Search PubMed⌕ Search

Biomedical subjects

A Okuda

Publications and source records attributed to A Okuda.

At least 127 records · Page 7Linked to original sources
PubMed1984

Purification and characterization of delta 4-3-ketosteroid 5 beta-reductase.

delta 4-3-Ketosteroid 5 beta-reductase was purified about 230-fold from 100,000 X g supernatant of rat liver homogenate using 7 alpha-hydroxy-4-cholesten-3-one as substrate throughout. The purified enzyme was electrophoretically homogeneous, and its molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 37,000 and that determined by gel filtration chromatography on calibrated Sephadex G-100 column was 37,200. The absorption spectrum of the purified enzyme showed only a peak at 276 nm due to aromatic amino acids, precluding the presence of a prosthetic group such as flavine in the molecule. The enzyme is highly labile in a low buffer concentration, but is markedly stabilized in the presence of 20% glycerol in 10 mM phosphate buffer. Higher buffer concentration such as 300 mM potassium phosphate buffer was also effective to prevent deterioration in the absence of glycerol, but the effect was somewhat lower compared to glycerol. The purified enzyme showed the activity toward a variety of substrates including testosterone, cortisol, cortisone, progesterone, 4-androstene-3,17-dione, 7 alpha-hydroxy-4-cholesten-3-one, and 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one. The optimal pH for the 5 beta-reduction of 7 alpha-hydroxy-4-cholesten-3-one was 7.4, and the cofactor required for the reaction was NADPH, while NADH revealed no effect. The enzyme activity was inhibited by p-chloromercuribenzoate, but its inhibition was prevented by the presence of a reduced form of glutathione.

Animals↗
PubMed1984

Cell growth and differentiation in vitro in mouse macrophages transformed by a tsA mutant of simian virus 40. II. Changes in the distribution of DNA content during the reversible transition between macrophage and nonmacrophage states in the cultures of tsA640-transformed macrophages.

Cultures of mouse macrophage cell lines transformed by wild-type or the tsA640 mutant of simian virus 40 (SV40) show a reversible phenotypic transition between the nonmacrophage (proliferating phase) and the macrophage (stationary phase) states (Takayama, 1980; Tanigawa et al., 1983). Distribution of DNA content in the cultures of the tsA640-transformed macrophage lines in the process of the phenotypic transition was determined by flow cytometry. Taking the mean DNA content of mouse peritoneal macrophages as 1 unit in the scale of fluorescence intensity in the flow cytogram, the transformed macrophages showed, at 33 degrees C, two peaks, one located around the 1.0-unit position (peak 1.0) and the other around the 1.6-unit position (peak 1.6), and a plateau distribution continuing to 3.2 units. Peak 1.0 was predominant in the stationary-phase culture, whereas peak 1.6 was predominant in the proliferating-phase culture. Almost the entire population of the strictly resting culture, which was obtained by culturing the stationary-phase culture for a further 5 days at nonpermissive temperature (39 degrees C), was phagocytic, and had accumulated at peak 1.0. Cells in peak 1.0 moved to peak 1.6 and to higher positions, after the strictly resting culture was sparsely reseeded and incubated at 33 degrees C. In contrast, the DNA content distribution of the successively proliferating cells, which were obtained by repeated passage of an extensively proliferating culture and none of which were phagocytic, was similar to that of proliferating hypotetraploid BALB/c3T3 fibroblasts with a G1 peak at 1.6 unit followed by a plateau containing S- and G2-phase cells. The peak 1.0 cell population appeared from the recloned population of the successively proliferating cells in company with the restoration of the culture condition-dependent phagocytic ability when cocultured with primary macrophages. Each peak in the flow cytogram reflected fairly well DNA content per cell as determined by other methods.

Animals↗
PubMed1984

Genetic analysis of control of proliferation in fibroblastic cells in culture. I. Isolation and characterization of mutants temperature-sensitive for proliferation or survival of untransformed diploid rat cell line 3Y1.

Mutants temperature sensitive for proliferation or survival were isolated from an untransformed diploid clone of fibroblastic rat cells (3Y1), according to an isolation protocol that selected for mutants defective at 38.5 degrees C (selection temperature) in undergoing the transition from quiescent to proliferating state while maintaining viability at 38.5 degrees C. Of the 108 temperature-sensitive clones isolated, 32 were examined for survival in sparse cultures at 39.8 degrees C (nonpermissive temperature) and classified into four classes. Results of temperature shift-up experiments suggest that functions defective in 11 of the 32 mutants are necessary not only for changing from the quiescent to proliferating state but also for maintenance of the proliferating state. Of the 32 mutants, 17 were assigned to eight complementation groups. Results of the physiological characterization of the representative mutants of each of the eight complementation groups are presented.

Animals↗
PubMed1984

Control in previous and present generations of preparation for entry into S phase and the relationship to resting state in 3Y1 rat fibroblastic cells.

In both the presence and absence of serum, 3Y1 rat fibroblastic cells synchronized at early S phase by aphidicolin entered M phase 6 h after removal of aphidicolin. However, in the second generation their entry into S phase in the presence of serum was delayed due to the deprivation of serum in the first generation. A similar delaying effect in the second generation was observed when the resting cells were stimulated by serum and then deprived of serum during a period of 8 h preceding mitosis. In both cases, the interval between mitosis and entry into S phase in the second generation was almost equal to that required for the resting cells to enter S phase when stimulated by serum. A similar delaying effect was also observed when the cells, synchronized at early S phase, were kept in suspension culture in the presence of serum for a period in the first generation. Results of a similar type of experiments using various combinations of growth factors showed that, when the G1 period in the second generation was shortened by exposure to growth factors in the first generation, and when the resting cells were stimulated to enter S phase, the same combination of growth factors was required. These and previous results suggest that the preparation for entry into S phase is controlled in both previous and present generations of 3Y1 cells.

Animals↗
PubMed1984

Abortive transformation of rat 3Y1 cells by simian virus 40: viral function overcoming inhibition of cellular proliferation under various conditions of culture.

Resting cultures of nonpermissive rat 3Y1 cells were infected with simian and T antigen expression and entry into S phase were examined under various conditions of culture. In the complete absence of serum from the medium or at an extremely high cell density, the cells delayed T antigen expression and entry into S phase, leaving the interval between the two events constant. Results using the viral mutants deleted in the coding region for the small t antigen ruled out the role of this antigen in induction of S phase. From these and other results presented, we conclude that the large T antigen induces S phase with the same efficiency under different conditions of cultures. We also present the evidence that the large T antigen function is required and is sufficient for entry into S phase in the second as well as in the first generation.

Animals↗
PubMed1983

Kinetic analysis of entry into S phase in resting rat 3Y1 cells stimulated by serum. Effects of serum concentration and temperature.

The kinetics of entry into S phase after stimulation of resting 3Y1 cells by serum was examined in relation to serum concentration, temperature and the time at which the serum was withdrawn or at which the temperature was shifted. The kinetics of entry into S phase could be represented not only by a lag phase followed by a negative exponential curve (fit 1), but also by a normal distribution of the reciprocals of the time required for cells to enter S phase (velocities) (fit 2). As the temperature was lowered below 37 degrees C, the exponential slope decreased and the lag period increased (fit 1), and both the mean velocity and its standard deviation decreased (fit 2). As the serum concentration decreased below 10%, the exponential slope decreased without change in the lag period (fit 1), and the mean velocity decreased with increase in the standard deviation (fit 2). The cells which did not enter S phase within 8 h on removal of serum, stopped or delayed entry into S phase. In this case the lag phase was not changed (11 h). When serum was removed just before the end of the lag phase, no effect was seen on the kinetic curve. When the temperature was shifted at any time, including after the lag phase, the characteristics of the kinetic curve (lag phase, synchrony) changed. These facts indicate that there is a serum-non-requiring, but temperature-dependent period before S phase. Most of the asynchrony in entry into S phase under conditions of low serum seems to be generated during the serum-requiring period presumably by the random transition to the state in which cells are committed to enter S phase or by the variability of reaction rates at unpredictable times due to undeterministic effects.

Animals↗
PubMed1983

Physiological function and kinetic mechanism of human liver alcohol dehydrogenase as 5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol dehydrogenase.

To test whether human liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) plays a role in the metabolism of cholesterol in man or not, a major isozyme of human liver alcohol dehydrogenase, beta 2 beta 2, was purified from homozygous atypical human livers, measuring both liver acetaldehyde reductase activity and that of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-al reductase (5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetraol:NAD+ 26-oxidoreductase, EC 1.1.1.161) in the course of purification, and it was found that both enzyme activities were accompanied with each other at any step. Both enzyme activities of the highly purified isozyme, beta 2 beta 2, were inhibited by a chelating agent, 1,10-phenanthroline, for Zn which resides in the active site of the enzyme, the pKiapp value of which was 4.1 for both activities. They were also inhibited by a known competitive inhibitor, isobutyramide, in the same fashion and the Ki values calculated from both activities were the same (2.0 mM). From these results it was suggested that both enzyme activities are catalyzed by the same active site of the same enzyme protein. Kinetic studies of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol dehydrogenase have shown that neither Theorell-Chance mechanism nor simple Ordered Bi Bi mechanism holds in the reaction; instead a mechanism which is asymmetric in both directions is operative.

Alcohol Dehydrogenase↗
PubMed1983

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture, clonal growth in low density culture, and stimulation to enter S phase in resting culture.

A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin, transferrin, epidermal growth factor, poly-D-lysine, bovine albumin, oleic acid, and bovine alpha-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetal bovine serum (FBS), and colonies, albeit of smaller sizes, did form. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking alpha-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding alpha-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.

Albumins↗
PubMed1982

Effects of serum deprivation on the initiation of DNA synthesis in the second generation in rat 3Y1 cells.

Rat 3Y1 cells arrested at early S by hydroxyurea traversed the remainder of S and G2 and completed mitosis after removal of the drug, irrespective of the absence of serum from the culture medium. When cells were deprived of serum for a period between early S and mitosis after removal of hydroxyurea, the cells delayed entry into S in the presence of serum in the second generation for the time length approximately equal to that of serum deprivation. When mitotic cells, which had been continuously exposed to serum after removal of hydroxyurea, were deprived of serum for the next 24 hours and then were reexposed to serum, the cells delayed entry into S for more than 24 hours (more than the time length of serum deprivation). On the other hand, the cells already deprived of serum between early S and G2 in the first generation were less delayed in entry into S after postmitotic 24-hour serum deprivation than were the cells exposed to serum between early S and G2 in the first generation. These results suggest that serum-dependent events continue to occur in the first generation for on-time entry into S in the next generation, and that these premitotic events (the potential for entry into S) decay if serum is absent for a long period of time after mitosis.

Animals↗