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K Kohmoto

Publications and source records attributed to K Kohmoto.

At least 37 records · Page 2Linked to original sources
PubMed1991

Activation of p34cdc2 protein kinase activity in meiotic and mitotic cell cycles in mouse oocytes and embryos.

p34cdc2 protein kinase is a universal regulator of M-phase in eukaryotic cell cycle. To investigate the regulation of meiotic and mitotic cell cycle in mammals, we examined the changes in phosphorylation states of p34cdc2 and its histone H1 kinase activity in mouse oocytes and embryos. We showed that p34cdc2 has three different migrating bands (referred to as upper, middle and lower bands) on SDS-PAGE followed by immunoblotting with anti-PSTAIR antibody, and that the upper and middle bands are phosphorylated forms since these two bands shifted to the lower one by alkaline phosphatase treatment. In meiotic cell cycle, only germinal vesicle (GV) stage oocytes had the three forms. The phosphorylated forms decreased gradually in oocytes up to 2 h after isolation from follicles, and thereafter the phosphorylation states did not change significantly until metaphase II. However, the histone H1 kinase activity oscillated, being activated at the first and second metaphase in meiosis and inactivated at the time of the first polar body extrusion. These results suggest that changes in phosphorylation states of p34cdc2 triggered its activation at the first metaphase, but not inactivation and reactivation at the first and second metaphase, respectively. In mitotic cell cycle, phosphorylated forms appeared at 4 h after insemination, increased greatly just before metaphase, and were dephosphorylated in metaphase. Histone H1 kinase activity was high only at metaphase. This kinase activation is probably triggered by dephosphorylation of p34cdc2.

Animals↗
PubMed1990

Thermodynamic analysis of the interaction of prolactin with its receptor in the rabbit mammary-gland microsomes.

The interaction of prolactin (PRL) with its membrane receptor depends markedly on temperature. Thermodynamic parameters for this reaction have been evaluated from data for time-course kinetics and equilibrium binding at multiple temperatures between 19 and 31 degrees C. The free-energy change with temperature and the van't Hoff plot were found to be linear. These suggest that there are minimal structure changes at the PRL-receptor contact site over this temperature range. The positive signs of the entropy and enthalpy of reaction, and of the entropy of activation (delta S++) for association, indicate that the hydrophobic bonding is the most significant force involved in PRL-receptor formation. The delta S++ for dissociation was negative, and the enthalpy of activation for dissociation was about 20.3 kJ.mol-1 larger than that for association, indicating that the PRL-receptor complex is further stabilized by contributions of hydrogen bonds and van der Waals contacts after the initial interaction. The free energy of activation for dissociation, at 25 degrees C was about 2.5-fold larger than that for association. This would cause slow dissociation of PRL from its receptor.

Animals↗
PubMed1989

Biosynthetic pathways of testosterone and estradiol-17 beta in slices of the embryonic ovary and testis of the chicken (Gallus domesticus).

To elucidate synthetic pathways of testosterone and estradiol-17 beta in embryonic gonads of the chicken, metabolism of various 14C-labeled steroids in slices of the left ovaries and paired testes of 15- and 9-day-old chicken embryos was examined. (1) Fifteen-day-old chicken embryos: From pregnenolone, more 17 alpha-hydroxypregnenolone was produced than progesterone in the ovary, while more progesterone was produced than 17 alpha-hydroxypregnenolone in the testis. From 17 alpha-hydroxypregnenolone, however, only dehydroepiandrosterone was detected as a product in both gonads. Dehydroepiandrosterone was converted mainly into androstenedione and its 5 beta-reduced derivatives by both gonads. Progesterone was converted into 5 beta-pregnane-3,20-dione more than into 17 alpha-hydroxyprogesterone by both gonads. Both gonads metabolized 17 alpha-hydroxyprogesterone, androstenedione, and testosterone predominantly into their corresponding 5 beta-reduced steroids, while production of androstenedione from 17 alpha-hydroxyprogesterone and of testosterone from androstenedione was limited. Estradiol-17 beta was produced from androstenedione and testosterone only by the ovary. (2) Nine-day-old chicken embryos: From pregnenolone, production of progesterone and 17 alpha-hydroxypregnenolone was similar in the ovary. On the other hand, in the testis, more progesterone was produced than 17 alpha-hydroxypregnenolone from pregnenolone. For delta 4-3-oxo steroids, strong activity of 5 beta-reductase was demonstrated in both gonads. From these results, both delta 4- and delta 5-pathways are involved in the formation of testosterone and then finally of estradiol-17 beta by the embryonic gonads of the chicken, and relative preference for the pathway seems to depend on sexes and embryonic ages. In addition, it is suggested that steroidogenesis in these embryonic gonads is characterized by marked activity of 5 beta-reductase, irrespective of sexes or ages.

17-alpha-Hydroxypregnenolone↗
PubMed1989

Host-Specific Effects of Toxin from the Rough Lemon Pathotype of Alternaria alternata on Mitochondria.

Host-specific toxin from the rough lemon (Citrus jambhiri Lush) pathotype of Alternaria alternata (ACR toxin) was tested for effects on mitochondria isolated from several citrus species. The toxin caused uncoupling of oxidative phosphorylation and changes in membrane potential in mitochondria from leaves of the susceptible host (rough lemon); the effects differed from those of carbonylcyanide-m-chlorophenylhydrazone, a typical protonophore. ACR toxin also inhibited malate oxidation, apparently because of lack of NAD(+) in the matrix. In contrast, the toxin had no effect on mitochondria from citrus species (Dancy tangerine and Emperor mandarin [Citrus reticulata Blanco], and grapefruit [Citrus paradisi Macf.]) that are not hosts of the fungus. The effects of the toxin on mitochondria from rough lemon are similar to the effects of a host-specific toxin from Helminthosporium maydis (HMT) on mitochondria from T-cytoplasm maize. Both ACR and HMT toxins are highly selective for the respective host plants. HMT toxin and methomyl had no effect (toxic or protective) on the activity of ACR toxin against mitochondria from rough lemon.

Journal Article↗
PubMed1988

Monoclonal antibody detection of prolactin-binding subunits in the rabbit mammary gland.

The structure of prolactin (PRL) receptor in the rabbit mammary gland was examined using a receptor-specific monoclonal antibody (MAb). The PRL receptor preparation used was purified by making use of a PRL-affinity column. MAb inhibited the binding of PRL to the receptor, in a dose-dependent manner and completely at a high concentration. Using the receptor directly labelled by 125I, the preparation was incubated with MAbs and the immune complex was collected by Pansorbin and examined by SDS/polyacrylamide-gel electrophoresis. The autoradiography showed that three species with apparent Mr values of 77,000, 41,000 and 25,000 specifically reacted with MAbs. The pattern changed little in the presence or absence of dithiothreitol. Western blot analysis showed that two species (Mr 77,000 and 41,000) reacted with MAb. Affinity labelling of the receptor with labelled PRL revealed three bands with Mr values of 96,000, 60,000 and 43,000 on SDS gels. The high-Mr complex (Mr greater than 200,000) was always present at the top of the gel. These results show that the mammary gland contains at least three PRL-binding subunits. The differences in Mr before and after PRL binding were close to the Mr of PRL. This would suggest that each PRL binding subunit reacts with one PRL molecule.

Animals↗
PubMed1988

Sexual differences of steroidogenic enzymes in embryonic gonads of the chicken (Gallus domesticus).

The left ovary and testis of 15-day-old embryos of the chicken were compared in the enzyme activities related to steroidogenesis. The activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase in the ovary was similar to that of the testis. Activities of 17 alpha-hydroxylase and C-17-C-20 lyase in the ovary were 2.5 and 2.6 times those in the testis. From the CO-induced difference spectrum, the content of cytochrome P-450 in the ovarian microsomes was estimated as 27.6 pmol/mg protein. However, no detectable amount of cytochrome P-450 was observed in the testicular microsomal fraction. The substrate (progesterone)-induced difference spectrum was appreciable only in the ovarian microsomes. The activity of microsomal NADPH-cytochrome c reductase in the ovary was significantly higher than that in the testis. The activities of 17 beta-hydroxysteroid dehydrogenase in both gonads were similar to each other, when androstenedione was used as substrate. However, its activity in the ovary was 1.4 and 3.1 times that in the testis, when dehydroepiandrosterone and estrone were used as substrate, respectively. Aromatase activity in the ovary was over 100 times that in the testis, as assessed by release of [3H]water from [1-3H]testosterone. Appreciable amounts of radioactive estradiol-17 beta and estrone were formed from [4-14C]testosterone and [7-3H]androstenedione, respectively, only by the ovarian tissue. 5 beta-Reductase activity in the ovary was 1.4 times that in the testis.

Aldehyde-Lyases↗
PubMed1988

Developmental changes of steroidogenic enzyme activities in the embryonic gonads of the chicken: the sexual difference.

Steroidogenic enzyme activities in the left ovary and the testes of 9- to 15-day-old chicken embryos were measured, and development of the activities was compared between sexes. Activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase in the ovary and in the testis was comparable, and did not change throughout the period examined. Activity of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in the ovary was similar to or higher than that in the testis, depending on substrates employed. In both gonads, 17 beta-HSD activity did not change or tended to decrease from 9 to 15 days of development. On the other hand, activities of 17 alpha-hydroxylase, C-17--C-20 lyase in the ovary were three to eight times those in the testis, and aromatase activity in the ovary was definitely higher than that in the testis at all stages examined. The activities of 17 alpha-hydroxylase, C-17--C-20 lyase, and aromatase significantly increased from 9 to 11 days only in the ovary. From 13 to 15 days, the activities of 17 alpha-hydroxylase and C-17--C-20 lyase markedly increased only in the testis. These results suggest that, in the gonads of developing chicken embryos, there are sexual differences in the regulation of 17 alpha-hydroxylase, C-17--C-20 lyase, and aromatase activities.

17-Hydroxysteroid Dehydrogenases↗
PubMed1987

Beneficial effect of serum on the fertilizability of mouse oocytes matured in vitro.

Mouse oocytes matured in vitro in chemically defined medium were not penetrated by spermatozoa. The time required for dissolution of the zona pellucida of such oocytes by alpha-chymotrypsin was much longer than that for ovulated oocytes. Addition of fetal calf serum to the medium for maturation of oocytes improved the incidence of sperm penetration and shortened the time of enzymic dissolution of the zona pellucida. These results suggest that the low rate of fertilization of oocytes matured in vitro is mainly due to qualitative changes of the zona pellucida, which could be overcome by a factor or factors in fetal calf serum.

Animals↗
PubMed1986

Separation of rabbit mammary-gland prolactin receptors by ion-exchange chromatography, h.p.l.c.-gel filtration and ultracentrifugation.

Rabbit mammary-gland prolactin (Prl) receptors in the microsomal fraction were solubilized in 7.5 mM-Chaps) or 1% Triton X-100 and analysed by ion-exchange chromatography using DEAE-Bio-Gel A. Prl receptors in the presence of 7.5 mM-Chaps were separated into two different fractions (Fr. A and B), both of which showed identical specificity of binding to peptide hormones as those in the Chaps or Triton extract. oPrl and human growth hormone (hGH) bound to the same site, but other non-lactogenic hormones (follicle-stimulating hormone, oGH, luteinizing hormone and insulin) failed to bind to the Prl receptors. The dissociation constant (Kd) for Prl binding to the receptors in Fr. A was about 50% of those in Fr. B, suggesting that the rabbit mammary gland contains two types of Prl receptors, one with a high, and one with a low, Kd for Prl binding. A decrease in the concentration of Chaps in the column buffer to 4 mM caused aggregation of the receptors in Fr. A. H.p.l.c.-gel filtration, using Shim pack 150 and 300 columns connected in series, separated the receptor as a protein with an Mr of 74,000 +/- 4,900 (mean +/- S.D.) in the presence of 5 mM-Chaps, or of 36,800 +/- 2,100 in the presence of 7.5 mM-Chaps. Sucrose-gradient-centrifugation analysis showed that the Prl-receptor complexes in the presence of 5 mM-Chaps were sedimented between gamma-globulin and bovine serum albumin (5.56 +/- 0.22 S). As the Chaps concentration was increased to 7.5 mM, a further peak of the Prl-receptor complexes (4.01 +/- 0.23 S) appeared below ovalbumin. The present data suggest that the binding subunit causes the monomeric subunit to aggregate with itself or with another specific associated protein of similar Mr.

Animals↗
PubMed1985

Metabolism of steroid hormones in vitro by follicular tissues of the Japanese quail.

The cell-free homogenates of the theca layers and granulosa layers of quail follicles were incubated at 39 degrees C with 14C-labeled steroids in the presence of NADPH. At the end of incubation, radioactive steroids were extracted and analyzed by thin-layer chromatography. When radioactive progesterone was employed as the substrate, 17 alpha-hydroxyprogesterone and androstenedione were obtained as the metabolites. 17 alpha-Hydroxylase activity, estimated from the amounts of these two metabolites, was high in the theca layers of the second largest (F2) and the third largest (F3) follicles. The theca layer of the largest follicle (F1) and the granulosa layers of all three follicles were essentially devoid of this enzyme activity. The activity of C17-20 lyase was estimated from the amount of androstenedione that was obtained as a sole metabolite in the incubation of radioactive 17 alpha-hydroxyprogesterone. This enzyme showed a tissue distribution similar to 17 alpha-hydroxylase. When radioactive androstenedione was used as the substrate, testosterone, 5 beta-androstane-3,17-dione, and 3 beta-hydroxy-5 beta-androstan-17-one were identified as the metabolites. 17 beta-Hydroxysteroid dehydrogenase activity, estimated from the amount of testosterone, was higher in the granulosa layers than in the theca layers. On the other hand, 5 beta-reductase activity, estimated from the sum of 5 beta-androstane-3,17-dione and 3 beta-hydroxy-5 beta-androstan-17-one, was almost equally distributed in the two layers. In order to investigate the changes in the enzyme activities during the ovulatory cycle, birds were killed at various times before the predicted ovulation of F1. When the 17 alpha-hydroxylase activity was estimated in the cell-free homogenates of the theca layers, peaks in the activity were observed 32, 42, 54, and 66 h before ovulation of F1. There was a small peak 18 h before ovulation, and activity then started to decrease. The change of C17-20 lyase activity during the cycle was completely parallel with that of 17 alpha-hydroxylase activity.

17-alpha-Hydroxyprogesterone↗
PubMed1985

Correlation between mammary prolactin receptors of lactating mice and litter weight.

Correlations between numbers and dissociation constants of mammary prolactin receptors of lactating mice and litter weight were examined. The apparent numbers and dissociation constants of mammary prolactin receptors were obtained from Scatchard plots with inhibition of iodine-125 prolactin binding by various concentrations of unlabeled prolactin. Litter weight measured 5 h after separation from the mother showed a nearly quadruple, almost linear increase during the first 15 d after birth. The dissociation constant for prolactin binding was fairly constant through lactation and did not correlate with either number of prolactin receptors or litter weight. The number of prolactin receptors in mammary cells increased rapidly in early lactation, reached a maximum at mid-lactation, began to decrease thereafter, and was correlated closely with litter weights on d 5 (.70) and 10 (.64) postpartum, suggesting that number of prolactin receptors represents the lactational potential of the lactating mouse.

Animal Population Groups↗
PubMed1984

Complete amino acid sequence of mouse prolactin.

The complete primary structure of mouse prolactin has been established on the basis of tryptic peptides from cyanogen-bromide-treated, S-carboxymethylated mouse prolactin and Staphylococcus-aureus-protease-cleaved overlaps, which were sequenced by manual liquid-phase and solid-phase Edman degradation. Three disulfide bonds were assigned to Cys-4-Cys-9, Cys-56-Cys-172, and Cys-189-Cys-197 by digestion of intact prolactin with S. aureus protease. One of the characteristics to date is replacement of Trp-89, which is commonly present among prolactin, growth hormone and choriomammotropin, by serine. It was suggested, by comparison with five other prolactins, five growth hormones and human choriomammotropin, that Asp-18, His-25, Ser-60 and Thr-63 are essential to lactogenic activity.

Amino Acid Sequence↗
PubMed1983

Electrophoretic separation of prolactin secreted from mouse pituitary glands in vitro.

Prolactin secreted from the mouse pituitary gland in organ culture was characterized after disc or SDS-polyacrylamide gel electrophoresis. Some pituitary glands were cultured with 3H-labelled leucine for 24 h to obtain radioactive prolactin. In disc electrophoresis, immunoreactive, receptor-bindable and 3H-incorporated prolactins formed a single band with the same relative mobility (0.5). Prolactin migrating at Rf = 0.5 was extracted and re-analysed by SDS electrophoresis. A single stained and radioactive band was observed at the same position with an apparent molecular weight of 23 000 regardless of denaturing in the presence of absence of dithiothreitol. No other band was detected. These results indicate that mouse prolactin synthesized and secreted in organ culture is homogeneous and that mouse prolactin is a single-chain molecule.

Animals↗
PubMed1982

Changes in Scatchard plots for insulin binding to mammary epithelial cells from cycling, pregnant, and lactating mice.

Insulin binding to its receptors was studied in collagenase-dissociated mammary cells prepared from mice that were cycling, pregnant, or lactating. Mammary cells (3 X 10(6)) were incubated in medium 199 (0.4 ml) supplemented with iodinated insulin (0.8-1 ng/ml) for 45 min at room temperature. The dissociation of iodinated insulin from its receptors was accelerated by the presence of native insulin. The specific binding of insulin at a physiological concentration (1 ng/ml) increased on day 0 of pregnancy and on day 3 of lactation. The Scatchard plots showed predominantly high affinity binding during estrous cycles and in early pregnancy, and low affinity binding in late pregnancy and lactation. Scatchard plots were further analyzed for evidence of negative cooperativity and of two independent receptors. The negative cooperativity model gave well fitted curves for unconstrained parameters. However, dissociation constants for insulin binding to empty sites were too high during lactation. The independent two-receptor model gave also well fitted curves for unconstrained parameters at each stage. The dissociation constants for high affinity and low affinity receptors, which were optimal throughout the entire stage, were 1.0 and 20.0 nM, respectively. There was no significant difference between these dissociation constants and any of unconstrained constants at each stage. When dissociation constants were constrained throughout entire stages, the number of receptors for high affinity binding increased on day 0 of pregnancy and in early lactation, and the number for low affinity binding showed one peak on day 3 of lactation. Changes in these receptors were independent. From these results, we propose that at least two species of insulin receptors are present in mouse mammary epithelial cells; the high affinity receptor may be associated with the stimulation of DNA synthesis.

Animals↗
PubMed1982

Influence of glucocorticoids on mammary prolactin receptors in pregnant mice after ovariectomy.

Regulation of mammary prolactin receptors by steroid hormones was investigated in ovariectomized mid-pregnant mice. Ovariectomy increased the number of mammary prolactin receptors per cell with no effect or a slight decrease in dissociation constant (Kd). The simultaneous removal of adrenals prevented this increase in numbers. A single injection of glucocorticoid (corticosterone or cortisol) in ovariectomized-adrenalectomized mice restored the number of prolactin receptors in mammary glands to the same level as that in ovariectomized controls without changing the Kd. Aldosterone, deoxycorticosterone and oestradiol did not affect the number of mammary prolactin receptors after ovariectomy-adrenalectomy. Serum concentration of prolactin was not influenced by the hormone manipulation except with injections of oestradiol or cortisol and apparently did not correlate with the number of prolactin receptors. These results indicated that glucocorticoids are required for the increase in the number of mammary prolactin receptors induced by ovariectomy in mid-pregnant mice.

Adrenalectomy↗
PubMed1981

A method for the iodination of insulin and its binding to dissociated mouse mammary cells.

The iodination of insulin was accomplished by a modification of the lactoperoxidase method. The use of a low concentration of hydrogen peroxide (1.5 ng/ul) followed by Sephadex gel filtration and purification on a cellulose column yielded iodoinsulin with an activity equal to that of native insulin in stimulation of glucose oxidation in rat epididymal fat cells and with high specific binding to collagenase-dissociated mouse mammary cells from pregnant and lactating mice. Other hormones tested did not displace the binding. Analysis of displacement curves and scatchard plots suggests that both the affinity and the number of sites for insulin binding differ between pregnant and lactating mammary cells.

Animals↗
PubMed1980

Specific binding of prolactin to the mammary gland and lactose synthesis in pregnant rats after removal of ovaries or ovaries and adrenals.

Ovariectomy performed at mid pregnancy increased the specific binding of 125I-prolactin to the mammary gland (expressed as cpm/mg DNA) in rats 40 hr after the operation. When adrenals were removed simultaneously at the time of ovariectomy, no increase in the specific binding was observed. An appreciable amount of lactose was found in all mammary glands of ovariectomized-adrenalectomized animals, though the content was less than half of that of the ovariectomized animals. The plasma level of prolactin in ovariectomized and ovariectomized-adrenalectomized rats was slightly higher than that in sham operated control rats, though the differences were not statistically significant. These results seem to indicate that the adrenal gland is necessary for the induction of the site of specific binding of prolactin in the mammary gland of the rat and that an increase in the circulating levels of prolactin does not seem to play an essential role in the initiation of milk synthesis in this species.

Adrenal Glands↗