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Biomedical subjects

K Matsushita

Publications and source records attributed to K Matsushita.

At least 19 recordsLinked to original sources
PubMed1998

Marked, sustained expression of a novel 150-kDa oxygen-regulated stress protein, in severely ischemic mouse neurons.

The 150-kDa oxygen-regulated protein (ORP150) first was described with reference to the central nervous system in cultured astrocytes subjected to dense hypoxia. Subsequently its transcript was found in macrophages within human aortic atherosclerotic plaques, suggesting a role in protecting cells under hypoxic stress. In a mouse model of permanent focal brain ischemia, we aimed to elucidate the constitutive cellular localization in vivo of ORP150 in the central nervous system as well as the sequential alteration in its mRNA and protein expression during this severe ischemic insult. Immunohistochemical study demonstrated that ORP150 protein normally is present predominantly in neurons. The 78-kDa glucose-regulated protein, which is another well-known stress protein retained in the endoplasmic reticulum, also was stained in neurons. During the first 3 h after ischemia, ORP150 antigenicity was markedly enhanced in severely damaged neurons, while the amount of the glucose-regulated protein was decreased. Preceding this change, orp150 mRNA was selectively induced in neurons undergoing postischemic cytoskeletal proteolysis, as early as 1 h after middle cerebral artery occlusion. These results indicated that ORP150 might be regulated by transcriptional level as for many stress proteins, but unlike previously described other stress proteins it was translated in the center of ischemic lesions despite nearly complete energy depletion. In this paper, the biological potentials of ORP150 protein in the setting of brain ischemia in vivo will also be discussed.

Animals↗
PubMed1998

Mutant isolation of the Escherichia coli quinoprotein glucose dehydrogenase and analysis of crucial residues Asp-730 and His-775 for its function.

Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli were obtained and characterized. Of these, significant mutants were further characterized by kinetic analysis after purification or by site-directed mutagenesis to introduce different amino acid substitutions. H775R and H775A showed a pronounced reduction of affinity for a prosthetic group, pyrroloquinoline quinone (PQQ), suggesting that His-775 may directly interact with PQQ. D730N and D730A showed low glucose oxidase activity without influence on the affinity for PQQ, Mg2+, or substrate, but D730R showed reduced affinity for PQQ. The spectrum of tryptophan fluorescence revealed that the local structure surrounding PQQ was not changed by D730N mutation. Based on these data, we assume that Asp-730 may occur close to PQQ and function as a proton (and also electron) donor to PQQ or acceptor from PQQH2. Substitutions of Gly-689, that are located at the end of a unique segment of GDH among homologous quinoprotein dehydrogenases, directed reduction of the affinity for PQQ or GDH activity. Therefore, the unique segment and Asp-730 may play a specific role for GDH, which might be related to the intramolecular electron transfer from PQQ to ubiquinone.

Amino Acid Sequence↗
PubMed1998

Human chorionic gonadotropin (hCG) inhibits cisplatin-induced apoptosis in ovarian cancer cells: possible role of up-regulation of insulin-like growth factor-1 by hCG.

Gonadotropins have been suggested to play a role in the development or progression of ovarian cancer, and we have previously reported the expression of luteinizing hormone/ human chorionic gonadotropin (LH/hCG) receptor in 40% of epithelial ovarian carcinomas. To examine the biological effect of LH/hCG on ovarian cancer cells, apoptosis induced by cisplatin with or without hCG treatment was investigated in 2 ovarian cancer cell lines, OVCAR-3 and SK-OV-3. Stimulation of cell proliferation by hCG was also studied. In addition, to analyze further the mechanism of hCG signaling involved in apoptosis-inhibition, we examined the expression of LH/hCG receptors and the regulation by hCG for apoptosis-inhibitory pathways, such as the bcl-2/bax system and the insulin-like growth factor-1 (IGF-1)/IGF-1 receptor (IGFR) system. hCG did not increase cell proliferation in either cell line. However, hCG treatment suppressed cisplatin-induced apoptosis by 58% in the OVCAR-3 cells, as shown by immunofluorescent staining and quantitation of DNA fragmentation. LH/hCG receptor mRNA was expressed only in OVCAR-3, and no apoptosis-inhibitory effect of hCG was observed in the SK-OV-3 cells that did not express the receptor. In the OVCAR-3 cells, hCG significantly increased mRNA expression of IGF-1, but did not change mRNA levels of bcl-2/bax. Our findings suggest that LH/hCG influences the chemosensitivity of ovarian cancer cells through an apoptosis-inhibitory signal possibly via up-regulation of IGF-1 expression.

Apoptosis↗
PubMed1998

Unique DNA binding mode of the N-terminal zinc finger of transcription factor Sp1.

Transcription factor Sp1 has three tandem repeats of a Cys2His2-type zinc finger motif and specifically binds to GC box DNA. Although the DNA binding mode of the three zinc fingers of Sp1 is predicted to be similar to that of Zif268, this model does not explain the DNA binding property of the N-terminal zinc finger (finger 1). To understand the DNA recognition mode of Sp1, we have performed detailed analyses for the contribution of finger 1 to the high-affinity binding to the GC box DNA and for the interaction mechanism between finger 1 and DNA. Results of electrophoretic analyses using finger-deleted mutants of Sp1 and GC box mutants in the finger-contacting subsite demonstrate that the contribution of finger 1 to the total DNA binding affinity is lower than that of the C-terminal finger 3 but is dispensable for the high-affinity binding. The DNA sequence selectivity of finger 1 at the 3'-portion of the GC box is lower than that of fingers 2 and 3 at the 5'-portion. Alanine scanning mutagenesis in the alpha-helix of finger 1 reveals that Lys-1 immediately preceding the helix is important for the recognition of the two guanine bases, but other putative key amino acids do not affect the DNA binding. These results demonstrate that (1) the contribution of finger 1 to the DNA binding affinity and the sequence selectivity of Sp1 is smaller than that of fingers 2 and 3 and (2) the interaction mechanism between finger 1 and DNA is different from the Zif268 model. DNA interaction of Sp1 finger 1 has also been discussed in connection with that of TFIIIA or WT1.

Amino Acid Sequence↗
PubMed1998

Expression of substance P receptor in the substantia nigra.

Since the substantia nigra receives abundant substance P innervations but lacks clear evidences about a presence of substance P receptors, expressions for mRNA and protein of substance P receptors were investigated in the rat to resolve this mismatch. Expression levels of substance P receptors mRNA in the substantia nigra pars compacta and reticulata were 37.7 and 24.1% of those in the striatum, respectively, by reverse transcription-polymerase chain reaction (RT-PCR). Substance P receptors mRNA was found in dopamine neurons of the substantia nigra pars compacta by single cell RT-PCR. Ca. 90% of dopamine neurons in the substantia nigra pars compacta were immunoreactive to anti-substance P receptor antibody in the colchicine treated rats. These are the first direct evidence for the existence of substance P receptors in dopamine neurons of the substantia nigra pars compacta.

Animals↗
PubMed1998

Intramolecular electron transport in quinoprotein alcohol dehydrogenase of Acetobacter methanolicus: a redox-titration study

Quinohemoprotein-cytochrome c complex alcohol dehydrogenase (ADH) of acetic acid bacteria consists of three subunits, of which subunit I contains pyrroloquinoline quinone (PQQ) and heme c, and subunit II contains three heme c components. The PQQ and heme c components are believed to be involved in the intramolecular electron transfer from ethanol to ubiquinone. To study the intramolecular electron transfer in ADH of Acetobacter methanolicus, the redox potentials of heme c components were determined with ADH complex and the isolated subunits I and II of A. methanolicus, as well as hybrid ADH consisting of the subunit I/III complex of Gluconobacter suboxydans ADH and subunit II of A. methanolicus ADH. The redox potentials of hemes c in ADH complex were -130, 49, 188, and 188 mV at pH 7.0 and 24, 187, 190, and 255 mV at pH 4.5. In hybrid ADH, one of these heme c components was largely changed in the redox potential. Reduced ADH was fully oxidized with potassium ferricyanide, while ubiquinone oxidized the enzyme partially. The results indicate that electrons extracted from ethanol at PQQ site are transferred to ubiquinone via heme c in subunit I and two of the three hemes c in subunit II. Copyright 1998 Elsevier Science B.V.

Journal Article↗
PubMed1998

Detection of human serum antibody to encapsulated strains of Staphylococcus aureus by enzyme-linked immunosorbent assay inhibition test.

A specific and rapid enzyme-linked immunosorbent assay (ELISA) inhibition test was employed for detection of immunoglobulins to Staphylococcus aureus (S. aureus) capsular polysaccharide in human serum. Cap-sular polysaccharide antigens obtained from Smith diffuse (capsular type 2), Reynolds (capsular type 5), or Becker (capsular type 8) strains of S. aureus were added to microplates coated with these strains. Seventy-four patients with open fractures (31 serum samples from those with staphylococcal infections, 10 serum samples from those with non-staphylococcal infections, and 33 serum samples from the non-infected group) and 28 serum samples from healthy controls were then added. The plates were incubated at 37 degreesC for 2 h and the ELISA was performed. The ELISA inhibition assay showed remarkable inhibition with the capsular type 2, 5, and 8 polysaccharides in the 33 serum samples from the non-infected group and in the 28 serum samples from the healthy controls, but low inhibition was observed with the 31 sera with staphylococcal infections. Positive immunoglobulin (Ig)G and IgM titers showed marked inhibition with this assay, but IgA titer were not seen in any samples. These results indicate that the quantitation of human serum antibody against S. aureus capsular polysaccharide by the ELISA inhibition assay is useful for the demonstration of protective activities against S. aureus.

Antibodies, Bacterial↗
PubMed1998

Heterogeneous distribution of K-ras-mutated epithelia in mucinous ovarian tumors with special reference to histopathology.

The carcinogenic process of epithelial ovarian carcinomas is still unknown, and both pathways of de novo carcinogenesis from the surface epithelium and malignant transformation of benign cystadenoma have been suggested. Especially in mucinous ovarian tumors, the transition from benign cystadenomas to tumors of low malignant potential (LMP) or carcinomas has been implicated. To elucidate this possibility, we analyzed the presence or absence of heterogeneity of the K-ras mutation corresponding to the histological heterogeneity in 71 epithelial ovarian tumors, including 31 mucinous tumors. K-ras mutation was identified in nine mucinous tumors (4 of 10 carcinomas, 4 of 14 LMP tumors, and 1 of 7 cystadenomas) and in two nonmucinous carcinomas. Microdissection of multiple sites with reference to their histological appearance showed the heterogeneous distribution of K-ras-mutated epithelia in two of the nine mucinous tumors. One mucinous carcinoma showed K-ras mutation in all of the histologically LMP and malignant portions, but not in benign portions. In another LMP tumor, all of the LMP portions and one of the benign portions showed K-ras mutation, whereas the other two benign portions had no mutation. In the remaining seven mucinous tumors with K-ras mutation, however, there was a homogeneous distribution of K-ras-mutated epithelia irrespective of their histological appearance. These findings suggest that the K-ras mutation occurs during the transformation from benign cystadenomas to LMP or malignant lesions, providing molecular genetic support for the hypothesis of the "adenoma-carcinoma sequence" in some mucinous ovarian tumors, but in other cases an alternative pathway may also be possible.

Adenocarcinoma, Mucinous↗
PubMed1998

IL-2-induced growth of CD8+ T cell prolymphocytic leukemia cells mediated by NF-kappaB induction and IL-2 receptor alpha expression.

The binding of interleukin-2 (IL-2) to its receptor on normal T cells induces nuclear expression of nuclear factor kappaB (NF-kappaB), activation of the IL-2 receptor (IL-2R) alpha chain gene, and cell proliferation. In the present study, the role of IL-2R signaling in the growth of CD8+ T cell prolymphocytic leukemia (T-PLL) cells has been investigated. Flow cytometry revealed that primary leukemia cells from a patient with CD8+ T-PLL expressed IL-2Ralpha and beta chains, and the cells showed a proliferative response and an increase in IL-2Ralpha expression on culture with exogeneous IL-2. Northern blot analysis failed to detect IL-2 mRNA, suggesting that IL-2 may act in a paracrine manner in vivo. Electrophoretic mobility-shift assays revealed that recombinant IL-2 increased NF-kappaB binding activity in nuclear extracts of the leukemia cells, and Northern blot analysis showed that IL-2 increased the abundance of mRNAs encoding the NF-kappaB components c-Rel and KBF1 in these cells. IL-2 binding analysis demonstrated that IL-2 markedly increased the number of low affinity IL-2Rs on the leukemia cells, without an effect on the number of high-affinity IL-2Rs. These results show that IL-2 is capable of inducing the nuclear expression of NF-kappaB in primary CD8+ T-PLL cells, and that this effect is mediated, at least in part, at a pretranslational level.

Blotting, Northern↗
PubMed1998

Alterations of Bcl-2 family proteins precede cytoskeletal proteolysis in the penumbra, but not in infarct centres following focal cerebral ischemia in mice.

Apoptosis has drawn attention in ischemic neuronal death recently. However, studies of apoptosis in cerebral ischemia have concentrated largely in DNA fragmentation, a late phase in apoptotic nuclei, at the expense of possible primary ischemic targets at the subcellular level and of upstream apoptotic signalling. To assess those issues, we used an intraluminal middle cerebral artery occlusion model in mice with or without reperfusion, and examined sequential changes of Bcl-2 family proteins modulating apoptotic signalling immunohistochemically and studied nuclear DNA fragmentation, to compare their chronology in relation to the development of infarct as detected by loss of microtubule-associated protein-2, an early marker of cytoplasmic damage. In the centre of the lesion, Bax protein increased and Bcl-2 and Bcl-x proteins decreased after loss of microtubule-associated protein-2 antigenicity occurred, but at the border of the lesion, the former changes preceded loss of microtubule-associated protein-2 antigenicity. Additionally, close morphologic analysis of DNA fragmentation in situ indicated that transient ischemia predominantly induced apoptotic cells but permanent ischemia produced necrosis of cells in the centre of the lesion. The contrasting cell death mechanisms, apoptosis and necrosis, are selectively involved in the pathology of cerebral ischemia, depending on its severity.

Animals↗
PubMed1998

Localization of the adenomatous polyposis coli tumour suppressor protein in the mouse central nervous system.

The adenomatous polyposis coli gene is mutated in familial adenomatous polyposis and in sporadic colorectal tumours. The adenomatous polyposis coli gene product is a 300,000 mol. wt cytoplasmic protein that binds to at least three other proteins; beta-catenin, a cytoplasmic E-cadherin-associated protein; hDLG, a human homologue of the Drosophila discs large tumour suppressor protein and glycogen synthase kinase 3 beta, a mammalian homologue of the Drosophila ZESTE WHITE 3 protein. The adenomatous polyposis coli gene is highly expressed in the brain, suggesting that it may be involved in nerve function. Here we show that adenomatous polyposis coli is localized in the pericapillary astrocytic endfeet throughout the mouse central nervous system. Adenomatous polyposis coli is also localized in the astrocytic processes in the cerebellar granular layer, and displays concentrated expression in the terminal plexuses of the basket cell fibres around Purkinje cells. Adenomatous polyposis coli is further expressed in neuronal cell bodies and/or nerve fibres in the olfactory bulb, hippocampus, brain stem, spinal cord and dorsal root ganglia. Adenomatous polyposis coli is demonstrated to be co-localized with beta-catenin and/or hDLG in neurons and nerve fibres, but not in astrocytes. From these results, adenomatous polyposis coli is suggested to participate in a signal transduction pathway in astrocytes which is independent of beta-catenin and hDLG, and also in regulation of neuronal functions in association with beta-catenin and hDLG.

Adaptor Proteins, Signal Transducing↗
PubMed1998

The carpal stretch test at the scapholunate joint.

The purpose of this study was to evaluate the principle of the carpal stretch test. Eleven upper extremities from 6 fresh cadaver specimens and 40 normal wrists from 20 volunteers were used in this study. Changes in intercarpal distances (carpal interval ratio of the radioscaphoid, radiolunate, and lunocapitate joints) and step-off deformities at the scapholunate joint seen on the posteroanterior view of an x-ray film were analyzed with and without the carpal stretch test. In the cadaver wrists, the dorsal capsulotomy and radioscapholunate ligament (stage 1), the palmar half of the scapholunate interosseous ligament (stage 2), the dorsal half of the scapholunate interosseous ligament (stage 3), and the radioscaphocapitate ligament (stage 4) were sequentially sectioned. The carpal stretch test was completed in each stage. Each carpal interval ratio increased as each stage was added. Step-off deformities at the scapholunate joint were seen in 6 of 11 wrists in stage 2 and in 10 wrists in stages 3 and 4 with the carpal stretch test. In the normal wrists, neither gender nor wrist laxity (Garcia's criteria) not influenced the results of the carpal stretch test. The carpal stretch test may be helpful in identifying occult and/or dynamic scapholunate dissociation.

Adult↗
PubMed1998

Expression of abnormal transcripts of the FHIT (fragile histidine triad) gene in ovarian carcinoma.

To elucidate the role of the FHIT (fragile histidine triad) gene in ovarian carcinogenesis, the expression of the gene was analysed by reverse transcription-polymerase chain reaction (RT-PCR) in 51 cases of ovarian carcinoma, 6 cases of borderline tumour and 4 cases of benign ovarian tumour. The concomitant expressions of normal and abnormal FHIT transcripts were detected in 39% of carcinomas and in 83% of borderline tumours, while benign tumours and normal ovarian tissues expressed only normal transcript. In addition, there were 4 (8%) cases of carcinoma lacking expression of normal FHIT transcript, all of which were in advanced stages (stage III-IV) and poorly differentiated. These results suggest that the expression of abnormal transcripts of the FHIT gene is a feature of ovarian malignant/borderline tumours and that the complete loss of normal FHIT expression is related to the progression of ovarian carcinoma in a subset of the cases. However, abnormal FHIT transcripts themselves were not associated with any clinicopathological parameters, such as clinical stage, histological subtype of tumour, grade of differentiation or outcome of the patient. Additionally, abnormal FHIT expression was not associated with the presence of loss of heterozygosity (LOH) at this locus, suggesting that abnormal FHIT transcripts are not derived from genetic alteration or that genetic alteration at this locus is complicated.

Acid Anhydride Hydrolases↗
PubMed1998

Protein kinase C mediates the signal for interferon-gamma mRNA expression in cytotoxic T cells after their adhesion to laminin.

A cytotoxic T-cell line, CTLL-2 cells, showed spreading after adhering to extracellular matrix proteins such as fibronectin (FN), laminin (LN) and hyarulonic acid (HA). The adhesion of CTLL-2 cells to LN was mediated by very late activation antigen-6 (VLA-6). Expression of interferon-gamma (IFN-gamma) mRNA was enhanced in CTLL-2 cells, also when they adhered to extracellular matrix proteins; and the enhanced IFN-gamma mRNA expression by adhering to LN was blocked by anti-alpha 6 antibody. Calphostin C, a protein kinase C (PKC) inhibitor, markedly inhibited the enhancement of IFN-gamma mRNA expression in a dose-dependent manner, which suggested that PKC acted as a second messenger in the IFN-gamma mRNA expression mediated by the interaction of VLA-6 with LN in CTLL-2 cells. Furthermore, confocal laser-microscopic analysis and Western blot analysis revealed that PKC-alpha was activated after CTLL-2 cells adhered to LN. PKC activity translocated from the cytosol fraction to the particulate fraction, after CTLL-2 cells adhered to LN. Altogether, we suggest that PKC plays an important role in the signal transduction for IFN-gamma mRNA expression after cytotoxic T cells adhere to LN.

Animals↗
PubMed1998

Sphingosylphosphorylcholine stimulates proliferation and upregulates cell surface-associated plasminogen activator activity in cultured human keratinocytes.

Of the various sphingolipid metabolites, including sphingosine, sphingosylphosphorylcholine (SPC), dimethylsphingosine, sphingosine-1-phosphate, N-acetylsphingosine, and skin-specific ceramides, only SPC accelerated cutaneous wound healing in full-thickness excision wounds in genetically healing-impaired diabetic (db/db) mice. A histologic examination revealed that SPC promoted not only granulation tissue formation, but also the re-epithelization of epidermal keratinocytes. As the direct effects of SPC on keratinocytes are completely unknown, we investigated the effects of SPC on normal cultured human keratinocytes. SPC concentration-dependently enhanced DNA synthesis in keratinocytes, with an increase in intracellular calcium concentrations due to the release of calcium ions from intracellular stores. SPC upregulated cell surface plasminogen activity, and at the same time increased the cell surface expression of urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator-receptor (uPA-R) in keratinocytes. Furthermore, SPC promoted the in vitro wound repair of cultured keratinocytes, which was partially blocked by an anti-uPA monoclonal antibody. Our results suggest that one of the mechanisms responsible for the SPC-mediated promotion of cutaneous wound healing seems to be an enhancement of re-epithelization caused by the direct stimulation of the proliferation of keratinocytes, and an activation of the uPA/uPA-R system, which enhances the migration of keratinocytes.

Animals↗