Nobuo Inoue

Analysis of the fibrin-polymerizing reaction using sodium dodecylsulfate-agarose gel electrophoresis

1. A new method of sodium dodecylsulfate gel electrophoresis, sodium dodecyl-sulfate-agarose gel electrophoresis, was developed. The new electrophoresis was shown to have a high and efficient resolving power for fibrin polymers and was also applicable to other proteins. 2. By sodium dodecylsulfate-agarose gel electrophoresis, the fibrin polymerizing reaction with activated fibrin stabilizing factor was analyzed in detail. The cross-linking between the gamma-chains was indicated to occur at an intermolecular level. The solubility of polymerized fibrin was suggested to be related mainly to the formation of the crosslinks between gamma-chains.

Nuclear DNA Ligase and Its Action on Chromatin DNA in Neuronal, Glial and Liver Nuclei Isolated from Adult Guinea Pigs

Abstract: DNA ligase activities were measured in neuron‐rich and glial nuclear preparations and liver nuclei isolated from adult guinea pigs. The enzymatic properties of cerebral and liver nuclear DNA ligases were studied with isolated nuclei and nuclear extracts. ATP (Km= 46–48 μM) and bivalent cation (Mg2+ or Mn2+) were required for the maximal activities in cerebral and liver nuclei. β‐Mercaptoethanol did not affect the activities, but N‐ethylmaleimide and p‐chloromercuribenzoate completely inhibited the activities. Deoxyadenosine‐5′‐triphosphate partially inhibited the activities in both cerebral and liver nuclei. An interdependent effect of Na+ and Mg2+ on the enzyme activities was observed. A high concentration (200 mM) of Na+ activated both enzymes and shifted to the acid side the optimal pH for both enzymes. DNA ligase was more easily extracted with lower concentrations of NaCl from liver nuclei than from cerebral nuclei, but the extraction curves from both nuclear species reached a plateau level (92% of total activities of nuclear enzymes) at 200 mM‐NaCl. Apparent Km for the substrate [32P]phosphoryl DNA was determined according to a modification of the Michaelis‐Menten equation, which was applied for the case where an unknown amount of substrate nicks in chromatin DNA coexisted with the nicks in exogenous substrate DNA. Neuronal and glial nuclear enzymes had similar Km values (about 20 μg of [32P]phosphoryl DNA/ml), but the liver nuclear enzyme had a higher Km value (54 μg of [32P]phosphoryl DNA/ml). The modified Michaelis‐Menten equation provided the amounts of nicks available as substrate in chromatin DNA of isolated nuclei. Neuronal and glial nuclei contained 1.5 and 0.29 pmol of nicks/μg of nuclear DNA, respectively, in contrast to an intermediate amount of nicks in liver nuclei (0.63 pmol/μg of nuclear DNA). DNA ligase activity in neuronal nuclei [312 units (fmol of 5′‐phosphomonoester converted into a phosphatase‐resistant form per min at 37°C) per μg of nuclear DNA] was 11‐fold higher than that in glial nuclei [28.7 units/μg of nuclear DNA]. Liver nuclei contained an intermediate activity [54.7 units/μg of nuclear DNA].

DNA synthesis in neuronal, glial and liver nuclei isolated from the adult guinea pig.

Abstract— [3H]Deoxythymidine‐5′‐triphosphate incorporation into P51 (51% neuronal nuclei: 49% glial nuclei), P3 (3% neuronal nuclei: 97% glial nuclei) and liver nuclear preparations, isolated from the adult guinea pig, was determined in the presence of the other three complementary deoxyribonucleo‐tides. The enzymic characteristics of the DNA synthesis reaction were studied and DNA polymerase contents were estimated in neuronal, glial and liver nuclei. (1) Cerebral and liver nuclei exhibited similar enzymic properties for DNA synthesis activities with a few discrepancies. (2) P51 nuclei synthesized DNA 2.4‐fold more actively than P3 nuclei. Liver nuclei carried out the most active DNA synthesis. The proportion of chromatin DNA available as template and primer was estimated by comparison with native calf thymus DNA. The available proportions found, in terms of the total chromatin DNA. were 2.39% for P51 nuclei, 1.38% for P3 nuclei and 37.6% for liver nuclei. (3) Exogenous native and heat‐denatured calf thymus DNA were utilized as template and primer by DNA polymerase in nuclei in different ways depending on the nuclear species. The enzyme was saturated with native DNA by elevating the concentration and the activity reached a plateau. Denatured DNA inhibited the activity at the higher concentrations. (4) From the enzyme activities at a saturation concentration of exogenous DNA, DNA polymerase contents were estimated: P51 nuclei, 39.2 ± 2.6 (s.e.m.) units (fmol of TMP incorporated/30 min at 31°C)/μg of nuclear DNA; P3 nuclei. 24.5 ± 1.6; and liver nuclei, 72.5 ± 8.1; the specific activity obtained on a protein basis was 1.55 times higher with P3 nuclei than with P51 nuclei. (5) Denatured DNA inhibited the nuclear DNA polymerase activity dependent on native DNA. The efficiency of inhibition was in the order: P3 > P51 > liver nuclei.

Plasmic degradation of bovine fibrinogen and non-cross-linked fibrins in solution and in gel form

1. Analysis of degradation processes of bovine fibrinogen by bovine plasmin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a study on the mode of changes of the properties related to clotting of digestion products as a function of time were performed. Gross features and patterns very similar to those which had been reported with human fibrinogen-plasmin systems were obtained. 2. Based on the molecular size of the degradation products and the mode of appearance and disappearance of the degradation products, the processes could tentatively be divided into three stages: stage 1, where fibrinogen (mol. wt 370 000) was degraded to produce fragments X1 (330 000) and X2 (290 000); stage 2, fragment X2 was degraded with appearance of Y (210 000) and D1 (140 000); stage 3, appearance of fragments D1, D2 (110 000), and D3 (100 000) sequentially and E (68 000) with concomitant disappearance of Y. 3. A microseparation method, which is a combination of dansylation and sodium dodecylsulfate-polyacrylamide gel electrophoresis, was devised to analyze the events of stage 1 in detail, and a molecular model for the process was proposed. 4. The plasmic degradation processes of bovine non-cross-linked fibrins in solution and in gel form were compared with that of fibrinogen and it was found that the state of the substrates, fibrins, could cause differences in the degradation patterns. With the former substrate, essentially the same sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns as those with fibrinogen were obtained. With the latter substrate, however, a distinct difference in the mode of degradation of beta chains was observed.

An experimental study on the backward extrusion of high-density polyethylene.

The backward extrusion of high-density polyethylene is experimentally investigated with relation to the effects of extrusion ratio R, semi-cone angle α of the punch nose, and extrusion rate V. The appearance of the extrudates was observed to be greatly affected by R, α and V ; it went bad as R or α increased, or V decreased. Dead material was formed only when α>75° and acted as a tip of the front end of the punch. Extrusion pressure P increased as R increased, but decreased as V increased : it was also affected by α. The tensile strength of the extrudates increased as R increased in the axial direction, but it was little affected by R in the hoop direction.

Effects of Hydrostatic Extrusion on the Thermal Properties of Amorphous Polymers

The feasibility of hydrostatic extrusion has been studied for various polymers: including polyimide, polysulfone, high-density polyethylene, low-density polyethylene, and polystyrene [1,2]. Recently, the hydrostatic extrusion of brittle amorphous polymers, poly(methyl methacrylate) (PMMA) and high-impact polystyrene (HIPS), was successfully performed by sheathing these polymers with rubber at ambient temperature, without back pressure [3,4]. Nakayama and Inoue [3] reported that the elongation of the PMMA as well as HIPS extrudates, when tested in tension under atmospheric pressure, exhibited a marked increase, and that this behavior was due to the rearrangement of the molecular chains in the direction of extrusion.