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S Shimizu

Publications and source records attributed to S Shimizu.

At least 379 records · Page 21Linked to original sources
PubMed1997

Dimethylsulfoxide (DMSO) treatment reduces infarction volume after permanent focal cerebral ischemia in rats.

Dimethylsulfoxide (DMSO) is a common vehicle used for many drugs used in neuroprotective experiments. DMSO has many biological effects, including antiinflammatory, antioxidant, and local anesthetic effects that could be neuroprotective. To determine if DMSO is neuroprotective in ischemia, DMSO (0, 0.01, 0.03, 0.1, 0.3 and 1.0 ml) was administered intraperitoneally 30 min prior to permanent middle cerebral artery (MCA) occlusion in the rat. Twenty-four hours after MCA occlusion, brains were removed and sectioned. Mean infarction volume was significantly reduced in rats treated with 0.1, 0.3 and 1.0 ml of DMSO compared to saline controls. There was no acute effect of drug treatment upon arterial blood gasses or mean blood pressure. These results suggest that DMSO is neuroprotective in focal cerebral ischemia. Investigators must use appropriate controls when DMSO is used as a vehicle.

Animals↗
PubMed1997

Endogenous production of tumour necrosis factor is required for manganese superoxide dismutase expression by irradiation in the human monocytic cell line THP-1.

Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that scavenges superoxide (O2-) ions. We studied the regulation of MnSOD gene expression by irradiation and the mechanisms in human monocytic cell line THP-1. We found that irradiation induced expression of the MnSOD gene through the autocrine mechanism, involving the production of tumour necrosis factor (TNF). Irradiation increased TNF production in THP-1 cells, and TNF increased the levels of MnSOD transcripts. Supernatant from irradiated THP-1 cells induced the expression of MnSOD mRNA, and anti-TNF antibody blocked the induction of MnSOD mRNA. Irradiation also increased the levels of MnSOD mRNA in other myelocytic cell lines, HL60 and KG-1, and the ovarian cancer cell line SK-OV-3. Moreover, increased levels of MnSOD mRNA were observed in mature myeloid cells, including macrophages and granulocytes, as well as in immature cells. However, irradiation did not increase the level of MnSOD mRNA in THP-1 cells with prolonged exposure to PMA. We also found that irradiation increased the rate of MnSOD transcription, and irradiation stabilized MnSOD mRNA in THP-1 cells. Our results indicate that the endogenous production of TNF is required, at least in part, for the induction of MnSOD mRNA expression by irradiation in THP-1 cells, and the increased levels of MnSOD transcripts on irradiation occur through a pathway involving protein kinase C activation. Our results also indicate that the increase in MnSOD mRNA caused by irradiation is regulated by both transcriptional and post-transcriptional mechanisms.

Autocrine Communication↗
PubMed1997

Identification of active sites in amidase: evolutionary relationship between amide bond- and peptide bond-cleaving enzymes.

Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification. With regard to the presumptive active site residue Cys203, a Cys203 --> Ala mutant enzyme still retained 11.5% of the original specific activity. In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase. Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity. Furthermore, Asp191 --> Asn substitution as well as Ser195 --> Ala substitution completely abolished the specific activity. It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-NH2) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two.

Amides↗
PubMed1997

Cytokine gene expression in pancreatic islet grafts in the rat.

We examined the production of cytokine message in allogeneic and syngeneic rat pancreatic islet grafts using specific primers and polymerase chain reaction. Freshly isolated islet preparations contained transcripts for interleukin (IL)-1alpha, IL-6, IL-10, and interferon-gamma (IFN-gamma) but not for IL-2. IL-1alpha in allogeneic grafts showed increased and consistently high expression from 1 to 7 days after transplantation, but the level in syngeneic grafts fell quickly to pretransplant levels. IL-2 and IFN-gamma transcripts were found in allogeneic grafts at 1, 3, 5, and 7 days after transplantation with a peak at day 5, but these cytokines were almost absent from syngeneic grafts. The peak of IL-6 expression was 1 day after transplantation in both syngeneic and allogeneic grafts, and then the level fell quickly. IL-10 was produced at approximately the same high level at all time points in both syngeneic and allogeneic grafts. The results show that freshly isolated islet preparations contain IL-1alpha, IL-6, IL-10, and IFN-gamma transcripts at the time of transplantation. The initial production of cytokines in islet grafts, especially IL-1, may explain phenomena such as graft nonfunction, rapid rejection, and lack of response to immunosuppression.

Animals↗
PubMed1997

Isolation and characterization of D-threonine aldolase, a pyridoxal-5'-phosphate-dependent enzyme from Arthrobacter sp. DK-38.

D-Threonine aldolase is an enzyme that catalyzes the cleavage of D-threonine into glycine and acetaldehyde. Its activity was found in several genera of bacteria such as Arthrobacter, Alcaligenes, Xanthomonas, and Pseudomonas, but not in yeasts or fungi. The enzyme was purified to homogeneity from one strain, Arthrobacter sp. DK-38. The enzyme appeared to consist of a single polypeptide chain with an apparent molecular mass of 51 kDa. This enzyme, as well as L-threonine aldolase, requires pyridoxal 5'-phosphate (pyridoxal-P) as a coenzyme. Unlike other pyridoxal-P enzymes, D-threonine aldolase also requires a divalent cation such as Co2+, Ni2+, Mn2+, or Mg2+ for its catalytic activity. The enzyme completely lost its activity in the absence of either pyridoxal-P or a divalent cation. A divalent cation was also essential for the thermal stability of the enzyme. The metal-free enzyme tends to become thermally unstable, resulting in the irreversible loss of its catalytic activity. The enzyme is strictly D-specific for the alpha-position, whereas it cannot distinguish between threo and erythro forms at the beta-position. Thus, D-threonine and D-allothreonine act as substrates of the enzyme, but their kinetic parameters are different; the Km and Vmax values are 3.81 mM and 38.8 micromol x min(-1) x mg(-1) toward D-threonine, and 14.0 mM and 102 micromol x min(-1) x mg(-1) toward D-allothreonine. respectively. The aldolase reaction is reversible, and the enzyme is therefore able to produce nearly equimolar amounts of D-threonine and D-allothreonine through C-C bond formation between glycine and acetaldehyde. The enzyme also acts, in the same manner, on several other D-beta-hydroxy-alpha-amino acids, including D-beta-phenylserine, D-beta-hydroxy-alpha-aminovaleric acid, D-beta-3,4-dihydroxyphenylserine, and D-beta-3,4-methylenedioxyphenylserine.

Acetaldehyde↗
PubMed1997

Inhibition of apoptosis by the actin-regulatory protein gelsolin.

Gelsolin is an actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. In addition to its actin-regulatory function, gelsolin has also been proposed to affect cell growth. Our present experiments have tested the possible involvement of gelsolin in the regulation of apoptosis, which is significantly affected by growth. When overexpressed in Jurkat cells, gelsolin strongly inhibited apoptosis induced by anti-Fas antibody, C2-ceramide or dexamethasone, without changing the F-actin morphology or the levels of Fas or Bcl-2 family proteins. Upon the induction of apoptosis, an increase in CPP32(-like) protease activity was observed in the control vector transfectants, while it was strongly suppressed in the gelsolin transfectants. Pro-CPP32 protein, an inactive form of CPP32 protease, remained uncleaved by anti-Fas treatment in the gelsolin transfectants, indicating that gelsolin blocks upstream of this protease. The tetrapeptide inhibitor of CPP32(-like) proteases strongly inhibited Fas-mediated apoptosis, but only partially suppressed both C2-ceramide- and dexamethasone-induced apoptosis. These data suggest that the critical target responsible for the execution of apoptosis may exist upstream of CPP32(-like) proteases in Jurkat cells and that gelsolin acts on this target to inhibit the apoptotic cell death program.

Actins↗
PubMed1997

Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells.

Multiple growth factors synergistically stimulate proliferation of primitive hematopoietic progenitor cells. A human myeloid cell line, KPB-M15, constitutively produces a novel hematopoietic cytokine, termed stem cell growth factor (SCGF), possessing species-specific proliferative activities. Here we report the molecular cloning, expression, and characterization of a cDNA encoding human SCGF using a newly developed lambdaSHDM vector that is more efficient for differential and expression cloning. cDNA for SCGF encodes a 29-kDa polypeptide without N-linked glycosylation. SCGF transiently produced by COS-1 cells supports growth of hematopoietic progenitor cells through a short-term liquid culture of bone marrow cells and exhibits promoting activities on erythroid and granulocyte/macrophage progenitor cells in primary semisolid culture with erythropoietin and granulocyte/macrophage colony-stimulating factor, respectively. Expression of SCGF mRNA is restricted to myeloid cells and fibroblasts, suggesting that SCGF is a growth factor functioning within the hematopoietic microenvironment. SCGF could disclose some human-specific mechanisms as yet unidentified from studies on the murine hematopoietic system.

Amino Acid Sequence↗
PubMed1997

Biosynthesis of furan fatty acids (F-acids) by a marine bacterium, Shewanella putrefaciens.

A mutant derived from Shewanella putrefaciens 8CS7-4 treated with N-methyl-N'-nitro-N-nitrosoguanidine was found to produce 15-20 mg of a furan fatty acid (F-acid), 10,13-epoxy-11-methyloctadeca-10,12-dienoic acid (F18), per liter of growth medium (10-15% of total fatty acids). Capillary gas chromatography-mass spectrometry and proton nuclear magnetic resonance analysis of the fatty acid methyl esters of the mutant revealed the presence of other F-acids, 8,11-epoxy-9-methylhexadeca-8,10-dienoic acid (F16), 6,9-epoxy-7-methyltetradeca-6,8-dienoic acid (F14), and methyl branched unsaturated fatty acids, 11-methyl-12E-octadecenoic acid (11-me-18:1) and 11-methyl-10E,12E-octadecadienoic acid (1-me-18:2). About 90% of F-acids were present in phospholipids, in which the F-acids were found to be exclusively linked at the sn-1 position. 11-me-18:1 and 11-me-18:2 were also detected in the sn-1 position. Firstly, 11-me-18:1 increased and reached a maximum at 12 h, and then decreased rapidly. Secondly, the 11-me-18:2 content reached a maximum at 24 h, when 11-me-18:1 was little detected, and then decreased. Finally, the amount of F18 began to increase after 20 h and reached a plateau at 36 h. These results suggest that 11-me-18:1 and 11-me-18:2 are precursors of F18.

Animals↗
PubMed1997

Purification and characterization of L-allo-threonine aldolase from Aeromonas jandaei DK-39.

L-allo-Threonine aldolase (L-allo-threonine acetaldehyde-lyase), which exhibited specificity for L-allo-threonine but not for L-threonine, was purified from a cell-free extract of Aeromonas jandaei DK-39. The purified enzyme catalyzed the aldol cleavage reaction of L-allo-threonine (K(m) = 1.45 mM, Vmax = 45.2 mumol min-1 mg-1). The activity of the enzyme was inhibited by carbonyl reagents, which suggests that pyridoxal-5'-phosphate participates in the enzymatic reaction. The enzyme does not act on either L-serine or L-threonine, and thus it can be distinguished from serine hydroxy-methyltransferase (L-serine:tetrahydrofolate 5,10-hydroxy-methyltransferase, EC 2.1.2.1) or L-threonine aldolase (EC 4.1.2.5).

Aeromonas↗
PubMed1997

Methylnitrosourea-induced tumorigenesis in MGMT gene knockout mice.

Gene targeting was used to obtain mice defective in the MGMT gene, encoding O6-methylguanine-DNA methyltransferase [Tsuzuki et al., Carcinogenesis (Lond.), 17: 1215-1220, 1996]. These MGMT-/- mice were most sensitive to the alkylating carcinogen, methylnitrosourea; when varied doses of methylnitrosourea were administered to 6-week-old mice and survivals at the 30th day were determined, LD50s of MGMT-/- and MGMT+/+ mice were 20 and 240 mg/kg of body weight, respectively. MGMT+/- mice were as resistant as MGMT+/+ mice, but some difference in survival time was noted when the two genotypes of mice were exposed to a relatively high dose of methylnitrosourea. A large number of thymic lymphomas, as well as lung adenomas, occurred in MGMT-/- mice exposed to methylnitrosourea at a dose of 2.5 mg/kg of body weight. In case of exposure to the same dose of drug, no or few tumors occurred in the MGMT+/+ and MGMT+/- mice. It appears that the DNA repair methyltransferase protein protected these mice from methylnitrosourea-induced tumorigenesis.

Animals↗
PubMed1997

Intracellular ATP levels determine cell death fate by apoptosis or necrosis.

Although apoptosis and necrosis are morphologically distinct manifestations of cell death, apoptosis and some necroses share common features in the death signaling pathway involving functional steps of death-driving interleukin 1beta-converting enzyme family proteases and anti-cell death protein Bcl-2. One evident physiological difference in cells undergoing apoptosis versus necrosis is in intracellular levels of ATP. In this study, we specifically addressed the question of whether apoptosis depends on intracellular ATP levels, since longer incubation under ATP-depleting conditions results in necrotic cell death. Incubation of cells in glucose-free medium with an inhibitor of mitochondrial F0F1-ATPases reduces intracellular ATP levels and completely blocks Fas/Apo-1-stimulated apoptosis. ATP supplied through glycolysis or oxidative phosphorylation restores the apoptotic cell death pathway. ATP depletion also leads to a block in Fas-induced activation of CPP32/Yama(-like) proteases, and when ATP is depleted after the activation of the proteases, subsequent apoptosis is significantly blocked. Thus, ATP-dependent steps exist both upstream and downstream of CPP32/Yama(-like) protease activation in apoptotic signal transduction. Treatment with the calcium ionophore induces apoptosis under ATP-supplying conditions but induces necrotic cell death under ATP-depleting conditions, indicating that ATP levels are a determinant of manifestation of cell death.

Adenosine Triphosphate↗
PubMed1997

The GLY1 gene of Saccharomyces cerevisiae encodes a low-specific L-threonine aldolase that catalyzes cleavage of L-allo-threonine and L-threonine to glycine--expression of the gene in Escherichia coli and purification and characterization of the enzyme.

The GLY1 gene of Saccharomyces cerevisiae is required for the biosynthesis of glycine for cell growth [McNeil, J. B., McIntosh, E. V., Taylor, B. V., Zhang, F-R., Tang, S. & Bognar, A. L. (1994) J. Biol. Chem. 269, 9155-9165], but its gene product has not been identified. We have found that the GLY1 protein is similar in primary structure to L-allo-threonine aldolase of Aeromonas jandiae DK-39, which stereospecifically catalyzes the interconversion of L-allo-threonine and glycine. The GLY1 gene was amplified by PCR, with a designed ribosome-binding site, cloned into pUC118, and expressed in Escherichia coli cells. The enzyme was purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The enzyme has a molecular mass of about 170 kDa and consists of four subunits identical in molecular mass. The enzyme contains 2 mol pyridoxal 5'-phosphate/4 mol of subunit as a cofactor, and its absorption spectrum exhibits maxima at 280 nm and 420 nm. The enzyme catalyzes the cleavage of not only L-allo-threonine to glycine but also L-threonine. We have termed the enzyme a low-specific L-threonine aldolase to distinguish it from L-allo-threonine aldolase.

Aeromonas↗
PubMed1997

The bphDEF meta-cleavage pathway genes involved in biphenyl/polychlorinated biphenyl degradation are located on a linear plasmid and separated from the initial bphACB genes in Rhodococcus sp. strain RHA1.

The bphACB genes responsible for the initial oxidation of the aromatic ring of biphenyl/polychlorinated biphenyls (PCB) to meta-cleavage product in Rhodococcus sp. RHA1 have been characterized. We cloned the 6.1 kb EcoRI fragment containing another extradiol dioxygenase gene (etbC) which was induced during the growth on ethylbenzene. The bphD, bphE and bphF encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase, 2-hydroxypenta-2,4-dienoate hydratase and 4-hydroxy-2-oxovalerate aldolase, respectively, were found downstream of etbC. The deduced amino acid (aa) sequence of RHA1 bphD and bphE had 27-33% and 32-38% identity, respectively, with those of the corresponding genes in Pseudomonas. BphE and BphF are closely related to the corresponding homoprotocatechuate meta-cleavage pathway enzymes of Escherichia coli C. The bphD and bphF were expressed in E. coli and the BphD activity was detected. The etbCphDEF genes were transcribed in biphenyl and ethylbenzene growing cells. Pulsed field gel electrophoresis (PFGE) analysis indicated that RHA1 contains three large linear plasmids. Southern blot analysis indicated that the meta-cleavage pathway for biphenyl/PCB catabolism in RHA1 is directed by the 390 kb plasmid borne bphDEF genes located separately from bphACB gene cluster on the 1100 kb plasmid.

Benzene Derivatives↗
PubMed1997

Loss of p53 is an early event in induction of brain tumors in mice by transplacental carcinogen exposure.

Experimental carcinogenesis studies using p53-deficient mice have suggested that loss of function of this tumor suppressor gene is generally not an early event but is rather related to tumor progression. However, the biological functions of p53 and the accumulating evidence of alteration in human tumors imply a possible role for loss of p53 in the initial stages of tumorigenesis. Ethylnitrosourea administration to p53-heterozygous pregnant mice resulted in rapid development of primary brain tumors, which are extremely rare in mice, in 70% of the p53-null offspring. Brain tumors also developed later in 4% of heterozygous mice, but they had lost the wild-type allele. Thus, loss of normal p53 gene expression is of direct significance to early events in brain tumorigenesis, and this tumor suppressor gene may protect embryos from DNA damage in the brain induced by transplacental carcinogen exposure.

Animals↗
PubMed1997

Mutations of the Ki-ras, p53 and APC genes in adenocarcinomas of the human small intestine.

In contrast to the origins of colorectal carcinomas, the mechanisms of carcinogenesis in the small intestine remain unclear. We therefore analyzed the mutational status of the Ki-ras, p53, and adenomatous polyposis coli (APC) genes in primary carcinomas of the small intestine and compared the mutation patterns with those established for colorectal cancers. DNA was extracted from 15 formalin-fixed, paraffin-embedded lesions. Codons 12, 13 and 61 of the Ki-ras gene, exons 5-8 of the p53 gene, and codons 1268-1569, which contain the mutation cluster region (MCR) of the APC gene, were amplified by means of PCR, subcloned and sequenced. Mutations of the Ki-ras and p53 genes were observed in 8 (53.3%) and 4 lesions (26.7%), respectively. The mutational frequency of the Ki-ras gene in the present series of small intestinal carcinomas was similar, while that of the p53 gene was slightly lower than the reported frequencies for colorectal carcinomas. Only one case showed a mutation of the APC gene, involving an insertional mutation of an adenine at codons 1554-1556 with formation of a stop codon immediately downstream. Since the occurrence of an APC mutation is considered an early event in colorectal carcinogenesis, our findings indicating an extremely low frequency of such changes in and around the MCR suggest that carcinomas of the small intestine arise via a genetic pathway distinct from that involved in the development of carcinomas of the colorectum.

Adult↗
PubMed1997

Imidase, a dihydropyrimidinase-like enzyme involved in the metabolism of cyclic imides.

Imidase, which preferably hydrolyzed cyclic imides to monoamidated dicarboxylates, was purified to homogeneity from a cell-free extract of Blastobacter sp. A17p-4. Cyclic imides are known to be hydrolyzed by mammalian dihydropyrimidinases. However, imidase was quite different from known dihydropyrimidinases in structure and substrate specificity. The enzyme has a relative molecular mass of 105 000 and consists of three identical subunits. The purified enzyme showed higher activity and affinity toward cyclic imides, such as succinimide (Km = 0.94 mM; Vmax = 910 micromol x min(-1) x mg(-1)), glutarimide (Km = 4.5 mM; Vmax = 1000 micromol min (-1) x mg (-1) and maleimide (Km = 0.34 mM; Vmax = 5800 micromol x min(-1)x mg(-1)), than toward cyclic ureides, which are the substrates of dihydropyrimidinases, such as dihydrouracil and hydantoin. Sulfur-containing cyclic imides, such as 2,4-thiazolidinedione and rhodanine, were also hydrolyzed. The enzyme catalyzed the reverse reaction, cyclization, but with much lower activity and affinity. The enzyme was non-competitively inhibited by succinate, which was found to be a key compound in cyclic-imide transformation in relation with the tricarboxylic acid cycle in this bacterium, suggesting that the role of imidase is to catalyze the initial step of cyclic-imide degradation.

Amidohydrolases↗
PubMed1997

A novel transporter involved in cobalt uptake.

Cobalt is an essential component of a low molecular-mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1. We have found a new gene, nhlF, in the DNA region sandwiched between nhlBA encoding L-NHase and amdA encoding amidase, which are involved in the degradation of nitriles. The product of nhlF, NhlF, shows a significant sequence similarity with those of hoxN from Alcaligenes eutrophus, hupN from Bradyrhizobium japonicum, nixA from Helicobacter pylori, and ureH from Bacillus sp., which are considered to be involved in nickel uptake into these cells. Sequence and hydropathy plot analyses have shown that NhlF encodes a 352-amino acid (aa) protein with eight hydrophobic putative membrane-spanning domains. nhlF expression in R. rhodochrous ATCC 12674 and Escherichia coli JM109 confers uptake of 57Co in their cells, but not of 63Ni. The expression of both nhlF and nhlBA in R. rhodochrous ATCC 12674 exhibited higher NHase activity than nhlBA expression. These findings together with the inhibitory effect by uncouplers (CCCP and SF6847) for the cobalt uptake suggest that NhlF mediates the cobalt transport into the cell energy-dependently finally to provide L-NHase.

Amino Acid Sequence↗