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Biomedical subjects

S Shimizu

Publications and source records attributed to S Shimizu.

At least 505 records · Page 28Linked to original sources
PubMed1995

Purification and characterization of an ATP-dependent amidohydrolase, N-methylhydantoin amidohydrolase, from Pseudomonas putida 77.

N-Methylhydantoin amidohydrolase, an ATP-dependent amidohydrolase involved in microbial degradation of creatinine, was purified 70-fold to homogeneity, with a 62% overall recovery, and was crystallized from Pseudomonas putida 77. The enzyme has a relative molecular mass of 300,000. It is a tetramer of two identical small subunits (M(r) 70,000) and two identical large subunits (M(r) 80,000). The enzyme requires ATP for the amidohydrolysis of N-methylhydantoin and vice versa. Mg2+, Mn2+ or Co2+, and K+, NH4+, Rb+ or Cs+, were absolutely required concomitantly for the enzyme activity as divalent and monovalent cations, respectively. The Km and Vmax values for N-methylhydantoin were 32 microM and 9.0 mumol.min-1.mg protein-1. The hydrolysis of amide compounds and coupled hydrolysis of ATP were observed with hydantoin, DL-5-methylhydantoin, glutarimide and succimide in addition to N-methylhydantoin. 2-Pyrrolidone, 2-oxazolidone, delta-valerolactam, 2,4-thiazolidinedione, 2-imidazolidone, D-5-oxoproline methyl ester, DL-5-oxoproline methyl ester, and naturally occurring pyrimidine compounds, i.e. dihydrouracil, dihydrothymine, uracil, and thymine, effectively stimulated ATP hydrolysis by the enzyme without undergoing detectable self-hydrolysis.

Adenosine Triphosphate↗
PubMed1995

Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium.

The occurrence of a hitherto unknown pathway involving the action of two enzymes, a nitrile hydratase and an amidase for the biosynthesis of indole-3-acetic acid was discovered in phytopathogenic bacteria Agrobacterium tumefaciens and in leguminous bacteria Rhizobium. The nitrile hydratase acting on indole-3-acetonitrile was purified to homogeneity through only two steps from the cell-free extract of A. tumefaciens. The molecular mass of the purified enzyme estimated by HPLC was about 102 kDa, and the enzyme consisted of four subunits identical in molecular mass. The enzyme exhibited a broad absorption spectrum in the visible range with absorption maxima at 408 nm and 705 nm, and it contained cobalt and iron. The enzyme stoichiometrically catalyzed the hydration of indole-3-acetonitrile into indole-3-acetamide with a specific activity of 13.7 mol per min per mg and a Km of 7.9 microM.

Journal Article↗
PubMed1995

Ca2+ release from mitochondria induces cytosolic enzyme leakage in anoxic liver.

The mechanisms of elevation of cytosolic Ca2+ concentration during anoxia causing cytosolic enzyme leakage were studied in perfused rat liver. Before anoxia the Ca2+ contents in mitochondria of perfused rat liver were varied by addition of noradrenaline and glucagon, lowering Ca2+ concentration in the perfusion medium, and addition of Ca2+ channel blocker, either singly or in combination. The amount of cytosolic enzyme leakage during anoxic perfusion positively correlated with the mitochondrial Ca2+ content just before anoxia and also with Ca2+ release from mitochondria during anoxia. However, the amount of cytosolic enzyme leakage during anoxic perfusion did not correlate with the Ca2+ concentration in the perfusion medium and microsomal Ca2+ content. These data support the hypothesis that in anoxic liver, the cytosolic Ca2+ concentration is elevated to a pathological level mainly by Ca2+ release from mitochondria, causing the cytosolic enzyme leakage.

Animals↗
PubMed1995

Purification and characterization of N-carbamoyl-L-amino acid amidohydrolase with broad substrate specificity from Alcaligenes xylosoxidans.

N-Carbamoyl-L-amino acid amidohydrolase was purified to homogeneity for the first time from Alcaligenes xylosoxidans. The enzyme showed high affinity toward N-carbamoyl-L-amino acids with long-chain aliphatic or aromatic substituents, and hydrolyzed those with short-chain substituents quite well. The enzyme hydrolyzed N-formyl- and N-acetylamino acids quickly and very slowly, respectively. The enzyme did not hydrolyze beta-ureidopropionate and ureidosuccinate. The relative molecular mass of the native enzyme was about 135,000 and the enzyme consisted of two identical polypeptide chains. The enzyme activity was significantly inhibited by sulfhydryl reagents and required the following divalent metal ions: Mn2+, Ni2+ and Co2+.

Alcaligenes↗
PubMed1995

Heat-inducible expression system for a foreign gene in cultured tobacco cells using the HSP18.2 promoter of Arabidopsis thaliana.

A system for the controlled expression of a foreign gene in cultured tobacco cells (Nicotiana tabacum, BY2) by temperature shift was constructed. A 925-base-pair (bp) DNA fragment containing the 5'-flanking region of a low-molecular-mass heat-shock protein gene (HSP18.2) of Arabidopsis thaliana was inserted upstream of the beta-glucuronidase reporter gene (GUS). The resulting HSP18.2-GUS construct was introduced into BY2 cells by electroporation or Agrobacterium-mediated transformation. Transient expression of the HSP18.2 promoter in protoplasts was very low regardless of the heat shock. Although expression of the HSP18.2-GUS chimeric gene in the stable transformants of BY2 was hardly detected in culture at 25 degrees C, the expression increased rapidly on the transcriptional level when the incubation temperature was shifted to 35-37 degrees C. The optimal temperature for heat-shock induction was 37 degrees C. After a 2-h incubation at 37 degrees C, GUS activity was about 1000-fold greater than that before heat shock. The amount of GUS mRNA was maximum 2 h after heat shock, and then decreased gradually.

Agrobacterium tumefaciens↗
PubMed1995

Purification and characterization of dihydroorotase from Pseudomonas putida.

Dihydroorotase was purified to homogeneity from Pseudomonas putida. The relative molecular mass of the native enzyme was 82 kDa and the enzyme consisted of two identical subunits with a relative molecular mass of 41 kDa. The enzyme only hydrolyzed dihydro-L-orotate and its methyl ester, and the reactions were reversible. The apparent Km and Vmax values for dihydro-L-orotate hydrolysis (at pH 7.4) were 0.081 mM and 18 mumol min-1 mg-1, respectively; and those for N-carbamoyl-DL-aspartate (at pH 6.0) were 2.2 mM and 68 mumol min-1 mg-1, respectively. The enzyme was inhibited by metal ion chelators and activated by Zn2+. However, excessive Zn2+ was inhibitory. The enzyme was inhibited by sulfhydryl reagents, and competitively inhibited by N-carbamoylamino acids such as N-carbamoylglycine, with a Ki value of 2.7 mM. The enzyme was also inhibited non-competitively by pyrimidine-metabolism intermediates such as dihydrouracil and orotate, with a Ki value of 3.4 and 0.75 mM, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates and that dihydroorotase plays a role in the control of pyrimidine biosynthesis.

Chelating Agents↗
PubMed1995

Effects of mild Trendelenburg on central hemodynamics and internal jugular vein velocity, cross-sectional area, and flow.

Despite widespread use of the Trendelenburg position, its autotransfusion effect remains controversial. Additionally, its adverse effect on cerebral circulation is not generally appreciated. The effects of a 10 degrees head-down tilt on central hemodynamics and flow through the internal jugular vein (IJV) were examined in ten healthy volunteers. Left ventricular end-diastolic volume (LVEDV) and cardiac output (CO) were calculated from two-dimensional echocardiograms. IJV velocity and cross-sectional area were determined by the pulsed Doppler system. Measurements were made with the subjects in the supine position and at 1 minute and 10 minutes after tilting. A significant increase (16%) in CO followed by the increase in LVEDV was observed at 1 minute after tilting, although these changes disappeared after 10 minutes of tilting. Mean arterial pressure at the heart level did not change during the maneuver. The IJV velocity decreased whereas the IJV cross-sectional area increased at 1 minute after tilting, but both factors returned to control level at 10 minutes after tilting. As a result, calculated IJV blood flow was unchanged throughout the period of tilt. Therefore, the mild trendelenburg position produces a transient autotransfusion effect in normovolemic patients. Our data also suggest that the Trendelenburg produces no adverse effect on cerebral circulation in patients with normal cerebral autoregulation.

Adult↗
PubMed1995

Increased F1/GAP-43 mRNA accumulation in gerbil hippocampus after brain ischemia.

To assess whether ischemia could induce GAP-43 mRNA expression, we performed in situ hybridization in gerbil brains that had been subjected to 5 min of global ischemia. In control dentate granule cells, little hybridization was detected in contrast to the intense signal generated by pyramidal neurons of the adult hippocampal formation. After ischemia, we detected a robust GAP-43 signal over hippocampal granule cells at 3 h of reperfusion, persisting through 7 days, and disappearing by 14 days. This demonstrated GAP-43 gene induction after ischemia, and suggests that GAP-43 may be involved in reactive events, including fiber sprouting and synaptic reorganization, that follow ischemia.

Animals↗
PubMed1995

Early gastric cancer: its surveillance and natural course.

The present status of screening for gastric cancer, and the natural course of the disease, are the concerns of this paper. The development of new diagnostic modalities has led to an increase in the percentage of early gastric cancers detected. Improvements in technology, and alterations in nutrient intake, are linked to the reduction in the mortality from gastric cancer that has taken place. Observation of the natural course of gastric cancer development shows that "early gastric cancer" is genuinely the early stage of gastric cancer, and not a term for a different disease phenomenon. A "malignant cycle" was shown to be implicated in the process of cancer development.

Endoscopy, Gastrointestinal↗
PubMed1995

Quantitative detection of ultraviolet light-induced photoproducts in mouse skin by immunohistochemistry.

UVB-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4)photoproducts [(6-4)photoproducts] in mouse skin DNA were quantitatively measured using an immunohistochemical approach with a computer-aided color image analyzer. The skins of the C3H/HeN mice were irradiated with ultraviolet B (UV-B, 280-320 nm), and processed to give conventional formalin-fixed, paraffin-embedded histologic sections. Routine immunohistochemistry clearly demonstrated a dose-dependent induction of both photoproducts. CPDs were detectable at doses > or = 125 J/m2, while for (6-4)photoproducts, the minimal dose at which they were detectable was 250 J/m2 in the present study. A time course study showed that the repair of (6-4)photoproducts was more rapid than that of CPDs, and that epidermal cells had a higher capacity for their removal than dermal cells. About half of the (6-4)photoproducts were excised within the first 24 h after the irradiation, and the process was essentially complete by 72 h. In contrast, there was no apparent removal (less than 10%) of CPDs in the first 24 h and they only completely disappeared from the epidermal cells at 120 h after irradiation. The effect of DNA dilution due to increased turnover of epidermal cells after UV-B irradiation was evaluated by quantitative immunohistochemical measurement of the time course of bromodeoxy-uridine (BrdUrd) incorporated into nuclei at 2 days post irradiation when the proliferation reaches a peak. The removal of photoproducts was more marked than the decrease in BrdUrd staining. Our results suggest that mouse skin cells can repair both (6-4)photoproducts and CPDs, but with considerably lower efficiency, especially in the latter case, then human or monkey skin cells.

Animals↗
PubMed1995

Correlation between a sandwich ELISA and an in-vitro bioassay for erythropoietin in human plasma.

A sandwich-type enzyme-linked immunosorbent assay (ELISA) for human erythropoietin (EPO) using two anti-EPO monoclonal antibodies has been compared with an in-vitro EPO bioassay based on CFU-E colony formation in fetal mouse liver cell cultures. In normal subjects and non-uraemic anaemic patients the plasma EPO values estimated with the ELISA correlated well with those by the bioassay, and also inversely with the values of blood Hb, PCV and RBC counts. Dose-response curves for plasma and standard recombinant human EPO in the ELISA were parallel to each other. These results further confirm the validity for the ELISA in measuring circulating human EPO.

Adult↗
PubMed1995

A topographical and ultrastructural study of sensory trigeminal nerve endings in the rat temporomandibular joint as demonstrated by anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP).

To extend our previous light microscopic observations concerning the distribution of trigeminal sensory nerves in the synovium of the rat temporomandibular joint, we investigated the detailed distribution and fine structure of sensory nerve endings at the light and electron microscopic level by the anterograde transport method using wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injected into the trigeminal ganglion. At the light microscopic level, HRP-labeled nerve fibers were observed in the joint capsule and peripheral portion of the disc. The anterior portion of the disc was more densely innervated than the posterior portion, while no nerves were found in the central portion. At the electron microscopic level, HRP reaction products were observed intra-axonally in the thinly myelinated (A delta) and unmyelinated (C) axons in the anterior portion of the joint capsule, and were also localized in the extracellular space surrounding the unmyelinated fibers and terminals. In the subsynovial layer of the synovial membrane, the majority of labeled axons located near blood vessels or among the collagenous fibrils were covered by Schwann cell sheaths, although some naked axon terminals without sheaths were also found. These unsheathed terminals contained mitochondria, small clear vesicles, and large granular vesicles, and were close to the synovial A and/or B cells near the joint cavity. The minimum distance between the terminals and synovial cells was 75 nm. This is the first demonstration of trigeminal sensory nerve terminals close to synovial lining cells or joint cavity and suggests that neuropeptides such as substance P may be released close to the synovial lining cells or joint cavity.

Animals↗
PubMed1995

A new enzymatic method for the measurement of creatinine involving a novel ATP-dependent enzyme, N-methylhydantoin amidohydrolase.

A new enzymatic method for the measurement of serum and urine creatinine is described. The method is based on a novel microbial creatinine degradation pathway via N-methylhydantoin [Shimizu et al., Clin. Chim. Acta, 185, 241-252 (1989)]. By using two novel enzymes, N-methylhydantoin amidohydrolase and N-carbamoylsarcosine amidohydrolase, as key enzymes, coupled with a colorimetric procedure for hydrogen peroxide detection, the creatinine level can be measured. The results obtained for human serum and urine show good correlation with those obtained by a standard chemical method based on the Jaffe reaction. The new method is simple and specific, and shows excellent sensitivity and reliability.

Adenosine Triphosphate↗
PubMed1995

Proliferative response and cytokine production of bovine peripheral blood mononuclear cells induced by the superantigens staphylococcal enterotoxins and toxic shock syndrome toxin-1.

The potential of staphylococcal enterotoxin A (SEA), B (SEB), C(SEC) and toxic shock syndrome toxin-1 (TSST-1) to act as superantigens by inducing polyclonal T-cell mitogenesis and cytokine production was tested on bovine peripheral blood mononuclear cells (PBMC). These four toxins were capable of inducing strong proliferative response of PBMC from calves over a broad dosage range (1 pg/ml to 1 microgram/ml) in vitro. The toxin-activated blast cells consisted of both CD4+ T-cells and CD8+ T-cells, but the T-cell proliferation depended upon the presence of monocytes. Treatment of monocytes with monoclonal antibody to major histocompatibility complex class II antigens substantially inhibited the toxin-induced T-cell proliferative response, but paraformaldehyde-fixation did not abrogate the accessory function. SEA, SEB, SEC and TSST-1, all induced the in vitro release of interleukin-2, interferon gamma and tumor necrosis factor alpha in a dose dependent manner. The results indicate that SEA, SEB, SEC and TSST-1 are capable of acting as superantigens by stimulating bovine T-cells as shown in the human and murine systems. The possible implications of these toxins in the immunopathogenesis of bovine mastitis caused by the infection with Staphylococcus aureus are discussed.

Animals↗
PubMed1995

[Age-related memory impairment and hippocampal damage in ddY male mice].

The relationship between behavioral change and hippocampal lesion was studied in ddY male mice, at 4.5, 20, 40 and 60 weeks of age. In passive avoidance response, the aging mice (40 and 60 weeks of age) showed shorter latency than young mice (4.5 weeks of age). The degenerated pyramidal cells were more numerous after 40 weeks of age in the CA3 than CA1. The mean incidence of the degenerated pyramidal cells in the CA3 was 20.8% at 60 weeks of age. In 71.9% of mice, impairment of passive avoidance response was associated well with hippocampal lesions. These results indicate that the hippocampus plays some role in the memory in mice. The ddY mice may be possible to use as models for reversible hippocampal lesion.

Aging↗