PubMed Health⌕ Search

Biomedical subjects

A Asano

Publications and source records attributed to A Asano.

At least 19 recordsLinked to original sources
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

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↗
PubMed1994

Immunohistochemical localization of interferon-gamma in normal human ovary.

There is increasing evidence that cytokines are important intraovarian non-steroidal regulators. The aim of the present study was to evaluate the presence in the human ovary of interferon (IFN)-gamma, a cytokine produced by T lymphocytes after mitogenic or antigenic stimulation. Very low levels of IFN-gamma (0.025-0.057 IU/ml) were found in follicular fluid of large spontaneously maturing follicles, in the ovarian vein (< 0.01-0.079 IU/ml) or peripheral blood (< 0.01-0.06 IU/ml). The avidin-biotin immunocytochemical technique, with appropriate monoclonal antibodies, was used to localize IFN-gamma-positive cells, human leukocyte antigen (HLA)-DR molecules, activated T cells, T helper/inducer cells and T cytotoxic/suppressor cells. IFN-gamma-positive cells were only detected in preovulatory follicles, associated with the follicular basal lamina, thecal vessels and interstitial tissue. In the same large follicles very few T helper/inducer cells were detected, but a high proportion of T lymphocytes expressed the CD8 phenotype in the theca, interstitial tissue and follicular cavity. No IFN-gamma-positive cells were observed in preantral and small antral follicles. The results indicate that the human ovary contains immunoreactive IFN-gamma, suggesting that the cytokine plays a paracrine role in human ovarian function.

Adult↗
PubMed1993

A novel transcript from a pseudogene for human glucocerebrosidase in non-Gaucher disease cells.

Human glucocerebrosidase (GC)-encoding cDNA clones were isolated from a promyelocytic HL-60 cDNA library and analyzed. A novel cDNA clone was found to originate from a gene referred to as a GC pseudogene. Using the polymerase chain reaction (PCR) with primers specific for the GC pseudogene, we found that all the human cell lines examined, HL-60, K-562, WI-38, HepG2 and HeLa, expressed a pseudogene transcript. In vitro translation of RNA synthesized by transcription of the pseudogene cDNA produced a polypeptide of approximately 30 kDa.

Amino Acid Sequence↗
PubMed1993

Identification of two novel mouse nuclear proteins that bind selectively to a methylated c-Myc recognizing sequence.

The c-Myc recognizes the sequence CACGTG (Blackwell, T. K., Kretzner, L., Blackwood, E.M., Eisenman, R. N., and Weintraub, H. (1990) Science 250, 1149-1151), and its binding is inhibited by methylation of the core CpG (Prendergast, G. C. and Ziff, E. B. (1991) Science 251, 186-189). We identified two novel nuclear proteins, MMBP-1 and MMBP-2, that bound specifically and under physiological salt condition to the c-Myc binding motif of which cytidine in the CpG sequence was methylated. MMBP-1 was about 42 kD and MMBP-2 was about 63 kD. MMBP-1 was found in specific cells, while MMBP-2 was found in all the cell lines tested, suggesting that MMBP-1 may modulate the role of MMBP-2 in tissue specific manner. We propose that the two proteins play a role in the regulation of c-Myc function through stabilizing or destabilizing the methylation state of the c-Myc binding motif.

Animals↗
PubMed1993

Brefeldin A inhibits muscle-specific gene expression during differentiation in C2C12 myoblasts.

In analyzing regulatory mechanisms underlying differentiation-dependent expression of muscle proteins, we have found that mouse C2C12 myoblast cells treated with Brefeldin A (BFA), which specifically blocks the intracellular transport of protein from endoplasmic reticulum (ER) to the Golgi apparatus, failed to differentiate. BFA reversibly inhibited myotube formation at a threshold concentration of 1.0 micrograms/ml. The same concentration of BFA completely blocked accumulation of secretory proteins, e.g., fibronectin into the culture medium, probably due to retention in ER. Interestingly, the induction of muscle creatine phosphokinase (MCK) activity was sensitive to BFA, although a constitutive expression of lactate dehydrogenase, one of the cytosolic housekeeping enzymes, was resistant to BFA. In the kinetic analysis of MCK induction, translation of existing MCK mRNA was not inhibited by BFA since MCK activity became resistant to BFA once levels of MCK transcript accumulated. The differentiation-dependent accumulation of MCK transcript was also suppressed by BFA treatment to approximately 30% of control, although neither general transcriptional activity in vivo nor stability of existing mRNAs was affected even in the presence of BFA. Moreover, induction of muscle regulatory genes, MyoD1 and myogenin, which are upstream of the MCK gene, were inhibited at their transcription level by BFA. Such an effect on muscle regulatory gene transcription by BFA suggests that transport of some factor(s) to the cell surface may be a prerequisite for the muscle-specific gene expression upon differentiation of C2C12 myoblasts.

Animals↗
PubMed1993

Age dependency of mitochondrial DNA decrease differs in different tissues of rat.

Mitochondrial DNA contents were measured using non-radioactive DNA probes for specific mtDNA sequences. The nuclear repetitive DNA sequence, LINE, was used as an internal standard marker. Age-dependent decreases in the mtDNA/nucDNA ratio in rat hepatic cells were confirmed with this method and, furthermore, a similar decrease was found in cardiac muscles. On the other hand, in thigh skeletal muscles such a decrease was not observed, slight increases being rather found in 18- to 24-month-old rats. The possible relationship of the decrease in mtDNA and the mechanism underlying animal senescence was discussed.

Aging↗
PubMed1993

Transitional expression of neural cell adhesion molecule isoforms during chicken embryonic myogenesis.

The neural cell adhesion molecule, NCAM, is known to be expressed in chicken muscle as at least three principal molecular forms (molecular masses of 155 kDa, 145 kDa, and 120 kDa). They are generated from a single gene by alternative splicing. To distinguish these molecular species and to investigate their expressions in muscle differentiation during chicken embryonic development, we prepared antipeptide antibodies against three different domains of the NCAM. The antibody named MSD<+> was designed to detect muscle-specific domain (MSD) which was inserted into a muscle-specific NCAM by alternative splicing. The locus encompassing the MSD insertion site was detected with the antibody named MSD<->, and cytoplasmic sites near the transmembrane region were detected with the antibody named CYT. Immunoblot analysis utilizing the peptide antibodies prepared here revealed that, of three NCAMs, two isoforms, 155 kDa and 120 kDa, were recognized with the antibody MSD<+> and the same 155-kDa and the other 145-kDa molecule were recognized with the antibody CYT. The antibody MSD<-> was capable of detecting all three isoforms of the NCAM. Expression of the 120-kDa form with MSD but lacking a cytoplasmic tail increased but that of the 145-kDa form with cytoplasmic tail but lacking MSD declined during embryonic day 5 to day 18. The 155-kDa form NCAM with both MSD and cytoplasmic tail was expressed specifically and transiently during embryonic day 11 to day 14 in chick muscle; this period coincides with the period of extensive myotube formation. Thus, this largest isotype seems to play an important role in muscle differentiation.

Amino Acid Sequence↗
PubMed1992

Quantitation of changes in mitochondrial DNA during aging and regeneration of rat liver using non-radioactive DNA probes.

By using DNA probes prepared from cloned cells which contain mitochondrial DNA (mtDNA) sequences in plasmids, the specific detection of mtDNA became possible in the presence of large excess of nuclear DNA by DNA-DNA hybridization. For this purpose, we prepared mtDNA probes labeled with non-radioactive substrate, which allowed a wider possibility of application. This method revealed that the contents of mtDNA in rat liver are strikingly decreased during aging. Furthermore, it was observed that although mtDNA content increased upon partial hepatectomy even in old rats, it decreased to the pre-operation level rather rapidly within 1 week after reaching peak in regenerated liver.

Aging↗
PubMed1991

The characteristic CD spectra of DNA from young rat liver.

The circular dichroism spectroscopy was used to study conformational changes of DNA upon aging. DNA from 8 weeks old rat liver showed A-form unexpectedly in physiological saline. The characteristic spectrum gradually changed upon aging and that of 30 months old rat liver DNA showed normal B-form.

Aging↗
PubMed1990

Direct evidence for the presence of methylmercury bound in the thyroid and other organs obtained from mice given methylmercury; differentiation of free and bound methylmercuries in biological materials determined by volatility of methylmercury.

Peroxidase in mouse thyroid was inhibited by mercuric chloride but not by methylmercury in in vivo and in vitro systems (Nishida, et al., J. Histochem. Cytochem., 37, 723 (1989)). To identify the reason for the difference, the present study was conducted to examine whether methylmercury is indeed bound within cells or tissues. Mice were given radioactive methylmercury by intubation for 18 d and the tissues were dissected out and vacuum-dried. With this procedure, free methylmercury was evaporated off and the bound mercury remained. The thyroid, liver, kidney and fats examined showed no loss of radioactivity under the vacuum, indicating that the mercury was bound to the thyroid, as well as the other tissues. Radioactive mercuric chloride was nonvolatile regardless of the presence or absence of the tissues. The preferential affinity of methylmercury for SH-containing materials was re-confirmed by this method.

Animals↗