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M Kobayashi

Publications and source records attributed to M Kobayashi.

At least 19 recordsLinked to original sources
PubMed1992

Molecular cloning of hippocalcin, a novel calcium-binding protein of the recoverin family exclusively expressed in hippocampus.

We have isolated a cDNA clone encoding a novel calcium-binding protein of the recoverin family from rat brain cDNA library. This clone (PCB11) has 588 nucleotides in the open reading frame including the termination codon, 174 nucleotides of the 5' leader and 800 nucleotides of the 3' noncoding region. The complete amino acid sequence deduced from the cDNA is composed of 195 residues, has a calculated molecular mass of 22,574 Daltons, and contains three putative calcium-binding domains of the EF-hand structure. The deduced amino acid sequence has a striking sequence homology to those of the retinal recoverin family (recoverin, visinin, P26, 23kD protein, S-modulin) and the brain-derived recoverin family (P23k, 21-kDa CaBP and neurocalcin). Northern blot, in situ hybridization, immunoblot and immunohistochemical analyses revealed that the protein is exclusively expressed in pyramidal layer of the hippocampus. The protein was therefore designated hippocalcin.

Amino Acid Sequence↗
PubMed1992

Carrier-mediated transport system for choline and its related quaternary ammonium compounds on rat intestinal brush-border membrane.

The characteristics of the intestinal transport system for choline were investigated using isolated brush-border membrane vesicles from rat small intestine. In spite of the diminutive lipid solubility, the uptake of choline by membrane vesicles reflected smooth permeation into intravesicular space rather than the binding to the membrane surface. Physiological conditions, present in the intact intestine, such as an inward-directed Na+ or H+ gradient and inside negative membrane potentials, didn't directly involve in choline transport across the brush-border membrane. Moreover, an outward-directed H+ gradient had no significant effect on the time course of choline transport. However, in the absence of a driving-force, the initial uptake of choline exhibited a saturable manner. A kinetic analysis of the initial uptake rate gave an apparent Km of 159 microM. Furthermore, unlabeled choline caused both cis-inhibition and trans-stimulation for labeled choline transport, suggesting the existence of a carrier-mediated transport system for choline, classified as so-called 'facilitated diffusion'. Since tetramethylammonium, acetylcholine, and N1-methylnicotinamide caused both cis-inhibition and trans-stimulation, they appear to be accepted as the substrate of choline carrier. On the other hand, quaternary ammonium compounds (QACs) such as those which possessed hydrophobic parts in their molecules exhibited only cis-inhibition. They also inhibited Na(+)-dependent D-glucose transport, indicating that they influenced various carrier-mediated transport systems non-specifically due to interaction with the membrane. These findings strongly suggest that the choline transport system on the brush-border membrane of rat intestine recognizes only small molecular QACs as its substrate.

Animals↗
PubMed1992

Different sensitivities of human esophageal cancer cells to multiple anti-cancer agents and related mechanisms.

Mechanisms responsible for drug resistance in human esophageal cancer cell lines were investigated. Three cell lines established from human esophageal carcinoma (TE-1, SH-1, and TH) showed different sensitivities to vindesine, vincristine, cisplatin (CDDP), etoposide (VP-16), and pepleomycin. Both SH-1 and TH cell lines were twofold to sevenfold more resistant to pepleomycin, vindesine, and vincristine than TE-1 was. SH-1 showed twofold more resistance to CDDP than either TE-1 or TH did, and TH and TE-1 showed a 3-fold or 1.5-fold more resistance, respectively, to VP-16 than SH-1 did. The accumulation of tritiated vincristine in SH-1 and TH was approximately 50% that in TE-1. Two multidrug resistance reversal agents, cepharanthine and a synthetic dihydropyridine analogue (NK-252; Nikken Chemicals, Saitama, Japan), potentiated the cytocidal actions of vindesine against SH-1, TH, and TE-1 cells, with no apparent expression of P-glycoprotein in the three cell lines. The glutathione S-transferase pi gene was expressed in all three cell lines. DNA topoisomerase II levels were lowest in TE-1, followed by SH-1 and TH, although the accumulation of tritiated VP-16 was less in both TH and SH-1 than in TE-1. Differential sensitivities to anti-cancer drugs appear to be mediated through pleiotropic mechanisms.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells.

Natural killer cell stimulatory factor (NKSF), or interleukin 12 (IL-12), is a 70-kD heterodimeric cytokine composed of two covalently linked chains, p40 and p35. NKSF/IL-12 has multiple effects on T and NK cells and was originally identified and purified from the supernatant fluid of Epstein-Barr virus (EBV)-transformed human B lymphoblastoid cell lines. We have produced a panel of monoclonal antibodies against both chains of NKSF/IL-12. Some of these antibodies have neutralizing activity, and several combinations of them have been used to establish sensitive radioimmunoassays detecting the free p40 chain, the free p35 chain, or the p70 heterodimer. Using these reagents, we have determined that most EBV-transformed human B lymphoblastoid cell lines constitutively produce low levels of the p70 heterodimer and an excess of the free p40 chain, whereas Burkitt lymphoma-derived, T, myeloid, and many solid tumor-derived cell lines produce neither. Production of both p40 and p70 is increased several-fold upon stimulation of the EBV-transformed cell lines with phorbol diesters. The ability of supernatant fluids from unstimulated and phorbol diester-stimulated cell lines to induce interferon gamma (IFN-gamma) production from T and NK cells, one of the effects of NKSF/IL-12, parallels the levels of production of the p70 heterodimer, known to be the biologically active form of NKSF/IL-12. Staphylococcus aureus Cowan I strain (SAC) and other stimuli induce accumulation of p40 mRNA and production of both p40 and p70 by peripheral blood mononuclear cells (PBMC). The producer cells appear to include both adherent cells and nonadherent lymphocytes, possibly B cells. The supernatant fluids from SAC-stimulated PBMC mediate the typical functions of NKSF/IL-12 (i.e., IFN-gamma induction, mitogenic effects on T/NK blasts, enhancement of NK cell cytotoxicity) at concentrations of p70 similar to those at which recombinant NKSF/IL-12 mediates the same functions. Moreover, these activities are significantly inhibited by anti-NKSF/IL-12 antibodies. The neutralizing anti-NKSF/IL-12 antibodies also inhibit 85% of the IFN-gamma production in response to SAC, an NKSF/IL-12 inducer, and approximately 50% of the IFN-gamma production in response to non-NKSF/IL-12-inducers such as IL-2, phytohemagglutinin, and anti-CD3 antibodies. These results indicate that induced or constitutively produced NKSF/IL-12 has a major role in facilitating IFN-gamma production by peripheral blood lymphocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Nitrilase from Rhodococcus rhodochrous J1. Sequencing and overexpression of the gene and identification of an essential cysteine residue.

The amino acid sequences of the NH2 terminus and internal peptide fragments of a Rhodococcus rhodochrous J1 nitrilase were determined to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 750-base DNA fragment thus amplified was used as the probe to clone a 5.4-kilobase PstI fragment coding for the whole nitrilase. The nitrilase gene modified in the sequence upstream from the presumed ATG start codon was expressed to approximately 50% of the total soluble protein in Escherichia coli. The predicted amino acid sequence of the nitrilase gene showed similarity to that of the bromoxynil nitrilase from Klebsiella ozaenae. The 5,5'-dithiobis(2-nitrobenzoic acid) modification of the nitrilase from R. rhodochrous J1 resulted in inactivation with the loss of one sulfhydryl group/enzyme subunit. Of 4 cysteine residues in the Rhodococcus nitrilase, only Cys-165 is conserved in the Klebsiella nitrilase. Mutant enzymes containing Ala or Ser instead of Cys-165 did not exhibit nitrilase activity. These findings suggest that Cys-165 plays an essential role in the function of the active site.

Amino Acid Sequence↗
PubMed1992

Protective effect of a selective endothelin receptor antagonist, BQ-123, in ischemic acute renal failure in rats.

To elucidate the pathophysiological role of endogenous endothelin (ET), we examined the effects of the newly synthesized ETA receptor-selective antagonist, BQ-123, on ischemic acute renal failure induced by bilateral clamping of renal artery and vein followed by reperfusion in rats. BQ-123, when given by i.v. infusion of 0.5 mg/kg per min for 2.5 h during the pre- and post-ischemic period, was found to prevent the decrease in creatinine clearance and increases in blood urea nitrogen, plasma creatinine and the fractional excretion of sodium. Morphological observation also showed an effect of BQ-123, i.e. prevention of proximal tubular (S3 segment) necrosis. At 2 h after the start of reperfusion, the ET-1 content in the kidney increased to its maximal level. At this time, the Ca2+ content in the mitochondrial fraction of the renal cortex increased, with a concomitant increase in blood urea nitrogen. However, these increases were limited by treatment with BQ-123. Thus, BQ-123 was effective to both prevent mitochondrial Ca2+ accumulation in the early phase of ischemic acute renal failure and protect proximal tubular cells from post-ischemic degeneration. We conclude that ET may be at least partially involved in the pathogenesis of tubular cell injury in this acute renal failure model.

Acute Kidney Injury↗
PubMed1992

Thrombin-induced shape change in human megakaryoblastic leukemic cells, MEG-01, is mediated by protein kinase C.

We investigated the intracellular processes of the shape change in human megakaryoblastic leukemic cells, MEG-01, by platelet agonists. Thrombin induced the formation of many pseudopods. This shape change was also induced by phorbol 12-myristate 13 acetate (TPA) and weakly by Ca2+ ionophore, A23187, but not by ADP, collagen, or epinephrine. Electron microscopy and FITC-labeled phalloidin staining revealed thick submembranous microfilament bundles in the pseudopods of the shape-changed cells by thrombin. Shape change was inhibited by cytochalasin B. Since Ca(2+)-dependent phosphorylation reactions play central role on the initiation of shape change of platelet, we examined the effects of protein kinase C (PKC) inhibitor, H-7, and myosin light chain (MLC) kinase inhibitor, ML-9, on the shape change of MEG-01 cells induced by thrombin, and observed that H-7 potently inhibited thrombin-induced shape change, while ML-9 did not. These results suggest that thrombin-induced reorganization of microfilaments and shape change of MEG-01 cells are mediated by PKC, but not by MLC kinase.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Myristoylation-dependent membrane targeting and release of the HTLV-I Gag precursor, Pr53gag, in yeast.

The unprocessed HTLV-I Gag precursor, Pr53gag, was synthesized in yeast, Saccharomyces cerevisiae. The synthesized Pr53gag was myristoylated, associated with the cellular membrane, and released into the culture medium. The released Pr53gag was pelleted by centrifugation at 100,000 x g for 2 h. Conversion of Gly2 to Ala allowed synthesis of a non-myristoylated soluble Pr53gag which was not released into the culture medium. These results suggest that the release of the HTLV-I Gag precursors Pr53gag, occurs in yeast in a myristoylation-dependent manner.

Amino Acid Sequence↗
PubMed1992

Primary structure of an aliphatic nitrile-degrading enzyme, aliphatic nitrilase, from Rhodococcus rhodochrous K22 and expression of its gene and identification of its active site residue.

Peptides obtained by cleavage of a Rhodococcus rhodochrous K22 nitrilase, which acts on aliphatic nitriles such as acrylonitrile, crotonitrile, and glutaronitrile, have been sequenced. The data allowed the design of oligonucleotide probes which were used to clone a nitrilase encoding gene. Plasmid pNK21, in which 2.05-kb sequence covering the region encoding the nitrilase was was placed under the control of the lac promoter, directed overproduction of enzymatically active nitrilase in response to addition of isopropyl beta-D-thiogalactopyranoside in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell extract showed that the amount of nitrilase was about 40% of the total soluble proteins, leading to the establishment of a simple purification of the nitrilase. The nucleotide sequence of the nitrilase gene predicts a protein composed of 383 amino acids (M(r) = 42,275), including only one cysteine. The amino acid sequence homology between the Rhodococcus nitrilase and the Klebsiella ozaenae bromoxynil nitrilase [Stalker et al. (1988) J. Biol. Chem. 263, 6310-6314] was 38.3%, and a unique cysteinyl residue (Cys-170) in the former nitrilase was conserved at the corresponding position in the latter nitrilase. Cys-170 of the Rhodococcus nitrilase was replaced by Ala or Ser by site-directed mutagenesis. Both mutations resulted in the complete loss of nitrilase activity, clearly indicating that this cysteinyl residue is essential for the catalytic activity.

Amino Acid Sequence↗
PubMed1992

Chemiluminescence in the crude extracts of soybean seedlings. Postulated mechanism on the formation of hydroperoxide intermediates.

It has been reported that weak chemiluminescence (CL) from crude extracts of soybean seedlings is remarkably enhanced with the addition of various aldehydes (Biochim. Biophys. Acta 1058, 209-216). The reactivity of certain emitter(s) with oxygen species was examined in the autoclaved extracts of seedlings. When samples were reduced by the addition of hydrosulfite, two different types of reactivities in CL were defined. One type showed an initial rapid increase and a subsequent fast decay in CL upon mixing with oxygen. This rapid increase in CL intensity was independent of the presence of aldehydes, and was significantly suppressed by SOD. However, the subsequent slow decay phase in CL was dependent on the presence of aldehydes. In the sample reduced more moderately by borohydride, the same slow decay of CL appeared upon mixing with acetaldehyde and oxygen. This second type of CL was not inhibited by active oxygen scavengers. Hydrogen peroxide added to unreduced (oxidized) samples also elicited CL. Three types of primary emitters may be oxidized to form transient hydroperoxide, and excited for light emission by slightly different ways: two of them are excited by abstraction of one atomic oxygen from the hydroperoxy intermediate with aldehyde or hydrogen peroxide, leading to formation of an excited hydroxide intermediate. The third is excited directly on the binding of superoxide anion to the reduced primary emitter.

Acetaldehyde↗
PubMed1992

Cell adhesion molecule L1 guides cell migration in primary reaggregation cultures of mouse cerebellar cells.

We demonstrate a new assay in vitro using reaggregated cerebellar neurons in order to test the role of the L1 molecule in migration events. Cells other than neurons were eliminated in these cultures. Cerebellar neuron reaggregates plated on L1 molecule substrates migrated at rates similar to those observed in vivo. This migration was blocked by anti-L1 antibody. These findings suggest that this culture could provide a useful system with which to directly study relationships between neuron migration and adhesion molecules.

Animals↗
PubMed1992

[A case of Cushing syndrome caused by ACTH producing atypical lung carcinoid tumor].

A forty-one-year-old male who was found to have lung carcinoid tumor showed clinical features of Cushing syndrome. At the age of 38 years during a regular check-up, a chest roentgenogram showed multiple nodular shadows in the bilateral lung, and he was admitted to hospital. By open lung biopsy, he was diagnosed as having malignant carcinoid or small cell carcinoma of the lung. The abnormal lung shadows did not respond to chemotherapy (CDDP + VDS). The clinical course after discharge was uneventful until the age of 41 years, when he was readmitted to our hospital because of brain metastasis. Physical examination revealed moon face and central obesity. Plasma ACTH level was high and dexamethasone suppression test showed no cortisol suppression on 8 mg dexamethasone administration. Therefore, it was thought that the patient had ACTH producing ectopic tumor which led to Cushing syndrome. We measured the molecular weight of ACTH by column chromatography and found he had a big ACTH (molecular weight about 22,000). Reexamining him clinically and histologically, we concluded that the patient had atypical carcinoid tumor in the lung which might produce ACTH causing Cushing syndrome with metastasis to the brain.

Adrenocorticotropic Hormone↗
PubMed1992

Requirement of N- and C-terminal regions for enzymatic activity of human T-cell leukemia virus type I protease.

The requirement of N- and C-terminal regions for the enzymatic activity of human T-cell leukemia virus type I (HTLV-I) protease was investigated using a series of deletion mutants. The activity was analyzed by autoprocessing of the protease itself or by processing of the gag p53 precursor. The deletional analyses indicated that Asp38-Gly152 with an additional Met-Pro sequence at the N-terminus was probably sufficient for the enzymatic activity, although the mature HTLV-I protease consists of Pro33-Leu157. A molecular model of HTLV-I protease, which was constructed by comparison with the structure of Rous sarcoma virus protease, predicted that Pro33-Leu37 and Gly143-Leu147 would form a beta-sheet. Our experimental results and the model structure suggest that (a) five amino acids in the N-terminal region (Pro33-Leu37), which are thought to be involved in the beta-sheet, are not crucial for the enzymatic activity; (b) Pro153-Leu157 is not necessary but Pro148-Gly152 is important for the enzymatic activity, in addition to Gly143-Leu147 involved in the beta-sheet.

Amino Acid Sequence↗
PubMed1992

Frequent loss of heterozygosity at the DCC locus in gastric cancer.

We examined 28 cases of surgically resected gastric cancer, excluding the diffuse type, for loss of heterozygosity (LOH) on 12 chromosomal arms using polymorphic DNA markers. LOH on chromosome 18q was detected in 61% (14 of 23) of the cases by the probes OLVIIA8, OLVIIE10, p15-65, SAM 1.1, and OS-4, and a putative common region showing LOH included the locus of the DCC tumor suppressor gene. LOH on chromosome 17p was also frequently found (8 of 19 or 42% of the cases) by the probes p10-3 and pHF12-1, and in 5 of these 6 cases the LOH on chromosome 17p was accompanied by LOH on chromosome 18q. On the other hand, the incidence of LOH was 30% or less using probes pHRnES, pHF12-65, p-c-mybE2.6, NJ3 3.2, pHF12-8, pHINS6.0, p9D11, hp2-alpha, pCMM6, and P1A5 on chromosomes 1q, 5, 6q, 7q, 9, 11p, 13q, 16q, 20, and 22q, respectively. LOH on chromosome 18q was frequent irrespective of the depth of tumor invasion, whereas the incidence of LOH on chromosome 17p was higher in the cases in which the tumor invaded beyond the muscularis propria than in those in which tumor invasion was limited to the submucosa and muscularis propria. These results suggest that LOH on chromosome 18q occurs at an earlier stage than LOH on chromosome 17p and that the inactivation of tumor suppressor genes located on chromosome 17p and 18q (e.g., the p53 and DCC genes) is critically involved in the development of the majority of gastric cancers. While alteration of the p53 gene is observed in various human cancers, that of the DCC gene is considered to occur more selectively in gastrointestinal cancers.

Chromosome Deletion↗
PubMed1992

Regulation of rat hepatic peroxisomal enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme by thyroid hormone.

Rat hepatic t protein that is negatively regulated by thyroid hormone in nuclear globulin extract was characterized by the antibodies. The following evidence indicated that t protein is a peroxisomal enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (bifunctional enzyme). 1. Both proteins had an identical molecular size, and were immunologically indistinguishable from each other. 2. The t protein was abundant in mitochondrial fraction which contained abundant peroxisomes. 3. The amount of the t protein was increased by a peroxisomal proliferator. 4. The activity of the peroxisomal bifunctional enzyme corresponded to the t protein in CM-Sephadex column chromatography. The amount of peroxisomal bifunctional enzyme was increased by thyroidectomy and decreased by 3,5,3'- triiodo-L-thyronine treatment in the whole homogenate of rat liver. These results indicate that the levels of peroxisomal bifunctional enzyme were regulated by thyroid hormone in vivo.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1992

Functional expression of a full-length cDNA coding for rat neural cell adhesion molecule L1 mediates homophilic intercellular adhesion and migration of cerebellar neurons.

Neural cell adhesion molecule L1 is postulated to be involved in cell-cell interaction, neurite elongation, fasciculation of axons, cell migration, and myelination. To determine the function of L1 directly, we have transfected rat L1 cDNA into mouse fibroblast L cells. Stable transformants expressing L1 showed uniform surface expression of the molecule without phenotypic changes. Dispersed L1-expressing transfectants aggregated with faster kinetics than control cells in a homophilic manner. Divalent cations were not required for this cell aggregation. L1-transfected cells markedly enhanced neuronal cell adhesion and migration in co-culture with rat cerebellar neurons. These results indicate that L1 is involved in a determinant step of neural development through molecular interactions.

Animals↗
PubMed1992

Selective inhibition of membrane fusion events in echinoderm gametes and embryos by halenaquinol sulfate.

Halenaquinol sulfate, a hydroquinone sulfate obtained from the sponge Xestospongia sapra, prevented cell membrane fusion events of echinaderm gametes but did not affect early embryonic development of fertilized eggs up to the gastrula stage. However, halenaquinol sulfate inhibited secretion of hatching enzyme, resulting in the formation of gastrulae that were surrounded by the fertilization envelope. Therefore, the use of halenaquinol sulfate offers a unique opportunity to analyze the role of secretory events in complex populations of cells without affecting other cellular functions.

Animals↗