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Biomedical subjects

H Suzuki

Publications and source records attributed to H Suzuki.

At least 19 recordsLinked to original sources
PubMed1992

Purification and characterization of DNA topoisomerase IV in Escherichia coli.

The subunits of topoisomerase IV (topo IV), the ParC and ParE proteins in Escherichia coli, were purified to near homogeneity from the respective overproducers. They revealed type II topoisomerase activity only when they were combined with each other. In the presence of Mg2+ and ATP, topo IV was capable of relaxing a negatively or positively supercoiled plasmid DNA or converting the knotted P4 phage DNA, whether nicked or ligated, to a simple ring. However, supercoiling activity was not detected. The topoisomerase activity was not detectable when the purified ParC and ParE proteins were combined with the purified GyrB and GyrA proteins, respectively. This is consistent with the result that neither a parC nor a parE mutation was compensated by transformation with a plasmid carrying either the gyrA or the gyrB gene. Simultaneous introduction of both the gyrA and gyrB plasmids corrected the phenotypic defect of parC and parE mutants. The results suggest that DNA gyrase can substitute for topo IV at least in some part of the function for chromosome partitioning. Antisera were prepared against the purified ParC, ParE, GyrA, and GyrB proteins and used to investigate cellular localization of these gene products. ParC protein was found to be specifically associated with inner membranes only in the presence of DNA. This result suggests that one of the functions of topo IV might be to anchor chromosomes on membranes as previously proposed for eukaryotic topoisomerase II.

Bacterial Proteins↗
PubMed1992

Identification of inducible calmodulin-dependent nitric oxide synthase in the liver of rats.

A calmodulin-dependent nitric oxide synthase was significantly induced in the liver of rats treated intravenously with heat-killed Propionibacterium acnes and 5 days later with Escherichia coli lipopolysaccharide. The apparent calmodulin-dependent and -independent isozymes were separated by Mono Q column chromatography after their partial purification by 2',5'-ADP-agarose affinity chromatography. Both enzymes had a molecular weight of 125,000 as determined by SDS-polyacrylamide gel electrophoresis and required NADPH, tetrahydrobiopterin, and dithiothreitol as cofactors. Their activities were completely inhibited by the specific nitric oxide synthase inhibitors NG-monomethyl-L-arginine and N omega-nitro-L-arginine at 80 and 800 microM, respectively. The peptide maps of these two isozymes with lysylendopeptidase and their reverse-phase column chromatographic profiles were indistinguishable. In the presence of bovine calmodulin, the purified calmodulin-dependent isozyme behaved as a calmodulin-independent isozyme on Mono Q column chromatography. The purified calmodulin-independent isozyme was converted to a calmodulin-dependent isozyme by EDTA and EGTA. Calmodulin blot analysis using 125I-calmodulin showed that the two isozymes bound calmodulin equally efficiently.

Amino Acid Oxidoreductases↗
PubMed1992

Escherichia coli gamma-glutamyltranspeptidase mutants deficient in processing to subunits.

Arginyl residues 513 and 571 of Escherichia coli K-12 gamma-glutamyl-transpeptidase (EC 2.3.2.2) were substituted with alanyl and glycyl residues, respectively, by oligonucleotide-directed in vitro mutagenesis. Both mutants were devoid of the enzymatic activity. On Western blot analysis, we found that both mutants accumulated a gamma-glutamyltranspeptidase precursor which was not processed into large and small subunits in the periplasmic space of Escherichia coli.

Alanine↗
PubMed1992

Studies of the relationship between cell proliferation and cell death. II. Early gene expression during concanavalin A-induced proliferation or dexamethasone-induced apoptosis of rat thymocytes.

Several data in the literature suggest that an intriguing relationship exists between cell proliferation and cell death. Accordingly, we studied the early expression of different genes in the same cells, i.e. rat thymocytes, undergoing cell proliferation upon stimulation with Concanavalin A or cell death following dexamethasone treatment. We showed that an early accumulation of c-fos, c-jun and c-myc mRNA occurred in both phenomena but with different kinetics. It can be speculated that the early induction of nuclear oncogenes is necessary to allow the later induction of other genes probably regulated at the transcriptional level by the AP-1 complex and/or by Myc protein. The accumulation of the transcript for another gene, i.e. poly(ADP-ribose)polymerase, an enzyme responsible for posttranslational modifications of several nuclear proteins, could instead be related to chromatin modifications occurring in both processes.

Animals↗
PubMed1992

Role of leucine residues in the C-terminal region of human interleukin-6 in the biological activity.

Site-directed mutagenesis of two sets of three periodic leucine residues which appear at every seventh position in the C-terminal region of human interleukin-6 (IL-6) was performed. Both receptor-binding and immunoglobulin (Ig)-induction activities of a triple mutant Leu168,175,182-->Val were only 1% compared with those of wild-type IL-6. However, the mutant Leu152,159,166-->Val had 13% receptor-binding and 2% Ig-induction activities of those of wild-type IL-6. In order to obtain more direct information on the receptor-binding region, we prepared two synthetic peptides. A significant binding activity was observed for the peptide Leu168-Met185, but not for the peptide Leu152-Arg169. These results indicate that leucine residues in the C-terminal region, especially Leu168, Leu175, and Leu182, play an important role in the receptor-binding and Ig-induction activities.

Amino Acid Sequence↗
PubMed1992

Involvement of DNA polymerase beta in proliferation of rat liver induced by lead nitrate or partial hepatectomy.

We have studied the expression pattern of DNA polymerase beta in two different models of in vivo cell proliferation. Both mRNA levels and enzyme activity of DNA polymerase beta markedly increased before and/or during DNA synthesis in proliferating hepatocytes in mitogen-treated and partially hepatectomized rats. The time-courses of the expression of the gene coding for DNA polymerase beta were significantly different in the two cell systems. A 5-fold increase in DNA polymerase beta mRNA was observed 8 h after lead nitrate administration, i.e. well before the onset of DNA synthesis. In the regenerative liver cells a 3-fold increase in the amount of mRNA was observed 24-48 h after partial hepatectomy, the event being coincident with extensive DNA synthesis. In both systems, the increase of mRNA levels was always paralleled by an increase in enzyme activity, suggesting that DNA polymerase beta activity may be regulated at a pre-translational level.

Animals↗
PubMed1992

Effective blocking of natural cytotoxicity of young rabbit serum on murine thymocytes by high concentration of glucose in complement-dependent cytotoxicity method.

By an accidental observation, the natural cytotoxicity of 21-day-old rabbit sera to murine thymocytes was found to be almost completely inhibitable by 4.5 mg/ml glucose. This enabled us to use unabsorbed young rabbit serum as a source of complement for antibody-mediated cytotoxic treatment of murine thymocytes. This glucose-sensitive natural cytotoxic activity was recovered in the immunoglobulin fraction purified on an affinity column, and was dependent on complement activity. The unabsorbed 21-day-old rabbit sera had much higher complement activity than those from 14-day-old rabbits and commercially available low background complement. Indeed, complete depletion of CD4+ cells from murine thymocytes and splenocytes was achieved using unabsorbed 21-day rabbit serum as a source of complement and a monoclonal antibody GK1.5 which has been claimed to be inefficient in complement-dependent killing. Owing to the increase in complement titer and circulating blood volume 14-21 days after birth, the use of 21-day-old rabbit sera assures a much more abundant supply of complement for the treatment of murine lymphocytes.

Animals↗
PubMed1992

Molecular cloning of a cDNA coding for neurofibromatosis type 1 protein isoform lacking the domain related to ras GTPase-activating protein.

Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder, and a gene linked to NF1 was recently identified. Its gene product (NF1 protein) contains a domain functionally related to mammalian ras GTPase-activating protein (GAP). Here, we cloned a cDNA coding for NF1 protein isoform lacking the region related to GAP from a oligo(dT)-primed cDNA library of human placenta. This cDNA carries the insert of about 2.4 kb, coding for a protein of 551 amino acid residues, which shares the same aminoterminal 547 residues with authentic NF1 protein. We show that NF1 mRNAs of about 2.9, 11, and 13 kb are expressed in human tissues, and that the isolated cDNA may represent the 2.9-kb transcript.

Amino Acid Sequence↗
PubMed1992

Existence of two pathways for the endocytosis of epidermal growth factor by rat liver: phenylarsine oxide-sensitive and -insensitive pathways.

The effect of phenylarsine oxide (PAO) on the internalization rate of epidermal growth factor (EGF) was investigated using perfused rat liver and isolated rat hepatocytes. In perfused liver, a tracer concentration of 125I-EGF alone or with excess unlabeled EGF (20 nM) was perfused and the internalization rate constants (kint) were measured. In the absence of PAO, kint values did not differ significantly for either dose condition. However, with the addition of PAO to the perfusate, the kint value dropped to 4% of that of the control at the low concentration of EGF, while dropping to only 40% of that of the control at the high concentration of EGF. These results suggest the existence of a PAO-insensitive internalization pathway having a kint value comparable with that of the other pathway. Similar EGF concentration-dependent inhibition of 125I-EGF internalization caused by PAO was ascertained using isolated rat hepatocytes. PAO also decreased the cellular ATP content in isolated hepatocytes. However, when we lowered the cellular ATP content with rotenone, the cell-surface binding and internalization of EGF were comparable with the control levels. We concluded that there exist dual pathways for the internalization of EGF and that excess doses of EGF lead to EGF internalization not only through a PAO-sensitive pathway but also through a PAO-insensitive pathway, whereas at a tracer dose of EGF, the internalization occurs mainly via the PAO-sensitive pathway.

Adenosine Triphosphate↗
PubMed1992

Inhibitory actions of MCI-154 on guinea-pig femoral artery and vein preparations.

In guinea-pig femoral artery and vein preparations, the effects of MCI-154 were investigated on: (1) membrane depolarizations produced by 29.6 mM [K+]0 (high-K) solution and noradrenaline (NA) and on e.j.p.s produced by perivascular nerve stimulation; (2) contractions produced by NA, high-K and perivascular nerve stimulation; and, (3) endothelium-dependent relaxation produced by acetylcholine (ACh). In both femoral artery and vein preparations, MCI-154 (up to 10(-5) M) did not change the resting membrane potential or the depolarization produced by high-K. In preparations of femoral vein but not femoral artery, MCI-154 reduced NA-induced depolarization. The contractions produced by NA and high-K were reduced by MCI-154, the former more than the latter. The actions of MCI-154 were more pronounced in the vein than in the artery. The excitatory junction potential and contractions produced by perivascular nerve stimulation in guinea-pig saphenous artery preparations were inhibited by MCI-154 (greater than 10(-7) M). ACh-induced relaxations of guinea-pig femoral artery preparations precontracted with high-K were not affected by MCI-154. It was concluded that MCI-154 is an antagonist at postjunctional alpha 2-adrenoceptors, and, at high concentrations, inhibits voltage-dependent Ca2+ influx in vascular smooth muscle cells. These actions may contribute to the hypotensive effect of this drug.

Animals↗
PubMed1992

Molecular cloning of cDNAs for human fibroblast nucleotide pyrophosphatase.

Nucleotide pyrophosphatase (NPPase) activity is markedly elevated in cultured skin fibroblasts from patients of Lowe's syndrome. cDNA clones encoding the NPPase were isolated using synthetic oligonucleotide probes designed on the basis of partial amino acid sequence of the enzyme purified from human placenta. The complete sequences of these clones yielded a merged sequence of 3508 bases. The polypeptide chain of the enzyme was deduced to comprise 873 amino acids with a calculated molecular weight of 99,703 and had the characteristics of a class II transmembrane protein. Ten potential N-glycosylation sites were detected in the protein. RNA blot analysis showed that human fibroblasts contain two minor mRNAs of 7.0 and 8.2 kb, respectively, in addition to a major 3.6-kb species that coincides with the merged cDNA in size. A computer search of a nucleotide sequence data-base revealed that plasma cell membrane glycoprotein PC-1, whose function was unknown at the time, is identical with the NPPase. Comparison of the amino acid sequence of the NPPase with the active site sequence of bovine 5'-nucleotide phosphodiesterase allowed the assignment of a putative active site domain to the central region of the COOH-terminal extracellular domain of the NPPase. The gene for human NPPase was localized to chromosome 6 at q22-q23 by in situ hybridization with a fragment of the NPPase cDNA.

Amino Acid Sequence↗
PubMed1992

Transmural pressure inhibits nitric oxide release from human endothelial cells.

We examined the effect of transmural pressure on histamine-stimulated nitric oxide release from cultured endothelial cells prepared from human umbilical cord veins. PO2 and pH were kept constant throughout the experiments. Various levels of transmural pressure and atmospheric pressure (40, 80, 120 and 160 mm Hg) were applied. Nitric oxide release was inhibited in a pressure-dependent manner. The inhibitory effects were reversible, and nitric oxide had no effect on the morphology of the cells. Our results suggest that transmural pressure-mediated inhibition of nitric oxide release contributes to pressure-induced vasoconstriction and reduced endothelium-dependent relaxation in patients with hypertension.

Atmospheric Pressure↗
PubMed1992

A specific method for determination of peroxisomal beta-oxidation activity in cultured human skin fibroblasts using a specific substrate, C9: a possible application for screening of peroxisomal disorders.

We developed a specific method for direct determination of peroxisomal beta-oxidation activity in cultured human skin fibroblasts. When control fibroblasts were incubated with N-(alpha-methylbenzyl)azelaamic acid (C9), a specific peroxisomal substrate, C5 and C7, the chain-shortened products, were detected with cell concentration and incubation time dependencies and no other products including C3 were detected. In glutaric aciduria type I and type II fibroblasts, the formation rates of C2 units liberated from C9 were almost similar to that in control cells. In contrast to these cell types, the fibroblasts from patient of Zellweger syndrome, in which peroxisomal beta-oxidation was impaired, showed no conversion of C9 to C5 and C7. The lack of the C2 units liberation in Zellweger fibroblasts was not due to an impairment of mitochondrial beta-oxidation and/or activation of C9 to C9-CoA derivative for subsequent beta-oxidation reaction, but rather, appeared to be due to the specific defect of peroxisomal beta-oxidation system. These results indicate that C9 is a useful substrate for the estimation of peroxisomal beta-oxidation activity in cultured human skin fibroblasts.

Cells, Cultured↗
PubMed1992

Human soluble IL-6 receptor: its detection and enhanced release by HIV infection.

By using a fluorescence sandwich ELISA for the quantification of soluble human IL-6R, normal human PBMC were found to be induced to release IL-6R into culture supernatant by stimulation with PHA. Furthermore, certain promonocyte cell lines and human T-cell leukemia virus I (HTLV-I)-positive cell lines produced sIL-6R into culture supernatants constitutively. However, this was not found with HTLV-I negative T cell lines and Burkitt's B cell line. In addition, generation of supernatant IL-6R of the promonocyte cell line was significantly increased 27-fold after PMA treatment and sevenfold after infection with HIV. The released IL-6R molecules were characterized as an apparent m.w. of 50 to 55 kDa by both size-exclusion HPLC and immunoprecipitation of the soluble protein with IL-6R-specific mAb followed by SDS-PAGE analysis. Furthermore, increased levels of serum IL-6R were detected in blood donors seropositive for HIV. Moreover, the released IL-6R could bind efficiently to purified rIL-6 on solid phase and suppressed the proliferative responses of PBMC. These results suggest that the release of soluble IL-6R might be linked to regulatory functions of immune responses induced by IL-6 stimulation during normal and human retrovirus-infected cell growth and differentiation.

Enzyme-Linked Immunosorbent Assay↗
PubMed1992

Malignant lymphoma of mucosa-associated lymphoid tissue arising in the thymus of a patient with Sjögren's syndrome. A morphologic, phenotypic, and genotypic study.

A 59-year-old woman with Sjögren's syndrome had an anterior mediastinal tumor. The tumor had epithelium-lined thymic cysts. Histologically, centrocyte-like (CCL) cells were present as clusters intermingling with small lymphocytes and plasma cells, invaded the epithelium, and formed characteristic lymphoepithelial lesions; the tumor was identified as malignant lymphoma arising in mucosa-associated lymphoid tissue (MALT). Within the tumor, trapped Hassall's corpuscles were recognized. Immunohistochemical staining demonstrated monotypic cytoplasmic kappa light chains in a small portion of the CCL cells. Furthermore, Southern blot hybridization studies showed rearrangements of immunoglobulin heavy chain, immunoglobulin kappa light chain, and T-cell receptor beta genes. The findings are consistent with thymic low-grade B-cell MALT lymphoma.

Cytoplasm↗
PubMed1992

Roles for interleukin-2 (IL-2) and IL-4 in the generation of allocytotoxic T cells in the primary and secondary responses in vitro.

Roles for interleukin-2 (IL-2) and IL-4 in the generation of murine allocytotoxine T lymphocytes (allo-CTL) in the primary and secondary responses were studied in vitro. The generation of allo-CTL in the primary response was inhibited by anti-IL-2 monoclonal antibody (mAb), but was not inhibited by anti-IL-4 mAb. On the other hand, the generation of allo-CTL in the secondary response was partially inhibited by either anti-IL-2 or anti-IL-4 mAb, and it was almost completely inhibited by the combination of two mAbs. CD8+ cell-depleted splenocytes produced IL-2, but not IL-4, in response to alloantigens in the primary response, and these cells produced both IL-2 and IL-4 in the secondary response. Both exogenous IL-2 and IL-4 induced functionally active allo-CTL in the primary response from CD4+ cell-depleted splenocytes when these cells were stimulated with T cell-depleted allogeneic cells. These results suggest that the allo-CTL induction in the primary response is IL:-2-dependent and secondary allo-CTL induction is both IL-2 and IL-4-dependent, because unprimed CD4+ T cells produce IL-2, but not IL-4, whereas primed cells produce both IL-2 and IL-4 in response to alloantigens.

Animals↗
PubMed1992

Marked enhancement of acyl-CoA synthetase activity and mRNA, paralleled to lipoprotein lipase mRNA, in adipose tissues of Zucker obese rats (fa/fa).

To clarify the role of acyl-CoA synthetase in development of obesity, the mRNA levels and activities were studied in Zucker fatty rats (fa/fa). In Zucker fatty rats compared with their lean littermates, marked enhancement of ACS were observed in adipose tissues. Obese/lean rats ratio of ACS activity and mRNA in abdominal subcutaneous fat (3.3- and 3.9-fold, respectively) were greater than in mesenteric fat (2.0- and 2.2-fold). The enhancement of ACS activity and mRNA in the liver of fatty rats (1.2- and 1.8-fold) were less than those in the adipose tissues. There were no enhancement of ACS activities and mRNA levels in heart tissue of the obese rats. LPL mRNA levels were also enhanced in adipose tissue of fatty rats and obese/lean ratio of LPL mRNA was also higher in abdominal subcutaneous fat than mesenteric fat (6.2- vs 3.1-fold). The larger obese/lean rats ratio of LPL and ACS parameters in abdominal subcutaneous fat than mesenteric fat may be related to the observation that the increase of subcutaneous fat weight was larger than that of mesenteric fat weight in fatty rats (21.1- vs 4.9-fold). Integrated enhancement of LPL and ACS gene expression in adipose tissue may play an important role in the development of obesity.

Adipose Tissue↗