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Biomedical subjects

K Nagata

Publications and source records attributed to K Nagata.

At least 451 records · Page 25Linked to original sources
PubMed1995

Morphological changes of BM-N4 cells induced by bombyxin, an insulin-related peptide of Bombyx mori.

Bombyxin is a brain secretory peptide of the silkmoth Bombyx mori whose amino acid sequence shows considerable sequence homology with vertebrate insulin-family peptides. We found that a Bombyx cell line, named BM-N4, showed morphological changes when bombyxin was added to the culture medium at doses as low as 10(-10) M. Bombyxin-treated cells displayed a series of morphological modifications: 1 to 2 weeks after introduction of bombyxin, the cells increased in size, then they trended to aggregate, or took a spindle shape. These changes of the cells were bombyxin specific, not induced by other vertebrate insulin-family peptides. To prove that these modifications of the cells are mediated by receptors on the cell surface of the cells, we performed a receptor binding assay using 125I-labeled bombyxin. Scatchard analysis of the binding assay indicated that this cell line has a single class of receptors for bombyxin with a Kd = 2.36 +/- 0.56 nM and each cell has 15,800 +/- 1400 binding sites on the cell surface.

Animals↗
PubMed1995

Tamoxifen directly stimulates the mineralization of human osteoblast-like osteosarcoma cells through a pathway independent of estrogen response element.

Tamoxifen, an estrogen antagonist, stimulated the mineralization of osteoblasts in vitro in a dose-dependent manner in the presence of inorganic phosphate. The mechanism of mineralization by tamoxifen was different from that by 1 alpha, 25-dihydroxyvitamin D3. In addition, tamoxifen did not activate estrogen-response-element-mediated transcription in osteoblasts. These findings suggest that tamoxifen directly stimulates the mineralization of osteoblasts through a pathway independent of the conventional estrogen-response-element-mediated gene expression.

Alkaline Phosphatase↗
PubMed1995

Replication factor encoded by a putative oncogene, set, associated with myeloid leukemogenesis.

DNA replication of the adenovirus genome complexed with viral core proteins is dependent on the host factor designated template activating factor I (TAF-I) in addition to factors required for replication of the naked genome. Recently, we have purified TAF-I as 39- and 41-kDa polypeptides from HeLa cells. Here we describe the cloning of two human cDNAs encoding TAF-I. Nucleotide sequence analysis revealed that the 39-kDa polypeptide corresponds to the protein encoded by the set gene, which is the part of the putative oncogene associated with acute undifferentiated leukemia when translocated to the can gene. The 41-kDa protein contains the same amino acid sequence as the 39-kDa protein except that short N-terminal regions differ in both proteins. Recombinant proteins, which were purified from extracts of Escherichia coli, expressing the proteins from cloned cDNAs, possessed TAF-I activities in the in vitro replication assay. A particular feature of TAF-I proteins is the presence of a long acidic tail in the C-terminal region, which is thought to be an essential part of the SET-CAN fusion protein. Studies with mutant TAF-I proteins devoid of this acidic region indicated that the acidic region is essential for TAF-I activity.

Amino Acid Sequence↗
PubMed1995

Stimulation of DNA transcription by the replication factor from the adenovirus genome in a chromatin-like structure.

Adenovirus (Ad) genome DNA is complexed with viral core proteins in the virus particle and in host cells during the early stages of infection. This DNA protein complex, called Ad core, is thought to be the template for transcription and DNA replication in infected cells. The Ad core functioned as template for DNA replication in the cell-free system consisting of viral replication proteins, uninfected HeLa nuclear extracts, and a novel factor, template activating factor-I (TAF-I) that we have isolated from uninfected HeLa cytoplasmic fractions. The Ad core did not function as an efficient template in the cell-free transcription system with nuclear extracts of uninfected HeLa cells. The addition of TAF-I resulted in the stimulation of transcription from E1A and ML promoters on the Ad core. TAF-I was required, at least, for the formation of preinitiation complexes. These observations suggest that, in addition to factors essential for transcription on naked DNA template, the factor such as TAF-I needed for replication of the Ad core is also required for transcription from the Ad genome in a chromatin-like structure.

Adenoviridae↗
PubMed1995

Transcriptional elements directing a liver-specific expression of P450/6 beta A (CYP3A2) gene-encoding testosterone 6 beta-hydroxylase.

The P450/6 beta A (CYP3A2) gene encoding a testosterone 6 beta-hydroxylase is expressed predominantly in liver and induced by the treatment of rats with various compounds. To understand the mechanism of the basal transcriptional activation of the CYP3A2 gene, the cis-acting elements in the proximal promoter region (-165 to -73) of the CYP3A2 gene were identified in this study. Nuclear extract from rat livers interacted with three sites, 6 beta A-A (-106 to -87), 6 beta A-B (-140 to -119) and 6 beta A-C (-163 to -145). These sites were detectable by DNase I footprinting and gel mobility shift assays and found to share nucleotide sequence similarity with each other (T(A/C)(A/C)N(A/G)AAG(G/T)(C/T)CA). Direct repeats of AGTTCA (-134 to -120) and AG(G/C)TCA (-162 to -148) are also detected in 6 beta A-B and 6 beta A-C sites, respectively. To elucidate the relationship of these sites with basal transcriptional activation of the CYP3A2 gene, varying lengths of the proximal promoter region (-164 to +41) fused to a CAT reporter gene were transfected in human hepatoma (HepG2) and mouse adrenal tumor (Y-1) cells. The relative level of CAT activity in HepG2 cells was slightly increased by the deletion of the 5'-portion from -164 to -111 bp, but was reduced to 14% of the control (the construct including from -110 to +41) by the deletion from -110 to -81 including the 6 beta A-A site. On the other hand, these deletions have no clear effect on the level of the activity in Y-1 cells. Substitution mutations at two nucleotides in the 6 beta A-A site resulted in the reduction of CAT activity in HepG2 cells to 12% of the activity in the wild-type construct. The interaction of an oligonucleotide corresponding to the 6 beta A-A site (-106 to -87) with liver nuclear factors was completely inhibited by the addition of a typical oligonucleotide for hepatocyte nuclear factor-4 (HNF-4) binding site (F. M. Sladek, W. Zhong, E. Lai, and J. E. Darnell, Jr., 1990, Genes Dev. 4, 2353-2365) but not of oligonucleotides corresponding to 6 beta A-B or 6 beta A-C sites. These results suggest an essential role of the binding of HNF-4 and/or HNF-4-related nuclear factors to the 6 beta A-A site on the basal transcriptional activation of the CYP3A2 gene in liver cells.

Animals↗
PubMed1995

Quercetin suppresses heat shock response by down regulation of HSF1.

A bioflavonoid quercetin suppressed the stress response in heat-shocked cells and we have investigated the inhibitory mechanism in this report. After treatment of cells with nonlethal concentrations of quercetin, the binding of heat shock factor (HSF) to the heat shock element (HSE) was inhibited as detected by gel shift assay. We examined whether quercetin inhibits heat shock response by inhibiting HSF trimer-formation and found it was not the case. Instead, pretreatment of the cells with quercetin caused a decrease in the level of HSF1, especially of the constitutively phosphorylated form. This decrease was more prominent in heat-shocked cells than in control cells. These data suggest that (1) quercetin does not affect the trimer formation of HSF1, and (2) the decline of the HSF1-HSE complex might be linked to the decrease of HSF1 levels caused by quercetin.

Blotting, Western↗
PubMed1995

Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells.

The biological action of adrenomedullin, a novel hypotensive peptide, on bovine aortic endothelial cells, was examined. The specific binding of adrenomedullin to these cells was observed, and adrenomedullin was found to induce intracellular cAMP accumulation in a dose-dependent manner. EC50 for the cAMP accumulation was about 100 times lower than the apparent IC50 for the binding assay. Adrenomedullin also induced increase of intracellular free Ca2+ in endothelial cells in a dose-dependent manner. The Ca2+ response to adrenomedullin was biphasic with an initial transient increase due to the release from thapsigargin-sensitive intracellular Ca2+ storage and a prolonged increase by influx through the ion channel on the plasma membrane. This intracellular free Ca2+ increase resulted from phospholipase C activation and inositol 1,4,5-trisphosphate formation, and seemed to cause nitric oxide synthase activation by monitoring intracellular cGMP accumulation. Both cAMP accumulation and Ca2+ increased responses to adrenomedullin were mediated by cholera toxin-sensitive G protein, but the two signal transduction pathways were independent. Thus, the results suggest that adrenomedullin elicits the hypotensive effect through at least two mechanisms, a direct action on vascular smooth muscle cells to increase intracellular cAMP and an action on endothelial cells to stimulate nitric oxide release, with both leading to vascular relaxation.

Adrenomedullin↗
PubMed1995

Age-related attenuation of HSP47 heat response in fibroblasts.

The collagen-binding heat shock protein of molecular weight 47,000 (HSP47), resident in the endoplasmic reticulum (ER), is assumed to play a specific role as a molecular chaperon in the processing of procollagen molecules. The present investigation of age-related alteration in the HSP47 heat response in cultured murine and human fibroblasts revealed expression in cells with a low population doubling level (PDL) derived from young mice and people more inducible by heat treatment than those from older mice and people. On the other hand, cells with a high PDL showed a very low heat response in terms of HSP47 expression regardless of the donor age. Northern blot analysis of HSP47 m-RNA indicated that the age related attenuation of HSP47 expression was regulated by transcriptional mechanisms. Furthermore, immunofluorescent analysis using a monoclonal antibody against the carboxylterminal propeptide of type I procollagen revealed far greater retention of procollagen molecules in the ER lumen of cells from old persons than in those from young persons. This was particularly prominent in heat-treated cells from old persons, indicating the possibility that the observed decrease in HSP47 heat response might cause blockage of procollagen transport to the Golgi and therefore secretion.

Aging↗
PubMed1995

Characterization of the 5'-flanking region and chromosomal assignment of the human brain natriuretic peptide gene.

Brain natriuretic peptide (BNP) is a cardiac hormone that occurs predominantly in the ventricle, and synthesis and secretion of BNP are greatly augmented in patients with congestive heart failure and in animal models of ventricular hypertrophy. In order to elucidate the molecular mechanisms underlying the human BNP gene expression in the heart, the human BNP gene was isolated from a size-selected genomic minilibrary. The 1.9-kb human BNP 5'-flanking region (-1813 to +110) contained an array of putative cis-acting regulatory elements. Various lengths of the cloned 5'-flanking sequences were linked upstream to the bacterial chloramphenicol acetyltransferase (CAT) gene, and their promoter activities were assayed. The 1.9-kb promoter region showed a high-level CAT activity in cultured neonatal rat ventricular cardiocytes. When the CT-rich sequences (-1288 to -1095) were deleted, the high-level activity was reduced to approximately 30%. The 399-bp BNP 5'-flanking region (-289 to +110) showed approximately 10% activity of the 1.9-kb region. Furthermore, using human-rodent somatic hybrid cell lines, the BNP gene was assigned to human chromosome 1, on which the atrial natriuretic peptide gene is localized. The present study leads to a better understanding of the molecular mechanisms for the human BNP gene expression in the heart.

Animals↗
PubMed1995

Discrepant 99mTc-ECD images of CBF in patients with subacute cerebral infarction: a comparison of CBF, CMRO2 and 99mTc-HMPAO imaging.

Three patients with subacute ischemic cerebral infarction examined by SPECT with 99mTc-ECD and PET within the same day showed signs of luxury perfusion in the subacute phase, which is between 9 to 20 days after the onset. A 99mTc-HMPAO SPECT study was also performed within 2 days of the ECD-SPECT study. ECD-SPECT images of three patients displayed a focal decreased uptake in the infarcted lesions, while in infarcted foci, there was almost equivalent or increased CBF compared to normal and unaffected areas, decreased CMRO2, and high HMPAO uptake. The ECD-SPECT results were similar to those of CMRO2 rather than CBF, though the HMPAO-SPECT image was similar to that of CBF. In one patient, HMPAO images revealed hyperfixation of the tracer. In the chronic phase and in the acute phase before 5 days after the onset, there were no discrepancies among the ECD-SPECT, CBF, HMPAO-SPECT, and CMRO2 images. These observations indicated that 99mTc-ECD is a good indicator of damaged brain tissues in subacute ischemic infarction. They also suggested that 99mTc-ECD is a potential agent with which to evaluate cerebral tissue viability in some pathological states of cerebrovascular disease. The characteristics may be suitable for confirming the effects of thrombolytic therapy in acute ischemia, because these conditions often show signs of luxury perfusion when the therapy is successful.

Aged↗
PubMed1995

Identification of heterotrimeric GTP-binding proteins in human megakaryoblastic leukemia cell line, MEG-01, and their alteration during cellular differentiation.

Various heterotrimeric GTP-binding proteins (G proteins) are possible to have important functions in hematopoietic cells. However, there has been no information regarding their expression in magakaryoblasts and/or megakaryocytes. In the present study, protein contents of seven G protein alpha subunits (Gs alpha, Gi2 alpha, Gi3 alpha, Gz alpha, G11 alpha, Gq alpha and G12 alpha) and beta subunit in a human megakaryoblastic leukemia cell line, MEG-01, were analyzed by immunoblotting. Immature MEG-01 cells expressed the alpha subunits of Gs, Gi2, Gi3, Gz, G11 and G12 at protein molecule level. During the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced differentiation process, the contents of Gi2 alpha and Gi3 alpha increased, whereas the protein levels of Gz alpha, Gs alpha, G11 alpha and G12 alpha were observed to hardly change. beta subunit was also observed to be present in immature MEG-01 cells and to increase continuously throughout the differentiation process. For the expression of Gi2 alpha and beta subunits, chronic TPA-treatment was required although Rac2, a low M(r) GTP-binding protein, was expressed abundantly by only 30 min-TPA-treatment followed by 3 day-culture.

Blood Platelets↗
PubMed1995

Two endogenous methyl-alpha-D-glucopyranoside uptake activities in Xenopus oocytes.

Endogenous methyl-alpha-D-glucopyranoside (AMG) uptake in Xenopus oocytes was measured with 14C-labeled AMG. Two AMG uptake activities, one Na(+)-dependent and the other Na(+)-independent, were observed in oocytes incubated with 2 mM AMG. However, only Na(+)-dependent uptake was observed at 0.1 mM AMG. Na(+)-dependent AMG uptake was attributed to the endogenous Na+/glucose co-transporter since it was inhibited by phlorizin. On the other hand, Na(+)-independent AMG uptake was inhibited by cytochalasin B or 2-deoxy-D-glucose. This suggests that AMG can be transported by the endogenous facilitated glucose transporter as well as by the endogenous co-transporter. In addition, a substrate range of the endogenous co-transporter was examined by competition experiments.

Animals↗
PubMed1995

Sodium channels and GABAA receptor-channel complex as targets of environmental toxicants.

Voltage-activated sodium channels and GABAA receptor-chloride channel complex are among the most important target sites of various environmental neurotoxicants. Pyrethroids keep the sodium channels open for prolonged periods of time leading to hyperexcitation of the entire nervous system. In rat cerebellar Purkinje neurons and dorsal root ganglion neurons, only about 1% of sodium channel population needed to be modified by the pyrethroid tetramethrin to increase the depolarizing after-potential to the level of the threshold membrane potential for generation of repetitive after-discharges. This concept of toxicity amplification is applicable to other chemicals that go through a threshold phenomenon to exert their effects. The potency of pyrethroids on neuronal sodium channels increased with lowering the temperature with a Q10 value of 0.2. The selective pyrethroid toxicity between mammals and insects can be quantitatively explained on the basis of the differences in 5 factors, i.e. the intrinsic sodium channel sensitivity, the sodium channel modification due to temperature difference, the reversibility of sodium channel, the detoxication of pyrethroids, and body size. These 5 factors are multiplied to approximately 2000 which is in the same order of magnitude as that of the difference in LD50. Dieldrin had a dual action on the GABAA receptor-chloride channel complex of rat dorsal root ganglion neurons. The initial transient potentiation of GABA-induced currents after application of dieldrin was followed by a suppression. Dieldrin-induced potentiation of current was observed only when the gamma 2 subunit was present in embryonic kidney cells (HEK-293) transfected with GABA receptor subunits. Dieldrin-induced suppression was observed in the presence and absence of the gamma 2 subunit. The dieldrin suppression of GABA-induced currents is deemed directly responsible for hyperactive symptoms of poisoning in animals.

Animals↗
PubMed1995

Stabilized enzymatic reagents for measuring glucose, creatine kinase and gamma-glutamyltransferase with thermostable enzymes from a thermophile, Bacillus stearothermophilus.

Stabilized enzymatic reagents for measuring some components in biological fluids have been successfully developed based on two kinds of thermostable enzymes derived from Bacillus stearothermophilus with separation of the reagent into two complementary solutions. The thermostable glucokinase produced was applied to the measurement of glucose and creatine kinase activity, while the alanine dehydrogenase produced was used for the measurement of gamma-glutamyltransferase activity. The enzymatic reagents were also stabilized by developing two separate reagents with an optimum pH for the main reagent components. The stability of the reagents in liquid form was examined at 10 degrees C. It was clearly shown that the reagents for measuring glucose and creatine kinase activity were stable and retained their full capability for accurate measurement in biological fluids for over one year. The alanine dehydrogenase product was stable for at least 40 days.

Alanine Dehydrogenase↗
PubMed1995

Differential effects of dobutamine and a phosphodiesterase inhibitor on early diastolic filling in patients with congestive heart failure.

OBJECTIVES: This study was designed to compare the influence of beta-adrenergic stimulation (dobutamine) and a selective phosphodiesterase inhibitor (MS-857) on left ventricular diastolic performance and Doppler transmitral flow velocity patterns in patients with congestive heart failure and to elucidate the mechanisms for changes in early diastolic filling induced by each agent. BACKGROUND: Both beta-adrenergic agonists and phosphodiesterase inhibitors act through the cyclic adenosine monophosphate pathway. However, it is controversial whether they have similar effects on diastolic performance. No previous studies have investigated the effects of these agents on Doppler-derived measurements of diastolic filling. We hypothesized that they would have different effects on early diastolic filling in patients with congestive heart failure. METHODS: Twenty patients with chronic congestive heart failure resulting from idiopathic dilated cardiomyopathy were randomized to receive intravenous infusion of dobutamine (5 micrograms/kg body weight per min, n = 10) or oral administration of MS-857 (15 mg, n = 10). Transmitral flow velocity patterns were obtained with simultaneous recordings of pressure-volume loops using a conductance catheter with a micromanometer tip before and after drug administration. RESULTS: Left ventricular filling pressure was reduced by both agents. Dobutamine decreased the time constant of isovolumetric relaxation and increased the difference between pulmonary artery wedge pressure at the peak of the v wave and left ventricular minimal pressure (10 +/- 3 to 12 +/- 4 mm Hg, p < 0.01) and peak early filling velocity (47 +/- 7 to 56 +/- 11 cm/s, p < 0.01). The diastolic pressure-volume relation showed a leftward shift in all patients. In contrast, MS-857 did not affect the time constant but maintained the pressure difference (9 +/- 3 to 8 +/- 3 mm Hg, p = NS) and peak early filling velocity (50 +/- 7 to 49 +/- 12 cm/s, p = NS). The diastolic pressure-volume relation after MS-857 showed a downward shift in most patients. CONCLUSIONS: These results indicate that beta-adrenergic stimulation and phosphodiesterase inhibitors have different effects on early diastolic filling through different mechanisms in patients with congestive heart failure.

Administration, Oral↗
PubMed1995

N-ras and p53 gene mutations are very rare events in multiple myeloma.

The frequencies of p53 and N-ras gene mutations were examined in multiple myeloma. DNA samples of 45 cases of multiple myeloma were obtained from stored bone marrow smears. All samples were analyzed for the N-ras gene at codon 12 by the allele specific restriction analysis method and at codon 61 by direct sequencing. DNA was screened for mutations by single-strand conformation polymorphism analysis for the p53 gene in exons 5 to 8. DNA fragments showing an altered electrophoretic pattern were further studied by direct sequencing to confirm the mutations. Ras mutations were found in two cases at codon 61 (4.4%). A point mutation of the p53 gene was detected in one of 45 cases (2.2%). These 3 cases with mutations were in an advanced stage. In conclusion, N-ras and p53 gene mutations may occur at a late stage of multiple myeloma, but the frequencies of the mutations are very low.

Adult↗
PubMed1995

Fluorescence-labeled synthetic glycopolymers: a new type of sugar ligands of lectins.

Radical copolymerization of a polymerizable dansyl derivative, N-2-propenyl-(5-dimethylamino)-1-naphthalene sulfonamide, with sugar monomers and acrylamide proceeded smoothly in aqueous solution in the presence of ammonium persulfate and N,N,N',N'-tetramethylethylenediamine and afforded a novel type of water-soluble glycopolymers having fluorescent side-chains. Fluorescence emission spectra of these polymeric sugar-ligands by excitation at 340 nm revealed maxima at 448 and 528 nm. When the glycopolymer carrying galactose residues was saturated with Ricinus communis agglutinin (RCA60), the fluorescence emission maxima at 448 and 528 nm were not shifted significantly, although the fluorescence intensities were decreased by 20 and 14%, respectively. Polymeric sugar-cluster effects drastically enhanced the association constants of galactose residues with RCA60 in the order of 10(8) M-1. The significance for efficient binding of galactose density on the glycopolymer was also demonstrated by using glycopolymers with different degrees of galactose branching.

Allyl Compounds↗