP) takes part mainly in regulation of DNA repair, thereby maintaining genomic stability in the nucleus. However, what role PARP plays in mitotic cells is not known. Centrosomes play an important role in maintaining the fidelity of chromosome distribution during cell division. Loss of these functions might cause chromosomal instability and aneuploidy. p53 and BRCA1 were recently found to localize to the centrosome at mitosis. We found that PARP is localized to the centrosomes and the chromosomes at cell-division phase and interphase by indirect immunofluorescence. Furthermore, by analysis of isolated centrosomes PARP protein was found to associate with the centrosomes during mitosis. These data suggest that PARP may be involved in maintenance of chromosomal stability.

Cell Line↗

PubMed2000

A staining procedure for micronucleus test using new methylene blue and acridine orange: specimens that are supravitally stained with possible long-term storage.

The micronucleus test has been widely used as an in vivo cytogenetic test. It employs two different kinds of supravital staining methods which use either new methylene blue (N) and Giemsa (G) or acridine orange (AO). We have developed a new staining procedure for the preparation of specimens supravitally stained with possible long-term storage, using both N and AO. This N/AO-staining method involves three steps; (1) combination of the target tissue or target cells with an equivalent volume of 0.5% solution of new methylene blue (N-staining step), (2) immediate smear of the mixture, followed by treatment with methanol for 10 min for fixation and removal of N and drying (referred to as fixed-decolorized specimens), and (3) staining with 0.007% solution of AO for 3 min, followed by washing with Sörensen's buffer (pH 6.8) and covering of specimens before observation (AO-staining step). To examine whether the N/AO-staining method is useful for the micronucleus test, comparisons were made between N-, N/AO-, and AO-stained specimens prepared supravitally from peripheral blood of rats with and without treatment of cyclophosphamide. The results indicate that N/AO-stained specimens can be supravitally observed after long-term storage with the same coloration and comparable frequencies of micronucleated reticulocytes with a positive response as AO-stained specimens, if the staining process is temporarily stopped before AO-staining (as fixed-decolorized specimens), or if the AO-staining step is repeated. The results also showed that separated reticulocyte types are supravitally stained in a similar fashion to N-stained specimens but not to AO-stained specimens, indicative of the preservation of the supravital feature of N-staining. Taken together these results suggest that the N/AO-staining procedure could offer an additional useful staining tool for the micronucleus test.

Acridine Orange↗

PubMed2000

Cloning, expression analysis, and chromosomal mapping of GTPBP2, a novel member of the G protein family.

We have identified a novel gene encoding a protein bearing GTP-binding motifs, the characteristics of GTP-binding proteins (G proteins). The deduced amino acid sequence exhibited the highest overall homology with GTPBP1 and its mouse orthologue GP-1. Hence, we named the gene GTPBP2. The mouse orthologue of this gene, Gtpbp2, showed 98% identity with GTPBP2 over the entire protein (the HGMW-approved nomenclature symbol is GTPBP2 and mouse orthologue is Gtpbp2). A phylogenetic analysis showed GTPBP2 and homologous G proteins (GTPBP1, AGP-1, and CGP-1) did not belong to major G protein families. They formed a distinct branch in the phylogenetic tree, suggesting that they constitute a novel G protein family. A 2. 9kb mRNA was predominantly detected in the testis along with various other organs. In situ hybridization analysis revealed that Gtpbp2 was predominantly expressed in spermatocytes and round-spermatids in the testis. These novel genes were localized to human chromosome 6p21.1-2 and mouse chromosome 17qC-D.

Amino Acid Sequence↗

PubMed2000

A glutathione S-transferase inducer from papaya: rapid screening, identification and structure-activity relationship of isothiocyanates.

We have developed a simple system for rapid detection and measurement of glutathione S-transferase placental form (GSTP1) that detoxify polycyclic aromatic hydrocarbons using the cultured rat normal liver epithelial cell line, (RL34) cells. Survey of fruit extracts for GST inducing ability identified both papaya and avocado as significant sources. Benzyl isothiocyanate (BITC) was isolated from papaya methanol extract as a principal inducer of GST activity. Further, the GST inducing ability of a total of 20 isothiocyanates (ITCs) and their derivatives was investigated. Some ITCs showed significant induction, and BITC was one of the most potent inducers among all compounds tested in the present study. The modification of isothiocyanate group (-NCS) or introduction of substituent group to the alpha-carbon modifies GST induction. Moreover, a significant correlation (P<0.01, r=0.913) between the GST activity enrichment and GSTP1 protein induction by ITCs was observed. We also indicated that phenethyl ITC and nitrophenyl ITC, potently inducing GST activity, but not inactive benzyl isocyanate, are potential inducers of intracellular reactive oxygen intermediates (ROIs). Our system of GSTP1 induction is appropriate for the chemical research such as screening and identification of novel type of inducers as well as the structure-activity relationship studies, providing mechanistic insight into essential structural elements for GSTP1 induction.

Animals↗

PubMed2000

Antiplatelet and anticancer isothiocyanates in Japanese domestic horseradish, Wasabi.

6-Methylsulfinylhexyl isothiocyanate (MS-ITC) was isolated from wasabi (Wasabia japonica, Japanese domestic horseradish) as a potential inhibitor of human platelet aggregation in vitro through our extensive screening of vegetables and fruits. In the course of an another screening for the induction of glutathione S-transferase (GST) activity in RL34 cells, MS-ITC was inadvertently isolated from wasabi as a potential inducer of GST. MS-ITC administered to rats or mice also showed both activities in vivo. As a result from elucidation of the platelet aggregation inhibition and the GST induction mechanisms of MS-ITC, the isothiocyanate moiety of MS-ITC plays an important role for antiplatelet and anticancer activities because of its high reactivity with sulfhydryl (RSH) groups in biomolecules (GSH, cysteine residue in a certain protein, etc.).

Animals↗

PubMed2000

Induction of glutathione S-transferase by prostaglandins.

Animals↗

PubMed2000

Acute hemodynamic effects of insulin-sensitizing agents in isolated perfused rat hearts.

Troglitazone has direct effects on the hemodynamics of the heart. We investigated the effects of other insulin-sensitizing agents (rosiglitazone, pioglitazone and JTT-501 (4-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]benzyl]-3, 5-isoxazolidinedione)) on the hemodynamics of the heart using isolated perfused rat hearts. Rosiglitazone significantly decreased heart rate and coronary perfusion pressure, and increased peak isovolumic left ventricular pressure, peak rate of rise of left ventricular pressure and peak rate of fall of left ventricular pressure. The effects of rosiglitazone, however, were milder than those of troglitazone. Neither pioglitazone nor JTT-501 had any effect on the heart. D-alpha-tocopherol, a structural component of troglitazone, did not exert any effect on the heart. The coronary vasorelaxant effect of troglitazone and rosiglitazone was significantly suppressed by indomethacin, but not by N(omega)-nitro-L-arginine methyl ester. In conclusion, only rosiglitazone, as well as troglitazone, exerted positive inotropic, positive lusitropic, negative chronotropic, and coronary vasorelaxant effects on the heart. The coronary vasorelaxant effect of troglitazone and rosiglitazone was mediated by prostaglandin production.

Animals↗

PubMed2000

[Treatment of renovascular hypertension using stent implantation in an elderly patient with NIDDM] [In Process Citation]

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Journal Article↗

PubMed2000

4-hydroxy-2-nonenal, the end product of lipid peroxidation, is a specific inducer of cyclooxygenase-2 gene expression.

Cyclooxygenase-2 (COX-2), an enzyme responsible for catalyzing the committed step in prostanoid biosynthesis, is the product of an immediate early gene capable of being upregulated by diverse stimuli. Based on the experimental evidence that oxidative stress is associated with the upregulation of COX-2, we evaluated the effect of the oxidized fatty acid metabolites on COX-2 induction in rat liver epithelial RL34 cells. Among the compounds tested, only 4-hydroxy-2-nonenal (HNE), a highly mutagenic and genotoxic aldehyde generated during oxidative stress, dramatically induced COX-2. Enhanced gene expression of COX-2 by treatment with HNE was evident as a drastic elevation of the mRNA level. We also found that intracellular glutathione status was strictly related to HNE-induced COX-2 expression. These findings suggest the presence of a signaling pathway in the cellular response mediated by locally produced lipid peroxidation products under oxidative stress.

Aldehydes↗

PubMed2000

Two different types of nonthrombogenic surfaces: PEG suppresses platelet adhesion ATP-independently but HEMA-St block copolymer requires ATP consumption of platelets to prevent adhesion.

Poly(ethylene glycol) (PEG) and a hydrophobic-hydrophilic microdomain structured block copolymer comprising poly(2-hydroxyethyl methacrylate) and polystyrene (HEMA-St) have been reported to show good blood compatibility owing to inhibition of platelet activation. By using a computer-assisted novel technique to analyze platelet behavior on the surfaces, we found two different mechanisms to prevent platelet adhesion. Platelets were prevented from adhesion and spreading on the microdomain surface and retained cell movement for a long time. The platelet movement velocity was not significantly different between PEG-grafted surfaces and HEMA-St block copolymer-cast surfaces. However, platelet motion was qualitatively different. Platelets on HEMA-St block copolymer-cast surfaces moved with rolling, spinning, and vibrating, whereas platelet movement was limited to oscillatory vibration on PEG-grafted surfaces. When platelets were treated with NaN(3), an adenosine triphosphate (ATP) synthesis inhibitor, before contacting the surfaces, platelets movement velocity was decreased only on HEMA-St block copolymer-cast surfaces. Such an inhibitory effect was hardly observed with platelets on PEG-grafted surfaces. We propose two different mechanisms to prevent platelet adhesion onto surfaces. One is ATP-independent as observed with PEG, and the other is ATP-dependent for HEMA-St block copolymer, where platelets consume ATP to prevent adhesion.

Adenosine Triphosphate↗

PubMed2000

Role of reactive aldehyde in cardiovascular diseases.

There is increasing evidence that aldehydes generated endogenously during the degradation process of biological molecules are involved in many of the pathophysiologies associated with cardiovasular diseases such as atherosclerosis and the long-term complications of diabetes. Major sources of reactive aldehydes in vivo are lipid peroxidation, glycation, and amino acid oxidation. Although the types of aldehydes are varied, the important aldehydes that can exert biological effects relevant to the pathobiology of oxidant injury are represented by 2-alkenals, 4-hydroxy-2-alkenals, and ketoaldehydes. These aldehydes exhibit facile reactivity with proteins, generating stable products at the end of a series of reactions. The protein-bound aldehydes can be detected as constituents not only in in vitro oxidized low-density lipoproteins but also in animal models of atherosclerosis and in human patients with increased risk factors or clinical manifestations of atherosclerosis, indicating that they could indeed be involved in the caldiovascular pathology. On the other hand, a number of reactive aldehydes have been implicated as inducers in generating intracellular oxidative stress and activation of stress signaling pathways, that integrate with other signaling pathways to control cellular responses to the extracellular stimuli.

Aldehydes↗

PubMed2000

Detection of lipofuscin-like fluorophore in oxidized human low-density lipoprotein. 4-hydroxy-2-nonenal as a potential source of fluorescent chromophore.

It has recently been shown that the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) forms a fluorescent hydroxyiminodihydropyrrole derivative with the epsilon-amino group of lysine residue. In this study, we raised a monoclonal antibody (mAb2C12) directed to the fluorophore-protein conjugate and found that the antibody was specific to the chromophore structure of the compound. Immunohistochemical analysis of atherosclerotic lesions from the human aorta showed that the fluorophore was indeed present in the lesions, in which intense positivity was primarily associated with macrophage-derived foam cells and thickening of the neointima of the arterial walls. Antigenic materials were also detected in the oxidatively modified low-density lipoprotein (LDL) with Cu(2+) and in the oxidatively modified bovine serum albumin with an iron/linoleic acid autoxidation system, indicating that the HNE, which originated from the peroxidation of polyunsaturated fatty acids, could be a potential source of the fluorescent chromophore in oxidized LDL.

Aged↗

PubMed2000

Heterogeneities in the biological and biochemical functions of Smad2 and Smad4 mutants naturally occurring in human lung cancers.

Smad family members are essential intracellular signaling components of the transforming growth factor-beta (TGF-beta) superfamily involved in a range of biological activities. The loss of sensitivity to TGF-beta is frequent in human lung cancers and inactivation of Smad family members are thought to play important roles in disruption of TGF-beta signaling. In the study presented here, we characterized the biological and biochemical functions of six Smad2 and Smad4 mutants, which we previously identified in human lung cancers. All mutant Smad2 and Smad4 were in fact found to be defective in transmitting growth inhibitory signals originating from TGF-beta and incapable of activating Smad/hFAST-1-mediated transcription. Transcriptional activation of plasminogen activator inhibitor type 1 (PAI-1) was impaired in four of the six mutants due to the defects in homo- and/or hetero-oligomerization with wild-type Smads. In contrast, the remaining two Smad mutants showed a modest reduction in the PAI-1 transcriptional activation and apparently retained the ability to oligomerize with wild-type Smads. Significant loss of growth inhibition and Smad/hFAST-1-mediated transcriptional activation by all of the six mutants suggested that Smad mutants are indeed functionally impaired Smad mutations and may play a role in lung tumorigenesis. Moreover, the present findings suggest that in addition to the impairment in the homo- and/or hetero-oligomerization, there may be an alternative mechanism producing disruption of TGF-beta signaling, involving hFAST-1-or possibly other transcriptional cofactor(s)-mediated transcriptional activation.

DNA-Binding Proteins↗

PubMed2000

Oxidative injury is present in Purkinje cells in patients with olivopontocerebellar atrophy.

To verify the presence of lipid peroxidation products in spinocerebellar degeneration (SCD), the cerebella from eight patients with olivopontocerebellar atrophy (OPCA) and six non-OPCA patients were immunohistochemically investigated with 4-hydroxy-2-nonenal (HNE) antibody. On average, 84.6% of Purkinje cells were positively or strongly positively immunostained in OPCA patients while only 15.5% were positive in non-OPCA patients. Other cells in the molecular and granular layers showed no obvious immunoreactivity. These data suggest that a lipid peroxidation product is present in Purkinje cells of OPCA patients and that oxidative stress may play an important role in the degeneration process of SCD.

Aged↗

PubMed2000

Cyclopentenone prostaglandins as potential inducers of phase II detoxification enzymes. 15-deoxy-delta(12,14)-prostaglandin j2-induced expression of glutathione S-transferases.

Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes, such as glutathione S-transferases (GSTs). In the present study, we developed a cell culture system that potently responds to phenolic antioxidants and found that antitumor prostaglandins (PGs) are potential inducers of GSTs. We screened primary hepatocytes and multiple cell lines for inducing GST activity upon incubation with the phenolic antioxidant (tert-butylhydroquinone) and found that rat liver epithelial RL34 cells most potently responded. Based on an extensive screening of diverse chemical agents on the induction of GST activity in RL34 cells, the J2 series of PGs, 15-deoxy-Delta(12,14)-prostaglandin J2 (15-deoxy-Delta(12,14)-PGJ2) in particular, were found to be potential inducers of GST. Enhanced gene expression of Class pi GST isozyme (GSTP1) by 15-deoxy-Delta(12,14)-PGJ2 was evident as a drastic elevation of the mRNA level. Hence, we examined the molecular mechanism underlying the 15-deoxy-Delta(12, 14)-PGJ2-induced GSTP1 gene expression. From functional analysis of various deletion mutant genes, we found that the 15-deoxy-Delta(12, 14)-PGJ2 reponse element was localized in a region containing a GSTP1 enhancer I (GPEI) that consists of two imperfect phorbol 12-O-tetradecanoylphorbol-13-acetate response elements. When the GPEI was combined with the minimum GSTP1 promoter, the element indeed showed an enhancer activity in response to 15-deoxy-Delta(12, 14)-PGJ2. Point mutations of either of the two imperfect 12-O-tetradecanoylphorbol-13-acetate response elements in GPEI completely abolished the enhancer activity. Gel mobility shift assays demonstrated that 15-deoxy-Delta(12,14)-PGJ2 specifically stimulated the binding of nuclear proteins including the transcription factor c-Jun, but not Nrf2, to GPEI. These results suggest that 15-deoxy-Delta(12,14)-PGJ2 induces the expression of the rat GSTP1 gene through binding of proteins, including c-Jun, to a specific GPEI.

Animals↗

PubMed2000

The hepatic vagal reception of intraportal GLP-1 is via receptor different from the pancreatic GLP-1 receptor.

Glucagon-like peptide-1 (7-36)amide (tGLP-1), a representative humoral incretin, released into the portal circulation in response to a meal ingestion, exerts insulinotropic action through binding to the tGLP-1 receptor known to be a single molecular form thus far. We previously reported that the hepatic vagal nerve is receptive to intraportal tGLP-1, but not to non-insulinotropic full-length GLP-1-(1-37), through a mechanism mediated by specific receptor to the hormone. In the present study, we aimed to examine how modification of the receptor function alters this neural reception of tGLP-1, by using the specific agonist, exendin-4, and the specific antagonist, exendin (9-39)amide, of the receptor at doses known to exert their effects on the insulinotropic action of tGLP-1. Intraportal injection of 0.2 or 4.0 pmol tGLP-1, a periphysiological and pharmacological dose, respectively, facilitated significantly the afferent impulse discharge rate of the hepatic vagus in anesthetized rats, as reported previously. However, unexpectedly, intraportal injection of exendin-4 at a dose of 0.2 or 4.0 pmol, or of even 40.0 pmol, did not facilitate the afferents at all. Moreover, intraportal injection of exendin (9-39)amide at 100 times or more molar dose to that of tGLP-1, either 5 min before or 10 min after injection of 0.2 or 4.0 pmol tGLP-1, failed to modify the tGLP-1-induced facilitation of the afferents. The present results suggest that the neural reception of tGLP-1 involves a receptor mechanism distinct from that in the well-known humoral insulinotropic action.

Animals↗

PubMed2000

Redox regulation of glutathione S-transferase induction by benzyl isothiocyanate: correlation of enzyme induction with the formation of reactive oxygen intermediates.

Here we report the molecular mechanism underlying the induction of glutathione S-transferase (GST) in rat liver epithelial RL34 cells treated with a cancer chemopreventive isothiocyanate compound, benzylisothiocyanate (BITC). BITC was found to significantly induce GST activity in RL34 cells. Northern and Western blot analyses demonstrated that BITC specifically enhanced the production of the class pi GST isozyme (GSTP1). Our studies demonstrated for the first time that the addition of BITC to the cells resulted in an immediate increase in the reactive oxygen intermediates (ROIs) detected by a fluorescence probe, 2',7'-dichlorofluorescin diacetate. The level of the ROIs in the cells treated with BITC (10 microM) was approximately 50-fold higher than those in the control cells. Furthermore, glutathione depletion by diethyl maleate significantly enhanced BITC-induced ROI production and accelerated the BITC-induced elevation of the GST activity, whereas pretreatment of the cells with glutathione inhibited both the ROI production and GST induction. The structure-activity relationship of the isothiocyanates also indicated that the ROI-producing activities closely correlated with their GST-inducing potencies. Moreover, the GSTP1 enhancer I-containing region was found to be essential for induction of the GSTP1 gene by intracellular ROI inducers such as BITC and diethyl maleate. These data suggest the involvement of the redox regulation on the induction of GSTP1 by BITC.

Animals↗

PubMed2000

Trans-4-hydroxy-2-nonenal, an aldehydic lipid peroxidation product, lacks genotoxicity in lacI transgenic mice.

In order to cast light on the significance of lipid peroxidation products for carcinogenesis, the lacI mutant frequency (MF), micronucleus induction and cell proliferation were analyzed in lacI transgenic mice treated with trans-4-hydroxy-2-nonenal (HNE), a typical example. Male mice were ip injected with HNE at doses of 0, 5 or 50 mg/kg bw and 48 h thereafter, peripheral blood was collected for analyzing micronucleus induction. After 14 days, the mice were sacrificed to allow tissue sampling for examination of lacI MF and cell proliferative activity. Sixty percent of the mice given 50 mg/kg HNE died within 5 days after the treatment, but no other mortalities were observed. Histopathologically, marked pulmonary hemorrhage was found in the 50 mg/kg HNE group mice that survived until day 14. Immunohistochemically, HNE-modified proteins were detected in their alveolar macrophages. The HNE treatment did not increase lacI MF in the liver, kidney and lung and no significant increase in micronucleus induction or cell proliferation in major organs was found in either treatment. Moreover, no tumors developed in the 5 mg/kg HNE-treated mice which survived until week 78. Our results thus indicate that HNE lacks in vivo genotoxicity in lacI transgenic mice even when lethal doses are applied.

Aldehydes↗