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S Shimizu

Publications and source records attributed to S Shimizu.

At least 793 records · Page 44Linked to original sources
PubMed1988

Kinetic evaluation of conversion of phosphatidylcholine to phosphatidylethanolamine by phospholipase D from different sources.

Transphosphatidylation from phosphatidylcholine (PC) to phosphatidylethanolamine (PE) was conducted by seven phospholipase D preparations from different sources, one of which was of cabbage origin and other six of Streptomyces origin. The reactions were carried out at 30 degrees C using mixture of ethyl acetate containing PC and buffer containing ethanolamine and phospholipase D. To obtain the activity ratio of transphosphatidylation and hydrolysis at various ratios of ethanolamine and water concentrations, the apparent rate constant ratios of transphosphatidylation and hydrolysis, (k'3/k3)app, were calculated, keeping the concentration of PC constant (17.8 mM). Among the seven enzymes examined, five showed good transphosphatidylation, in which 100% of PC could be converted to PE at ethanolamine concentrations ranging from 0.3 to 1.0 M, while two showed poor transphosphatidylation activity. At higher ethanolamine concentrations, the reaction rate was decreased due to substrate inhibition. Hydrolytic reactions were conducted at 30 degrees C with respective enzymes by using mixture of ethyl acetate containing PC or PE and buffer containing phospholipase D. The ratio of (kcat/Km)PE/(kcat/Km)PC was calculated to determine the substrate specificity of various phospholipase D enzymes. The values of (k'3/k3)app varied with the origin of the enzymes, whereas the values of (kcat/Km)PE/(kcat/Km)PC were not so different. The results obtained show that (k'3/k3)app is a good parameter to select an enzyme and that the timing of stopping the reaction is also important to avoid the hydrolysis of the product.

Biological Transport↗
PubMed1988

A novel fungal enzyme, NADPH-dependent carbonyl reductase, showing high specificity to conjugated polyketones. Purification and characterization.

A novel enzyme which specifically catalyzes the reduction of conjugated polyketones was purified to homogeneity from cells of Mucor ambiguus AKU 3006. The enzyme has a strict requirement for NADPH and irreversibly reduces a number of quinones such as p-benzoquinone, alpha-naphthoquinone and acenaphthenequione. The enzyme also reduces polyketones such as isatin and ketopantoyl lactone, and their derivatives. The apparent Km values for isatin and ketopantoyl lactone are 49.9 microM and 714 microM, respectively. The reduction of ketopantoyl lactone proceeds stereospecifically to yield L-(+)-pantoyl lactone. The pro-S (A) hydrogen at C-4 of NADPH is transferred to the substrate. The enzyme is not a flavoprotein and consists of two polypeptide chains with an identical relative molecular mass of 27,500. Quercetin, dicoumarol and some SH reagents inhibit the enzyme activity. 3-Methyl-1,2-cyclopentanedione and 1,3-cyclohexanedione are uncompetitive inhibitors with Ki values of 80.9 microM and 64.5 microM, respectively, to ketopantoyl lactone.

4-Butyrolactone↗
PubMed1988

Effect of recombinant human interferon-alpha A/D on in vivo murine tumor cell growth.

We investigated the effect of human recombinant interferon-alpha A/D A/D-IFN), which is known to delay the growth of murine tumor cells, on the growth of S1 and R1 subline cells of murine Meth A fibrosarcoma in the peritoneal cavity of mice. In vitro growth of S1 cells was sensitive to, and that of R1 cells was resistant to, the direct effect of A/D-IFN, as with murine natural IFN-alpha/beta, which was used originally to isolate these sublines. In vivo, however, the growth of not only S1 cells but also R1 cells was suppressed by the administration of A/D-IFN, and the survival time of tumor-bearing mice was prolonged. Although A/D-IFN had a direct effect on S1 cells in vivo, R1 cells were susceptible only to the indirect effect via the host cells. Macrophages (M phi) harvested from the peritoneal cavity of A/D-IFN-treated mice bearing ascitic R1 cells were very effective in suppressing the in vitro growth of R1 cells; those from non-R1-bearing A/D-IFN-treated mice were less effective. The results of in vitro experiments indicate that M phi are very probably activated by the synergism of A/D-IFN and M phi diameter-activating factor(s) produced by lymphoid cells in tumor-bearing mice.

Animals↗
PubMed1988

Gelatinases of metastatic cell lines of murine colonic carcinoma as detected by substrate-gel electrophoresis.

Gelatinases were detected in the conditioned medium of murine colonic carcinoma cells by SDS-polyacrylamide gel electrophoresis using gels copolymerized with gelatin. Several gelatinase activities differing in molecular weight were detected but the major activities migrated with molecular weights of 60,000 and 95,000. The enzymes did not hydrolyze bovine serum albumin or casein, and required calcium for activity. All of the gelatinase activities were inhibited by EDTA, 1,10-phenanthroline and dithiothreitol but not by N-ethylmaleimide and phenylmethylsulfonyl fluoride. The 95,000 dalton gelatinase was separated from the 60,000 dalton gelatinase by affinity chromatography on Ricinus communis agglutinin-agarose, and the former activity was markedly increased in highly metastatic cell lines as compared with its activity in poorly metastatic cell lines.

Animals↗
PubMed1988

Fungal mycelia as a novel source of eicosapentaenoic acid. Activation of enzyme(s) involved in eicosapentaenoic acid production at low temperature.

Several filamentous fungi belonging to the genus Mortierella were found to produce large amounts of 5,8,11,14,17-cis-eicosapentaenoic acid (EPA) in their mycelia only when grown at low temperature (12 degrees C), i.e., not at physiological growth temperature (20-28 degrees C). The results of experiments with cell-free extracts suggested that this unique phenomenon is due to activation of enzyme(s) involved in EPA formation at low temperature. Mortierella alpina 1S-4 produced 0.3 g/l of EPA (27 mg/g dry mycelia). This high productivity show the practical significance of these novel EPA producers.

Adenosine Triphosphate↗
PubMed1988

Effects of high O2 affinity of blood on oxygen consumption (VO2) of dog gracilis muscle at varying O2 delivery.

To clarify the influence of high O2-affinity blood on oxygen consumption by peripheral tissue, we perfused the dog gracilis muscle alternately with normal and low P50 blood at constant CaO2 and varying blood flows. VO2 was plotted against O2 delivery which was a product of CaO2 and blood flow. VO2 of the resting dog gracilis muscle was O2 delivery-dependent below a critical O2 delivery of 0.40 ml/min.100g, but became delivery-independent above this level. This relationship was essentially the same as that observed in rat skeletal muscle (Kolár and Janský, 1984), and was independent of the O2 affinity of blood, both qualitatively and quantitatively, although the PVO2 was significantly lower in the perfusions with the high affinity blood than in those with normal blood. The average PVO2 value by which the delivery-dependent and -independent VO2 regions were separated decreased from 33 Torr in the normal perfusions to 20 Torr in the high O2 affinity perfusions. In conclusion, the elevation of blood O2 affinity does not induce any reduction in VO2, and thus hypoxia, in resting skeletal muscle under normoxic conditions when O2 delivery is not below the critical level.

Animals↗
PubMed1988

Wide variation of myoglobin contents in gizzard smooth muscles of various avian species.

We determined myoglobin contents of gizzards (muscular stomach) and breast muscles in 34 avian species by a modification of Reynafarje's spectrophotometric procedure. The birds were apparently differentiated into two groups in respect of the gizzard, one with a high myoglobin content (7.74 +/- 1.81 mg/g muscle) and the other with a low (1.54 +/- 0.41 mg/g). In the former group of 15 species all but one were herbivorous, and all but one were carnivorous or else omnivorous in the latter group of 19 species. The myoglobin level was considered to closely correlate with mechanical performance and therefore oxygen demands of the gizzards. It might also be relevant to a circulatory situation during the tonic contractions of this organs.

Animals↗
PubMed1988

Enzyme-linked immunosorbent assay using solubilised antigen for detection of antibodies to Anaplasma marginale.

An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against Anaplasma marginale. A marginale bodies were separated from parasitised erythrocytes by a modified nitrogen decompression method, sonicated, then solubilised with Triton X-100 and used as the ELISA antigen. In this ELISA system the required amount of antigen protein was 16.2 ng for each well. In the course of experimental infections, of calves, significant antibody levels were detected by ELISA and the complement fixation test (CFT) at almost the same time. Antibodies against A. marginale were detectable for longer periods using the ELISA than using the CFT. Sera from calves infected with Babesia bigemina, B. bovis, B. ovata, Theileria sergenti and Eperythrozoon wenyoni gave no reaction; however, antisera against A. centrale did react with the A. marginale ELISA antigen.

Anaplasma↗
PubMed1988

Endotoxin-specific chromogenic assay for plasma in pregnant women, umbilical cords, neonates and children.

The concentration of endotoxin (Et) was measured by means of a new endotoxin-specific assay (Endospecy) in 15 paired samples of maternal and umbilical cord (UC) blood, and 38 neonates and 5 children. The Et levels in maternal and UC blood with normal pregnancy and delivery were 0.8 +/- 0.4 and 1.3 +/- 0.6 pg/ml, respectively. In some cases of premature rupture of the membrane, Et in maternal and UC blood was slightly increased. The level in non-infectious neonates was 6.7 +/- 5.1 pg/ml and that in children less than 10 pg/ml. However, patients with infections showed a high Et level, which decreased on antibiotic treatment. In addition, the pattern of the changes in Et in cerebrospinal fluid (CSF) did not parallel that in plasma. Therefore, in perinatal high-risk babies, the probability of affection with endotoxemia should be considered, but the effects of endotoxin on the central nervous system are presumably indirect because of the low permeability of Et through the blood-CSF-barrier.

Adult↗
PubMed1988

A new approach to the prevention of hypertensive cardiovascular diseases by controlling the proliferation of vascular smooth muscle cells.

Cholera toxin subunit B suppressed the proliferation of cultured vascular smooth muscle cells from the thoracic aorta of Wistar-Kyoto rats (WKY), stroke-resistant spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP). Since cholera toxin subunit B did not stimulate cyclic AMP accumulation in vascular smooth muscle cells, the effect of cholera toxin subunit B might be due to another mechanism. Cholera toxin subunit B bound to the surface of vascular smooth muscle cells and was rapidly incorporated into them. The morphological structure of vascular smooth muscle cells was transformed from the synthetic type to the non-synthetic type, in which microfilaments and intermediate filaments were abundantly formed, while rough endoplasmic reticulum was decreased after CTB treatment.

Animals↗
PubMed1988

Immortalization of murine leukocytes by oncogenes. II. Phenotypic characterization of transformants immortalized by v-src or Ha-ras oncogenes: expression of B220, a B-cell lineage specific antigen.

In the course of study to obtain murine dendritic cell lines using oncogenic retroviruses, we have established several immortalized cell lines with characteristics different from those of dendritic cells. The transformants were mainly round nonadherent cells, capable of growing in soft agar, and negative for nonspecific esterase activity. Profiles of cell surface antigens were examined by indirect immunofluorescence technique. The cell lines were positive for Fc receptor (2.4G2), J11d (J11d.2), and B220 (RA3-3A1/6.1) antigens and negative (or dull positive in small percentages) for Ia (M5/144.15.2), IL-2 receptor (3C7), Thy-1 (B5-5), Mac-1 (M1/70.-15.11.5), and macrophage (F4/80) antigens. They were negative for both surface and cytoplasmic immunoglobulins. Several clones were established from these transformant cell lines and cell surface antigens were examined. Antigenic profiles of these clones were very similar to those of the parental cell lines. Some of these clones, however, seemed to increase their Ia antigen expression. The results suggest that the transformants originated from early B-lineage cells.

Animals↗
PubMed1988

Intergeneric Protoplast Fusion between Ruminococcus albus and an Anaerobic Recombinant, FE7.

Intergeneric protoplast fusion between Ruminococcus albus, a cellulolytic, gram-positive, anaerobic bacterium (Pc Sm Km), and an anaerobic recombinant, FE7 (Pc Sm Km), having lignin-related compound-degrading activities, was performed under strictly anaerobic conditions to introduce cellulase genes into strain FE7. The fusion frequency varied with different selected markers from 3.0 x 10 to 3.3 x 10. Two fusants, obtained from a synthetic medium with selective pressures of penicillin and streptomycin and with cellooli-gomer as the sole carbon source, were gram-negative rods. One of them, named FE7R2, showed 45 to 47% of the beta-glucosidase and cellobiosidase activities of its parent R. albus and still maintained a level of degradation activity against dehydrodivanillin, a lignin-related compound, of up to 87% of that of the parent strain FE7. To verify that the cellulolytic activities expressed in the fusant FE7R2 originated from R. albus cellulase genes, the beta-glucosidase gene of R. albus was cloned into Escherichia coli HB101 with plasmid pBR322. Cells bearing a recombinant plasmid, pRAII, produced high enzyme activities against both p-nitrophenyl-beta-d-glucoside and p-nitrophenyl-beta-d-cellobioside and could degrade cellobiose to glucose. Southern blot results showed that the cloned DNA fragment could hybridize with chromosomal DNAs of both R. albus and FE7R2, but did not with the chromosomal DNA of FE7, indicating that the beta-glucosidase gene fragment was introduced into the chromosome of FE7R2 from R. albus via the protoplast fusion. The fusant FE7R2 could utilize simultaneously both cellobiose and dehydrodivanillin. These results gave evidence that the fusion product FE7R2 is a recombinant strain between its parents R. albus and FE7. This recombinant has stably kept the above properties for about 2 years.

Journal Article↗
PubMed1988

Isolation and characterization of an anaerobic dehydrodivanillin-degrading bacterium.

A novel, strictly anaerobic, gram-negative, non-spore-forming, fusiform, rod-shaped bacterium having high dehydrodivanillin (DDV)-degrading activity was isolated from cow ruminal fluid. This strain degraded a range of six main lignin-related compounds such as DDV, ferulic acid, dehydrodiisoeugenol, guaiacoxyacetic acid, vanillin, and veratrylglycerol-beta-guaiacyl ether to the extent of 14 to 83% within 2 days under strictly anaerobic conditions. As DDV degradation intermediates, three aromatic compounds (dehydrodivanillic acid, vanillic acid, and 5-carboxyvanillic acid) and two alicyclic compounds (cyclohexanecarboxylic acid and cyclohexanol) were detected by thin-layer, high-performance liquid, and gas chromatography and mass spectrometry. The addition of 1% glucose and peptone in a synthetic medium stimulated growth of the strain but slowed down DDV degradation. The presence of 0.1% yeast extract increased both cell growth and DDV degradation. The growth yield in defined medium was 151.5 g (dry weight) of cells per mol of DDV utilized. Characterization of the strain indicated that it was distinct from known Fusobacterium and Clostridium species. The bacterium was easily induced to form protoplasts after treatment with either penicillin or lysozyme. The frequencies of protoplast formation and regeneration in the strain were 94 and 18%, respectively.

Animals↗
PubMed1988

Cloning of the cellulase gene from Ruminococcus albus and its expression in Escherichia coli.

The gene for cellulase from Ruminococcus albus F-40 was cloned in Escherichia coli HB101 with pBR322. A 3.4-kilobase-pair HindIII fragment encoding cellulase hybridized with the chromosomal DNA of R. albus. The Ouchterlony double-fusion test gave a single precipitation line between the cloned enzyme and the cellulase from R. albus. The size of the cloned fragment was reduced by using HindIII and EcoRI. The resulting active fragment had a size of 1.9 kilobase pairs; and the restriction sites EcoRI, BamHI, PvuII, EcoRI, PvuII, and HindIII, in that order, were ligated into pUC19 at the EcoRI and HindIII sites (pURA1). Cellulase production by E. coli JM103(pURA1) in Luria-Bertani broth was remarkably enhanced, up to approximately 80 times, by controlling the pH at 6.5 and by reducing the concentration of NaCl in the broth to 80 mM.

Aerobiosis↗
PubMed1988

Escherichia coli Spheroplast-Mediated Transfer of pBR322 Carrying the Cloned Ruminococcus albus Cellulase Gene into Anaerobic Mutant Strain FEM29 by Protoplast Fusion.

Intergeneric protoplast fusion between Escherichia coli HB101 with pBR322 carrying the cloned o-(carboxymethyl)cellulase (CMCase) gene of Ruminococcus albus (Pro Leu Ap Km) and an anaerobic mutant strain, FEM29 (Trp His Ap Km), with dehydrodivanillin-degrading activity was performed in the presence of 40% polyvinyl alcohol 300 under aerobic and anaerobic conditions to transfer the cloned cellulase gene into the mutant. The mutant FEM29 had a unique property. When it was incubated in liquid medium with 1% glucose and sucrose, protoplasts could be produced autogenously and regenerated on the agar slant. E. coli spheroplasts formed from a plasmid-amplified overnight culture after 10 min of treatment with lysozyme (20 mug/ml) in a hypertonic solution (0.01 M Tris hydrochloride [pH 7.5], 0.4 M mannitol). Protoplast regeneration rates of FEM29 and HB101 were 30 and 83%, respectively, on the agar-yeast extract medium. Ap Km fusants were obtained at high frequency: 1.7 x 10 anaerobically and 8.2 x 10 aerobically. These fusants showed 23 to 57% of CMCase and dehydrodivanillin-degrading activities, respectively, as compared with parental strains. All the fusants isolated were gram-negative rods with main phenotypes such as urease and catalase activities as in HB101 and esterase and chymotrypsin activities as in FEM29. Southern hybridization experiments suggested that pBR322 with the cloned CMCase gene existed autonomously in the fusant cells. This is the first report describing transfer of pBR322 with a cloned cellulase gene into an anaerobic mutant by polyvinyl alcohol-mediated fusion with an E. coli spheroplast.

Journal Article↗
PubMed1988

Identification and characterization of an estrogen-inducible glycoprotein (uterine secretory protein-1) synthesized and secreted by rat uterine epithelial cells.

An estrogen-inducible 97-kilodalton (kDa) secretory glycoprotein, designated as uterine secretory protein (USP)-1, synthesized by rat uterine epithelial cells was identified and characterized. Uterine luminal fluid (ULF) proteins were labeled by direct administration of [35S]methionine into uterine lumen of rats. Incorporation of [35S]methionine into ULF proteins was negligible in ovariectomized rats. However, when 17 beta-estradiol (E2) was administered in the ovariectomized rat as sc paraffin pellets, a marked increase of [35S]methionine incorporation was noted after 2 days of treatment, showing that the de novo synthesis and secretion of ULF proteins were induced by E2. Six estrogen-inducible polypeptides (130, 110, 97, 65, 42, and 39 kDa) were identified in the analysis of the labeled ULF proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography. Four of these polypeptides (110, 65, 42, and 39 kDa) were adsorbed by the immobilized antibody against rat serum proteins, indicating that these polypeptides are antigenically similar to serum proteins. Two polypeptides (130 and 97 kDa) not adsorbed by the antibody column were suggested to be uterine-specific secretory proteins. The 97-kDa protein (USP-1) was further purified by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis and electroelution. Reactivity of purified USP-1 to various lectins and carbohydrate composition analysis suggested that USP-1 possesses biantennary N-linked complex-type carbohydrate chain with fucose. Rabbit polyclonal antibody which can specifically immunoprecipitate [35S]methionine-labeled USP-1 was developed. Dot blot enzyme immunoassay showed that 4-day E2-treated ULF contains 78.3 +/- 24.1 (+/- SE) micrograms USP-1/mg protein. Immunohistochemical staining of rat uterine tissue showed that this protein localized only in the epithelial cells treated with estrogen (E2 and diethylstilbestrol). Testosterone, progesterone, and dexamethasone failed to induce synthesis and secretion of USP-1 as assessed by dot blot enzyme immunoassay, immunoprecipitation of [35S]methionine-labeled ULF proteins, and immunohistochemical staining of uterine tissue. The present result, for the first time, revealed that estrogen can induce synthesis and secretion of specific secretory protein which could be the useful marker to analyze the molecular mechanism of estrogen action in rat uterine epithelial cells.

Animals↗