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Biomedical subjects

M Shimizu

Publications and source records attributed to M Shimizu.

At least 505 records · Page 28Linked to original sources
PubMed1997

Phosphatidylethanol inhibits phosphatidylinositol-phospholipase C activity in a competitive manner with phosphatidylinositol-4,5-bisphosphate.

The effect of phosphatidylethanol (PEth) on phosphatidylinositol-phospholipase C (PLC) activity was investigated in rat cerebral cortex. PEth decreased PLC activity in both the membrane and the cytosol of the cortex in a concentration-dependent manner, varying from 6 to 400 microM, and PLC activity was almost entirely inhibited at concentrations of PEth over 200 microM (90% inhibition). The IC50 of PEth in the cytosol was 25.2 microM and was 22.1 microM in the membrane. Preincubation of the cytosol with anti-PLC-beta 1, anti-PLC-gamma 1 or anti-PLC-delta 1 antibodies did not prevent the decrease in PLC activity. These results suggest that PEth caused the decrease in PLC activity without isozyme specificity. The sensitivity of PLC to Ca2+ in the cytosol and membrane was not changed by PEth, suggesting that PEth may act on PLC at a site different from that of Ca2+ activation. In higher concentrations of the PLC substrate, PEth did not inhibit PLC activity, indicating that PEth inhibited PLC activity in a substrate competitive manner. Neomycin, which binds to negatively charged phospholipids such as phosphatidylinositol-4,5-bisphosphate (PIP2) and thus causes inhibition of PLC activity, attenuated the PEth-induced decrease in PLC activity. This result suggests that the inhibitory action of PEth on PLC may be related to the fact that PEth is a negatively charged phospholipid similar to PIP2.

Animals↗
PubMed1997

Fluorimetric assay of 1 alpha,25-dihydroxyvitamin D3 in human plasma.

The first fluorimetric method for assaying plasma 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is described. Lipid extracts from human plasma were successively purified by a Bond Elut NH2 column and a normal-phase HPLC column, and the 1,25-(OH)2D3 fractions obtained were fluorescence-labelled with 4-[2-(6,7-dimethoxy-4-methtyl-3-oxo-3,4-dihydroquinoxalyl)et hyl]-1, 2,4-triazoline-3,5-dione (DMEQ-TAD). The fluorescent adducts of 1,25-(OH)2D3 were analyzed by HPLC on a reversed-phase column, after extensive elimination of the degradation products of excess reagents, by a Bond Elut PSA column followed by a normal-phase HPLC column. The intra- and inter-assay coefficients of variation were within 10% in both assays. The analytical recoveries of standard 1,25-(OH)2D3 added to plasma were quantitative. The present fluorimetric assay was compared with the established radioreceptor assay (RRA) used in measuring plasma 1,25-(OH)2D3 concentration. The regression analysis of the data afforded a relationship y = 1.049x - 2.657 and a correlation coefficient (r) of 0.900.

Adult↗
PubMed1997

Transcriptional repression at a distance through exclusion of activator binding in vivo.

The yeast repressor Rme1p acts from distant binding sites to block transcription of the chromosomal IME1 gene. Rme1p can also repress the heterologous CYC1 promoter when Rme1p binding sites are placed 250-300 bp upstream of CYC1 transcriptional activator binding sites (UAS1 and UAS2). Here, in vivo footprinting studies indicate that Rme1p acts over this distance by preventing the binding of the CYC1 transcriptional activators to UAS1 and UAS2. Inhibition of activator binding by Rme1p has the same genetic requirements as repression: both depend upon sequences flanking the Rme1p binding sites and upon Rgr1p and Sin4p, two subunits of the RNA polymerase II-associated Mediator complex that are required for normal nucleosome density. Thus Rme1p may alter chromatin to prevent binding of transcriptional activators to distant DNA sequences.

CCAAT-Binding Factor↗
PubMed1997

Effect of capsianoside, a diterpene glycoside, on tight-junctional permeability.

Previous work (Hashimoto et al., (1994) Biosci. Biotech. Biochem. 58, 1345) revealed that a sweet pepper extract enhanced the tight-junctional (TJ) permeability of a human intestinal Caco-2 cell monolayer. In the present study, the substance which modulated the TJ permeability was chromatographically purified from the extract. The active substances were identified as capsianosides A-F, diterpene glycosides. Treatment of the cells with capsianoside F, the most active compound, decreased the cellular G-actin content by 40% and increased the F-actin content by 16%. The effect of capsianoside F was significantly suppressed by disturbing the cytoskeletal structure with cytochalasin D at a low dose (50 ng/ml). These results suggest that capsianosides affected the cytoskeletal function by modulating the reorganization of actin filaments, by which the TJ structure and permeability were changed. The possible involvement of a PKC inhibition in the mechanism of an increase in TJ permeability is also suggested.

Actins↗
PubMed1997

Preliminary X-ray studies of a new crystal form of PHO4-DNA complex.

A new crystal form of PHO4 DNA-binding domain-DNA complex was obtained by the truncation of the N-terminal segment. This diffracts to a higher resolution than any forms reported previously. The present crystal belongs to orthorhombic space group P2(1)2(1)2(1), with unit-cell dimensions a=53.51, b=68.30, c= 108.77 A. The crystal contains one protein dimer-DNA complex in the asymmetric unit.

Journal Article↗
PubMed1997

High-performance liquid chromatographic separation of microcystins derivatized with a highly fluorescent dienophile.

Microcystins are potent hepatotoxins produced by cyanobacteria, and are also tumor promoters as well as potent inhibitors of the catalytic subunits of protein phosphatases 1 and 2A. In order to establish a physicochemical method for individual detection and determination of trace amounts of microcystins, we developed a derivatization method for fluorescence (FL) and chemiluminescence (CL) detection, in which a highly fluorescent dienophile, DMEQ-TAD (4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl) ethyl]-1,2,4-triazoline-3,5-dione), was used as the labeling reagent. DMEQ-TAD reacted smoothly with the conjugated diene of the Adda moiety to give 2 stereoisomers of the adducts. As a result of the extensive experiments, the following reaction conditions were optimized for the labeling: sample amount, 10 micrograms; reaction solvent, DMF:acetonitrile (1:1); reaction time, 15 minutes; reaction temperature, 70 degrees C; amount of DMEQ-TAD used relative to that of microcystin, 80 equivalent. The resulting 6 adducts from microcystins-LR, -YR, and -RR can be separated from one another using the following reversed phase HPLC conditions in combination with a clean-up using ODS silica gel: column, Cosmosil 5C18-AR (150 x 4.6 I.D. mm); mobile phase, methanol:0.05M phosphate buffer (pH 3) (1:1); flow rate, 1.0 ml/min; detection, FL lambda ex 370 nm, lambda em 440 nm. The detection limits of the DMEQ-TAD derivatives were estimated to be 100 and 500 pg for LR, and 65 and 2,500 pg for RR using FL and CL detections, respectively; and the detection behavior was different from that of the Dns-Cys derivatives, which were more sensitive to CL than FL.

Bacterial Toxins↗
PubMed1997

In vivo but not in vitro stimulations rescue of the defective class switching to IgG or IgA in B-cells of immunodeficiency with hyper IgM.

In an X-linked immunodeficiency with hyper IgM (X-HIM), several mutations of genes coding for CD40 ligand are responsible for B-cell defects. However, it is controversial whether the defective class switching from IgM to IgG or IgA but not IgE is rescued by in vitro stimulations or not. In six X-HIM patients, signaling through CD40 induced IgE but not IgG or IgA secretion in vitro. When transferred into SCID mice, however, B-cells of X-HIM patients secreted as much IgG and IgA as those of healthy controls. Moreover, sIgG and sIgA expressions were also induced by the in vivo stimulation. These results support that B-cells of X-HIM patients are defective of in vitro class switching from IgM to IgG or IgA and show that the in vivo stimulations rescue the defective class switching and suggest that the class switching of IgE and that of IgG or IgA might be regulated quite differently.

Animals↗
PubMed1997

Disopyramide concentrations in human plasma and saliva: comparison of disopyramide concentrations in saliva and plasma unbound concentrations.

OBJECTIVE: This study was performed to investigate whether it is possible to use saliva instead of blood usually used for therapeutic drug monitoring (TDM) of disopyramide. METHODS: Six healthy male volunteers ingested 200 mg of disopyramide base, and the disopyramide concentrations in saliva and plasma (total and unbound) were determined by the HPLC. RESULTS: Disopyramide concentration-time profiles for the saliva were nearly equal to those for the plasma unbound concentrations. A large variation for absorption time of the drug was observed among the subjects. Disopyramide concentrations (Cs) in saliva did not correlated well with plasma total concentrations (Cp), r = 0.799, but did well with unbound concentrations (Cpu), r = 0.969, for the 3-12 h period on the elimination phase. The mean ratio of disopyramide concentrations in the saliva against the plasma unbound concentrations was almost constant (1.02(0.10), CV = 9.7%) for the period. The pharmacokinetic parameters (tmax, t1/2, AUC, AUMC and MRT values) for disopyramide calculated from the saliva data were nearly equal to those from the unbound data. CONCLUSION: Disopyramide concentrations in saliva correlated well with plasma unbound concentrations on the elimination phase.

Adult↗
PubMed1997

The role of granzyme B-expressing CD8-positive T cells in apoptosis of keratinocytes in lichen planus.

Epidermal basal cell injury with colloid body formation is a characteristic feature of lichen planus. Infiltrated cells are thought to be responsible for the epidermal injury. Ultrastructural findings of colloid bodies are typical of apoptosis. Granzymes in cytotoxic T lymphocytes are involved in apoptosis probably together with perforin. Based on this background, we analyzed the role of granzyme B in the mechanisms of epidermal injury in lichen planus. On electron microscopy, basal and suprabasal cells showed condensed chromatin and fragmented nuclei which are typical morphological features of apoptosis. Nuclei of colloid bodies were positively stained by the in situ nick end labeling technique indicating that colloid bodies are subsequently formed in the process of apoptosis. Immunohistochemical staining showed CD8-positive infiltrating cells to contain granzyme B. Cells undergoing exocytosis also contained granzyme B. By immunoelectron microscopy, granzyme B molecules were observed to be secreted from a lymphocyte to an apoptotic keratinocyte. These findings suggest that granzyme B-positive CD8 cells seem to induce apoptosis of keratinocytes in lichen planus.

Adult↗
PubMed1997

Immunochemical and immunohistochemical study of the 27- and 29-kDa calcium-binding proteins and related proteins in the porcine tooth germ.

Our previous report identified 27- and 29-kDa calcium-binding proteins in porcine immature dental enamel. In this study we revealed that the N-terminal amino acid sequences of the two proteins were identical: LLANPXGXIPNLARGPAGRSRGPPG. The sequence matches a portion of the amino acid sequence of the porcine sheath protein, sheathlin. Porcine tooth germs were investigated immunochemically and immunohistochemically using specific antibodies raised against synthetic peptide that included residues 13-25 of this sequence. The affinity-purified antibodies reacted with several proteins extracted from newly formed immature enamel in immunochemical analyses, especially protein bands migrating at 62, 35-45, 29, and 27 kDa in SDS-polyacrylamide gels. The largest protein detected was a weak band near 70 kDa. In immunochemical analyses of proteins extracted from the inner (old) immature enamel, the antibody reacted faintly with the 27- and 29-kDa proteins. In immunohistochemical preparations, the Golgi apparatus and secretory granules of the secretory ameloblast, and the surface layer of immature enamel showed immunoreactivity. The immunoreactivity of immature enamel just beneath the secretory face of the Tomes' process was intense. No immunoreactivity was found in the Golgi apparatus of the maturation ameloblast. These results suggest that the 70-kDa protein, whose degradation might be very fast, is the parent protein of the 27- and 29-kDa proteins.

Ameloblasts↗
PubMed1997

Fine structure and distribution of lymphatic vessels in the human dental pulp: a study using an enzyme-histochemical method.

The existence of lymphatic vessels in the human dental pulp and their distribution were established by light and electron microscopy using an enzyme-histochemical method. The distinction between lymphatic and blood vessels was made by light microscopy on cryostat sections of undecalcified and decalcified teeth using 5'-nucleotidase(5'-Nase)-alkaline phosphatase double staining. On the tissue surface, 5'-Nase-positive lymphatic vessels were highlighted with good contrast and resolution by backscattered electron imaging using scanning electron microscopy. By transmission electron microscopy, dense granular precipitations resulting from the 5'-Nase reaction were seen on the luminal surface of the lymphatic endothelial cells as well as in the area at the basal side, but were absent in the blood vessels. These lymphatic vessels were more numerous in the central part than in the peripheral odontoblastic layer.

5'-Nucleotidase↗
PubMed1997

Roles of a neuronal cell-surface molecule, neuropilin, in nerve fiber fasciculation and guidance.

Neuropilin is a cell-surface glycoprotein that was first identified in Xenopus tadpole nervous tissues and then in chicken and mouse. The primary structure of neuropilin is highly conserved among these vertebrate species. The extracellular part of the molecule is composed of three domains referred to as a1/a2, b1/b2, and c, each of which is expected to be involved in molecular and/or cellular interactions. Neuropilin can mediate cell adhesion by heterophilic molecular interaction. In all vertebrate species examined, the neuropilin protein is restricted to axons of particular neuron classes, and at stages when axon growth is active. The gain and loss of function of neuropilin in developing mouse embryos causes defasciculation and incorrect sprouting of nerve fibers. These findings suggest that neuropilin serves in a variety of neuronal cell interactions by binding to a variety of molecules, and that it plays essential roles in nerve fiber fasciculation and guidance.

Animals↗
PubMed1997

Interleukin-2 modulates the responsiveness to angiotensin II in cultured vascular smooth muscle cells.

Preincubation with interleukin-2 (IL-2), a T cell-derived cytokine, enhanced the increase in intracellular Ca2+ ([Ca2+]i) induced by angiotensin II (AII) in vascular smooth muscle cells (VSMC). IL-2 itself did not affect the basal [Ca2+]i level or the maximal response of [Ca2+]i increase induced by AII. Furthermore, IL-2-induced enhancement was not observed in the absence of extracellular Ca2+, suggesting that IL-2 enhances Ca2+ influx induced by AII. IL-2 also enhanced the stimulation of DNA synthesis induced by AII, although IL-2 alone did not stimulate DNA synthesis. Genistein, an inhibitor of protein tyrosine kinases, significantly inhibited IL-2-induced enhancement of both Ca2+ influx and DNA synthesis induced by AII. A neutralizing antibody against heparin-binding epidermal growth factor-like growth factor (HB-EGF) partially inhibited IL-2-induced enhancement of DNA synthesis induced by AII. These findings suggest that autocrine HB-EGF is partially involved in the mechanism of IL-2-induced enhancement of DNA synthesis. On the other hand IL-2 stimulated both glycosaminoglycan (GAG) and prostacyclin syntheses and enhanced the stimulation of both GAG and prostacyclin syntheses induced by AII. Therefore, IL-2 may play important roles in the pathogenesis of atherosclerosis and vascular disease by modulating the responsiveness to AII in VSMC.

Angiotensin II↗