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Biomedical subjects

J H Clark

Publications and source records attributed to J H Clark.

At least 181 records · Page 10Linked to original sources
PubMed1982

Alteration of rumen fermentation, milk fat synthesis, and nutrient utilization with mineral salts in dairy cows.

The ability of mineral salts to alter rumen fermentation, rumen fluid dilution rate, milk fat synthesis, and nutrient utilization was investigated in dairy cows fed a high-concentrate, milk-fat depressing diet. Four rumen-fistulated Holstein cows were in a 4 X 4 Latin square design. Treatments consisted of: 1) basal (25% corn silage: 75% concentrate on a dry matter basis), 2) basal + 2.0% sodium chloride, 3) basal + 2.0% sodium bicarbonate, and 4) basal + 2.4% limestone. Addition of limestone to the basal diet reduced dry matter intake but increased efficiency of dietary nutrient utilization for milk synthesis. Sodium bicarbonate increased synthesis of milk fat. Sodium chloride also tended to increase milk fat synthesis whereas limestone had no effect. Milk yields (kg/day) and milk fat (%) for the four treatments were 1) 29.5, 2.40; 2) 29.3, 2.66; 3) 28.9, 3.26; and 4) 29.2, 2.32. Rumen fluid pH, dilution rate (%/hour), and molar percentage of acetate and propionate were: 1) 5.98, 10.3, 49 and 39; 2) 6.02, 12.4, 55 and 32; 3) 6.16, 12.2, 58 and 25; and 4) 5.92, 10.7, 51 and 38. Limestone was totally ineffective in altering ruminal pH, fluid dilution rate, molar percentages of acetate and propionate, and synthesis of milk fat. Improved feed efficiency for milk production after addition of limestone was related to an increase in starch digestion compared to the basal ration (95 versus 88%).

Animals↗
PubMed1982

Estrogen binding sites in the nucleus of normal and malignant human tissue: optimization of an exchange assay for the measurement of specific binding.

An exchange assay has been validated and used to measure the total concentration of estrogen receptors in the nuclei of cells from human breast tumors and myometrium. Tissue homogenates were centrifuged through 1.2 M sucrose pads to separate crude chromatin from soluble ane membranous fractions. The yield of DNA by this procedure was approximately 80%. Total binding sites were measured by incubating the sedimented pellets with tritiated estradiol at 30 degrees for 60 min and then precipitating the receptor-steroid complexes with protamine sulfate. Saturation analysis by this procedure provides evidence for the presence of a specific, nuclear estrogen-binding site in addition to the established estrogen receptor in both these tissues.

Breast Neoplasms↗
PubMed1982

Estrogen binding sites in the nucleus of normal and malignant human tissue: characteristics of the multiple nuclear binding sites.

The studies presented here describe the effects of the nonionic detergent, Tween 80 and the reducing agent, dithiothreitol (5 mM), on the quantitation of specific nuclear estrogen binding sites, both estrogen receptors (ER) and type II estrogen binding sites (EBS), in human breast cancer. Neither of these agents had any significant effect on the apparent Kd of the amount of ER measured; however, both had a dramatic effect on the amount of type II EBS measured. Two antiestrogens, tamoxifen and nafoxidine, were also tested for their ability to bind to ER and type II EBS in human breast tumors. Both of the antiestrogens were capable of competing with estradiol for ER and type II EBS. We also present a series of 25 individual human breast cancer specimens which were analyzed for cytoplasmic ER and progesterone receptor by sucrose density gradient centrifugation and for nuclear ER and type II EBS by the sucrose pad assay. Sixty-four % (16 of 25) of the tumors contained nuclear ER and type II EBS when analyzed by the sucrose pad assay. The concentration of type II EBS was strongly correlated to the concentration of cytoplasmic progesterone receptor.

Breast Neoplasms↗
PubMed1981

Nuclear association states of rat uterine oestrogen receptors as probed by nuclease digestion.

The solubilization of oestrogen receptors from uterine nuclei by micrococcal nuclease and deoxyribonuclease I was examined after the injection of oestradiol or Nafoxidine into castrated female rats. At 1h after an injection of oestradiol, 30% (0.18pmol/mg of DNA) of the nuclear oestrogen receptors was solubilized by 5 min of mild digestion with either nuclease. No further receptor release occurred, although DNA hydrolysis continued throughout a 20min interval. The limitation in receptor solubilization was not due to an artifact of digestion conditions or insufficient nuclease concentrations. Similar patterns of receptor solubilization and DNA hydrolysis were obtained with both nucleases whether the animals had been injected with oestradiol 1h before death or if the uteri from uninjected animals were incubated with [(3)H]oestradiol for 1h in vitro. When uterine nuclei were digested with these enzymes 12h after the animal was injected with oestradiol there was little change in the quantity of nuclease-sensitive sites (0.11pmol/mg of DNA); however, the quantity of nuclease-resistant sites decreased 10-fold. These values correspond quantitatively to the changes in salt-resistant and salt-extractable sites observed over a 12h interval after oestradiol treatment. Nuclease digestion of uterine nuclei obtained 16h after Nafoxidine treatment gave a pattern qualitatively and quantitatively similar to that observed 1h after oestradiol treatment, a result consistent with the agonist/antagonist action of this compound. An analysis by sucrose-density-gradient centrifugation of the time course of nuclease-dependent receptor solubilization indicated that the solubilized receptors were not associated with discrete nucleosomal fragments. We believe that these data indicate that only a portion of the receptors translocated to the nucleus become associated with chromatin, and this association may occur on regions of chromatin that are preferentially susceptible to nucleolytic cleavage.

Animals↗
PubMed1981

An oestrogen receptor activator protein in rat uterine cytosol.

Earlier reports of oestrogen receptor binding to DNA indicated that a protein which does not bind oestrogen might be involved in the conversion of the 4S oestradiol receptor to the 5S form but no direct evidence for the existence of this protein was presented. This receptor transformation or activation step is thought to be a requirement for events in the nucleus. We considered it likely that such a protein might be similar to the cytoplasmic proteins which stimulate eukaryotic RNA polymerases. These are basic proteins which are not adsorbed to DEAE cellulose and have a sedimentation coefficient of approximately 3S. To examine this possibility, we fractionated unlabelled uterine cytosol by DEAE-cellulose chromatography. Unlabelled cytosol was used to avoid the possibility that such a factor might be retained on the column as a component of the receptor--oestradiol complex. Here we present evidence of a protein present in uterine cytosol which forms a complex with the 4S form of the oestrogen receptor to produce a 5S-activated form. This activated form binds to uterine nuclei, DNA-cellulose and native calf thymus DNA. The cytosol protein, which we call receptor activation factor, does not bind oestradiol, has a sedimentation coefficient of approximately 3S and does not bind to DEAE-cellulose.

Animals↗
PubMed1981

Nuclear bodies as structural indicators of estrogenic stimulation in uterine luminal epithelial cells.

Preliminary evidence has indicated that the number of nuclear bodies in uterine luminal epithelial cells of the immature rat may be related to the duration of nuclear retention of the estrogen receptor complex (Clark et al., 1978). To test this hypothesis, an ultrastructural analysis of nuclear and cytoplasmic differentiation was performed at 4, 12, 24, 48, and 72 hr after a single injection of estradiol or nafoxidine (synthetic estrogen agonist/antagonist) into 21 day female rats. Variations in nuclear and cytoplasmic differentiation and in the frequency of occurrence of nuclear bodies (simple and complex) were determined and compared with established biochemical changes in the concentration of nuclear estrogen receptor and RNA polymerase activity (Clark et al., 1978). Following nafoxidine there is sustained elevation of the nuclear concentration of the estrogen receptor as well as RNA polymerase I and II activities over the entire 72-hr period. From 4 to 72 hr the height of the luminal epithelial cell as well as the frequency of nuclear bodies increase at linear rates. Through steady expansion of the cytoplasmic membrane system (RER) and Golgi) the relatively undifferentiated epithelial cells of the control uterus are converted progressively into ones equipped for protein secretion. At 72 hr the effects of an estradiol implant resemble closely those observed after a single injection of nafoxidine; these include sustained nuclear receptor occupancy, elevated RNA polymerase activity, epithelial hypertrophy, and high frequency of nuclear bodies. However, after a single injection of estradiol, the luminal epithelial cells become slightly but significantly taller than the control cells and remain close to this size from 24 to 72 hr.; the frequency of nuclear bodies decreases linearly from 4 to 72 hr to fall below the control level. In addition, limited cytoplasmic autolysis is evident from 24 to 72 hr. A single injection of estradiol results in short-term nuclear receptor occupancy and elevated RNA polymerase activities which return to control levels by 24 hr. This collective evidence offers further support to the hypothesis that the duration of nuclear occupancy by the estrogen receptor is reflected in the size of the nuclear body populations in these epithelial target cells. Also during hyperestrogenization, epithelial hypertrophy is accompanied by steady formation of nuclear bodies.

Animals↗