Search PubMed⌕ Search

Biomedical subjects

R G MacDonald

Publications and source records attributed to R G MacDonald.

69 records · Page 4Linked to original sources
PubMed1983

Conditions for the measurement of nuclear estrogen receptor at low temperature.

This study was undertaken to determine optimum conditions for the extraction and measurement of uterine nuclear estrogen receptor at low temperature. We measured the influence of glycerol, 0.5 M KCl, 10 mM pyridoxal 5'-phosphate, and 0.5 M NaSCN on the dissociation of estradiol from the receptor at 0 degree C. The half-time (t 1/2) of estradiol dissociation from the receptor in 0.5 M KCl nuclear extracts containing 30% glycerol was very slow (greater than 250 h). Exclusion of glycerol from the extract (Tris buffer) increased the dissociation rate (t 1/2 = 35 h). The inhibitory effect of glycerol on estradiol dissociation kinetics predominated over the mild stimulatory effect of KCl; and both effects were independent of the electrical conductivity of the buffer. When pyridoxal phosphate was added to a nuclear KCl extract (barbital buffer) lacking glycerol, dissociation of the estrogen-receptor complex increased such that the t 1/2 decreased from 20 to 7.6 h; the receptor extracted from nuclei with 10 mM pyridoxal phosphate exhibited these same rapid dissociation kinetics. The t 1/2 of estradiol dissociation from the receptor at 0 degree C in the presence of 0.5 M NaSCN was 5.6 h. Following extraction of uterine receptor by KCl, pyridoxal phosphate, or NaSCN, we measured the number of estradiol binding sites at each of two incubation temperatures: 30 degree C for 1 h and 0 degree C for 24 h. We verified that unoccupied receptor was measured reliably in KCl extract during incubation at 0 degrees C in the presence of glycerol. Total receptor can be determined using either pyridoxal phosphate extract or NaSCN extract at low temperature. However, the number of sites recovered in either pyridoxal phosphate or NaSCN extract was twice the number obtained with the KCl procedure at elevated temperature. It is noteworthy that pyridoxal phosphate and NaSCN increased the number of sites when added directly to nuclear KCl extract, and the effect of pyridoxal phosphate and NaSCN was reversed by treatment with L-lysine and dialysis against KCl, respectively. Thus, the lower receptor recovery with the KCl procedure is not due to the inability of KCl to extract these sites from the nucleus but rather is ascribable to the assay procedure itself. Although total receptor can be measured at low temperature with either NaSCN or pyridoxal phosphate, the pyridoxal phosphate method can be used to assay nuclear progesterone receptor in the same extract.

Animals↗
PubMed1983

Localization of estrogen receptor regulatory factor in the uterine nucleus.

The objective of this study was to determine whether progesterone-induced estrogen receptor regulatory factor (ReRF) is a component of hamster uterine nuclei or a cytoplasmic contaminant of the nuclear fraction. Crude uterine nuclei were prepared in low salt buffer and were purified by either the hexylene glycol method or an isotonic sucrose-Triton X-100 procedure. ReRF activity was measured as a net percentage increase in estrogen receptor inactivation after incubation of a 0.5 M KCl nuclear extract at 36 degrees C for 30 min. Progesterone treatment (5 mg/kg, s.c. in oil, 2 h) increased net receptor inactivation to the same extent (9-12% net increase) in extract made from each nuclear preparation. Extensive washing of nuclei caused a progressive decline in recovery of ReRF activity, in parallel with the behavior of KCl-extractable nuclear protein and estrogen receptor. These results demonstrate that ReRF is a loosely bound component of the uterine nucleus.

Animals↗
PubMed1983

Progesterone modulation of specific protein synthesis in the decidualized hamster uterus.

Progesterone-dependent synthesis of specific proteins was studied on Days 6, 7 and 8 of pseudopregnancy in the decidualized hamster uterus. Progesterone withdrawal was induced by removal of subcutaneous steroid implants from ovariectomized animals; hamsters retaining the implants served as controls. Eight hours after hormone withdrawal, myometrial and deciduomal tissues were labeled with [35S]methionine during incubation for 1 h in vitro, and proteins were separated by two-dimensional gel electrophoresis followed by autoradiography of the dried gels. Analysis of the two-dimensional gel patterns (maps) revealed both increases and decreases in autoradiographic spot intensity comparing progesterone-withdrawn samples to controls, indicating that progesterone both suppressed and stimulated gene expression in the decidualized uterus. Protein synthesis in the deciduoma was more responsive to hormone withdrawal than that in the myometrium; e.g., 45 changes on Day 7 vs. 26 changes, respectively. The sets of proteins in each tissue responding to progesterone withdrawal through changes in synthesis have been termed "domains." The progesterone domains shifted from day to day during decidualization in size and degree of overlap (changes in protein synthesis common to the domain on different days). Although the degree of overlap was similar in cytosol from both tissues, marked differences were apparent in the overlap of responses in the nucleus. Overlap was extensive in the deciduomal nucleus where six protein spots increased intensity following progesterone removal on each day. In contrast, little overlap was seen between the progesterone domains in the myometrial nucleus. These findings emphasize the differences between these two tissues in their responses to progesterone withdrawal, yet five proteins (one cytosolic, four nuclear) responded similarly to hormone removal in both tissues. These studies show that: 1) progesterone domination of the uterus during pseudopregnancy alters synthesis of many proteins in both the deciduoma and myometrium; 2) progesterone effects on specific protein synthesis during decidualization occur in both positive and negative directions; 3) the proteins modulated by progesterone and, by extension, the genes affected by this hormone are predominantly different in the deciduoma and myometrium, and 4) progesterone modulation of specific protein synthesis in the decidualized uterus is time-dependent, such that the domains of response vary daily in composition, size and overlap.

Animals↗
PubMed1982

Glucocorticoids inhibit precursor incorporation into protein in splenic lymphocytes by stimulating protein degradation and expanding intracellular amino acid pools.

In the presence of tracer concentrations of extracellular leucine (5 microM), treatment of rat splenic lymphocyte suspensions in vitro with 1 microM dexamethasone for 2.5-4 h caused a 30-35% inhibition of [3H]leucine incorporation into protein. As the extracellular leucine concentration was raised to 5mM, this inhibition was progressively reduced to 0-12%. This phenomenon correlated with a marked dependence on extracellular leucine concentration of the dexamethasone-dependent enlargement of free intracellular leucine pools in splenic lymphocytes: a 123% increase in pool size with tracer extracellular leucine; a 10% increase with 5 mM leucine. Varying extracellular leucine had no effect on: (1) nuclear [3H]dexamethasone binding by the cells; (2) the concentration of dexamethasone needed for half-maximal inhibiton of [3H]leucine incorporation; (3) the time course of onset and maximal expression of the hormonal inhibition of [3H]leucine incorporation; or (4) the magnitude of dexamethasone-dependent inhibition of [3H]uridine incorporation into RNA by these cells. There was no detectable effect of dexamethasone on uptake and retention of [3H]leucine by the cells regardless of the extracellular leucine concentration. Treatment of splenic lymphocytes for 4 h in vitro with 1 microM dexamethasone caused a small shift of ribosomes from larger aggregate polysomes to smaller forms. Thus, glucocorticoid-induced inhibition of amino aicd incorporation in splenic lymphocytes is a multicomponent response, of which an actual decrease in protein synthesis is only a small part. Enlargement of free intracellular amino acid pools, probably resulting from increased protein degradation, is the major contributing factor to the hormonal inhibition of amino acid incorporation.

Amino Acids↗
PubMed1982

Reduced sulfhydryl groups are required for activation of uterine progesterone receptor. Possible involvement of an inhibitor of activation.

The dependence of uterine progesterone receptor activation on sulfhydryl groups was studied using binding to DNA-cellulose as a measure of activated receptor. Dithiothreitol, thioglycerol, or other sulfhydryl reducing reagents stimulated receptor activation 2- to 3-fold. This effect was produced under mild conditions, i.e. after a 22-h incubation at 0-3 degrees C with 50 mM dithiothreitol. Other characteristics of the sulfhydryl-dependent stimulation of receptor activation, such as pH-dependence and sensitivity to the sulfhydryl blocking agent iodoacetamide, suggest that the sulfhydryl groups essential for receptor activation are different from those involved in steroid binding. Progesterone receptor activation by sulfhydryl reduction was a reversible process, dependent on addition or removal (by dialysis) of the reducing agent. Optimal receptor activation (up to 75% of total receptor) occurred when cytosol was diluted with buffer in the presence of dithiothreitol, suggesting that dissociation of either a receptor subunit or an inhibitory factor present in cytosol may also be involved in the activation process. This putative inhibitor appears to have a Mr greater than or equal to 30,000 since it is not removed from cytosol by dialysis or gel filtration. These results emphasize the importance of sulfhydryl groups or a disulfide bridge, perhaps associated with the DNA-binding domain of the receptor, in a key regulatory step in the mechanism of steroid hormone action: activation and subsequent binding of the steroid-receptor complex to DNA or chromatin.

Animals↗
PubMed1982

Progesterone-induced estrogen receptor-regulatory factor is not 17 beta-hydroxysteroid dehydrogenase.

These studies were done to determine if the progesterone-induced estrogen receptor-regulatory factor (ReRF) in hamster uterus is 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), i.e. that rapid loss of nuclear estrogen receptor (Re) might be due to enhanced estradiol oxidation to estrone catalyzed by 17 beta-HSD. Treatment of proestrous hamsters with progesterone (approximately 25 mg/kg BW) for either 2 h or 4 h had no effect on 17 beta-HSD activity measured as the rate of conversion of [6,7-3H]estradiol to [3H]estrone by whole uterine homogenates at 35 degrees C. During this same time interval, progesterone treatment increased the rate of inactivation of the occupied form of nuclear Re as determined during a 30 min incubation of uterine nuclear extract in vitro at 36 degrees C. Since we previously demonstrated that such in vitro Re-inactivating activity represents ReRF, the present studies show that ReRF is not 17 beta-HSD or a modifier of that enzyme.

17-Hydroxysteroid Dehydrogenases↗
PubMed1981

Glucocorticoid regulation of two serine hydrolases in rat splenic lymphocytes in vitro.

A quantitative assay employing binding of [3H] diisopropylfluorophosphate ([3H]DFP) and SDS-polyacrylamide gel electrophoresis was used to measure serine hydrolases in cell-free extracts from rat splenic lymphocytes. After labeling with [3H]DFP at pH7, six major serine hydrolases are detected on 10% gels, having molecular weights of 78, 55, 34, 30, 28, and 17 (.10(-3)). When labeled at pH4, only four activities are measured, with Mr of 79, 55, 33 and 17(.10(-3)). Incubation of splenic lymphocytes for 8 h in vitro with 1 microM dexamethasone followed by [3H]-DFP labeling at pH 7 produces a 91% increase in the 17000 [3H]DFP. Hormone treatment for 8 h with subsequent labeling at pH 4 results in a 15% increase in the largest (78000) species, as well as 73% increase in the 17000 enzyme, compared with lysates from cells incubated without steroid. These effects are not observed after only 4 h of glucocorticoid exposure. Dexamethasone treatment for 8 h does not produce a decrease in any of these serine hydrolases, nor is there an apparent induction of new enzymes (i.e., having a molecular weight different from the preexisting species). Studies examining the effect of protease inhibitors on the [3H]DFP capacity of these proteins, show that the 17000 enzyme is sensitive to the protease inhibitor, pepstatin A, as well as the sulfhydryl reagents dithiothreitol and N-ethylmaleimide. These results suggest that this dexamethasone-responsive enzyme is a protease which requires a free thiol group for optimal activity. These findings are discussed with regard to the mechanism of glucocorticoid action in lymphocytes.

Animals↗
PubMed1981

Uterine progesterone receptor: stabilization and physicochemical alterations produced by sodium molybdate.

Incubation of hamster uterine cytosol with millimolar concentrations of sodium molybdate prior to addition of labeled steroid increased recovery of progesterone receptor 2-fold. This stabilizing effect of molybdate was also manifest on gel electrophoresis of the receptor. In the absence of molybdate, no specific [3H]progesterone binding was detectable on polyacrylamide gel electrophoresis. But, in the presence of 5 mM sodium molybdate, a [3H]progesterone-binding species was clearly evident on the gels. The radioactivity associated with this binding was displaceable by unlabeled progesterone but not by cortisol and depended on the concentration of [3H]progesterone employed, suggesting that this binding species is a progesterone receptor. Molybdate treatment produced a small increase in receptor size on low-salt sucrose gradients (from 6-7 to S to 7.5 S). There was no effect of molybdate of receptor sedimentation in the presence of high salt (0.3 M KCl). Further analysis of this phenomenon by gel filtration suggested that this molybdate-mediated increase in receptor size was due to receptor aggregation. In low-salt buffers, molybdate treatment markedly increased the proportion of receptors contained in large aggregates (Stokes radius greater than 8.0 nm). Again, this effect was abolished in the presence of high salt. In conjunction with receptor stabilization, molybdate prevented binding of uterine progesterone receptor to DNA--cellulose. These findings suggest that sodium molybdate stabilizes the unliganded, unactivated form of the receptor. Moreover, these effects seem to be mediated through a direct interaction of molybdate with the receptor, one which results in receptor aggregation.

Animals↗
PubMed1981

Progesterone-induced estrogen receptor-regulatory factor in hamster uterine nuclei: preliminary characterization in a cell-free system.

In vitro studies have demonstrated a progesterone-induced activity associated with the uterine nuclear fraction which resulted in the loss of nuclear estrogen receptor. Uterine nuclear suspension or nuclear KCl (0.5 M) extract from control and progesterone-treated (30 min or 2h) hamsters were incubated at 37 C for 0, 15, or 30 min in Tris-glycerol buffer. Preparations from progesterone-treated hamsters showed an accelerated reduction of total estrogen receptor which was primarily due to preferential loss of occupied receptor. This progesterone-dependent stimulation of estrogen receptor loss was absent when nuclear extract was prepared in phosphate buffer rather than Tris buffer. In addition, sodium molybdate and sodium metavanadate (both at 10 mM) inhibited this activity in nuclear extract. These observations support the hypothesis that progesterone modulation of estrogen action may be accomplished by induction (or activation) of an estrogen receptor-regulatory factor (Re-RF), and this factor may in turn act to eliminate the occupied form of estrogen receptor from the nucleus, perhaps through a hypothetical dephosphorylation-inactivation mechanism.

Animals↗
PubMed1977

Erythrocyte 2,3-diphosphoglycerate and associated haematological parameters during the menstrual cycle and pregnancy.

Erythrocyte 2,3-diphosphoglycerate (2,3-DPG) and associated haematological parameters were measured throughout the normal menstrual cycle, in each trimester of pregnancy and on the third day post partum. There was no significant change during the menstrual cycle but the 2,3-DPG concentration was increased early in pregnancy, and thereafter gradually decreased. Although other metabolic changes are likely to contribute to the increase in 2,3-DPG concentration, it is concluded that 'functional anaemia', secondary to increased oxygen demand, is a more important stimulus than the decrease in haemoglobin concentration. The implications of a decreased oxygen affinity of haemoglobin, secondary to the rise in 2,3-DPG concentration, are discussed in relation to anaemia and blood transfusion in pregnancy.

Adolescent↗