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Biomedical subjects

N Shimizu

Publications and source records attributed to N Shimizu.

At least 19 recordsLinked to original sources
PubMed1992

Hepatocyte growth factor is linked by O-glycosylated oligosaccharide on the alpha chain.

The glycosylation site and the structure of O-glycosylated oligosaccharide of recombinant human HGF were investigated. N-acetylgalactosamine (GalNAc) in the alpha chain suggested the presence of O-glycosylated oligosaccharide. Sugar analysis and amino acid sequence analysis of peptide fragments produced by limited degradation revealed that O-glycosylated oligosaccharide linked to Thr445 of the alpha chain. The molecular weight of the oligosaccharide was determined with ion spray mass spectrometry. From these studies, the structure of the O-glycosylated oligosaccharide on the alpha chain of HGF was concluded as [formula: see text].

Amino Acid Sequence↗
PubMed1992

Concurrent measurement of serotonin metabolism and single neuron activity changes in the lateral hypothalamus of freely behaving rat.

To further investigate the activity of serotonin neurons in relation to feeding behavior, the metabolic activity of the serotonergic system and single neuron activity changes in the lateral hypothalamic area (LHA) were investigated concurrently in freely behaving rats. The extracellular concentration of 5-hydroxyindoleacetic acid (5-HIAA), a metabolic product of serotonin in the LHA, began to increase concomitantly with the early stage of nocturnal eating. The increased 5-HIAA returned to the basal level within 3 or 4 h. In conjunction with the increase in serotonin metabolism, activity of 12 out of 30 LHA neurons (40%) increased, whereas it decreased in 7 (23%), and in 11 (37%) it showed no change. An intracerebroventricular injection of lisuride suppressed the increased activity in 7 of the 12 neurons, but had no effect on the others. These results suggest that the concurrent increase in serotonin metabolism and neuron activity changes in the LHA may occur in the early portion of the nocturnal eating period, and may be important in controlling feeding behavior.

Animals↗
PubMed1992

DNA topoisomerase I phosphorylation in murine fibroblasts treated with 12-O-tetradecanoylphorbol-13-acetate and in vitro by protein kinase.

The phosphorylation of DNA topoisomerase I in quiescent murine 3T3-L1 fibroblasts treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was characterized by in vivo labeling with [32P] orthophosphate and immunoprecipitation with a scleroderma anti-DNA topoisomerase I autoantibody. DNA topoisomerase I phosphorylation was stimulated 4-fold by 2 h of TPA treatment (TPA at 100 ng/ml maximally enhanced phosphorylation). Purified DNA topoisomerase I was phosphorylated in vitro in a Ca2+ and phospholipid-dependent fashion by types I, II, and III protein kinase C. The phosphorylation reaction was stimulated by TPA and had an apparent K(m) of 0.4 microM. DNA topoisomerase I was phosphorylated in vivo and in vitro predominantly at serine. The major tryptic phosphopeptides from DNA topoisomerase I in TPA-treated fibroblasts and phosphorylated by protein kinase C comigrated in thin-layer electrophoresis. The half-life of incorporated phosphate on DNA topoisomerase I was 40 min in both TPA-treated and control cells. These results suggest that phosphorylation is a mechanism for activating DNA topoisomerase I in fibroblasts treated with TPA and that protein kinase C functions in the phosphorylation.

3T3 Cells↗
PubMed1992

cis-acting elements responsible for muscle-specific expression of the myosin heavy chain beta gene.

The 5' flanking region of the rabbit myosin heavy chain (HC) beta gene extending 295 bp upstream from the cap site provides muscle-specific transcriptional activity. In this study, we have identified and functionally characterized cis-acting elements that regulate the muscle-specific expression within this region. By using linker-scanner (LS) mutants between -295 bp and a putative TATA box, we found five distinct positive cis-acting sequences necessary for transcription: element A, the sequences between -276 and -263, which contains a putative M-CAT motif in an inverted orientation; B, the sequences between -207 and -180; C, the sequences between -136 and -127; D, the sequences between -91 and -80; and E, a TATA consensus sequence at -28. The fragment containing both A and B elements dramatically enhanced the expression of the chloramphenicol acetyltransferase (CAT) gene driven by a heterologous promoter in differentiated muscle cells, whereas fragments containing either A or B elements alone had little or no effect in either muscle or nonmuscle cells. Therefore, these two elements appear to act cooperatively in determining a high level of muscle- and stage-specific expression. Unlike the typical enhancer element, this region functions in an orientation-dependent manner. In contrast, the fragment containing C and D elements activates the heterologous promoter in both muscle and nonmuscle cells in an orientation-independent manner.

Animals↗
PubMed1992

[Long-term treatment of acromegaly and gigantism with octreotide (SMS 201-995)].

Twenty-one patients with active acromegaly and two patients with pituitary gigantism were treated with the long-acting somatostatin analogue octreotide (100-600 micrograms/day, sc, two or three times daily or 300-1500 micrograms daily by intermittent sc infusion) for 9-63 months. There was rapid clinical improvement. The fasting plasma GH levels were significantly suppressed (less than 50% of the values before treatment) in 17 patients and were normalized (less than 5 ng/ml) in 6 patients (27.3%). Plasma IGF-I levels were lowered by 50% and were normalized in 7 out of 18 cases. The effect of octreotide on pituitary tumor size was evaluated in 13 patients. In 4 cases, the shrinkage of the pituitary tumor was detected by computed tomographic scans and/or magnetic resonance imaging studies. The drug was generally well tolerated. However, there were probably newly formed gallstones in two patients during the therapy. Our study suggests that octreotide is an effective and relatively safe new approach for treating active acromegaly and gigantism.

Acromegaly↗
PubMed1992

cDNA cloning and chromosome assignment of the gene for human brain 14-3-3 protein eta chain.

We present the nucleotide sequence of a cDNA clone of mRNA encoding human 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases and an endogenous inhibitor of protein kinase C. The 1,730-nucleotide sequence of the cloned cDNA contains 191 bp of a 5'-noncoding region, the complete 738 bp of coding region, and 801 bp of a 3'-noncoding region containing three canonical polyadenylation signals. The 14-3-3 protein eta chain cDNA encoded a polypeptide of 246 amino acids with a predicted molecular weight 28,196. The predicted amino acid sequence of human 14-3-3 protein eta was highly homologous to that of previously reported bovine and rat 14-3-3 proteins with only two amino acid differences. The sequence carries structural features as putative regions responsible for activation of tyrosine and tryptophan hydroxylases and for inhibition of Ca2+/phospholipid-dependent protein kinase C. Northern blot analysis demonstrated widespread expression of the 14-3-3 protein eta chain in cultured cell lines derived from various human tumors. These findings suggest the conservative functions of the 14-3-3 protein among species. Spot blot hybridization analysis with flow-sorted chromosomes showed that the human 14-3-3 protein eta chain gene is assigned to chromosome 22.

14-3-3 Proteins↗
PubMed1992

Outcome of patients with lung cancer detected via mass screening as compared to those presenting with symptoms.

We performed lung cancer resection in 721 patients between 1980 and 1989. Cancers were detected via mass screening programs by annual chest X-ray examination in the majority of cases. We evaluated the surgical results in patients with tumors detected by mass screening and compared them to those in whom the malignancy was detected by symptoms. Lesions in the mass screened group were T1 to T2 tumors in 90% of the cases, and NO in 73%. Stage I disease accounted for 65.3% in the mass screened group. The overall 5-year survival rate was 56.2% in the mass screened group, which was significantly better than the 25.3% for the symptom group (P less than 0.001). The surgical results in the lung cancer cases detected by the mass screening program had better results than the cases who presented with symptoms.

Aged↗
PubMed1992

Transsternal thoracotomy for bilateral pulmonary metastasis.

Advances in the surgical treatment of primary malignancies and the recent chemotherapy have led to an expansion of the surgical treatment of metastatic lung tumors. However, multiple pulmonary metastases are often found and may affect both lungs. It is difficult to reach tumors in the posterior parts of the lung when using a common midsternal approach, especially lesions located in the left lower lobe. We performed transsternal simultaneous bilateral thoracotomy on 10 patients with bilateral lung tumors (9 bilateral metastatic pulmonary tumors and 1 bilateral primary lung cancer). This procedure provides a wide operative field and is an effective method of thoracotomy for patients with bilateral lung tumors. In future, this method should be more actively performed for patients in whom it is indicated.

Adolescent↗
PubMed1992

Six strains of human immunodeficiency virus type 1 isolated in Japan and their molecular phylogeny.

Five strains of human immunodeficiency virus type 1 (HIV-1) were isolated from five Japanese hemophilia patients. Two isolates, HIV-1[GUN-1] and HIV-1[GUN-2], were from brother patients with hemophilia B and the other three isolates, HIV-1[GUN-3], HIV-1[GUN-4], and HIV-1[GUN-5], were from hemophilia A patients. Another HIV-1 strain, HIV-1[GUN-6], was isolated from a Canadian male homosexual with AIDS. The restriction endonuclease cleavage maps of the proviral genomes of these six HIV-1 strains revealed that they were apparently different from each other. The phylogenetic trees constructed using restriction maps and nucleotide sequences were quite similar, indicating that phylogenetic analyses of Japanese HIV-1 isolates can be done using restriction maps of the proviruses. Phylogenetic analyses showed that they were more closely related to HIV-1s which had been reported to be isolated from homosexual patients in the United States than those isolated from African patients. In particular, GUN-1 and GUN-2 isolates were on the branch of a San Francisco isolate, ARV2, while GUN-5 and GUN-6 isolates were on the branch of HTLV-IIIB-related isolates.

AIDS-Related Complex↗
PubMed1992

Methylation status of ribosomal RNA gene clusters in the flow-sorted human acrocentric chromosomes.

Southern blot analysis of the human acrocentric chromosomes that were flow-sorted from B-lymphoblastoid cell line GM130B revealed that the sensitivity of the ribosomal RNA (rDNA) gene clusters to the restriction enzyme NotI differs among these rDNA-containing chromosomes: the rDNA clusters of Chromosomes (Chr) 13, 14, and 15 are much more sensitive to NotI digestion than those of Chrs 21 and 22 in this particular cell line. Detailed analysis by use of methylation-sensitive enzymes HpaII and HhaI and methylation-insensitive enzyme MspI confirmed the significant variation in the methylation status of rDNA clusters among these chromosomes. Quantitative analysis by fluorescent in situ hybridization (FISH) indicated that copy number of rDNA varies among individual chromosomes, but the average copy number in the acrocentric Chrs 21 and 22 is significantly greater than that of the Chrs 13, 14, and 15 in GM130B cells. Similar analysis reveals that the methylation status of rDNA clusters in another B-lymphoblastoid cell line GM131 was different from that of GM130B. These data together indicate that the copy number and methylation patterns of rDNA clusters differ among individual acrocentric chromosomes in a given cell line, and they are different among cell lines.

Blotting, Southern↗
PubMed1992

Detection of variation in the ribosomal RNA gene clusters by a modified fluorescence in situ hybridization method.

Physical mapping of genes by fluorescence in situ hybridization (FISH) has become routine using fluorescein isothiocyanate (FITC) for probe detection and propidium iodide (PI) for chromosome staining. We have modified this conventional FISH method in a way that utilizes Texas red (TR) for signal detection and quinacrine mustard (QM) for chromosome banding. Using this Texas red and quinacrine (TRQ) method, we were able to identify individual acrocentric chromosomes with varying degrees of ribosomal RNA gene clusters. Two acrocentric chromosomes were found to carry extremely small number of rRNA gene copies as compared to the other eight counterparts in human diploid lymphoblastoid cell line GM00130B. Thus, the TRQ method allows one to probe for a specific sequence while identifying individual chromosomes and will be powerful for the chromosomal localization of various genes.

Cell Line↗
PubMed1992

Stimulation by interleukin-1 of interleukin-6 production by human periodontal ligament cells.

Interleukin-1(IL-1), a cytokine present in the gingiva and crevicular fluid of patients with periodontitis and in the periodontal ligament (PDL) of experimentally moved teeth, has multiple biological activities, including the ability to elicit bone resorption. Interleukin-6, also found in the gingiva of patients with periodontitis, may induce osteoclastic bone resorption through an effect on osteoclastogenesis. Here IL-6 production and its gene expression in response to recombinant IL-1 beta were examined in primary cultures of PDL cells. IL-1 beta stimulated IL-6 production by these cells in a dose- and time-dependent manner; this increase in IL-6 production was much higher than that in human gingival fibroblasts. In situ hybridization, using a synthetic oligonucleotide DNA probe of the IL-6 gene, revealed that most PDL cells expressed IL-6 mRNA in response to IL-1 beta treatment. The finding that IL-6 is produced by PDL cells and is regulated by IL-1 beta has revealed a potentially important mechanism for controlling alveolar bone resorption.

Cells, Cultured↗
PubMed1992

Regulation of arginase production by glucocorticoid in three human gastric cancer cell lines.

Gastric cancer tissues have high levels of glucocorticoid receptors (GR) and arginase. To investigate the interrelation of glucocorticoid, GR and arginase, three human gastric cancer cell lines (AZ-521, NUGC-3, KATO-III) were treated with hydrocortisone in the presence or absence of a glucocorticoid antagonist RU38486. GR were found to be present in all three lines, and hydrocortisone significantly increased the production of total arginase in all 3 lines. The induction of arginase production by hydrocortisone was inhibited by RU38486. These findings suggest that the regulation of arginase production by hydrocortisone in gastric cancer cells is mediated through GR.

Arginase↗
PubMed1992

A role for protein kinase C in the growth of human erythroid progenitor cells.

We searched for a possible role for protein kinase C in the growth of human erythroid progenitor cells, using pharmacologic approaches. Two protein kinase C inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine, dose-dependently inhibited the growth of immature erythroid progenitor cells (BFU-E) induced by interleukin 3 (IL-3) plus erythropoietin (Ep) or granulocyte macrophage colony-stimulating factor (GM-CSF) plus Ep whereas a weaker analog of H-7, N-(2-guanidinoethyl)-5-isoquinoline sulfonamide (HA-1004), had no effect on the number of BFU-E. These three compounds had no effect on the growth of mature erythroid progenitor cells (CFU-E) stimulated by Ep. The culture of accessory cell-depleted bone marrow demonstrated that the effects of these compounds on colony formation do not appear to be mediated by accessory cells. The potential of these compounds to inhibit the GM-CSF-dependent growth of KG-1 cells correlated well with the extent of their inhibitor of protein kinase C activities from KG-1 cells. Thus, the protein kinase C system is apparently involved in the growth of BFU-E, supported by IL-3 or GM-CSF. The growth signal for CFU-E transduced by Ep may be achieved through other systems.

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

Structure and expression of human and rat D2 dopamine receptor genes.

D2 dopamine receptor may be related with the pathogenesis of Parkinson's disease and schizophrenia. Furthermore, the antipsychotic drugs have high affinity for D2 dopamine receptor. We carried out the cloning of the genomic DNA for human D2 dopamine receptor and clarified the structure of this gene. Our isolated gene spans about 15 kbp and consists of seven exons interrupted by six introns. However, putative first exon was not yet identified. Spot blot hybridization analysis of cell sorter fractionated human chromosomal DNA with D2 receptor genomic DNA revealed the localization of this gene in the chromosome 11 fraction. We analyzed human genomic DNA by Southern blot hybridization with D2 dopamine receptor genomic DNA as a probe, but so far we could not find RFLP. Northern blot analyses of brain RNA of several animals and rat brain RNA after various treatments were carried out. Developmental changes of D2 dopamine receptor mRNA were observed in the rat brains.

Aging↗
PubMed1992

Electrophysiological study of neurotropin-induced responses in guinea pig hypothalamic neurons.

To investigate the direct actions of neurotropin (NSP, a nonproteinaceous extract from inflamed skin of rabbits which is in therapeutic use), intracellular recordings were made from neurons of the ventromedial hypothalamic nucleus (VMH) and lateral hypothalamic area (LHA) in slices of guinea pig brain. In the VMH, NSP, applied by perfusion (0.1-3.0 NU/ml), caused dose-dependent depolarization in 29 of 48 neurons (60%) tested. No change in membrane resistance was observed during the depolarization, which hypothesized that the NSP-induced depolarization might be mediated through the inactivation of the Na-K pump. The NSP-induced depolarization persisted even after the elimination of synaptic activity by perfusion with Ca(2+)-free and high Mg2+ Ringer solution. NSP hyperpolarized the cell membrane of three neurons (6%) while two neurons (4%) showed biphasic responses; transient depolarization followed by long-lasting hyperpolarization. Membrane potential of the remaining 14 neurons was not changed by application of NSP. Of 14 LHA neurons tested for NSP effects, eight (57%) were depolarized, three (21%) were hyperpolarized, and one showed a biphasic response. The present results suggest that NSP significantly modulates hypothalamic neuron activity, and the central modulation of autonomic functions by NSP might be mediated through hypothalamic neurons.

Animals↗