Retinol inhibition of ornithine decarboxylase induction and G1 progression in Chinese hamster ovary cells.
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Biomedical subjects
Publications and source records attributed to D H Russell.
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Polyamines have been implicated as markers of cell kinetic parameters. Putrescine and spermine were elevated in the urine of women with normal pregnancies. Particularly striking was the over 75-fold increase in spermine excretion. Putrescine was more than twofold that found for normal women. In several patients followed serially during pregnancy to assess the temporal pattern of the urinary excretion of polyamines, it was found that the highest levels of all three polyamines occurred at 12 weeks of gestation. In amniotic fluid, putrescine was higher prior to 30 weeks of gestation whereas spermidine was significantly higher at or beyond 30 weeks of gestation. Spermine was relatively high in both groups. Studies are ongoing to assess whether alterations from these normally elevated patterns will occur in the fluids of pregnant women at high risk for abnormal fetal development.
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Free and conjugated [14C]spermidine were measured in plasma samples from normal individuals and cystic fibrosis patients. Within 4 minutes, the 14C-labeled material in the plasma from normal individuals was 70 percent conjugated compared to no detectable conjugation by cystic fibrosis patients. Further, the patients excreted only 11 to 13 percent of the [14C]spermidine in their urine within 72 hours whereas normal excretion was 60 to 76 percent. In both cases, the labeled material was in a conjugated form.
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Cyclic AMP as well as the specific activity of cyclic AMP-dependent protein kinase decreased from the first two hours after Chinese hamster ovary cells in plateau phase were stimulated to proliferate by tripsinization of confluent cultures and dilution in fresh media. From two to five hours after this stimulation, the cyclic AMP level and the specific activity of cyclic AMP-dependent protein kinase increased two-fold. There was a 40--50% increase in the degree of activation of cyclic AMP-dependent protein kinase during this same time interval. In plateau cultures prior to being stimulated to proliferate, type I cyclic AMP-dependent protein kinase was the predominant soluble form of these enzymes. At five hours after release from plateau, the predominant type of cyclic AMP-dependent protein kinase was type II. However, there was also a significant amount of type I present at this time. Types I and II cyclic AMP-dependent protein kinases were differentially detectable during the cell cycle of Chinese hamster ovary cells synchronized by mechanical selection of metaphase cells following colcemid treatment. During mitosis, type I kinase was predominant with only a small amount of type II activity detectable. The amount of activity of type I then progressively decreased as cells entered G1. During early G1, there was no detectable activity of type II kinase, but its activity increased from mid to late G1 and then decreased during the S phase. These data show a tight temporal relationship between the levels of cyclic AMP, the total cellular pool of type I and II cyclic AMP-dependent protein kinases, and the degree of activation of these kinases as cells traversed G1 toward S phase. These data suggest that the expression of each type of kinase may be important for the regulation of substrate phosphorylation during the cell cycle.
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The total amounts of type I and type II cytoplasmic cyclic AMP-dependent protein kinase activities were measured in various tissues of intact rats and rats subjected to castration, hypophysectomy, or adrenalectomy. After castration, the total amount of type I activity decreased rapidly in classifically steroid-responsive tissues such as the ventral prostate and levator ani muscle and less rapidly in the liver. After hypophysectomy and adrenalectomy, type I activity in the liver decreased to the same extent as after castration. Type I activity could be maintained in the ventral prostate and levator ani muscle at control levels by the daily injection of dihydrotestosterone. Furthermore, after post-castration regression of the prostate for 3 days, three daily subcutaneous injections of dihydrotestosterone resulted in a complete restoration of type I activity toe the intact level. The amount of type II activity was not altered by any of the experimental ablations. This study provides evidence linking steroid action to the ability of steroid-responsive tissues to maintain a substantial activity of type I cyclic AMP-dependent protein kinase.
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A single dose of growth hormone (10 mg/kg, i.p.) was injected into male weanling rats (50--60 g), and the temporal changes in cyclic AMP concentration, protein kinase activation, and ornithine decarboxylase activation were measured in the liver and adrenal gland. The level of cyclic AMP did not change significantly from control values in either liver or adrenal following growth hormone administration. Cyclic AMP-dependent protein kinase(s); however, was markedly activated in liver and adrenal within 30 min. Protein kinase remained activated for more than 4 hr in the liver, while activation of protein kinase in the adrenal returned to control value within 2 hr. Ornithine decarboxylase activity was elevated 20-fold in liver within 4 hr of injection and was increased 7- to 8-fold in be adrenal within l hr. These observations are discussed with regard to the generality of the role of cyclic AMP as the second messenger for target-specifici trophic hormone action and the significance of protein kinase activiation as an index of the cyclic nucleotide involvement in the growth response.
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Forty-two percent of collagen sponges tested as an intravaginal barrier contraceptive method developed malodor when retained for 5 days. Only 4% developed odor when the sponge was removed within 24 hours after intercourse, rinsed, and reinserted. While sexually active volunteers found odor in 37% of the sponges, odor formed only in 4% of the sponges worn by sexually inactive users. No difference in the rate of odor formation was found when neutral pH (7.0) and acid pH (3.4) collagen sponges were tested, although we believe that a pH 3.4 is too acid and promotes odor formation. The optimal pH of the sponge should be 4.5 to 5.5. Malodor was efficiently extracted from sponges by washing in acid milieu of tap water and vinegar or 0.1 M acetate buffer, pH 4.0. Alkali extraction procedures were ineffective, and lukewarm water was slightly less effective than acid extraction of odor. At the time of malodor development, the high content of polyamines (putrescine, spermine, spermidine) in the ejaculate decreased to undetectable values. We conclude that the ejaculate is the major source of malodor formation in intravaginally worn collagen sponges. Removal, rinsing optimally in vinegar solution, and reinsertion within 24 hours after intercourse reduces the chance of malodor formation.
The administration of [14C]putrescine or [14C]spermidine (i.v., 100 muCi) to normal volunteers or patients with advanced cancer resulted in alpha-phase half-lives of 40 and 30 sec and beta-phase half-lives of 30 and 60 min, respectively. No significant differences were found between the plasma decay curves of normals and those of cancer patients. Urinary excretion was similar with both groups excreting approximately 45% of [ 14C]putrescine within 24 hr and 60 to 76% of [14C]spermidine within 48 hr. Dowex chromatography indicated that more than 90% of the radiolabel in the urine was in a conjugated form. Plasma conjugation studies of [14C]putrescine and [14C]spermidine in both groups indicated near-total conjugation of the radiolabel within 4 to 5 min of i.v. injection. Since putrescine and spermidine are markers of disease activity, characterization of the conjugates will be important for the development of rapid, specific tests of altered disease activity in response to multimodality therapy.
Polyamine concentrations were evaluated in normal human prostatic tissue as well as hyperplastic prostate. Normal tissues had high concentrations of putrescine and spermine with intermediate spermidine concentrations, whereas there was a dramatic increase in the spermine concentration in patients with hypertrophy of the prostate. Although not highly significant, spermidine concentrations were elevated slightly in benign hyperplasia, whereas the putrescine content was decreased compared to normal tissue. Polyamine concentrations were measured also in human kidney tumors and corresponding healthy kidney tissue. The concentration of spermidine in renal carcinomas was significantly elevated when compared to histologically normal areas of the same kidney. The spermine concentration of the tumor was generally lower but not highly significant (p less than 0.01). These data suggest that polyamines are accumulated above normal levels in pathological conditions such as benign hyperplasia of the prostate and renal carcinoma. In both cases, spermidine turnover rate may be influenced by carcinogenesis.