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A Asano

Publications and source records attributed to A Asano.

At least 19 recordsLinked to original sources
PubMed1999

Immortal brown adipocytes from p53-knockout mice: differentiation and expression of uncoupling proteins.

Brown adipose tissue (BAT) is the specific site for metabolic heat production in mammals. To establish a novel immortal brown adipocyte cell line, the stromal-vascular fraction containing preadipocytes was obtained from interscapular BAT of mice deficient of a tumor-suppressor gene p53. The p53-deficient cells, tentatively named as HB2 cells, could be cultured in vitro after repeated passages and differentiated into adipocytes in the presence of insulin, T3 and/or troglitazone, expressing some adipocyte-specific genes and accumulating intracellular lipid droplets. The mRNA level of uncoupling protein 1 (UCP1), a mitochondrial protein specifically present in brown adipocytes, was undetectable in HB2 preadipocytes, but increased after adipose differentiation. In HB2 adipocytes, UCP1 mRNA expression was markedly activated after stimulation of the beta-adrenergic receptor pathway. The mRNA of UCP2 and UCP3, recently cloned isoforms of UCP1, were also detected in HB2 adipocytes, but their levels were not influenced by adrenergic stimulation. Thus HB2 cells seem useful for in vitro studies of BAT and UCP functions.

Adipocytes↗
PubMed1998

A cis-acting regulatory element that affects the alternative splicing of a muscle-specific exon in the mouse NCAM gene.

The pre-mRNA encoding the neural cell adhesion molecule (NCAM) is spliced to generate NCAM isoforms containing the muscle-specific domain (MSD) during myogenesis. Utilizing chimeric NCAM minigenes, we searched for cis-acting elements that contribute to the alternative selection of exon MSDb, one of the four exons encoding MSD, and identified an intronic cis-element located downstream of exon MSDb. The cis-element acted as a negative regulator for the selection of exon MSDb in nonmuscle fibroblasts but not in myoblasts, that are already destined to differentiate into muscle cells. The suppressive effect of this cis-element on the selection of exon MSDb was released in the process of myogenesis. When MyoD was co-expressed with a minigene containing this element in fibroblasts, the suppressive effect of the cis-element was released as the cells underwent differentiation. We propose that this cis-element contributes at least as one of the regulatory elements in the differentiation state-dependent selection of MSD exons in vivo.

Alternative Splicing↗
PubMed1998

Premorbid psychosocial behavior in demented patients.

We investigated the premorbid behavioral characters of demented patients in terms of life style, type A behavior, life events and coping behavior. We adopted the case control study. Significant differences were not found in the inventory scores of life style, passive coping behavior and type A behavior between dementia group and normal controls. However, the life events occurred significantly frequent in the vascular dementia group and the score of positive coping behavior was significantly low in dementia group. The results suggest that those life events and positive coping behavior might be related to the onset of dementia.

Adaptation, Psychological↗
PubMed1998

[Hemophilia B].

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Animals↗
PubMed1997

Trans-regulation of myogenin promoter/enhancer activity by c-ski during skeletal-muscle differentiation: the C-terminus of the c-Ski protein is essential for transcriptional regulatory activity in myotubes.

c-ski gene product is a nuclear protein with myogenesis-promoting and transforming activities. We have analysed the effects of c-ski transfection on the promoter/enhancer activity of the upstream region of the myogenin gene during in vitro myogenesis using CAT reporter assay. When co-transfected with c-ski into myogenic C2C12 cells, promoter/enhancer activity was efficiently suppressed in proliferating cells, but the myogenesis-induced increase in activity was potentiated approximately ten times more (150-fold in the ski-transfected cells) than the ordinary increase (12-fold in the mock) 48 h after induction of differentiation. In non-myogenic 10T1/2 cells, c-ski transfection caused persistent suppression of promoter/enhancer activity in both proliferating and growth-arrested (i.e. myogenesis-inducing) conditions. Thus the ski-dependent potentiation of myogenin gene transcriptional activity appears to be specific for myogenesis. The C-terminal region (amino acids 595-663) of the c-Ski protein was essential for the potentiating activity in myotubes. Other members of the ski-gene family, snoN and snoA, were ineffective in transactivation, possibly because of the defect in the corresponding C-terminal region. c-Ski protein underwent a mobility shift on SDS/PAGE after in vitro myogenesis which may explain the conversion of the activity from suppressive in myoblasts to potentiating in myotubes. Deletion analysis of the upstream region of the myogenin gene revealed that a responsive element to c-ski in myotubes is located at a distinct site upstream of the basal promoter/enhancer region.

Animals↗
PubMed1997

Adrenergic activation of vascular endothelial growth factor mRNA expression in rat brown adipose tissue: implication in cold-induced angiogenesis.

Cold exposure produces adaptive hyperplasia and growth of brown adipose tissue (BAT), the major site of non-shivering thermogenesis in rodents, associated with increased angiogenesis in this tissue. Vascular endothelial growth factor (VEGF), one of the most potent angiogenic factors, was found to be expressed abundantly in BAT of the rat. When rats were exposed to cold at 4 degrees C, the VEGF mRNA level in BAT was increased by 2-3-fold in 1-4 h, but returned to the basal level within 24 h. VEGF expression in other tissues such as heart, kidney and lung did not change after cold exposure. The cold-induced increase in VEGF mRNA was abolished by surgical sympathetic denervation, but mimicked by administration of noradrenaline or a beta3-adrenoceptor agonist CL316,243, indicating the critical role of the beta-adrenergic pathway in VEGF expression in BAT. Among three isoforms of VEGF, the mRNA of a short form (VEGF120) lacking heparin-binding activity was preferentially increased after cold exposure and treatment with the adrenergic agonists. These results suggest that cold exposure activates the sympathetic nerves and leads to a rapid increase in synthesis of VEGF in BAT, which in turn stimulates the proliferation of surrounding vascular endothelial cells.

Adipose Tissue, Brown↗
PubMed1997

Inhibition of mitochondrial protein synthesis impaired C2C12 myoblast differentiation.

Various factors are required for the regulation of muscle cell differentiation. In an attempt to elucidate the mechanism underlying myogenesis, we examined the possible contribution of mitochondria to terminal differentiation of murine myoblast cell line, C2C12, using a specific inhibitor for mitochondrial protein synthesis, tetracycline. Tetracycline impaired myotube formation and induction of muscle creatine kinase activity which was specifically observed in differentiated myocytes. Transcript levels of muscle-specific proteins, creatine kinase and troponin-I were also significantly suppressed in a dose-dependent manner. However, those proteins with myogenic regulatory factors, MyoD and myogenin, and common proteins including glycolytic enzymes were not affected. Cellular viability, mitochondrial transcription, and mitochondrial proliferation were confirmed not to be impaired by tetracycline treatment. These results suggest that mitochondrial stress may affect regulation of differentiation-specific gene expression. This system may contribute to an understanding of mechanisms for differentiation inhibition caused by inhibitors of mitochondrial protein synthesis that have also been observed in other kinds of cells.

Adenosine Triphosphate↗
PubMed1996

A transient increase of snoN transcript by growth arrest upon serum deprivation and cell-to-cell contact.

To analyze the possible involvement of c-ski and c-sno during the course of in vitro myogenesis, expression of their transcripts during differentiation of a murine muscle cell line (C2C12) was monitored by competitive reverse transcription-polymerase chain reaction (RT-PCR). The transcripts of c-snoN were temporarily increased 25-fold above basal level at 12 h prior to the onset of transcription of muscle-specific gene, e.g. myogenin and muscle creatine kinase, whereas c-ski was expressed invariably. The transient increase of c-snoN was blocked when myogenesis was interrupted by the presence of fetal calf serum in culture medium, probably due to growth factors being included; basic fibroblast growth factor (b-FGF) blocked the transient increase whereas epidermal growth factor (EGF) did not, consistent with the inhibitory effect of b-FGF and no effect of EGF on myotube formation of C2C12. In fibroblastic C3H10T1/2 cells, snoN exhibited a similar transient increase of transcript when growth arrested under the same conditions as for in vitro myogenesis, indicating that the expression of snoN is not sufficient to induce the onset of muscle differentiation and an unknown factor involved in myogenic cells is necessary. The transient increase of snoN transcript may represent a common entrance step of cells into the G0 phase where muscle differentiation is substantiated, considering that it was observed upon growth arrest of fibroblastic C3H10T1/2 cells and prior to the elevation of MCK in C2C12 but undetected when entry into G0 was blocked by b-FGF.

Animals↗
PubMed1995

Isolation of a novel cDNA that encodes a protein localized to the pre-acrosome region of spermatids.

We have identified a novel cDNA clone, named AZ1, obtained from a cDNA library of mRNA prepared from C3H10T1/2 cells that had been transiently exposed to 5-azacytidine, a potent demethylating reagent. The amount of transcript increased with 5-azacytidine treatment of C3H10T1/2 cells and the transcript was highly expressed in mouse testis. As the mutant mouse jsd/jsd, which has a defect in germ cell maturation, barely expressed the transcript, the message was expected to be expressed specifically in spermatocytes. The mRNA was detected at significant levels in the testes from mice aged 16 days after birth, suggesting that its expression started at the pachytene spermatocyte stage. The elucidated nucleotide sequence contained a 2841-nucleotide open reading frame, and the expected amino acid sequence had a molecular mass of 107,254 Da. Specific antibodies raised against the fusion protein including glutathione S-transferase revealed an approximately 130-kDa band of a translation product in testis and in cultured cells transfected with AZ1 cDNA in the expression vector on Western-blot analysis. The protein was localized to the pre-acrosome region of round and elongated spermatids. However, it was not detected at a more advanced stage of spermatids, i.e. just before their release from Sertoli cells. This protein may play an important role in spermatogenesis.

Acrosome↗
PubMed1995

Overexpression of DNA methyltransferase in myoblast cells accelerates myotube formation.

We overexpressed mouse DNA methyltransferase in murine C2C12 myoblast cells and tested the isolated clones for their ability to differentiate. Significant numbers of the clones showed distinct myotubes 24 h after the isolated transformants had been induced to differentiate, whereas the parent C2C12 cells did not form myotubes at this time point. Transfection of the vacant vector or the plasmid containing the reverse-oriented DNA methyltransferase cDNA did not provide significant numbers of transformants with the accelerated differentiation phenotype, suggesting that the effect is caused by the expression of DNA methyltransferase. The expressions of skeletal muscle myosin and creatine kinase in clones that showed the accelerated differentiation-phenotype were also induced about 24 h earlier and at higher levels relative to the parent C2C12 or the control cells, indicating that the entire process of myogenesis had been accelerated. All the methyltransferase-transfected clones, regardless of their phenotypes, demonstrated about threefold higher DNA methyltransferase activity and higher methylation levels than those of the clones transfected with vector alone or the reverse-oriented plasmid. At the early stage of transfection of the sense-oriented plasmid, high de novo methylation activities were detected. We consider it likely that this high de novo methylation activity is the reason for the high methylation levels and the accelerated myotube formation of the clones transfected with the sense-oriented plasmid. In some transformants which showed the accelerated differentiation phenotype, MyoD1 was already fully expressed under the growth conditions while, in control cells, MyoD1 was expressed at low levels. This elevated level of MyoD1 transcription could account for the accelerated myotube formation observed in the transformants. The methylation state of the HpaII sites in exon 1 through exon 2 of the MyoD1 gene and the expression of the MyoD1 transcript are positively correlated.

Animals↗
PubMed1995

Expression of several muscle-specific genes during differentiation of cultured quail pineal body cells under artificial (high NaCl) conditions.

A change in cell differentiation of pineal body cells to cells with myogenic features can be induced when such cells are cultured with stepwise increases in the NaCl concentration up to a 125 mM NaCl excess. Further differentiation can be achieved by changing the culture medium to Cosmedium (serum-free) with the same high NaCl concentration. This system is a good candidate as an in vitro model for the commitment of pre-myoblastic cells to myogenic cells, and for studies on early phases of myogenic differentiation, since the myogenic fate is not selected under conventional isotonic culture conditions. Our first step was to analyze the expression schedule of an intermediate filament protein, desmin, as an early stage marker of muscle differentiation. Desmin expression was detected at a low level in mononucleated cells during early differentiation stages (two days in medium with a 50 mM excess of NaCl), and a higher order of expression was observed in later stages in 20-40% of cells corresponding to myotubes. Expression of qmf1 and qmf2 mRNA, quail counterparts of MyoD and myogenin, respectively, was detected in two different stages: in the pineal body of 9-day-old embryos and in myogenically differentiated pineal cells, by Northern blotting and RT-PCR analysis. When pineal cells were transferred to the culture system, qmf1 and qmf2 expression was not present in isotonic medium and no myogenic differentiation occurred under these conditions. However, they reappeared and increased during the final stages of myogenic differentiation in the high NaCl medium. Furthermore, by use of muscle-specific domain (MSD)-specific antibodies we found the expression of a muscle-specific neural cell adhesion molecule (NCAM) species at the late stages of differentiation. These results show that the gene expression and mRNA splicing patterns of the cells changed from pineal body-specific to muscle-specific. On the basis of these results, we conclude that this system is appropriate for studying the differentiation of non-muscle cells to muscle cells.

Animals↗
PubMed1995

Roles of prostaglandins D2 and E2 in interleukin-1-induced activation of norepinephrine turnover in the brain and peripheral organs of rats.

Possible roles of prostaglandins (PGs) in interleukin-1 (IL-1)-induced activation of noradrenergic neurons were examined by assessing norepinephrine (NE) turnover in the brain and peripheral organs of rats. An intraperitoneal injection of human recombinant IL-1 beta accelerated NE turnover in the hypothalamus, spleen, lung, diaphragm, and pancreas. A similar increase in NE turnover was also observed after intracerebroventricular injection of corticotropin-releasing hormone (CRH). Pretreatment with indomethacin (cyclooxygenase inhibitor) abolished the IL-1-induced, but not the CRH-induced, increase in hypothalamic and splenic NE turnover. To elucidate which eicosanoid-cyclooxygenase product(s) is responsible for accelerating NE turnover, PGD2, PGE2, PGF2 alpha, U-46619 (stable thromboxane A2 analogue), or carbacyclin (stable prostacyclin analogue) was administered intracerebroventricularly. Among them, PGE2 was the only eicosanoid effective in increasing NE turnover in spleen, whereas PGD2 was effective in the hypothalamus. The stimulative effect of PGD2 was abolished by pretreatment with intracerebroventricular injection of a CRH antiserum. These results suggest that the action of IL-1 is mediated through PGD2 production to activate the noradrenergic neurons in the hypothalamus, and through PGE2 production to increase sympathetic nerve activity in spleen.

Animals↗
PubMed1995

Isolation and expression of a chicken DNA methyltransferase cDNA.

A 0.5 kb fragment of chicken DNA methyltransferase cDNA was PCR-amplified using a set of degenerate primers. A clone harboring a 5 kb insert was isolated from a cDNA library by screening with the PCR-amplified cDNA fragment as a probe. The elucidated nucleotide sequence gave a 4,614 nucleotide open reading frame, and the predicted protein was highly homologous to the mouse and human DNA methyltransferases, especially in the amino acid sequence of the catalytic domain in the carboxyl-terminal region. The cysteine-rich region and Lys-Gly repeat first found in the mouse sequence were also conserved in chicken. However, about 250 amino acid residues in the amino-terminal portion of chicken DNA methyltransferase diverged from the amino-terminus of the mouse or human sequence. Northern blot analysis showed that the message of chicken DNA methyltransferase was expressed at high levels in the testis, in the lung and in Marek's virus-transformed chicken T-lymphoma cells. Expression of the chicken DNA methyltransferase in COS1 cells demonstrated that the enzyme is a so-called maintenance-type methylase. When poly(dG-dC)-poly(dG-dC) was used as the methyl acceptor, to provide a measure of de novo methylase activity, the Km value for S-adenosyl L-methionine was about 5 microM, which was 10 times higher than that when poly(dI-dC)-poly(dI-dC) was used. The affinity of DNA methyltransferase for S-adenosyl L-methionine in catalyzing de novo-type methylation activity was lower than that in catalyzing maintenance-type activity, though it was still high enough for the enzyme to work as a de novo-type methylase under physiological conditions.

Amino Acid Sequence↗
PubMed1995

A novel DNA binding protein that recognizes the methylated c-Myc binding motif.

We detected a novel nuclear protein, MMBP-3, that bound to the c-Myc binding motif (CACGTG) in which deoxycytidine in the CpG sequence was methylated. MMBP-3 was partially purified by chromatography on heparin-agarose and hydroxyapatite, followed by affinity adsorption to a matrix coupled to the methylated binding motif. Its binding to the methylated c-Myc binding motif was specific, although it also recognized the unmethylated motif weakly. MMBP-3 was further found to recognize only one of two differently hemimethylated forms of the double-stranded c-Myc binding motif. MMBP-3 activity was detected in proliferating C2C12 and C3H/10T1/2 cells, and down regulated when the growth of these cells was inhibited. We propose that MMBP-3 plays a role in regulating the c-Myc function by recognizing the methylation state of the c-Myc binding motif in a growth-dependent manner.

Animals↗
PubMed1995

Temporally distinctive changes of alternative splicing patterns during myogenic differentiation of C2C12 cells.

It is well known that skeletal muscle differentiation is accompanied by the appearance of many muscle-specific components and that some of these components are generated through muscle-specific alternative splicing. It is not clear, however, in what manner, including timing, the system that regulates the muscle-specific splicing reactions is constructed during the process of myogenic differentiation. We simultaneously examined the changes in several splicing patterns for the neural cell adhesion molecule (NCAM), beta-tropomyosin, and M-type pyruvate kinase genes during myogenic differentiation of cultured myoblasts using the reverse transcription-polymerase chain reaction method. The NCAM glycosylphosphatidylinositol anchor form increased in preference to the transmembrane form immediately after the induction of differentiation, while the selection of NCAM MSD1 (muscle-specific domain 1) exons started and abruptly increased at about the time when cell-fusion appeared. M2-type pyruvate kinase was gradually substituted for the M1-type molecule. Skeletal muscle-type beta-tropomyosin was predominantly selected even in myoblasts in the growth medium. As a result, each transcript of these genes independently showed a temporally distinctive pattern of change in isoform selecting during the myogenic differentiation of C2C12 cells. These observations suggest that some independent regulation of alternative splicing reactions should occur during myogenic differentiation.

Alternative Splicing↗
PubMed1995

[Clinicopathological studies of anti-HCV P1P4 core antibody].

Anti-P1P4 core antibody, derived from a Japanese hepatitis C virus clone, was evaluated clinicopathologically in serum samples from 40 blood donors positive for anti-HCV antibody by 2nd generation assay and in 37 patients with HCV chronic hepatitis treated with interferon. The presence of anti-P1P4 antibody was highly correlated with the presence of HCV-RNA in the blood donors. In the patients with chronic hepatitis, more than a 50% reduction in P1P4 antibody titer after interferon therapy suggested the disappearance of HCV-RNA from the blood. Thus, anti-P1P4 antibody was useful in evaluating the virological effects of interferon therapy. However, clinically and pathologically, the titer of P1P4 antibody did not indicate the grade of liver inflammation.

Hepacivirus↗
PubMed1994

Selective inhibition of a step of myotube formation with wheat germ agglutinin in a murine myoblast cell line, C2C12.

Myoblast cells, C2C12, which is an established cell line from satellite cells of skeletal muscle of C3H mouse, start to fuse and form multinucleated cells (myotubes) and begin to express creatine phosphokinase and myosin, when culture medium is changed from the growth medium to the differentiation medium. Among the 12 lectins that we tested, wheat germ agglutinin apparently suppressed the myotube development judged by phase-contrast microscopy, but did not affect the induction of creatine phosphokinase activity. The addition of N-acetylglucosamine or N,N',N"-triacetylchitotriose, which is a specific ligand for wheat germ agglutinin, to the differentiation medium, recovered this apparently suppressive effect of wheat germ agglutinin on the myotube development. Tachypleus tridentatus (Japanese horseshoe crab) lectin that specifically recognizes N-acetylneuraminic acid, one of the sialic acids, showed no effect on the myotube development. It was suggested that wheat germ agglutinin suppressed the process through recognizing N-acetylglucosamine containing sugar. Surprisingly, even in the presence of wheat germ agglutinin, the ratio of mononucleated cell numbers to the genomic DNA content, which represents the fusion level, decreased after incubation in the differentiation medium, indicating that even when wheat germ agglutinin was present in the medium, cell fusion, which is the initial step of the myotube formation, occurred. Immunostaining with anti-skeletal muscle myosin antiserum confirmed that the myosin expressing cells actually fused and formed multinucleated cells. Their shape, however, was thin compared to that in the absence of wheat germ agglutinin. We propose that the membrane fusion step to form myotubes is composed of two distinct steps in C2C12; one fusion step is to form long and thin myotubes from mononucleated cells and the other one is to develop fat myotubes. Wheat germ agglutinin specifically inhibits the latter fusion step.

Agglutinins↗