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Biomedical subjects

N Sugano

Publications and source records attributed to N Sugano.

At least 145 records · Page 8Linked to original sources
PubMed1985

An endodeoxyribonuclease activity in nuclei from rat-ascites hepatoma.

The DNA in nuclei from rat-ascites hepatoma (AH) was rather resistant to endogenous endonucleolytic attack (autodigestion), compared with that in nuclei from normal rat liver (RL). In contrast, by micrococcal nuclease, the DNA in AH nuclei was cleaved in the same manner as in RL nuclei. A 0.6 M NaCl extract was prepared from RL or AH nuclei and subjected to Sephadex G-100 filtration. The resulting-nuclease fraction was separated further into two nuclease fractions, I and II, by CM-Sephadex column chromatography. The activity ratio of II to I was 7.1 for the RL and 2.0 for the AH nuclei. Moreover, the activity of fraction II from the AH nuclei was rather low, compared with that from the RL nuclei. Regenerating-liver nuclei from the normal rat were also assayed in the same way. The results obtained were very similar to those from the AH nuclei. In addition, each of fractions, I and II, cleaved pBR322 DNA of superhelical form; in other words, each had endonucleolytic ability.

Animals↗
PubMed1985

The association of a magnesium-dependent endonuclease activity with a nucleosome fraction from rat-liver nuclei.

The nucleosomes released by the incubation (autodigestion) of rat-liver nuclei were fractionated by sucrose-density gradient centrifugation, and subjected to nuclease assay with heat-denatured 3H-DNA from Escherichia coli as an exogenous substrate. With increasing incubation time, the nuclease activity was enhanced and localized in the mono/tetra-, hexa/hepta-, and long-chain oligonucleosome fractions. In contrast, independent of the nucleosome size, the activities of 0.35 M NaCl-soluble fractions from them were found to be almost equal in terms of specific activity (dpm/nucleosomal DNA). Such nuclease activity was not detected in the sucrose gradient (top region) lacking nucleosomes and/or chromatin. When the chromatin was dialyzed against a 0.35 M NaCl buffer and then fractionated in a sucrose gradient containing 0.35 M NaCl, most of the nuclease activity was solubilized into the above top region. On gel filtration of the mononucleosome fraction in the 0.35 M NaCl buffer, the nuclease activity was eluted at the position of 36,000 daltons. This nuclease cleaved heat-denatured DNA more rapidly than the native DNA in the presence of Mg2+, and had the ability to make both single-strand nicks and double-strand cuts in pBR322 DNA; in other words, it had an endonucleolytic activity. Moreover, four different classes of mononucleosomes were fractionated by electrophoresis of the nucleosomes released by autodigestion of the nuclei. These mononucleosomes also showed nuclease activity with the heat-denatured DNA. Thus, the present studies suggest that an Mg2+-dependent endonuclease of about 36,000 daltons is associated with the nucleosome particle(s) in rat-liver nuclei.

Animals↗
PubMed1984

Effect of doublet impulse sequences on the transient and steady responses in the computer-simulated nerve cell.

The effects of doublet impulse sequences of the excitatory axon on the output response as firing probability (pr.) in the computer-simulated nerve cell were examined. A simple model was formulated to simulate the nerve cell, including the property that the resetting potential is influenced by the final membrane potential in the previous stage before firing. The relationship between input sequences with alternating long and short interval at the same mean rate and the transient and steady responses of the nerve cell was investigated. In this simulation, three summarized results were obtained: i) The responses were very sensitive to changing small size of excitatory post-synaptic potential (EPSP), especially in the firing stage of the transient state. ii) In the transient state, the size of characteristic area of responses was depending upon the size of absolute refractory period (ARP). The rise for shorter intervals was faster than that for longer intervals, agreeing well with part of the experimental results from the crayfish claw opener muscles. The transient responses were almost finished before the fifth firing. iii) In the steady state, the doublet impulse sequences usually produced the minimum response or valley-like response at which the doublet interval Td was 20 and/or 25 ms. These effects related to the characteristic areas in the transient responses.

Computers↗
PubMed1983

Effect of doublet impulse sequences in the crayfish claw opener muscles and the computer-simulated neuromuscular synapse.

The effects of doublet impulse sequences of the excitatory motor axon on the movement of the claw opener muscles in the crayfish were examined. The excitatory motor axon was stimulated electrically with various patterns of doublet impulse sequences generated by a digital computer. Doublet impulse sequences of stimulation produced a larger sustained movement than an uniform impulse sequences at the same mean rate of stimulation. The movement was largest when the interval between the impulses of a doublet was about 5 ms. This interval generated a movement amplitude 25% greater than that for the uniform impulse sequence. A simple model was formulated to stimulate the neuromuscular synapse of the claw opener muscle. The relationship between stimulation sequences with alternating long and short intervals and responses (firing probabilities) of the neuromuscular synapse at the same mean rate was investigated. The responses was classified into two typical types which are noneffective Type I and effective Type II to the absolute refractory period (ARP). The characteristics which are larger responses with short intervals in Type I and reduction of responses in the ARP region of Type II formed a plateau peak of the experimental results. By incorporating the reduction of end-plate potential (EPP) as a property of nonlinear rule for temporal summation into the model, it was shown that Type I response is maximal with a plateau peak at short interval, agreeing well with the experimental results from the claw opener muscles.

Animals↗
PubMed1983

Association of benzo[a]pyrene with H1 histone in rat-liver nuclei.

Rat-liver nuclei were incubated with microsomal fraction, NADPH and [3H]benzo[a]pyrene ([3H]BP). From the incubated nuclei, H1 histone was extracted and purified. The purified histone was acid-hydrolyzed; the hydrolysate was filtered on a column of Sephadex LH-20. The 4 radioactive fractions thus obtained by methanol gradient were about 44% of the total radioactivity. By thin-layer chromatography, 1 ninhydrin-positive and fluorescent spot was detected in each of the 2 radioactive fractions. The gas chromatographic and mass spectrometric analyses suggest that 1 of these 2 fractions contains a BP-diol epoxide-lysine conjugate.

Animals↗
PubMed1983

Resistance of H1 histone to proteolytic attack in chromatin from rat-ascites hepatoma.

From rat-liver and ascites-hepatoma chromatins, NaCl-soluble fractions were prepared. The 0.35 M NaCl-soluble fraction from the hepatoma (AH) chromatin contained much non-histone protein of high-molecular weight, compared with the fraction from the rat-liver (RL) chromatin. The 0.35 M NaCl-insoluble, but 2 M NaCl/5 M urea-soluble fraction was composed mainly of 5 classes of histones. These histones were quantitatively not different between AH and RL chromatins. However, H1 histone was rather protease-resistant in AH chromatin, but not in RL chromatin. The proteolytic capacity was also lower in AH chromatin. In addition, in the micrococcal-nuclease digest of AH nuclei, the oligonucleosomes were considerably retained even by long-time digestion, but not in that of RL nuclei.

Animals↗
PubMed1982

Associations of benzo[a]pyrene with rat-liver chromatin and the chromatin protein.

Rat-liver nuclei were prepared in the course of time after the i.p. injection of [G-3H]benzo[a]pyrene ([3H]BP). The nuclei were lysed in the hypotonic buffer and centrifuged at 4000 X g. The recovery of the radioactivity of resulting supernatant (chromatin) was thus 91% at 24 h, 68% at 48 h and 74% at 168 h after the i.p. injection. The incorporation into nucleosome-oligomer fraction was always much more than into those of monomer and DNA-rich fractions. The preferential incorporation was found in the fraction which was enriched in non-histone chromatin proteins (NHCPs) of 49 000-55 000 daltons. This fraction steadily raised the incorporation level until at 168 h after the i.p. injection. In contrast, the levels of histone and DNA fractions were always very low. The significant incorporation was observed in the fraction which was composed of five classes of histones and low molecular-weight NHCPs (less than 30 000 daltons), despite the very low incorporation into the histone fraction. The fluorographic analysis revealed the predominant incorporations at the positions of molecular weight of 65 000, 52 000 and 44 000 daltons. In addition, the incorporations were clearly observed at the positions of 59 000, 49 000, 45 000, H1 histone, A24 protein and another one. On the other hand, these fractions were, at the final preparation steps, subjected to either dialysis of SDS-phenol treatment and/or acetone precipitation. The total recovery of radioactivity was thus 21% at 24 h, 32% at 48 h and 52% at 168 h after the i.p. injection. These results suggest that the chromatin contains considerable amounts of water-soluble, phenol and/or acetone-soluble BP-conjugates in the early period after the i.p. injection.

Animals↗
PubMed1979

Isolation and characterization of chromatin subfractions from rat liver.

Rat-liver chromatin was digested with micrococcal nuclease at low ionic strength in the presence of a low concentration of CaCl2. The nuclease digest was successfully separated into three fractions, P1, P2, and P3, by gel filtration on a column of Sepharose 2B. P1 fraction was shown to be a mixture of long fragments of partially digested chromatin by the sedimentation profile or by electrophoresis of DNA. P2 fraction contained four histones H2A, H2B, H3, and H4 in almost equal amounts, together with nonhistone protein of low molecular weight. The DNA was composed of three or four fragments less than 300 base pairs long. From the Kav value of the P2 fraction, the average size was estimated to be about 240 base pairs. On analytical ultracentrifugation, this fraction exhibited a monophasic boundary and a sedimentation value of 13.7S. P3 fraction contained nonhistone proteins which showed a molecular weight larger than that of H1 histone. The size of DNA was estimated to be less than 50 base pairs from the Kav value. Based on these results, the P2 fraction was concluded to consist of nucleosome monomer enriched in nonhistone proteins. The P3 fraction is presumably the nuclease-sensitive or internucleosome portion, which contains small amounts of nonhistone proteins.

Animals↗
PubMed1979

Two ribonucleases H from cultured plant cells.

Two forms of enzyme with ribonuclease H (RNase H) [EC 3.1.4.34] activities, have been partially purified from cultured plant cells, strain GD-2, derived from carrot root. One is an Mn2+-dependent RNase H, and the second is an Mg2+-dependent RNase H. These enzymes degrade RNA specifically in RNA-DNA hybrid structures. They were eluted at around 0.2 M and 0.4 M potassium chloride in phosphocellulose chromatography, and were further purified using blue Sepharose. Mg2+-dependent RNase H exhibits maximal activity at pH 9.0, and requires 10 to 15 mM Mg2+ for maximal activity, whereas the Mn2+-dependent enzyme is most active at pH 8.0, is maximally active at an Mn2+ concentration of 0.4 mM, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. The enzymes liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini. The apparent molecular weight of the Mg2+-dependent RNase H was estimated to 18--20 X 10(4) and that of the Mn 2+- dependent RNase H was estimated to be 14 x 10(4) by gel filtration.

Cells, Cultured↗
PubMed1978

Ribonuclease H activity in cultured plant cells.

Ribonuclease H (RNAase H) was extracted from cultured plant cells, strain GD-2 and characterized. RNAase H activity in logarithmical growing cells is much higher than that of stationary cells, and the response of RNAase H activity was very similar to that of DNA polymerase after culture. The activities of RNAase, DNAase, phosphodiesterase and alkaline phosphatase decrease parallel with the increase in growth, and increase to stationary phase, contrasting with those of DNA polymerase and RNAase H.

Cell Division↗
PubMed1975

Protein kinase in cultured plant cells.

A protein kinase (EC 2.7.1.37) which phosphorylates histones was purified partially from the soluble fractions of cultured plant cells. The optimum pH was 7.5 to 9.0. The activity wasnot stimulated by exogeneous cyclic AMP. It was thermolabile and completely dependent on the presence of Mg2+ or Mn2+ for activity. p-Chloromercuribenzoate inactivated this enzyme and this inactivation was overcome by mercaptoethanol.

Cyclic AMP↗
PubMed1975

Physicochemical properties of chromatin digested with trypsin.

Chromatin was prepared from calf thymus and digested with trypsin. Some physicochemical properties of chromatin were examined in connection with the time-course of the tryptic digestion. As the tryptic digestion proceeded, chromatin showed increases in viscosity and susceptibility to DNAase II and exhibited considerable alteration in thermal denaturation. A monophasic melting profile was found in the trypsic (digested) chromatin, but a biphasic one in the native (undigested) chromatin. The melting temperature descended from 78.2 degrees C for the native chromatin to 70.2 degrees C for the chromatin after only 10 min and further to 65.3 degrees C after 180 min tryptic digestions. The molar percent of total basic amino acid or lysine plus arginine in the chromatin increased with the time-course of the tryptic digestion whereas that of the total hydrophobic amino acid decreased. The molar ratio of hydrophobic amino acids to basic amino acids thus descended from 1.46 for the native chromatin to 1.05 for the chromatin after a 180-min tryptic digestion. These findings suggest that the neutral or hydrophobic portions in chromatin protein might play a role in the maintenance of the tertiary structure of chromatin.

Amino Acids↗