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Biomedical subjects

H Kobayashi

Publications and source records attributed to H Kobayashi.

At least 289 records · Page 16Linked to original sources
PubMed2000

Post-translational reduction of cell surface expression of insulin receptors by cyclosporin A, FK506 and rapamycin in bovine adrenal chromaffin cells.

Long-term (>/=3 h) treatment of cultured bovine adrenal chromaffin cells with cyclosporin A (CsA) decreased cell surface (125)I-insulin binding by 62% in a concentration (IC(50)=18 microM)- and time (t(1/2)=16 h)-dependent manner, but did not change the K(d) value. FK506 (1 microM) or rapamycin (3 microM) treatment reduced (125)I-insulin binding. Western blot analysis showed that CsA treatment decreased insulin receptor (IR) beta-subunit level (t(1/2)=15 h) in membrane fraction, but did not alter total cellular levels of IR precursor and IR beta-subunit. Internalization rate of cell surface IR measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network, was comparable between non-treated and CsA-treated cells. Thus, CsA, FK506 and rapamycin inhibit peptidyl prolyl cis-trans isomerase activities of cyclophilin and FK506-binding protein, and down-regulate IR presumably by reducing cell surface externalization of IR.

Animals↗
PubMed2000

Suppression of urokinase expression and invasiveness by urinary trypsin inhibitor is mediated through inhibition of protein kinase C- and MEK/ERK/c-Jun-dependent signaling pathways.

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, interacts with cells as a negative modulator of the invasive cells. Human ovarian cancer cell line, HRA, was treated with phorbol ester (PMA) to evaluate the effect on expression of urokinase-type plasminogen activator (uPA), since the action of uPA has been implicated in matrix degradation and cell motility. Preincubation of the cells with UTI reduced the ability of PMA to trigger the uPA expression at the gene level and at the protein level. UTI-induced down-regulation of PMA-stimulated uPA expression is irreversible and is independent of a cytotoxic effect. Down-regulation of uPA by UTI is mediated by its binding to the cells. We next asked whether the mechanism of inhibition of uPA expression by UTI was due to interference with the protein kinase C second messenger system. An assay for PKC activity demonstrated that UTI does not directly inhibit the catalytic activity of PKC and that PMA translocation of PKC from cytosol to membrane was inhibited by UTI, indicating that UTI inhibits the activation cascade of PKC. PMA could also activate a signaling pathway involving MEK1/ERK2/c-Jun-dependent uPA expression. When cells were preincubated with UTI, we could detect suppression of phosphorylation of these proteins. Like several types of PKC inhibitor, UTI inhibited PMA-stimulated invasiveness. We conclude that UTI markedly suppresses the cell motility possibly through negative regulation of PKC- and MEK/ERK/c-Jun-dependent mechanisms, and that these changes in behavior are correlated with a coordinated down-regulation of uPA which is likely to contribute to the cell invasion processes.

Cell Movement↗
PubMed2000

Origin of ferromagnetic exchange interactions in a fullerene-organic compound.

Organic ferromagnets, which exhibit exchange interactions between unpaired electrons in pi-orbitals, are rare, and the origin of ferromagnetism in these compounds has so far remained unexplained. Tetrakis(dimethylamino)ethylene-fullerene[60] (TDAE-C60) shows a transition to a ferromagnetic state with fully saturated s = 1/2 molecular spins at the relatively high Curie temperature (for organic materials) of 16 K (ref. 4). It has been suggested that the orientations of the C60 molecules may be important for ferromagnetism in this material, but in the absence of structural data at low temperatures there has been little progress towards understanding these microscopic interactions. Here we report the results of a comparative structural study of two different magnetic forms of TDAE-C60 crystals at low temperatures, correlating the structural properties--in particular, the intermolecular orientations--with the magnetic properties. We find that both ferromagnetism and spin-glass-like ordering are possible in this material, and depend on the orientational state of C60 molecules. This resolves the apparent contradictions posed by different macroscopic measurements, and opens the way to a microscopic understanding of pi-electron ferromagnetic exchange interactions in organic materials.

Journal Article↗
PubMed2000

High-performance liquid chromatographic method for determination of DDT and its degradation products in rat plasma, liver and brain: validation and application to a pharmacokinetic study.

A sensitive and reliable high-performance liquid chromatographic (HPLC) method, using a solid-phase extraction (SPE), was established and validated for determination of p,p'-DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] and its metabolite p,p'-DDE [1,1-(2,2-dichloroethanylidene)-bis(4-chlorobenzene)] in rat plasma, liver and brain. After being diluted with water, plasma, liver and brain samples were applied to a solid-phase extraction C18 cartridge. The extraction containing p,p'-DDT and p,p'-DDE from the cartridge were cleaned-up using a Florisil Sep-Pak cartridge. The samples were analyzed by HPLC using UV detection at 238 nm. The limit of detection for p,p'-DDT and p,p'-DDE was 0.1 mg kg(-1) liver or brain and 0.1 mg l(-1) plasma. For six replicate samples at 40, 4 and 0.2 mg kg(-1), intra-day precision values were within 4.9% for plasma, 6.4% for liver, and 9.7% for brain. Inter-day precision values at 4 mg kg(-1) were within 8.2% for plasma and tissues. The method performances were shown to be selective for p,p'-DDT and p,p'-DDE, and linear over the range 0.04-12 mg kg(-1) (mg L(-1) for plasma). The absolute recoveries of p,p'-DDT and p,p'-DDE in rat plasma and tissues were over 92%. The method was proved to be applicable to the pharmacokinetic study of DDT in rats after a single oral administration.

Animals↗
PubMed2000

Adrenomedullin receptors in rat cerebral microvessels.

To characterize the sites of action of adrenomedullin (AM) in the cerebral microvasculature, we studied the effect of AM on cyclic AMP (cAMP) level as well as expression of AM and its receptor in the rat cerebral microvessels. The microvessels were prepared from rat cerebral cortex by albumin flotation and glass bead filtration technique. AM and calcitonin gene-related peptide (CGRP) increased cAMP level in the microvessels in a concentration-dependent manner. The effect of AM was more than 100 times more potent than that of CGRP. The accumulation of cAMP by AM was inhibited by AM[22-52], an AM receptor antagonist, but not by CGRP[8-37], a CGRP receptor antagonist, suggesting that AM increased cAMP accumulation by acting on receptors specific to AM. [125I]AM binding to the microvessels was displaced by AM and less potently by AM[22-52]. The displacing potencies of CGRP and CGRP[8-37] were very weak. mRNAs for AM as well as calcitonin-receptor-like receptor and receptor-activity-modifying protein 2 which form a receptor specific to AM, were highly expressed in the microvessels. These results provide biochemical and pharmacological evidence that AM is produced in and acts on the cerebral microvessels in an autocrine/paracrine manner and is involved in regulation of cerebral microcirculation.

Adrenomedullin↗
PubMed2000

alpha(1)-Adrenergic receptor subtypes in rat cerebral microvessels.

To identify alpha(1)-adrenergic receptor subtypes involved in the regulation of cerebral microcirculation, we studied alpha(1)-adrenergic receptor subtypes expressed in the rat cerebral microvessels. The microvessels were prepared from rat cerebral cortex by albumin flotation and glass bead filtration techniques. [(125)I]HEAT binding to the cerebral microvessels was displaced by low concentrations of 5-methylurapidil, a selective antagonist for alpha(1A)-receptors, and modified Scatchard analysis of the data revealed that half of alpha(1)-receptors is alpha(1A)-subtype, and that alpha(1B)- and/or alpha(1D)-receptors are also present. The K(i) value of the high-affinity component for 5-methylurapidil was 3.90+/-1.08 nM, which is comparable with the value obtained in the rat cerebral cortex (2.17+/-0.88 nM). Reverse transcription and polymerase chain reaction experiments showed that mRNAs of alpha(1A)- and alpha(1B)-receptors, but not alpha(1D)-receptors, were expressed in the cerebral microvessels. These results suggest that alpha(1)-receptors involved in the regulation of cerebral microvessel function are alpha(1A)- and alpha(1B)-receptor subtypes.

Adrenergic alpha-Antagonists↗
PubMed2000

Suppression of urokinase-type plasminogen activator expression from human ovarian cancer cells by urinary trypsin inhibitor.

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, efficiently inhibits tumor cell invasion and metastasis. We examined the effect of UTI on urokinase-type plasminogen activator (uPA) expression in ovarian cancer cell lines, HOC-I and HRA. By Northern blot, Western blot, ELISA, and zymographic analyses, we demonstrated that UTI inhibited the expression of uPA mRNA and protein in these cells in a time- and dose-dependent manner, independent of whether induction was triggered by phorbol ester. Monoclonal antibody 4G12, which inhibits UTI binding to the cells, produced a dose-dependent abrogation in UTI-mediated down-regulation of uPA expression. These data suggest that UTI significantly down-regulates tumor cell uPA mRNA expression and protein secretion, and that UTI binding to the cells is necessary to exert the UTI's action.

Down-Regulation↗
PubMed2000

Involvement of BK(Ca) channels in the relaxation of detrusor muscle via beta-adrenoceptors.

Detrusor muscle relaxes upon activation of beta-adrenoceptors on smooth muscle cells. However, the mechanism of relaxation following the stimulation of beta-adrenoceptors remains unclear. In order to clarify the mechanism, we investigated the involvement of ion channels in bladder relaxation. In guinea-pig isolated bladder strips precontracted by high-K(+), isoproterenol caused concentration-dependent relaxation. The relaxation caused by isoproterenol (1 microM) was larger in 30 mM K(+) than in 120 mM K(+) (54.2+/-8.0% and 18.2+/-4.1% of papaverine-induced relaxation, respectively, n=4). Iberiotoxin (100 nM) inhibited the isoproterenol-induced relaxation (vehicle 69.5+/-8.0% vs. iberiotoxin 24.9+/-6.2%, respectively, n=5). Whole-cell patch-clamp recording revealed that isoproterenol as well as forskolin increased the iberiotoxin-sensitive K(+) currents, and this increase was abolished by protein kinase inhibitor. These results suggest that the isoproterenol-induced relaxation of guinea-pig bladder smooth muscle is mainly mediated by facilitation of BK(Ca) channels subsequent to the activation of the cAMP/protein kinase A pathway.

Animals↗
PubMed2000

Defining promiscuous MHC class II helper T-cell epitopes for the HER2/neu tumor antigen.

It is accepted that both helper and CTLs play a critical role in immune antitumor responses. Thus, the design of effective immune-based therapies for cancer relies in the identification of relevant tumor-associated antigens (TAAs) capable of eliciting strong helper and cytotoxic T-cell responses against tumor cells. The product of the HER2/neu oncogene is considered as a prototype TAA, because it is found overexpressed in a large variety of malignancies, whereas normal cells only produce low levels of this product. Several cytotoxic T-cell epitopes for HER2/neu have been identified that enable the design of peptide-based therapeutic vaccines for tumors expressing this TAA. Nevertheless, it is expected that inclusion of peptide epitopes capable of eliciting HER2/neu-specific T helper responses into these vaccines may enhance their effectiveness in the clinic. We describe here a strategy to identify helper T-cell epitopes for HER2/neu that focuses on peptides predicted to bind to numerous histocompatibility alleles (promiscuous epitopes), which would encourage their use in therapeutic vaccines for the general cancer patient population. Following this approach, we successfully identified several peptides that elicited T helper (CD4+) proliferative responses to peptides derived from HER2/neu. Most of the T-cell responses appeared to reflect a low affinity for antigen, which could be the result of immune tolerance because HER2/neu is expressed in low levels in normal cells and possibly including lymphocytes and monocytes. Interestingly, one of these peptides, HER2(883), was recognized by T cells in the context of either HLA-DR1, HLA-DR4, HLA-DR52, and HLA-DR53, indicating a high degree of histocompatibility promiscuity. Furthermore, T cells that reacted with peptide HER2(883) could also recognize antigen-presenting cells that process HER2/neu recombinant protein. These results may be relevant for the design of more effective therapeutic vaccines for tumors expressing the HER2/neu oncogene product.

Amino Acid Sequence↗
PubMed2000

Detection of PTEN nonsense mutation and psiPTEN expression in central nervous system high-grade astrocytic tumors by a yeast-based stop codon assay.

We have developed a new yeast-based assay for the detection of PTEN nonsense mutation, and applied it to a total of 42 astrocytic tumors. The assay utilizes homologous recombination of PCR-amplified PTEN cDNA samples to a yeast vector which expresses an in-frame PTEN::ADE2 chimera protein. An allele of nonsense mutation in the sample PTEN mRNA gives a truncated chimera protein in a yeast cell, resulting in the formation of a red colony. The assay and subsequent sequence analysis demonstrated nonsense mutations as red colonies of more than 10% in one of 10 anaplastic astrocytomas and six of 18 glioblastomas, but none in six pilocytic astrocytomas or in eight astrocytomas. Sequence analysis of white colonies showed one missense mutation in a glioblastoma. Interestingly, four of seven nonsense mutations were frame-shifts due to exon skipping. In addition, pink colonies were found in one of six pilocytic astrocytomas, three of eight astrocytomas, two of 10 anaplastic astrocytomas, and 10 of 18 glioblastomas. Sequence analysis of the pink colonies revealed a sequence similar to those reported as psiPTEN/PTH2. By testing mRNA and genomic DNA, it was found to be a processed pseudogene which was transcribed. The psiPTEN expression was complementary to PTEN mutation, for 14 of 18 glioblastomas showed either PTEN mutation or psiPTEN expression and only one case showed both PTEN mutation and psiPTEN expression (P<0.046), suggesting a pathological role of psiPTEN expression as an alternative to PTEN mutation in glioblastomas.

Astrocytoma↗
PubMed2000

Inhibition by neuroprotective drug NS-7 of nicotine-induced 22Na(+) influx, 45Ca(2+) influx and catecholamine secretion in adrenal chromaffin cells.

In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited nicotine-induced 22Na(+) influx via nicotinic receptors (IC(50)=15.5 microM); the suppression by NS-7 was observed in the presence of ouabain, an inhibitor of Na(+),K(+)-ATPase, and was not attenuated upon the washout of NS-7. NS-7 decreased nicotine-induced maximum influx of 22Na(+) without altering the EC(50) value of nicotine. Also, NS-7 diminished nicotine-induced 45Ca(2+) influx via nicotinic receptors and voltage-dependent Ca(2+) channels (IC(50)=14.1 microM) and catecholamine secretion (IC(50)=19.5 microM). These results suggest that NS-7 produces noncompetitive and long-lasting inhibitory effects on neuronal nicotinic receptors in adrenal chromaffin cells, and interferes with the stimulus-secretion coupling.

Adrenal Glands↗
PubMed2000

Crystal structure of Streptomyces olivaceoviridis E-86 beta-xylanase containing xylan-binding domain.

Xylanases hydrolyse the beta-1,4-glycosidic bonds within the xylan backbone and belong to either family 10 or 11 of the glycoside hydrolases, on the basis of the amino acid sequence similarities of their catalytic domains. Generally, xylanases have a core catalytic domain, an N and/or C-terminal substrate-binding domain and a linker region. Until now, X-ray structural analyses of family 10 xylanases have been reported only for their catalytic domains and do not contain substrate-binding domains. We have determined the crystal structure of a family 10 xylanase containing the xylan-binding domain (XBD) from Streptomyces olivaceoviridis E-86 at 1.9 A resolution. The catalytic domain comprises a (beta/alpha)(8)-barrel topologically identical to other family 10 xylanases. XBD has three similar subdomains, as suggested from a triple-repeat sequence, which are assembled against one another around a pseudo-3-fold axis, forming a galactose-binding lectin fold similar to ricin B-chain. The Gly/Pro-rich linker region connecting the catalytic domain and XBD is not visible in the electron density map, probably because of its flexibility. The interface of the two domains in the crystal is hydrophilic, where five direct hydrogen bonds and water-mediated hydrogen bonds exist. The sugar-binding residues seen in ricin/lactose complex are spatially conserved among the three subdomains in XBD, suggesting that all of the subdomains in XBD have the capacity to bind sugars. The flexible linker region enables the two domains to move independently and may provide a triple chance of substrate capturing and catalysis. The structure reported here represents an example where the metabolic enzyme uses a ricin-type lectin motif for capturing the insoluble substrate and promoting catalysis.

Amino Acid Motifs↗
PubMed2000

Identity of urinary trypsin inhibitor-binding protein to link protein.

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, directly binds to some types of cells via cell-associated UTI-binding proteins (UTI-BPs). Here we report that the 40-kDa protein (UTI-BP(40)) was purified from the cultured human chondrosarcoma cell line HCS-2/8 by UTI affinity chromatography. Purified UTI-BP(40) was digested with trypsin, and the amino acid sequences of the peptide fragments were determined. The sequences of six tryptic fragments of UTI-BP(40) were identical to subsequences present in human link protein (LP). Authentic bovine LP and UTI-BP(40) displayed identical electrophoretic and chromatographic behavior. The UTI-binding properties of UTI-BP(40) and LP were indistinguishable. Direct binding and competition studies strongly demonstrated that the NH(2)-terminal fragment is the UTI-binding part of the LP molecule, that the COOH-terminal UTI fragment (HI-8) failed to bind the NH(2)-terminal subdomain of the LP molecule, and that LP and UTI-BP(40) exhibited significant hyaluronic acid binding. These results demonstrate that UTI-BP(40) is identical to LP and that the NH(2)-terminal domain of UTI is involved in the interaction with the NH(2)-terminal fragment of LP, which is bound to hyaluronic acid in the extracellular matrix.

Aggrecans↗
PubMed2000

Multilineage differentiation of Cbfa1-deficient calvarial cells in vitro.

We characterized calvaria-derived cells of Cbfa1-deficient mice to determine their stages of differentiation. In long-term culture, Cbfa1-deficient calvarial cells did not acquire osteoblastic phenotypes, but numerous adipocyte foci appeared with an increase in the expression of marker genes for adipocyte differentiation. In culture with BMP-2, Cbfa1-deficient calvarial cells still failed to generate bone nodules but differentiated into chondrocytes and further to terminal hypertrophic chondrocytes, and adipocyte foci were decreased. Cbfa1-deficient calvarial cells transplanted into the peritoneal cavity of athymic mice using BMP-2-coated diffusion chambers generated cartilage but not bone. These data indicate that Cbfa1-deficient calvarial cells completely lack the ability to differentiate into mature osteoblasts and Cbfa1 has an inhibitory function in adipocyte differentiation. As Cbfa1-deficient calvarial cells were enriched with immature mesenchymal cells, which can differentiate into adipocytes and chondrocytes, it is suggested that Cbfa1 plays an essential role in determining the lineage of multipotential mesenchymal precursor cells.

Adipocytes↗
PubMed2000

Identification and characterization of a Kunitz-type protease inhibitor in ascites fluid from patients with ovarian carcinoma.

Urinary trypsin inhibitor (UTI; Mr 40 kDa) is a Kunitz-type protease inhibitor that efficiently inhibits cell-associated trypsin and plasmin activities. The aim of this study is to examine the expression pattern of UTI in the human ovarian carcinoma ascites fluid by Western blotting, zymography, immunoprecipitation, immunohistochemistry, biochemical and gene analyses and animal experiments. We have identified and characterized the 40 kDa immunoreactive UTI (UTI(40)) and 8 kDa degradation fragment (UTI(8)) in ascites fluid. The levels of UTI(40) and UTI(8) are elevated in ascites fluid taken from patients with ovarian carcinoma relative to paired plasma samples. The UTI(40) and UTI(8) were identified immunologically by the reactivity with 2 different anti-UTI antibodies recognizing different epitopes of the UTI molecule, functionally by its ability to bind trypsin and structurally by its apparent molecular mass with and without deglycosylation treatment. The purified polypeptides have been sequenced and were identical with sequences obtained from UTI and the carboxyl-terminal domain of UTI, respectively. However, UTI mRNA was not detected in the ovarian carcinoma tissue and ovarian carcinoma cell lines examined. Based on extravasation experiments using intravenously injected biotinylated inter-alpha-trypsin inhibitor (IalphaI; a precursor of UTI), we conclude that UTI(40) and UTI(8) found in the ascites fluid may result from (i) the extravasation of plasma proteins such as IalphaI into the peritoneal cavity via hyperpermeable vessels and (ii) the subsequent degradation of IalphaI and UTI(40) by tumor cell-associated trypsin-like enzymes.

Adenocarcinoma, Clear Cell↗
PubMed2000

Microcoaxial electrode for in vivo nitric oxide measurement.

Nitric oxide (NO) is a gaseous mediator involved in various physiological phenomena, such as vasorelaxation and neurotransmission. Investigation of local cellular responses of NO production in vivo and in vitro requires a measurement method with a high spatial resolution. For selective NO measurement, we therefore developed a microcoaxial electrode whose tip diameter is less than 10 microm. Calibration using various concentrations of NO (0.1-1.0 microM) showed that the electrode has good linearity (r = 0.99) and its detection limit is 0.075 microM (S/N = 3). We verified the applicability of this electrode to in vivo and in vitro local measurement NO released from bovine aortic cultured endothelial cells (BAECs) stimulated by acetylcholine (ACh). After the addition of ACh, a transient increase in NO concentration was detected by the electrode. In the presence of NG-nitro-L-arginine methyl ester (L-NAME), a putative NO synthase inhibitor, NO release (peak NO concentration) from RAECs was significantly less than that in the absence of L-NAME (0.18 +/- 0.04 microM vs 0.47 +/- 0.13; P < 0.01). After removal of L-NAME, NO release partially recovered (0.39 +/- 0.10 microM). In conclusion, the microcoaxial electrode was successfully applied to direct and continuous NO measurement in biological systems.

Calibration↗