Biomedical subjects
Publications and source records attributed to Y Ohta.
In female rats given a single injection of 1.25 mg 5 alpha-dihydrotestosterone during neonatal life, reactivity of the vaginal mucosa to hormones was permanently altered, so that the rats, when adult, could show vaginal cornification under the hormonic conditions supportive of development of deciduomata in the uterus.
The hydroxylase activities observed in extracts of Pseudomonas putida ORC after growth on orcinol and resorcinol as sole source of carbon have been purified to homogeneity. Both enzymes were shown to be flavoproteins and to contain approximately 1 mol of FAD for each polypeptide chain, S20,W values for each enzyme are 4.1 +/- 0.1 and are independent of the presence of their aromatic substrates. Molecular weight determinations under native (approximately 68000) and denaturing (approximately 70000) conditions indicated that they are monomeric. The visible absorption spectra identical but the circular dichroic spectra of the two proteins can be distinguished. Although each protein catalyzes the NAD(P)H and O2-dependent hydroxylation of both orcinol and resorcinol, the efficiency of the transformations of the substrates by the two enzymes is radically different; furthermore resorcinol hydroxylase is much more versatile in the aromatic compounds it can utilize as substrates and effectors. Other properties of the enzymes which clearly establish their own identity include their serological characteristics and amino acid composition; the latter property is particularly evident when the quantities of valine and alanine residues are compared. The synthesis of each enzyme is also under different regulatory constraints, being controlled by the substrate used for growth.
The murine-virus-infected erythroleukemia cell system provides an opportunity to examine regulatory mechanisms controlling cytodifferentiation. A cloned cell line (DR10c3) resistant to the erythropoiesis-inducing effect of dimethylsulfoxide (Me2SO) was isolated from the Me2SO-sensitive line DS19. DR10c3 is characterized as follows: (1) the uptake of [3H]Me2SO is similar to that in DS19; (2) cell growth with and without Me2SO is similar to that of DS19; (3) resistance is relatively stable; (4) the karyotype of DR10c3 reveals an average loss of five chromosomes per cell, but is otherwise similar to that of DS19; (5) total protein and globin synthesis by cells cultured 4 days with or without Me2SO is similar to these syntheses in DS19 cultured without Me2SO; (6) virtually no globin mRNA is detectable after 3 days in Me2SO, as assayed both by RNA-complementary DNA hybridization and by the heterologous cell-free protein-synthesizing system; (7) other polar compounds, N-methylpyrrolidinone, 1-methyl-2-piperidone, N, N-dimethylacetamide, and N-methylacetamide, induce erythroid differentiation in DR10c3, and the accumulation of alpha- and beta-globin chains is indistinguishable from that in DS19; and (8) the concentration optima for induction of differentiation by all these compounds are identical for DR10c3 and DS19.
Female mice of the C57 Black/Tw strain given 5 daily injections with 100 microng testosterone (T) or 5 alpha-dihydrotestosterone (DHT) from the day of birth showed estrogen-independent persistent proliferation and cornification of the vaginal epithelium in adulthood. The vaginal epithelium of the mice was essentially similar to that of the controls in histological structure during or shortly after neonatal injections of the androgens. In T- and DHT-mice aged over 20 days, however, a marked proliferation with or without superficial cornification took place in the epithelium lining the proximal and middle parts of the vagina (Müllerian vagina), while neither proliferation nor cornification occurred in the epithelium of the distal vagina (urogenital sinus vagina). On the second day of postnatal life in mice given a single injection with T on the day of birth, the mitotic activity in the epithelium of the middle vagina was heightened, but it dropped to the control level on the third day and remained low until 20 days. By contrast, the mitotic rates in the epithelium of the rest of the vagina in T-mice and of all parts of the vagina in DHT-mice were approximately the same as in the controls until 20 or 30 days. The mitotic rates in the epithelium of the Müllerian vagina were markedly elevated in T-mice at 20 days of age and DHT-mice at 30 days, and thereafter remained almost unchanged until 60 days of age. These results were different from the findings in mice given neonatal injections with the dose of estradiol-17 beta (E) capable of estrogen-independent vaginal cornification (Iguchi et al., 1976). The present finding seem to indicate that the mechanism involved in the induction of estrogen-independent vaginal changes by neonatal administration of androgen (T, DHT) is different from that following neonatal treatment with estrogen (E), although androgen and estrogen act directly on the vaginal epithelium of neonates.
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Orcinol hydroxylase (EC 1.14.13.6), which catalyzes the first reaction of orcinol catabolism in Pseudomonas putida 01, has been purified to homogeneity, and crystallized. Orcinol hydroxylase catalyzes the hydroxylation of orcinol with equimolar consumption of O2 and NADH (or NADPH) to 2, 3, 5-trihydroxytoluene, which is nonenzymically oxidized to a quinone. The visible absorption spectrum of the enzyme shows maxima at 373 and 454 nm and a shoulder at 480 nm. FAD can be dissociated from the protein. Reconstitution of enzymic activity was achieved with FAD, and to a limited extent by FMN. The enzyme has a molecular weight of 63,000 to 68,000 and contains 1 mol of FAD per mol of protein. K-m values for the three substrates orcinol, NADH, and O2 are 0.03, 0.13, and 0.07mM, RESPECTIVELY. The molecular activity of the crystalline enzyme is 1560 min minus 1. In the absence of orcinol, NADH is only slowly oxidized with formation of H2O2. Several analogs of orcinol also serve as substrates for hydroxylation, namely resorcinol, 4-methylresorcinol, and 4-bromoresorcinol. Other analogs, m-cresol, m-ethylphenol, 4-ethylresorcinol, and phloroglucinol, mimic orcinol as effectors, in that they (a) accelerate electron flow from NADH to the flavin and (b) decrease the apparent K-m for NADH but not to the same extent as the substrates that are hydroxylated. The latter compounds are not hydroxylated. Instead H2O2 accumulates as the only product of O2 reduction. The enzyme therefore behaves either as a hydroxylase or an oxidase. The ratio of hydroxylase to oxidase activities of the enzyme is decreased by an increase in the temperature of incubation; at 60 degrees the reaction with orcinol is almost 50% uncoupled from hydroxylation. The apparent K-m values for the effectors are in good agreement with the D-D values obtained for orcinol, resorcinol, and m-cresol. K-D values were obtained by measurement of the effector-induced perturbations of the visible absorption spectrum of the flavoprotein by difference absorption spectroscopy. The circular dichroism spectrum of orcinol hydroxylase is also altered in the presence of orcinol. The participation of the flavin in the over-all reaction is demonstrated by its rapid reduction under anaerobic conditions by NADH in the presence or orcinol, resorcinol, or m-cresol. Subsequent introduction of oxygen restores the oxidized form and yields H2O2 when m-cresol is the effector, but not when orcinol is the effector. Transfer of reducing equivalents from the reduced flavoprotein to free FAD may also occur. Reduction of orcinol hydroxylase by NADH in the absence of an effector is 10-4-fold slower than in the presence of an effector. The minimal structural requirements for effectors appear to be a 1,3-dihydroxy or 1-alkyl-3-hydorxybenzene, but only the former are substrates for hydroxylation.
In Experiment 1, female rats were given a single subcutaneous injection of 1.25 mg 5alpha-dihydrotestosterone (DHT) or its propionate (DHTP) on day 5 of postnatal life. All of them showed regular estrous cycles as adults like untreated control animals. At about 60 days of age, the rats were ovariectomized and given 7 daily injections of 2 mg progesterone (P) plus 0.2 mug estradiol-17beta (ED). Uterine trauma applied on the 4th day of P-ED injections resulted in well developed deciduomata in all animals by the day after the last injection. This made a sharp contrast to the failure of female rats receiving testosterone propionate (TP) neonatally to give a positive response under similar experimental conditions (Takewaki and Ohta, 1974). The mean weight of traumatized horns was significantly larger in DHTP-treated rats (but not in DHT-treated rats) than in controls. In Experiment 2, rats were ovariectomized on day 4 and given a dose of 1.25 mg DHT or DHTP on day 5. Controls were ovariectomized on day 4 but not given any steroid on the next day. A series of 7 daily injections of 2 mg P plus 0.2 mug ED was started at about 60 days of age, after the animals had received 3 daily injections of 0.2 mug ED or 30 daily injections of 0.1 mug ED. Incidence of deciduomata following uterine traumatization was markedly lowered only in animals treated with DHTP neonatally and given 0.1 mug ED for 30 days as adults, no significant differences being found in both incidence and size of deciduomata among the other groups. It was suggested that the effects of neonatal steroid administration on uterine responsiveness in adulthood are specific to the steroid. The previous conclusion that persistent estrus in androgen-sterilized rats plays a part in the reduction of uterine responsiveness was confirmed. An exposure of rats to estrogen for a prolonged postpuberal period was without effect, unless the animals had received enough androgen neonatally.
When rats ovariectomized at 3 days of age and given a single injection of 1.25 mg testosterone propionate on the next day became 60 days old, they were given 3 daily injections of 0.2 mug estradiol-17 beta followed by 7 daily combined injections of 2 mg progesterone and 0.2 mug estradiol. Incidence of deciduomata in reaction to uterine trauma applied on the 4th day of the progesterone-estradiol injections was almost as high as that in neonatally ovariectomized, non-androgenized rats, but the response was significantly smaller in size in androgenized rats than in non-androgenized animals. If females similarly operated on were given injections of 0.1 mug estradiol for 30 days prior to 7 daily injections of progesterone-estradiol, deciduoma formation in androgenized rats was markedly reduced in both incidence and size of the response. In non-androgenized group, deciduoma formation was not significantly affected by chronic administration of estradiol.Accordingly, it is likely that, although androgen injected during neonatal life is responsible for the reduction of uterine responsiveness in androgen-sterilized rats (Takewaki and Ohta, 1974) continued exposure of the uterus to estrogen may play a co-operative role in the event.
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