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Biomedical subjects

H Nagano

Publications and source records attributed to H Nagano.

At least 217 records · Page 12Linked to original sources
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

Changes in deoxyribonucleoside triphosphate pools in the starfish oocyte during maturation and early embryogenesis.

dNTP pools are quite low in immature oocytes of the starfish, expand during the 1-methyladenine-induced maturational process and thereafter reach a maximal level (approx. 35, 20, 15 and 5 fmoles/egg for dTTP, dCTP, dATP and dGTP, respectively) which is maintained in overmatured eggs. Maturing oocytes were inseminated at the stage just before extrusion of the first polar body and determination of dNTP pools during early embryogenesis showed the same expansion pattern as that of the 1-methyladenine-treated oocytes. Therefore, the increase in dNTP pools during early embryogenesis is dependent on 1-methyladenine (1-MA) but independent of fertilization. Aphidicolin, a specific inhibitor of eukaryotic DNA polymerase alpha, has no effect on dNTP pool size in 1-methyladenine-treated oocytes, but causes considerable expansion of dNTP pools in fertilized eggs which cleave achromosomally in the presence of the drug.

Animals↗
PubMed1983

Specific inhibitors of eukaryotic DNA synthesis and DNA polymerase alpha, 3-deoxyaphidicolin and aphidicolin-17-monoacetate.

Of several phytotoxins isolated from culture filtrates of Phoma betae Frank PS-13, an incitant of leaf spot disease of sugar beet, three have been identified as aphidicolin, 3-deoxyaphidicolin and aphidicolin-17-monoacetate. Aphidicolin is a selective inhibitor of eukaryotic DNA polymerase alpha (Ikegami et al. (1978) Nature 275, 458-460). Consequently, we studied the action mechanism of 3-deoxyaphidicolin and aphidicolin-17-monoacetate. These aphidicolin analogues markedly inhibited the in vivo DNA synthesis of sea urchin embryos and HeLa cells but not RNA and protein syntheses. Only DNA polymerase alpha, not DNA polymerase beta and gamma, was inhibited by these drugs. The mode of action of these analogues on DNA polymerase alpha from the sea urchin was competitive inhibition with respect to dCTP with Ki values of 0.44 micrograms/ml for deoxyaphidicolin and 0.89 micrograms/ml for aphidicolin monoacetate, respectively. None of the other three dNTPs competed with these drugs. A similar inhibitory mode was observed using the enzyme from HeLa cells and toad oocytes. These drugs at a concentration of 2 micrograms/ml caused a delay in the cleavage of fertilized eggs of the sea urchin and decomposition before blastulation, indicating the possibility of achromosomal cleavage because of the absence of DNA synthesis. Based on the above, it is concluded that these analogues can be used as other inhibitors of eukaryotic DNA synthesis and DNA polymerase alpha.

Animals↗
PubMed1983

Inhibition of DNA polymerases of sea urchin by palmitoyl coenzyme A.

Palmitoyl CoA noncompetitively inhibited the activities of DNA polymerase alpha and gamma, prepared from sea urchin germ cells, with Ki values of 28 microM and 116 microM, respectively. Myristoyl CoA also inhibited DNA polymerase alpha and gamma, while coenzyme A, short chain fatty acyl CoA's, Na-myristate and Na-palmitate failed to inhibit the enzymes. It was concluded that both the long hydrocarbon chain and CoA moiety of long chain fatty acyl CoA's are necessary for inhibition of DNA polymerase activity. DNA polymerase beta was not inhibited by long chain fatty acyl CoA's.

Acyl Coenzyme A↗
PubMed1983

Localization of DNA polymerase alpha on the nuclear membrane in sea urchin embryos.

Subnuclear localization of DNA polymerase alpha was studied in sea urchin embryos. Blastula nuclei treated with EDTA and potassium phosphate released subnuclear components bearing most of the nuclear DNA polymerase alpha. These components were suggested to be a part of nuclear membrane based on their buoyant densities (1.177 and 1.136 g/cm3) in isopyknic centrifugation and the nuclear pore-like structure. Contamination with DNA and endoplasmic reticulum membrane to the subnuclear components was shown to be negligible. These results suggested that DNA polymerase alpha associates with nuclear membrane of sea urchin embryos. Nuclear membrane deprived of DNA polymerase alpha was able to associate with nuclear DNA polymerase alpha from blastulae and the cytoplasmic enzyme of unfertilized eggs efficiently, but not with the cytoplasmic enzyme of gastrulae. This result suggests that the nuclear membrane is originates from the endoplasmic reticulum with which DNA polymerase alpha associates in unfertilized eggs.

Animals↗
PubMed1983

Mitochondrial DNA synthesis in overmatured eggs of the starfish.

DNA synthesis was studied during the overmaturational stage of starfish eggs. Some DNA synthesis took place in the overmature eggs of the starfish. The DNA synthesis was resistant to aphidicolin. Judging from its circularity and small molecular size, the DNA synthesized is of mitochondrial origin.

Animals↗
PubMed1983

Isolation and characterization of DNA polymerases from mature oocytes of the starfish, Asterina pectinifera.

Four distinctive DNA polymerases have been isolated from mature oocytes of the starfish, Asterina pectinifera, and characterized. The N-ethylmaleimide-resistant DNA polymerase activity was separated from the other three DNA polymerases by hydroxyapatite column chromatography. The enzyme, whose approximate sedimentation coefficient was 3.3S, effectively utilized poly(dA)-oligo(dT) as a template-primer and was completely inhibited by 10 microM 2', 3'-dideoxythymidine-5'-triphosphate but was not inhibited by aphidicolin. Therefore, this enzyme was classified as a eukaryotic DNA polymerase-beta. Three species of the N-ethylmaleimide-sensitive DNA polymerase were separated with a phosphocellulose column. Two (5.5S and 6.5S) of the enzymes eluted from the column preferred activated DNA as a template-primer. Both of the DNA polymerases were inhibited by aphidicolin but not by 2',3'-dideoxythymidine-5'-triphosphate. Judging from the above results, these two DNA polymerases were classified as eukaryotic DNA polymerase-alpha. The third N-ethylmaleimide-sensitive enzyme (approximately 7S) eluted from the phosphocellulose column utilized poly(rA)-oligo(dT) as well as poly(dA)-oligo(dT) as template-primers. The enzyme activity was resistant to aphidicolin but sensitive to 2',3'-dideoxythymidine-5'-triphosphate. This enzyme resembles eukaryotic DNA polymerase-gamma. Thus, it is concluded that the four species of DNA polymerase found in starfish oocytes resemble eukaryotic DNA polymerase-alpha, -beta, and -gamma.

Animals↗
PubMed1983

Reconstitution of endogenous DNA synthesis in isolated nuclei and template activity of chromatin with respect to mode of inhibition by aphidicolin.

We succeeded in reconstituting the endogenous nuclear DNA synthesis of the sea urchin. Endogenous DNA synthesis of isolated nuclei was reconstituted by mixing the salt-treated nuclei (chromatin exhibiting essentially no endogenous DNA synthesis) and the salt extract containing DNA polymerase-alpha. DNA synthesis in this reconstitution system showed a level of activity and a mode of inhibition by aphidicolin similar to those of the original isolated nuclei (noncompetitive with respect to dCTP). On the other hand, the inhibitory mode was competitive with respect to dCTP in DNA synthesis in the reconstituted system obtained from the chromatin and purified DNA polymerase-alpha, indicating that some other factor(s) in addition to DNA polymerase-alpha is necessary for the reconstitution with reference to the inhibitory mode of aphidicolin. We also studied the template activity of the chromatin. When chromatin was used as a template, inhibition by aphidicolin of DNA polymerase-alpha was noncompetitive and uncompetitive with respect to the template at high and low concentrations, respectively. Treatment of chromatin with 5 M urea gave urea-treated chromatin (nonhistone protein-deprived chromatin) and the extract (mainly nonhistone protein fraction). Inhibition by aphidicolin of DNA polymerase-alpha was uncompetitive with respect to the urea-treated chromatin. However, when chromatin reconstituted from the urea-treated chromatin and the extract was used as a template, the inhibitory mode by aphidicolin was similar to that with original chromatin, indicating that the nonhistone protein fraction contained factor(s) which modified the inhibitory mode of aphidicolin. Thus, the inhibitory mode of aphidicolin is a useful parameter for monitoring the resolution and reconstitution of endogenous DNA synthesis of isolated nuclei.

Animals↗
PubMed1983

Congenital hydrocephalus revealed in the inbred rat, LEW/Jms.

We studied the development of congenital hydrocephalus found in a colony of an inbred strain of Wistar-Lewis rats (LEW/Jms) at various intervals after birth. The disorder was transmitted as a simple recessive mendelian character. Hydrocephalic neonates were recognized 2 days after birth by stretching of the skin over the head. Death usually occurred between 10 and 20 days of age. The findings suggested the possibility of a disturbance of cerebrospinal fluid circulation resulting from primary occlusion of the 3rd or lateral ventricles during embryological development. In later phases, the hydrocephalus was aggravated by obliteration of the subarachnoid space and by stenosis of the aqueduct occurring secondary to compression of these structures from increased pressure within the brain. In some animals, external hydrocephalus occurred as a result of rupture of the occipital pole and the establishment of a direct communication between the lateral ventricles and the subdural space. We looked for antibodies against viruses that have been known to produce hydrocephalus in experimental animals and obtained entirely negative results.

Animals↗
PubMed1983

Lipoprotein metabolism in diabetics treated with diet, oral hypoglycemic drug and insulin.

In view of the high incidence of hyperlipidemia and the low sialic acid content in the membranes of diabetics, we analyzed the percentage composition of apolipoprotein CII, known as an activator of lipoprotein lipase, and a subspecies of apolipoprotein CIII, an inhibitor of lipoprotein lipase, in triglyceride-rich lipoproteins. CIII can be sub-divided into three groups, CIII0, CIII1 and CIII2, according to sialic acid content by isoelectric focusing gel. In 82 diabetics, serum lipids and lipids in various lipoprotein fractions differed according to treatment, (diet, oral hypoglycemic drug or insulin). CIIIo/CII showed a positive correlation to plasma triglyceride and cholesterol. In the group receiving oral medication (N = 20), CIIIo/CII vs HDL-cholesterol showed a positive correlation, whereas CIII2/CII vs plasma triglyceride showed an inverse correlation. In the insulin group (N = 25), the percentage of CIIIo in VLDL apo C subspecies was inversely correlated with plasma cholesterol. In 38 diabetics whose HbA1 was also examined, CIIIo/CII increased with elevation of HbA1. CIIIo/CII in diabetics with HbA1 higher than 10% was significantly high compared with the index in other diabetics. The percentage of CIII1 in VLDL apo C subspecies was correlated to HbA1 level positively in the diet group but inversely in the insulin group. These results suggest that lipoprotein metabolism in diabetics may vary according to treatment and the sialylation of apolipoprotein may play an important role in determining the severity of this disease.

Aged↗
PubMed1983

[Evaluation of tissue concentrations of FT-207 and 5-FU in head and neck cancer].

FT-207 was administered to 30 patients with squamous cell carcinoma of the head and neck. After FT-207 administration tumor concentration of FT-207 and 5-FU was measured using a chemical assay method. Differences of FT-207 and 5-FU in total doses, administration routes (oral, intrarectal, and intravenous), tumor sites (larynx, maxillary sinus, tongue, neck and hypopharynx), and types of differentiation (well-differentiated, moderately-differentiated, and poorly-differentiated) were studied and the following results were obtained: 1. In FT-207 tumor concentration study, no significant difference was observed in any group. 2. 5-FU tumor concentration increased in higher total administration doses and in advanced differentiated type groups as well as in the non-irradiated group. 3. 5-FU tumor concentration was measured by T/N ratio; oral and intravenous administration methods showed 4.5 (P less than 0.05) and 2.4 (P less than 0.10) respectively. In larynx tumor the highest concentration of 3.9 (P less than 0.05) was obtained among various tumors. The value of 4.0 (P less than 0.05) was yielded in the well-differentiated type, which was significantly higher compared to that of other differentiation types. Overall T/N ratio (5-FU concentration in the tumor/5-FU concentration in the normal tissue) was 2.7 (P less than 0.025).

Adult↗
PubMed1982

Developmental profiles of substance P and GABA contents in the substantia nigra of rat.

The developmental changes of concentrations of substance P and GABA in the substantia nigra at various postnatal ages of the rat were investigated. Both substance P and GABA were present in measurable amount at birth and continuously increased with age thereafter. When the two curves were compared, GABA seemed to develop slightly faster than substance P in the substantia nigra. Therefore, in contrast to previous reports, there was no discontinuously rapid increase in the concentrations of these transmitter candidates during normal postnatal development of the rat substantia nigra.

Animals↗
PubMed1982

The inhibitory mode of aphidicolin on DNA synthesis in NaCl-treated HeLa cell nuclei.

Isolated HeLa cell nuclei were treated with NaCl at various concentrations and inhibition by aphidicolin of DNA synthesis in the treated nuclei was studied. The inhibition was either noncompetitive or of the mixed type with respect to each dNTP when the nuclei were treated with NaCl at concentrations lower than 0.08 M. However, aphidicolin was a competitive inhibitor with respect to dCTP and a non-competitive or mixed type inhibitor with respect to the other 3 dNTPs when they were treated with NaCl at concentrations higher than 0.1 M. These results suggest the presence of nuclear factor(s) responsible for the changes in the inhibitory mode of aphidicolin on endogenous nuclear DNA synthesis.

Aphidicolin↗