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W Hashimoto

Publications and source records attributed to W Hashimoto.

At least 19 recordsLinked to original sources
PubMed1997

Microbial system for polysaccharide depolymerization: enzymatic route for gellan depolymerization by Bacillus sp. GL1.

A bacterium-producing polysaccharide lyase (gellan lyase) was isolated from soil samples and identified to be Bacillus sp. The lyase was purified from the culture fluid of the bacterium (designated Bacillus sp. GL1) grown in the presence of gellan as a carbon source. The purified gellan lyase depolymerized deacetylated gellan and gave a single oligosaccharide product with a molecular weight of 646, which was determined by fast atom bombardment mass spectrometry. The structure of the product was determined by the combination of mass spectrometry, HPLC analysis, and high-resolution proton nuclear magnetic resonance spectroscopy to be a tetrasaccharide of glucuronyl-glucosyl-rhamnosyl-glucose with unsaturated glucuronic acid at the nonreducing terminal. When incubated in cell extracts of Bacillus sp. GL1, the tetrasaccharide was first converted to trisaccharide without unsaturated glucuronyl residue, and the trisaccharide was then converted to hydrolyzed monosaccharides glucose and rhamnose. These results show that, in the bacterium Bacillus sp. GL1, gellan is first depolymerized to give a tetrasaccharide, a repeating unit in gellan molecule, by an extracellular gellan lyase and then the tetrasaccharide is hydrolyzed to monosaccharides by successive actions of intracellular exoglycosidases.

Bacillus↗
PubMed1997

Analysis of low temperature inducible mechanism of gamma-glutamyltranspeptidase of Escherichia coli K-12.

Escherichia coli K-12 cultured at 20 degrees C has more gamma-glutamyltranspeptidase (GGT: EC 2.3.2.2) activity than that cultured at 37 or 42 degrees C. On Western blot analysis, E. coli K-12 cells cultured at 20 degrees C produced more GGT protein than those cultured at 37 degrees C. mRNA of the GGT gene (ggt) in the cells was also measured and it was found that the level of ggt mRNA at 20 degrees C was 10-fold higher than that at 37 degrees C. When the ggt promoter was replaced by a tac promoter, GGT activity at 37 degrees C from the tac promoter was 5-fold higher than that at 37 degrees C from the ggt promoter, though there was less difference in GGT activity between both grown at 20 degrees C. The ggt mRNA at 20 degrees C was found to be more stable than that at 37 degrees C. These results suggested that the higher GGT activity in E. coli K-12 cells grown at 20 degrees C was due to a higher level of GGT protein at 20 degrees C caused by higher level of ggt mRNA at 20 degrees C because of a low-temperature dependent ggt promoter as well as the stability of ggt mRNA at 20 degrees C.

Base Sequence↗
PubMed1996

Cytotoxic gammadelta or alphabeta T cells with a natural killer cell marker, CD56, induced from human peripheral blood lymphocytes by a combination of IL-12 and IL-2.

Populations of cytotoxic CD3+CD56+ cells were selectively expanded when monocyte-depleted human PBL (M(-) PBL) were cultured with 20 U/ml IL-12 and 100 U/ml IL-2. In a majority of cases, CD4-CD8- as well as CD8+ gammadelta T cells with CD56 Ag were induced, but in some cases CD4+CD56+ alphabeta T cells were selectively increased. Determination of which will increase, gammadelta or alphabeta T cells, apparently is dependent upon individuals. When M(-) PBL were stimulated with IL-12 and IL-2 in the presence of immobilized anti-CD3 Ab, CD8+CD56+ alphabeta T cells increased exclusively. When M(-) PBL were cultured by IL-2 alone, CD3+CD56- cell expansion was predominant while CD3+CD56+ cells remained a minor population. Cytotoxic activity of M(-) PBL cultured with a combination of IL-12 and IL-2 is much greater than when cultured with IL-2 alone. The cytotoxicity of activated M(-) PBL was abrogated by the depletion of either CD3+ or CD56+ cells irrespective of their gammadelta or alphabeta phenotypes. These types of cells are preferentially present in the livers of humans. The results revealed that, under certain conditions, IL-12 synergizes with IL-2 to induce potent cytotoxic T cells with CD56 Ag of humans, and suggest that these cells in the human liver are functionally similar to NK1+ alphabeta T cells in murine liver.

Biomarkers↗
PubMed1996

Liver NK1.1+ CD4+ alpha beta T cells activated by IL-12 as a major effector in inhibition of experimental tumor metastasis.

We demonstrate herein evidence that IL-12-activated alpha beta T cells with intermediate TCR (NK1+ TCRint cells) in the liver inhibit metastases in the lung as well as in the liver metastases of i-v. injected tumors. IL-12 administration enhanced NK1 expression of NK1+ TCRint cells (NK1high) and increased CD4 weakly positive (CD4low) TCRint cells, while both CD4+ TCRint cells and double-negative TCRint cells were proportionally diminished. Accordingly, the major parts of NK1high TCRint cells are CD4low cells, and most of these cells are V beta 8+ cells. The cytotoxic assays of IL-12-stimulated hepatic mononuclear cells after treatment with respective Abs and complement in vitro and after sorting revealed that CD4low NK1high TCRint cells are cytotoxic effectors. When IL-12-stimulated hepatic mononuclear cells (but not splenocytes) were transferred into tumor-preinjected mice, EL-4 cell metastases in the liver as well as 3LL cell metastases in the lung were inhibited. The antimetastasis of hepatic mononuclear cells transfer was abrogated by the depletion of NK1+ cells, CD3+ cells, or CD4+ cells but not CD8+ cells before transfer. Moreover, transfer of these cells of nude mice into tumor-preinjected mice also inhibited metastases in both organs. Although NK1+ TCRint cells are nearly absent in the hepatic vein blood, a significant proportion of NK1high TCRint cells appeared by IL-12 administration. These results demonstrate that IL-12-stimulated liver NK1high TCRint cells, including extrathymic ones, are major effectors against tumor metastasis and suggest that the cells migrate and inhibit lung metastases.

Animals↗
PubMed1996

LPS induces NK1.1+ alpha beta T cells with potent cytotoxicity in the liver of mice via production of IL-12 from Kupffer cells.

We recently reported that systemic administration of IL-12 into mice activates NK1.1+ alpha beta T cells with intermediate TCR (NK1+TCRint) and induces strong MHC-unrestricted cytotoxicity in C57BL/6 mice. In the present report, we examined the effect of LPS on Kupffer cells and NK1+TCRint, cells in C57BL/6 mice. Administration of LPS, as well as synthetic lipid A analogue (ONO-4007), but not detoxified LPS, induces the increase of NK1 expression of NK1+TCRint cells (NKlhighTCRint) and the acquisition of strong MHC-unrestricted cytotoxicity of these cells against NK-sensitive and NK-resistant targets as does IL-12 administration. LPS as well as ONO-4007 induced IL-12 mRNA in hepatic mononuclear cells, mainly in plastic-adherent Kupffer cells. LPS-induced cytotoxicity of hepatic mononuclear cells was greatly reduced by in vivo injections of anti-IL-12 Ab, to a lesser extent by anti-IFN-gamma Ab, but not by anti-IL-1 nor anti-TNF-alpha Ab. Pretreatment of mice with LPS induced inhibition of hepatic metastases of i.v. injected EL4 cells in C57BL/6 euthymic and athymic mice and this antimetastasis was inhibited by injection of anti-IL-12 Ab. This antimetastatic effect of LPS in the liver was also observed in different strains of mice and tumors, In contrast to IL-12, however, LPS was not so effective when administered after tumor inoculation. These results revealed that LPS (lipid A) stimulates NK1+TCRint cells through IL-12 production from Kupffer cells and suggest that bacterial components, probably including those from intestine, are activators of Kupffer cells and NK1+TCRint, cells in the liver. It is also suggested that the host condition as well as LPS-induced cytokines other than IL-12 may affect antitumor effect induced by LPS in the liver.

Animals↗
PubMed1996

Pit structure on bacterial cell surface.

The yellow-pigmented bacterium isolated from a ditch was a gram negative rod with a G+C content of 63 mol%, and was classified in the genus Sphingomonas. Electron microscopy revealed that the bacterial cell surface was covered with many large plaits. When grown in a medium containing a polysaccharide as an essential nutrient, a pit of 0.02-0.1 micrometers in diameter was formed on the cell surface, and a thin section showed the rearrangement of the plaits and the presence of a region where the cell membrane sinks into the cytosol. The dependence of the pit formation on the presence of macromolecule may predict the existence of a direct uptake mechanism for macromolecules through a mouth-like pit, possibly in endocytosis fashion. The confirmation of the pit structure is the first such finding in the history of microbiology and may provide a new insight into the cell morphology and biochemistry of macromolecule transport in microbial cell system.

Alginates↗
PubMed1996

Interleukin-12 induces cytotoxic NK1+ alpha beta T cells in the lungs of euthymic and athymic mice.

We recently reported that interleukin-12 (IL-12) stimulated hepatic NK1.1 Ag+ alpha beta T cells with intermediate T-cell receptor (TCR; NK1+ TCRint cells) and enhanced their NK1 expression (NK1high TCRint), and that these cells acquire strong major histocompatibility complex (MHC) unrestricted cytotoxicity in C57BL/6 mice, both +/+ and nu/nu. In the present study, we find that although murine lung normally has few NK1+ TCRint cells, NK1high TCRint cells are induced in+/+ and nu/nu mice after systemic administration of IL-12; these cells exhibit strong MHC unrestricted cytotoxicity against NK-sensitive and -resistant targets. A small number of NK1high TCRint cells was also found in peripheral blood after increased amounts of IL-12 were administered. Cytotoxicity tests in vitro revealed that the cytotoxic activity of the lung mononuclear cells (MNC) of C57BL/6 mice induced by IL-12 was abrogated by the depletion of either NK1+ or CD3+ cells, but not of CD8+ cells, as reportedly was the case of hepatic MNC, suggesting that NK1high TCRint cells are an antimetastatic population not only in the liver but also in the lung of mice. IL-12 injection into mice markedly elevates serum interferon-gamma (IFN-gamma) levels. However, although IL-12-induced cytotoxicity of NK1high TCRint cells was significantly reduced by anti-IFN-gamma antibody injection (which decreased serum IFN-gamma to an undetectable level), the appearance of NK1high TCRint cells in the lung and liver was not so affected. These results suggest that IFN-gamma is an important mediator of the cytotoxicity of NK1high TCRint cells but is not an essential factor for induction of these cells. We also added data showing that IL-12 has a broad antimetastatic effect against various liver and lung metastatic tumours intravenously injected into several strains of mice, including NK-deficient bg/bg mice. It can be considered that, in addition to NK cells, CD8+ cytotoxic T cells and gamma delta T cells, NK1+ TCRint cells can be categorized as one of the cytotoxic effector populations. These novel type cells distinct from regular T cells may play an important role in monitoring intra- and perivascular areas.

Animals↗
PubMed1996

A preliminary description of the crystal structure of gamma-glutamyltranspeptidase from E. coli K-12.

The structure of GGT [EC 2.3.2.2] from E. coli K-12 was studied at 3 A resolution by X-ray crystallography. Initial protein phases were calculated using two kinds of Pb2+ derivatives. The phases were refined by non-crystallographic 2-fold symmetry electron density averaging combined with solvent flattening and histogram matching. The GGT molecule has overall dimensions of 60 x 50 x 40 A. There are two antiparallel beta-pleated sheets consisting of 6 and 7 beta-strands. The two beta-sheets form a wall-like structure. Twelve short alpha-helices were detected, of which the maximum length appears to be four helix turns.

Crystallography, X-Ray↗
PubMed1996

Crystallization and preliminary X-ray diffraction studies of N-acyl-D-glucosamine 2-epimerase from porcine kidney.

N-Acyl-D-glucosamine 2-epimerase from porcine kidney, which was cloned and expressed in Escherichia coli, was crystallized by the vapor-diffusion method, using polyethylene glycol and ammonium acetate as precipitants. The crystals were resistant to X-ray radiation damage and diffracted to more than 2.0 A resolution. The diffraction pattern indicated that the crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit-cell dimensions of a = 78.1, b = 97.2, and c = 100.7 A. It is supposed that the asymmetric unit consists of two N-acyl-D-glucosamine 2-epimerase molecules. Collection of data on the native crystals indicated that they are suitable for X-ray structural analysis.

Animals↗
PubMed1996

Purification and characterization of microbial gellan lyase.

Gellan lyase was purified from the culture fluid of soil samples incubated in a medium containing gellan as a sole carbon source. The enzyme was a monomer with a molecular mass of 140 kDa and was most active at pH 7.5 and 45 degrees C. The enzyme was highly specific to gellan and lowered the viscosity of the polymer.

Carbohydrate Sequence↗
PubMed1995

Cytotoxic NK1.1 Ag+ alpha beta T cells with intermediate TCR induced in the liver of mice by IL-12.

Systemic administration of IL-12 greatly reduced the hepatic metastases of i.v.-injected liver metastatic EL4 tumor cells in C57BL/6 +/+ and nu/nu mice. Cytotoxic assay in vitro revealed that administration of IL-12 greatly enhanced cytotoxicity of hepatic mononuclear cells (MNC) against various NK- sensitive and -resistant tumor targets, including EL4 cells, whereas only slight or moderate augmentation of the cytotoxicity was observed in splenocytes in normal and nude mice. After IL-12 administration, hepatic MNC increased in number and showed vigorous proliferation in vitro. Hepatic MNC of control C57BL/6 +/+ mice contain alpha beta T cells with intermediate TCR (TCRint) as well as alpha beta T cells with bright TCR, whereas hepatic MNC of nu/nu mice have only TCRint cells. These TCRint cells are found to be NK1.1 Ag+ (NK1+ TCRint). Systemic administration of IL-12 into normal and nude mice markedly augments the NK1 expression of NK1+ TCRint cells (NK1high TCRint), which is comparable to or brighter than that of NK cells in the liver, whereas alpha beta T cells with bright TCR or gamma delta T cells in the liver are NK1-. Depletion of either NK1.1+ or CD3+ cells, but not CD8+ cells, of hepatic MNC from IL-12-treated normal mice by respective Abs and C in vitro abrogate their cytotoxicity. These results revealed that TCRint cells are potent cytotoxic effector cells and suggest that NK1high TCRint cells are the main antimetastatic population in the liver, and that TCRint cells are functionally different from regular T cells with bright TCR.

Animals↗
PubMed1995

Effect of site-directed mutations on processing and activity of gamma-glutamyltranspeptidase of Escherichia coli K-12.

gamma-Glutamyltranspeptidase [EC 2.3.2.2] of Escherichia coli K-12 is thought to be synthesized from a single precursor polypeptide into a heterodimeric form through post-translational processing. Cells of a gamma-glutamyltranspeptidase-overproducing transformant of E. coli K-12 were fractionated and the localization of the enzyme was examined by Western blot analysis. The periplasmic fraction only contained the mature form of gamma-glutamyltranspeptidase, membrane fraction only contained the precursor of gamma-glutamyltranspeptidase, and no precursor of gamma-glutamyltranspeptidase was detected in the cytoplasmic fraction. Amino acid residues at the cleavage site for processing into the large and small subunits were substituted by site-directed mutagenesis. The processing phenotypes of six mutants were examined by Western blot analysis, and their gamma-glutamyltranspeptidase activities were measured. Mutations at the N-terminal amino acid residues of the small subunit (Thr-391, Thr-392, and His-393) prevented the maturation of the enzyme and the immature mutants exhibited no enzymatic activity. A mutation at the C-terminal residue of the large subunit (Gln-390) had less effect on the processing and enzymatic activity. These results suggest that the sequence of threonyl-threonyl-histidinyl residues at the N-terminal of the small subunit is very important for the processing of E. coli K-12 gamma-glutamyltranspeptidase and this processing is essential to the expression of gamma-glutamyltranspeptidase activity of E. coli K-12.

Amino Acid Sequence↗
PubMed1995

Subunit association of gamma-glutamyltranspeptidase of Escherichia coli K-12.

gamma-Glutamyltranspeptidase [EC 2.3.2.2] of Escherichia coli K-12 consists of one large subunit and one small subunit, which can be separated from each other by high-performance liquid chromatography. Using ion spray mass spectrometry, the masses of the large and the small subunit were determined to be 39,207 and 20,015, respectively. The large subunit exhibited no gamma-glutamyltranspeptidase activity and the small subunit had little enzymatic activity, but a mixture of the two subunits showed partial recovery of the enzymatic activity. The results of native-polyacrylamide gel electrophoresis suggested that they could partially recombine, and that the recombined dimer exhibited enzymatic activity. The gene of gamma-glutamyltranspeptidase encoded a signal peptide, and the large and small subunits in a single open reading frame in that order. Two kinds of plasmid were constructed encoding the signal peptide and either the large or the small subunit. A gamma-glutamyltranspeptidase-less mutant of E. coli K-12 was transformed with each plasmid or with both of them. The strain harboring the plasmid encoding each subunit produced a small amount of the corresponding subunit protein in the periplasmic space but exhibited no enzymatic activity. The strain transformed with both plasmids together exhibited the enzymatic activity, but its specific activity was approximately 3% of that of a strain harboring a plasmid encoding the intact structural gene. These results indicate that a portion of the separated large and small subunits can be reconstituted in vitro and exhibit the enzymatic activity, and that the expressed large and small subunits independently are able to associate in vivo and be folded into an active structure, though the specific activity of the associated subunits was much lower than that of native enzyme. This suggests that the synthesis of gamma-glutamyltranspeptidase in a single precursor polypeptide and subsequent processing are more effective to construct the intact structure of gamma-glutamyltranspeptidase than the association of the separated large and small subunits.

Amino Acid Sequence↗
PubMed1993

Crystallization and preliminary X-ray analysis of gamma-glutamyltranspeptidase from Escherichia coli K-12.

gamma-Glutamyltranspeptidase (EC 2.3.2.2) from Escherichia coli K-12 has been purified and crystallized by means of vapor diffusion in hanging drops. Two kinds of crystals on cell dimensions were found for X-ray diffraction analysis, one from ammonium sulfate and the other from polyethylene glycol 6000 as precipitants. The crystals of the orthorhombic form grown in the presence of 15% polyethylene glycol and 20 mM sodium acetate buffer were chosen for further analysis. The crystals belonged to space group P2(1)2(1)2(1), with cell dimensions of a = 128.1, b = 129.9 and c = 79.2 A, and two molecules constitute an asymmetric unit. These crystals diffracted to 2.0 A resolution and were suitable for X-ray crystallographic studies.

Bacterial Proteins↗
PubMed1993

Escherichia coli K-12 can utilize an exogenous gamma-glutamyl peptide as an amino acid source, for which gamma-glutamyltranspeptidase is essential.

Escherichia coli K-12 can utilize a gamma-glutamyl peptide as an amino acid source, for which gamma-glutamyltranspeptidase (EC 2.3.2.2) is essential. We suggest that the gamma-glutamyl linkage of a gamma-glutamyl peptide is hydrolyzed by gamma-glutamyltranspeptidase located in the periplasmic space, and the released amino acid is taken up and utilized by E. coli.

Amino Acids↗
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

Differential age-change in the numbers of CD4+CD45RA+ and CD4+CD29+ T cell subsets in human peripheral blood.

Peripheral blood mononuclear cells were obtained from people ranging in age from newborn to 102 years old and analyzed by dual color flow cytometer in terms of number and percentage of various subsets of T cells, B cells and natural killer cells (CD3, 4, 5, 8, 11b, 19, 20, 21, 25, 29, 45RA and 56). Numbers of T cells (CD3+ or CD5+ cells) significantly declined at the 3rd decade as compared with those of younger people, stayed at a relatively constant level between the 3rd and the 7th decade and gradually declined thereafter. In T cell subsets, both CD4 and CD8 positive positive cells decreased with age, but a decrease was more pronounced in the latter, showing an age-related increase of CD4/CD8 ratio. The most interesting finding was a contrasting age-change in two subsets of CD4+ T cells; i.e. a subset of suppressor inducer T cells (CD4+CD45RA+ naive cells) decreased with age, while a subset of helper inducer T cells (CD4+CD29+ memory cells) increased with age. CD20+ B cells also decreased with age in a manner similar to that observed in T cells. Natural killer cells (CD56) showed an increase in numbers with age. The relationship between these changes in various subsets of peripheral blood leukocytes and the age-related decline in immune functions has been discussed.

Adolescent↗