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T Yagura

Publications and source records attributed to T Yagura.

At least 19 recordsLinked to original sources
PubMed1992

Immunolocalization of Ku-proteins (p80/p70): localization of p70 to nucleoli and periphery of both interphase nuclei and metaphase chromosomes.

Distribution on both nuclei and metaphase chromosomes of Ku-proteins, recognized by autoantibodies from a patient with systemic lupus erythematosus, has been studied using a specific monoclonal antibody (mAbH6) that recognizes p70, one Ku-protein. Observation with either a conventional fluorescent microscope or a confocal laser scanning microscope revealed mAbH6-stained p70 antigen localized on both nuclear periphery and nucleoli of human interphase cells. The specific staining of nucleoli with mAbH6 has been confirmed using isolated nucleoli from rat liver in which the staining was seen as fine granules surrounding nucleolar DNA. During mitosis p70 antigen moved away from association with the nuclear envelope region to localization on the periphery of condensed chromosomes with no apparent staining of chromosome interior. The p70 antigen was copurified with DNA fragments by immunoaffinity column chromatography using mAbH6. The mAbH6 staining of both nuclear periphery and nucleoli was lost upon digestion with DNase I at low concentrations. These results suggest that p70 antigen is connected with these nuclear structures through DNA.

Amino Acid Sequence↗
PubMed1992

Novel monoclonal antibody mAb G3A5 recognizes 138-kDa glycoprotein localized on the Golgi membrane.

Partially purified Golgi membranes of HeLa cells were used as antigen to produce a novel monoclonal antibody (mAb G3A5). The mAb G3A5 specifically labeled Golgi apparatus of human and monkey cultured cells as ascertained by indirect immunofluorescence but did not stain those of bovine or mouse cells. Treatment with nocodazole and brefeldin A (BFA) induced fragmentation and redistribution of the staining. Western immunoblot analysis showed that mAb G3A5 was directed against a single polypeptide with an apparent molecular mass of 138-kDa (p138 antigen). The p138 antigen is an integral membrane protein of the Golgi apparatus, as assessed by several assays: protease protection, salt wash and flotation in sucrose density gradient centrifugation. The p138 antigen was purified using immunoaffinity chromatography. The apparent molecular mass of the p138 antigen decreased by 2 to 4 kDa after treatment with the peptide: N-glycosidase F, while digestion with ENDO F or Neuraminidase did not have this effect. Thus, p138 antigen is a glycoprotein containing asparagine-linked carbohydrates.

Animals↗
PubMed1990

Multivariable analysis of serum 3,5,3'-L-triiodothyronine concentration in patients of diabetes mellitus by blood glucose level and body weight.

Serum thyroid hormone concentrations, 1-thyroxine (T4), free T4 and 3,5,3'-l-triiodothyronine (T3) were measured in 213 patients of diabetes and analyzed their correlation with metabolic parameters, hyperglycemia and body weight. Haemoglobin A1 (HbA1) was used as an index of hyperglycemia. Body weight was expressed by relative body weight (body weight/standard weight). Among the thyroid hormones, only T3 had significant correlation with HbA1 and body weight (r = -0.476, P less than 0.01 and r = 0.369, P less than 0.01, respectively). Multivariable analysis of serum T3 by HbA1 and relative body weight gave the following regression equation. Serum T3 (ng/dl) = 108 + 0.362 x relative body weight (%) - 3.88 x HbA 1 (%). Though relative body weight had inverse correlation with HbA1, the contribution of the two metabolic parameters to the serum T3 was independent from each other. Our results confirm the previous reports that low T3 in diabetes correlates with severity of hyperglycemia and we report for the first time that serum T3 of diabetic patients has positive correlation with body weight, probably due to still available carbohydrate in spite of disturbances in the metabolism.

Adult↗
PubMed1990

Structure of DNA polymerase alpha-primase complexes from mammalian cells analyzed by using monoclonal antibodies.

The molecular masses of two of the four DNA polymerase alpha-primase complex subunit peptides from various mammalian cells have been compared through the use of specific monoclonal antibodies. One monoclonal antibody (E4) binds to 77-kDa peptide from HeLa cells and cognate peptides from other mammalian cells (monkey, mouse, bovine, Indian muntjac, and hamster). Another monoclonal antibody (A5) binds the 180-kDa type peptide and its degradation product (160-kDa peptide) of the mammalian DNA polymerase alpha-primase complexes. Neither of these antibodies reacts with DNA polymerase alpha-primase complex from chicken cells. Comparative immunoblot analysis indicates that the molecular masses of the two main peptides of DNA polymerase alpha-primase complex isolated from the various mammalian sources are in excellent agreement with each other, except for the 77-kDa type peptide from bovine and Indian muntjac cells which was found to be significantly smaller (68 kDa) in these cases. The small molecular mass of bovine 77-kDa type peptide is not attributable to the action of a protease which may be present in the extract of bovine cells.

Animals↗
PubMed1989

Intracellular localisation of a subunit of human DNA polymerase alpha affecting primase activity recognised by monoclonal antibody (HDR-854-E4) and its application to distinction between proliferative and non-proliferative lesions.

We have successfully established one murine hybridoma that secretes a monoclonal antibody specific for the 77,000 subunit of human DNA polymerase alpha. The results of immunochemical studies, using HDR-854-E4 monoclonal antibody (MAb) and immunoperoxidase detection methods, demonstrate intranuclear and intracytoplasmic localisation of the subunit in all the human culture cell lines tested. The immunoperoxidase reaction product exhibits a diffuse pattern of distribution within the cytoplasm and nucleoplasm, but nucleoli are clearly negative. In cultured cell lines, HeLa and KATO III, more than 95% of the cells are positive, suggesting that the subunit antigens persist throughout the mitotic cycle. No subunit antigen was recognised in resting mononuclear cells (MNC). Immuno-electron microscopic examination of HeLa cells confirms and extends these observations. We have further examined the expression level of the subunit antigen in various normal and cancerous tissues. Strong reaction was observed in proliferating normal and cancer cells such as cancer cells from the gastrointestinal (GI) tract, thyroid, malignant lymphoma, breast, cells in the germinal centres of lymph nodes, epithelial cells in the GI tract and nephrogenic zones in fetal kidney. Finally, we utilised this antibody as a diagnostic tool in biopsies of the thyroid and GI tract. Thyroid cancer was stained positively with this antibody, while follicular adenoma was not. Gastric cancer was stained strongly and adenomatous polyp and hyperplastic polyp were stained moderately. This antibody is not only specific and powerful for application of a novel approach to the complex biochemical mechanisms of mammalian DNA replication, but also useful for distinction between proliferative and non-proliferative lesions.

Antibodies, Monoclonal↗
PubMed1988

Transient neonatal hyperthyroidism and maternal thyroid stimulating immunoglobulins.

Serum thyroid stimulating hormone binding inhibitor immunoglobulins (TBII) and thyroid stimulating antibody (TSAb) concentrations were measured in three pregnant women with hyperthyroidism and then in their infants. The results suggested that TBII concentrations in infants in the neonatal period or from mothers during the third trimester of pregnancy showed a good correlation with the development of neonatal hyperthyroidism.

Adult↗
PubMed1987

Immunochemical detection of a primase activity related subunit of DNA polymerase alpha from human and mouse cells using the monoclonal antibody.

A hybrid cell line (HDR-854-E4) secreting monoclonal antibody (E4 antibody) against a subunit of human DNA polymerase alpha was established by immunizing mice with DNA replicase complex (DNA polymerase alpha-primase complex) prepared from HeLa cells. The E4 antibody immunoprecipitates DNA replicase complex from both human and mouse cells. The E4 antibody neutralizes the primase activity as assessed either by the direct primase assay (incorporation of [alpha-32P]AMP) or by assay of DNA polymerase activity coupled with the primase activity using unprimed poly(dT) as a template. The E4 antibody does not neutralize DNA polymerase alpha activity with the activated calf thymus DNA as a template. Western immunoblotting analysis shows that the E4 antibody binds to a polypeptide of 77 kilodaltons (kDa) which is tightly associated with DNA polymerase alpha. The 77-kDa polypeptide was distinguished from the catalytic subunit (160 and 180 kDa) for DNA synthesis which was detected by another monoclonal antibody, HDR-863-A5. Furthermore, it is unlikely that the 77-kDa peptide is the primase, since we found that the E4 antibody also immunoprecipitates the mouse 7.3S DNA polymerase alpha which has no primase activity, and Western immunoblotting analysis shows that the 77-kDa polypeptide is a subunit of the 7.3S DNA polymerase alpha. Furthermore, after dissociation of the primase from mouse DNA replicase by chromatography on a hydroxyapatite column in the presence of dimethyl sulfoxide and ethylene glycol, the 77-kDa polypeptide is associated with DNA polymerase alpha, and not with the primase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Activity levels of mouse DNA polymerase alpha-primase complex (DNA replicase) and DNA polymerase alpha, free from primase activity in synchronized cells, and a comparison of their catalytic properties.

To asses the possible roles of the two active forms of mouse DNA polymerase alpha: primase--DNA-polymerase alpha complex (DNA replicase) and DNA polymerase alpha free from primase activity (7.3S polymerase), in nuclear DNA replication the correlation of their activity levels with the rate of nuclear DNA replication was determined and a comparison made of their catalytic properties. The experiments using either C3H2K cells, synchronized by serum starvation, or Ehrlich culture cells, arrested at the S phase by aphidicolin, showed DNA replicase to increase in cells in the S phase to at least six times that of the G0-phase cells but 7.3S polymerase to increase but slightly in this phase. This increase in DNA replicase activity most likely resulted from synthesis of a new enzyme, as shown by experiments using a specific monoclonal antibody, aphidicolin and cycloheximide. Not only with respect to the presence or absence of primase activity, but in other points as well the catalytic properties of these two forms were found to differ; DNA replicase preferred the activated calf thymus DNA with wide gaps of about 100 nucleotides long as a template-primer, while the optimal gap size for 7.3S polymerase was 40-50 nucleotides long. Size analysis of the products synthesized on M13 single-stranded circular DNA with a single 17-nucleotide primer by DNA replicase and 7.3S polymerase suggested the ability of DNA replicase to overcome a secondary structure formed in single-stranded DNA to be greater than that of 7.3S polymerase.

Animals↗
PubMed1986

Size difference in catalytic polypeptides of two active forms of mouse DNA polymerase alpha and separation of the primase subunit from one form, DNA replicase.

There are two active forms of DNA polymerase alpha in mouse cells. One form (DNA replicase) is a DNA polymerase associated with primase activity and the other form (7.3 S polymerase) has no primase activity (Yaugar, T., Kozu, T. and Seno, T. (1982) J. Biol. Chem. 257, 11121-11127). The primase activity was dissociated from partially purified DNA replicase by hydroxyapatite column chromatography in buffer containing dimethyl sulfoxide and ethylene glycol. Nearly homogeneous primase, consisting of a 58 kDa polypeptide was obtained by glycerol gradient sedimentation and DEAE-cellulose column chromatography. Experiments on the effect of proteinase treatment and measurement of the molecular weight of the catalytic polypeptide of DNA replicase after its dissociation from the primase polypeptide indicated that the primase is not part of the DNA polymerase molecule, but an independent protein associated with DNA polymerase alpha, and that the latter is a 115 kDa catalytic polypeptide. The other form of DNA polymerase alpha, 7.3 S polymerase, consists of a 72 kDa catalytic polypeptide. Thus, the two forms of mouse DNA polymerase alpha have partially, if not completely, different catalytic polypeptide structures, suggesting that the 7.3 S polymerase is not simply formed from DNA replicase by dissociation of the primase subunit.

Animals↗
PubMed1985

Increased cyclic nucleotide phosphodiesterase (PDE) and calmodulin activities in soluble fraction of Graves' thyroid: analysis of increase in Ca+2 dependence of PDE activities.

Phosphodiesterase (PDE) and calmodulin (CaM) activities were studied in soluble fractions of normal and Graves' thyroid tissue. Normal and Graves' thyroid tissues were obtained at thyroid surgery. PDE activities were assayed with cAMP and cGMP as substrates. CaM activity was measured as the ability to activate bovine thyroid CaM-dependent PDE. cAMP and cGMP PDE activities were increased 1.5- and 2.2-fold above normal in Graves' thyroid, respectively. The major cause of the increase in enzyme activities was their higher Ca+2 dependence. CaM activity also was 1.6-fold increased in Graves' thyroid, although it probably does not contribute to the increase in the Ca+2 dependence of PDE activities because of its relative sufficiency vs. PDE in normal thyroid. Three forms of cAMP and cGMP PDE activities were eluted from Sephadex G-200 columns, with mol wt of 280,000, 140,000, and 80,000-100,000, respectively. The first peak had little or no CaM dependence, the second peak had moderate or dominant CaM dependence, and the third peak revealed weak or dominant CaM dependence. The increase in the CaM-dependent form of the third peak of PDE activity in Graves' thyroid tissue may explain the increase in Ca+2 dependence of PDE activities.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1983

Novel form of DNA polymerase alpha associated with DNA primase activity of vertebrates. Detection with mouse stimulating factor.

With a specific stimulating factor of mouse DNA replicase for its detection, a novel form of DNA polymerase alpha (DNA replicase) associated with DNA primase activity was partially purified from several vertebrates, i.e. the cherry salmon Oncorhyncus masou, the frog Xenopus laevis, the chick, and human (HeLa cells). Activity similar to DNA replicase was also partially purified from embryos of the sea urchin Anthocidaris crassispina. In all vertebrates examined, two forms of DNA polymerase alpha were separated by chromatography on ion-exchange columns; one form (DNA replicase) was associated with DNA primase activity and could utilize unprimed single-stranded DNAs as template, and the other could not utilize unprimed single-stranded DNAs. The sedimentation coefficient of the former, the novel form, obtained from each vertebrate in a glycerol gradient at high ionic strength was slightly larger than that of the other form which had no primase activity, except in the case of chick embryos where the sedimentation coefficients of the two forms were almost the same. The initiator RNA synthesized with the DNA primase activity associated with DNA replicase obtained from salmon, chick, HeLa cells, and sea urchin was 8 to 10 nucleotides long. The stimulating factor obtained from Ehrlich ascites cells has been found to stimulate both the activities of DNA primase and DNA polymerase in DNA replicase obtained from all the vertebrates examined, when unprimed single-stranded DNA was used as template, while the factor failed to stimulate both the activities of the enzyme of sea urchin embryos. This factor thus should be an effective tool in studies on the mechanism of vertebrate DNA replication.

Animals↗
PubMed1983

Mechanism of stimulation by a specific protein factor of de novo DNA synthesis by mouse DNA replicase with fd phage single stranded circular DNA.

Mouse DNA replicase is a functional multienzyme complex consisting of DNA polymerase and DNA primase. The DNA and initiator RNA syntheses by DNA replicase with single stranded DNA as template are stimulated by a stimulating factor (T. Yagura, T. Kozu and T. Seno, 1982, J. Biochem. (Tokyo).91, 607-618). The action mechanism of the stimulating factor on this novel DNA synthesis with fd phage single stranded circular DNA as template was studied. The stimulating factor directly stimulated initiator RNA synthesis but did not change the length of either initiator RNA (8 to 10 nucleotides long) or the product DNA (300 to 1,000 nucleotides long). Kinetic studies and analysis of the products by neutral agarose gel electrophoresis show that the stimulating factor increased the affinity of DNA replicase for template DNA without changing the apparent Km values for deoxy- and ribonucleotide substrates. Thus, in combination with a sufficient amount of the stimulating factor, DNA replicase quantitatively converted the template DNA to the position of double-stranded circular replicative form II DNA, as shown by agarose gel electrophoresis.

Animals↗
PubMed1983

Tight association of DNA primase with a subspecies of mouse DNA polymerase alpha.

Evidence was obtained for tight association of DNA primase activity with a subspecies of mouse DNA polymerase alpha by study with immunoadsorption assay using two monoclonal antibodies specific for human DNA polymerase alpha that have been shown to react with mouse murine myeloma DNA polymerase alpha (Tanaka, S., Hu, S.-Z., Wang, T.-S.-F. & Korn, D. (1982) J. Biol. Chem. 257, 8386-8390). This result was supported by the finding that ethidium bromide at concentrations of less than 20 microM somewhat stimulated the syntheses of DNA and initiator RNA on unprimed poly(dT) by the novel subspecies of DNA polymerase alpha, but strongly inhibited DNA synthesis with poly(dT) X oligo(rA), suggesting that the conversion of synthesis from initiator RNA to DNA is continuous. Furthermore, the results of neutralization assay with the antibodies and experiment using aphidicolin suggested that the primase site is functionally distinguishable from the catalytic site of DNA polymerase activity.

Animals↗
PubMed1983

Size maturation process of nascent DNA intermediates into chromosomal-sized DNA in Tetrahymena pyriformis macronuclear DNA replication.

An analysis was made of the size maturation process of nascent DNA intermediates in macronuclear DNA replication of Tetrahymena pyriformis. The first discrete size class of nascent intermediates larger than Okazaki fragments were replicon-sized DNA (about 2 X 10(7) D single-stranded (ss) DNA) and accumulated in cells treated with cycloheximide. On removal of cycloheximide, the replicon-sized intermediates were converted to middle-sized intermediates (about 10 X 10(7) D ssDNA) and then merged into chromosomal-sized DNA. As indicated by either aphidicolin inhibition or the technique of the photolysis of bromodeoxyuridine (BrdU)-substituted DNA with long-wave ultraviolet light, four to eight replicon-sized intermediates were joined together to form a middle-sized intermediate after rapid sealing by DNA synthesis of the late-replicating regions located between adjacent replicon-sized intermediates. The late-replicating regions may represent the short gaps or terminal regions where DNA synthesis was retarded by cycloheximide, since the size of late-replicating regions was suggested to be shorter than the replicon size by DNA fiber autoradiography. Therefore, it is probable that four to eight completed replicons are joined as a group such as a replicon cluster, as has been reported in DNA replication of other eukaryotic cells.

Animals↗
PubMed1983

Detection of DNA replicase activity in rat thymoma cells.

A DNA polymerase which was active with unprimed poly(dC) as a template was detected in the DNA polymerase alpha fraction from rat thymoma tissue. The activity was dependent on rGTP and associated with ribonucleotide polymerizing activity. The activity was partially sensitive to aphidicolin and dideoxy GTP and resistant to alpha-amanitin. Thus, this is a rat "replicase" which is very similar to that found in mouse cells. The replicase activity with poly(dT) or phage fd single stranded circular DNA was greatly enhanced by the mouse replicase-stimulating factor beyond the species difference.

Animals↗
PubMed1982

A new unextracted-sample radioimmunoassay method for hepatic endogenous nuclear L-tri-iodothyronine content. Validity of its use in determining nuclear receptor binding characteristics.

Endogenous l-tri-iodothyronine content in an hepatic nuclear extract was measured by a new unextracted-sample radioimmunoassay method using 8-anilinonaphthalene-1-sulphonic acid to inhibit the l-[(125)I]tri-iodothyronine binding to the nuclear l-tri-iodothyronine receptor within the extract. For this method, the lower sensitivity limit was 3.125 pg/tube, the recovery of added l-tri-iodothyronine was 90-120%, and the between-assay coefficient of variation was 10%. The amount of endogenous l-tri-iodothyronine was 10-40 pg/0.2 ml of hepatic nuclear extract from euthyroid rats, compared with less than 3.125 pg/0.2 ml from thyroidectomized rats. The results obtained by this new method were compared with a Sephadex G-25 column extracted-sample radioimmunoassay method and showed a good agreement. The values for the endogenous l-tri-iodothyronine content were utilized to correct for the l-tri-iodothyronine concentration within the binding assay mixture in order to accurately determine by Scatchard analysis the binding characteristics of the nuclear l-tri-iodothyronine receptor. The validity of the correction for endogeneous l-tri-iodothyronine was demonstrated by using a nuclear extract from a thyroidectomized rat which was preincubated with a small known amount of l-tri-iodothyronine before determining the nuclear l-tri-iodothyronine receptor binding characteristics. When the Scatchard plots were corrected for the preincubated dose, the results obtained were similar to true values, but they were falsely lower when not corrected. It is concluded that the necessity and validity of using endogenous l-tri-iodothyronine corrections in the Scatchard analytical computations of the nuclear l-tri-iodothyronine receptor binding characteristics has been demonstrated, being particularly more important for affinity constant than maximum binding capacity.

Anilino Naphthalenesulfonates↗