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Biomedical subjects

N Taniguchi

Publications and source records attributed to N Taniguchi.

At least 289 records · Page 16Linked to original sources
PubMed1995

Alterations of superoxide dismutase iso-enzyme activity, content, and mRNA expression with aging in rat skeletal muscle.

The alterations of superoxide dismutase iso-enzyme (Cu,Zn-SOD and Mn-SOD) activities, contents, and mRNA expressions with aging were studied in rat soleus muscle (SO) and extensor digitorum longus muscle (EDL). The activity and content of Cu,Zn-SOD in both muscles were significantly higher in old rats (24 months old) than in young rats (4 months old), whereas those of Mn-SOD showed no difference between young and old rats. After normalization to citrate synthase (CS) activity, however Mn-SOD/CS ratio in SO also showed the age-related increase. Moreover, the activities of other major antioxidant enzymes, glutathione peroxidase (GPX) and catalase (CAT), indicated age-related increases only in SO. As for the expressions of mRNAs for SOD iso-enzymes, that of Cu,Zn-SOD in either muscle showed no significant change with aging, unlike its activity and content, although that of Mn-SOD was decreased with aging only in EDL. Thus, aging appeared to raise the level of antioxidant enzyme system in rat skeletal muscle. However, the resistance of Cu,Zn-SOD and Mn-SOD to oxidative stress accompanied by aging was different, the former being obviously greater than the latter. Such changes also differed in muscle fiber type suggesting that fast-twitch fibers are more susceptible to age-related oxidative stress than slow-twitch fibers.

Aging↗
PubMed1995

Involvement of Ser-451 and Ser-452 in the catalysis of human gamma-glutamyl transpeptidase.

The serine residue required for catalysis of gamma-glutamyl transpeptidase was identified by site-specific mutagenesis of the conserved serine residues on the basis of sequence alignment of the light subunit of human, rat, pig and two bacterial enzymes. Recombinant human gamma-glutamyl transpeptidases with replacements of these serine residues by Ala were expressed using a baculovirus-insect cell system. Substitutions of Ala at Ser-385, -413 or -425 yielded almost fully active enzymes. However, substitutions of Ala at Ser-451 or -452 yielded enzymes that were only about 1% as active as the wild-type enzyme. Further, their double mutant is only 0.002% as active as the wild type. Kinetic analysis of transpeptidation using glycylglycine as acceptor indicates that the Vmax values of Ser-451 and -452 mutants are substantially decreased (to about 3% of the wild type); however, their Km values for L-gamma-glutamyl-p-nitroanilide as donor were only increased about 5 fold compared to that of the wild type. The double mutation of Ser-451 and -452 further decreased the Vmax value to only about 0.005% of the wild type, while this mutation produced only a minor effect (2-fold increase) on the Km value for the donor. The kinetic values for the hydrolysis reaction of L-gamma-glutamyl-p-nitroanilide in the mutants followed similar trends to those for transpeptidation. The rates of inactivation of Ser-451, -452 and their double mutant enzymes by acivicin, a potent inhibitor, were less than 1% that of the wild-type enzyme. The Ki value of the double mutant for L-serine as a competitive inhibitor of the gamma-glutamyl group is only 9 fold increased over that of the wild type, whereas the Ki for the serine-borate complex, which acts as an inhibitory transition-state analog, was more than 1,000 times higher than for the wild-type enzyme. These results suggest that both Ser-451 and -452 are located at the position able to interact with the gamma-glutamyl group and participate in catalysis, probably as nucleophiles or through stabilization of the transition state.

Amino Acid Sequence↗
PubMed1995

Elevation of aldose reductase gene expression in rat primary hepatoma and hepatoma cell lines: implication in detoxification of cytotoxic aldehydes.

Aldose reductase and aldehyde reductase are members of the aldo-keto reductase superfamily, and participate in the reduction of a wide range of carbonyl compounds. We have purified aldose reductase from rat lens and raised antiserum against it in rabbits. Immunoblot analyses using this antibody showed that a significant amount of aldose reductase was expressed in cell lines derived from hepatomas while it was negligible in normal hepatocytes. Elevated expression of aldose reductase was also observed in cancerous lesions of 3'-methyl-4-dimethyl-aminoazobenzene (3'-Me-DAB)-induced hepatocarcinomas. Expression of aldose reductase mRNA was confirmed in these cells by Northern-blot analysis, suggesting that the induction occurred at the stage of gene transcription. The level of aldehyde reductase, however, did not change in cancerous tissue or in the cell lines. The viability of hepatoma cells in the presence of 3-deoxyglucosone and glyceraldehyde was decreased by an aldose reductase inhibitor, ONO-2235 (5-[1Z,2E)-2-methyl-3-phenylpropenylidene]-4-oxo-2-thioxo -3- thiazolidineacetic acid). Taken together, induction of aldose reductase gene expression during hepatocarcinogenesis may render cancer cells resistant to various toxic carbonyl compounds produced during metabolism or administered as anti-cancer drugs.

Aldehyde Reductase↗
PubMed1995

Suppression of lung metastasis of B16 mouse melanoma by N-acetylglucosaminyltransferase III gene transfection.

The beta 1-6 structure of N-linked oligosaccharides, formed by beta-1,6-N-acetylglucosaminyltransferase (GnT-V), is associated with metastatic potential. We established a highly metastatic subclone, B16-hm, from low metastatic B16-F1 murine melanoma cells. The gene encoding beta-1,4-N-acetylglucosaminyltransferase (GnT-III) was introduced into the B16-hm cells, and three clones that stably expressed high GnT-III activity were obtained. In these transfectants, the affinity to leukoagglutinating phytohemagglutinin was reduced, whereas the binding to erythroagglutinating phytohemagglutinin was increased, indicating that the level of beta 1-6 structure was decreased due to competition for substrate between intrinsic GnT-V and ectopically expressed GnT-III. Lung metastasis after intravenous injection of the transfectants into syngeneic and nude mice was significantly suppressed, suggesting that the decrease in beta 1-6 structure suppressed metastasis via a mechanism independent of the murine system. These transfectants also displayed decreased invasiveness into Matrigel and inhibited cell attachment to collagen and laminin. Cell growth was not affected. Our results demonstrate a causative role for beta 1-6 branches in invasion and cell attachment in the extravasation stage of metastasis.

Agglutination↗
PubMed1995

Inactivation of glutathione peroxidase by nitric oxide. Implication for cytotoxicity.

S-nitro-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, inactivated bovine glutathione peroxidase (GPx) in a dose- and time-dependent manner. The IC50 of SNAP for GPx was 2 microM at 1 h of incubation and was 20% of the IC50 for another thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase, in which a specific cysteine residue is known to be nitrosylated. Incubation of the inactivated GPx with 5 mM dithiothreitol within 1 h restored about 50% of activity of the start of the SNAP incubation. A longer exposure to NO donors, however, irreversibly inactivated the enzyme. The similarity of the inactivation with SNAP and reactivation with dithiothreitol of GPx to that of glyceraldehyde-3-phosphate dehydrogenase, suggested that NO released from SNAP modified a cysteine-like essential residue on GPx. When U937 cells were incubated with 100 microM SNAP for 1 h, a significant decrease in GPx activity was observed although the change was less dramatic than that with the purified enzyme, and intracellular peroxide levels increased as judged by flow cytometric analysis using a peroxide-sensitive dye. Other major antioxidative enzymes, copper/zinc superoxide dismutase, manganese superoxide dismutase, and catalase, were not affected by SNAP, which suggested that the increased accumulation of peroxides in SNAP-treated cells was due to inhibition of GPx activity by NO. Moreover, stimulation with lipopolysaccharide significantly decreased intracellular GPx activity in RAW 264.7 cells, and this effect was blocked by NO synthase inhibitor N omega-methyl-L-arginine. This indicated that GPx was also inactivated by endogenous NO. This mechanism may at least in part explain the cytotoxic effects of NO on cells and NO-induced apoptotic cell death.

Animals↗
PubMed1995

Human gamma-glutamyl transpeptidase mutants involving conserved aspartate residues and the unique cysteine residue of the light subunit.

Mutant human gamma-glutamyl transpeptidases with amino acid substitutions on the light subunit at the Asp residues conserved among several species, and at the unique cysteine residue (Cys-454), were prepared and expressed in a baculovirus insect cell system. Replacement of Asp-423 by Ala or Glu led to major loss of enzyme activity, consistent with the conclusion that Asp-423 is essential for activity. A mutant in which Cys-454 was replaced by Ala was fully active, indicating that the unique light subunit thiol is not required for catalysis. Kinetic analysis of the hydrolysis reaction of L-gamma-glutamyl-p-nitroanilide indicated that the decreased activity of Asp-423 mutants is the consequence of an extremely high substrate Km value, which is more than a 1000-fold greater than that for the wild-type enzyme, whereas the Vmax is decreased only less than 90-fold. The results suggest that Asp-423, and to a lesser extent Asp-422, interact electrostatically with the alpha-amino group of the gamma-glutamyl donor substrate. Although further studies are required to evaluate the possibility that the reaction involves function of a charge (or proton) relay system, the present work suggests that the gamma-glutamyl moiety of the substrate binds electrostatically to specific groups on the enzyme; this facilitates gamma-glutamyl enzyme formation.

Aspartic Acid↗
PubMed1995

Transforming growth factor beta up-regulates expression of the N-acetylglucosaminyltransferase V gene in mouse melanoma cells.

beta-1,6-N-acetylglucosaminyltransferase V (GnT-V) (EC 2.4.1.155) that catalyzes beta-1,6 branching in asparagine-linked oligosaccharides is activated on viral or oncogenic transformation and is associated with tumor metastasis. To study the molecular mechanisms involved in regulation of expression of the GnT-V gene, we cloned cDNA and genomic DNA for the enzyme (Saito, H., Nishikawa, A., Gu, J., Ihara, Y., Soejima, Y., Sekiya, C., Niikawa, N., and Taniguchi, N. (1994) Biochem. Biophys. Res. Commun. 198, 318-327). We found that transforming growth factor beta (TGF beta) specifically induced GnT-V expression in mouse melanoma cells. The activity of GnT-V was increased 24 h after the addition of TGF beta and remained at high levels up to 72 h. Northern blot analysis showed that the mRNA levels of GnT-V were consistent with the increased activity. To further investigate the nature of the induction, mRNA stability and transcriptional activity were assayed. The enhancement of the GnT-V mRNA expression resulted from prolonged mRNA stability, not from increased transcription. Consequently, elevated mRNA levels were observed even 72 h after the addition of TGF beta. Lectin blot analysis involving leukoagglutinin showed newly synthesized beta-1,6 branching structures in the sugar chains of a protein of approximately 130 kDa at 48 h after TGF beta treatment. These results suggested that TGF beta caused changes in the sugar chains of proteins in melanoma cells by up-regulating GnT-V expression.

Animals↗
PubMed1995

Different sites of acivicin binding and inactivation of gamma-glutamyl transpeptidases.

Acivicin is a potent inhibitor of gamma-glutamyl transpeptidase (EC 2.3.2.2), an enzyme of importance in glutathione metabolism. Acivicin inhibition and binding are prevented by gamma-glutamyl substrates and analogs (e.g., serine plus borate), consistent with the previous postulate that acivicin and substrates bind to the same enzyme site. Inactivation of rat kidney transpeptidase by acivicin leads to its binding as an ester to Thr-523. The pig enzyme, which has Ala-523 in place of Thr-523, is inhibited by acivicin with esterification at Ser-405. The human enzyme has Thr-524 (corresponding to Thr-523 in rat); its inactivation leads to esterification of Ser-406 (corresponding to Ser-405 in rat and pig). Hydroxylamine treatment of the acivicin-inactivated enzymes restores activity and releases the acivicin-derived threo-beta-hydroxyglutamate moiety. The findings indicate that there are significant structural differences between the active site region of the rat enzyme and the active site regions of the human and pig. Human mutant enzymes in which Thr-524 and Ser-406 were replaced by Ala, separately and together, are enzymatically active, indicating that these amino acid residues are not required for catalysis. However, esterification of these residues (and of another near the active site) effectively blocks the active site or hinders its function. Acivicin can bind at enzyme sites that are close to that at which gamma-glutamylation occurs; it may bind at the latter site and then be transesterified to another enzyme site.

Alanine↗
PubMed1995

Heme requirement for production of active endothelial nitric oxide synthase in baculovirus-infected insect cells.

We have cloned cDNAs encoding endothelial nitric oxide synthase (ecNOS) from a human fetal liver cDNA library. Overproduction of ecNOS in a baculovirus/insect cell expression system in conventional medium yielded a large amount of ecNOS protein localized in particulate components, but ecNOS activity was low. This activity was increased by addition of hemin to 2.5-fold. While a precursor for heme biosynthesis increased the activity, inhibitors of heme biosynthesis reduced the ecNOS activity to 50% without affecting the level of enzyme. After extraction of cells with 1% Triton X-100, ecNOS protein was purified by column chromatography. The resultant ecNOS required supplementation with cofactors for activity, but it did not require hemin. Binding of a protoporphyrin IX heme was confirmed by a pyridine hemochrome assay.

Amino Acid Oxidoreductases↗
PubMed1995

High expression of UDP-N-acetylglucosamine: beta-D mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) in chronic myelogenous leukemia in blast crisis.

The activity and mRNA expression of UDP-N-acetylglucosamine: beta-D mannoside beta-1,4-N-acetylglucosaminyl transferase III (GnT-III: EC 2.4.1.144) were investigated in hematological malignancies. GnT-III activity was elevated in patients with chronic myelogenous leukemia (CML) in blast crisis and patients with multiple myeloma (MM), as compared to normal healthy subjects and patients with other hematological malignancies including CML in chronic phase. The GnT-III transcript was the same size in leukemic cells from various hematological diseases and cell lines, while expression of the transcript was not found to correlate significantly with enzyme activity, implying that post-translational modification might regulate the activity of GnT-III. Southern-blot analysis showed no significant variation in the structure and position of the GnT-III genome, indicating that the gene is present as a single copy without isoforms. Furthermore, analyses by immunoprecipitation and Western blot revealed that high GnT-III activity in KU812 cell, a CML cell line, resulted in an increase in E4-PHA binding to CD45, a major surface glycoprotein of the leukocyte, indicating that more bisecting GlcNAc was added to CD45 catalyzed by elevated GnT-III.

Acetylglucosamine↗
PubMed1995

Cell spreading in Colo 201 by staurosporin is alpha 3 beta 1 integrin-mediated with tyrosine phosphorylation of Src and tensin.

Staurosporin, a broad-spectrum kinase inhibitor, induced cell spreading in a human colon cancer cell line, Colo 201. On collagen and laminin, cell spreading was induced in more than 90% of the cells and was dependent on very late activation antigen-3, as shown by an antibody inhibition assay. Cell spreading required divalent cations and showed the order of preference Mn2+ > Mg2+ > Ca2+. On fibronectin, only about 30% of the cells were observed to spread, and spreading occurred via a non-integrin, RGD-independent pathway. Staurosporin-induced spreading was inhibited by treatment with tyrosine kinase inhibitors herbimycin A and methyl 2,5-dihydroxycinnamate. Despite the presence of staurosporin, seven proteins (220, 175, 150, 98, 62, 58, and 45 kDa) showed increased levels of tyrosine phosphorylation in association with cell adhesion. Two of these (58 and 220 kDa) were identified by immunoprecipitation as Src product and tensin, respectively. Flow cytometric analysis showed that the Colo 201 cells expressed the alpha 2, alpha 3, alpha 6, and beta 1 chains of integrin, but expression of these chains was not influenced by staurosporin. Immunofluorescence microscopy revealed that the alpha 3 chain, diffusely expressed on the cell surface in the absence of staurosporin, was concentrated at focal adhesion plaques after staurosporin treatment. Neither alpha 2 nor alpha 6 was focalized by the treatment.

Alkaloids↗
PubMed1995

In vivo glycation of aldehyde reductase, a major 3-deoxyglucosone reducing enzyme: identification of glycation sites.

We have reported that the enzyme which reduces 3-deoxyglucosone (3-DG), a major intermediate and a potent cross-linker in the Maillard reaction, is identical with aldehyde reductase [Takahashi, M., Fujii, J., Teshima, T., Suzuki, K., Shiba, T., & Taniguchi, N. (1993) Gene 127, 249-253]. The enzyme purified from normal rat liver was found to be partially glycated as judged by binding to a boronate column and reactivity to anti-epsilon-hexitol lysine IgG. Sites of in vivo glycation of rat liver aldehyde reductase were identified by sequencing of digested peptides labeled with NaB[3H]4 and by mass spectrometry. The major glycated sites were lysines 67, 84, and 140. The glycated enzyme had low catalytic efficiency (kcat/Km) as compared to the nonglycated form. In streptozotocin-induced diabetic rats, the glycated form was significantly increased in kidneys. Because the enzyme plays a role in detoxifying 3-DG formed through the Maillard reaction in vivo, glycation of aldehyde reductase and reduction of its activity may result in the metabolic imbalance under diabetic conditions.

Aldehyde Reductase↗
PubMed1995

Diphtheria toxin binds to the epidermal growth factor (EGF)-like domain of human heparin-binding EGF-like growth factor/diphtheria toxin receptor and inhibits specifically its mitogenic activity.

The membrane anchored form of human heparin-binding epidermal growth factor-like growth factor (HB-EGF) acts as the diphtheria toxin (DT) receptor. Transfection of human HB-EGF cDNA into mouse LC cells, L cells stably expressing DRAP27, conferred sensitivity to DT, but transfection of mouse HB-EGF cDNA did not. To define the essential regions of HB-EGF that serve as the functional DT receptor, we examined the sensitivity to DT and DT binding of cells expressing several human/mouse HB-EGF chimeras. It was found that DT binds to the EGF-like domain of the human HB-EGF. However, mouse HB-EGF does not serve as a functional DT receptor due to non-conserved amino acid substitutions in this domain. In addition, CRM197, a non-toxic mutant of DT, inhibited strongly the mitogenic activity of the secreted form of human HB-EGF, but not of mouse HB-EGF and other EGF receptor-binding growth factors. These results confirmed further that DT interacts with the EGF-like domain of HB-EGF and that this interaction is specific for human HB-EGF.

Amino Acid Sequence↗
PubMed1995

Expression of an active glycosylated human gamma-glutamyl transpeptidase mutant that lacks a membrane anchor domain.

A mutant of human gamma-glutamyl transpeptidase (EC 2.3.2.2, a membrane-bound enzyme of importance in glutathione metabolism) that differs from the wild type by deletion of the putative signal peptide/anchor domain (amino acid residues 1-27) was expressed in insect cells using a baculovirus system. In contrast to the wild-type enzyme--which, as expected, was mainly cell-associated--the mutant enzyme was secreted into the medium. The mutant and wild-type enzymes were purified and found to exhibit virtually identical catalytic properties. The mutant enzyme was glycosylated and processed into two subunits, as found for the wild-type enzyme. Brefeldin A inhibited secretion of the mutant enzyme and led to its accumulation in cells. The findings indicate that gamma-glutamyl transpeptidase can be targeted to the endoplasmic reticulum in a manner that does not involve function of an amino-terminal "signal/anchor" domain and that this domain is involved primarily in a membrane anchoring function. Another region of the enzyme may function as a signal domain.

Amino Acid Sequence↗
PubMed1995

Differential protective action of cytokines on radiation-induced apoptosis of peripheral lymphocyte subpopulations.

It is established that soluble factors involved in cell growth can prevent apoptosis of hematolymphoid cell lines in factor-deprived situations. The present study investigates the possible protective effects of various cytokines on radiation-induced apoptosis of apparently quiescent lymphocyte subpopulations. The exposure to gamma-irradiation resulted in appreciable apoptotic changes in all of lymphocyte subpopulations. Natural killer (NK) cells were the most radiosensitive, whereas CD8+ T and B cells showed weaker susceptibility to radiation and CD4+ T cells were relatively radioresistant. The radiation-induced apoptosis in NK cells was significantly inhibited by IL-2. In addition to IL-2, IL-4 and IL-7 rescued both CD4+ and CD8+ T cells from radiation-induced cell death. The viability of B cells was maintained by the presence of IL-4 but not others in culture. Furthermore, we conclude that the protective effect by each cytokine on radiation-induced apoptosis might be partly attributed to enhancement of cellular expression of bcl-2 protein.

Adult↗
PubMed1995

Leucocytosis as a marker of organ damage induced by chronic strenuous physical exercise.

The effects of strenuous physical exercise on the serial changes in the haematological, biochemical and hormonal markers were investigated. A group of 14 soldiers, aged 24-36 years, took part in a military training course for about 13 weeks. After severe exercise stress, an increase (90%) in the number of peripheral blood leucocytes was observed. The degree of leucocytosis showed a close correlation with the values of some serum parameters, such as concentrations of aspartate aminotransferase (AST; r = 0.747), lactate dehydrogenase (LD; r = 0.748), blood urea nitrogen (r = 0.756), creatine kinase (CK; r = 0.637), manganese-superoxide dismutase (Mn-SOD; r = 0.508), alanine aminotransferase (ALT; r = 0.542) and uric acid (r = 0.538), and concentrations of urinary parameters, such as vanilmandelic acid (r = 0.429) and free cortisol (r = 0.437). The subjects showing prominent leucocytosis over 9500 cells.microliters-1 exhibited a lower concentration of serum cholinesterase than those who showed milder leucocytosis. The serum Mn-SOD concentration was closely correlated with the serial changes in serum concentrations of AST, ALT, LD and CK, indicating exercise-induced muscle and liver damage. The change in peripheral leucocyte number was assumed to be diagnostically informative and may be a prognostic marker, reflecting organ damage and restoration after strenuous physical exercise.

Adult↗