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T G Muldoon

Publications and source records attributed to T G Muldoon.

At least 37 records · Page 2Linked to original sources
PubMed1987

Receptor-mediated interrelationships between progesterone and estradiol action on the anterior pituitary-hypothalamic axis of the ovariectomized immature rat.

The ovariectomized immature rat was used as a model for analysis of action of progesterone as a modulator of receptor-mediated functional responsiveness in the anterior pituitary and hypothalamus. In response to estrogen exposure, cytosolic progesterone receptors appear rapidly, rise in concentration to a peak at 12 h, then fall to a plateau level well above control, which is maintained for at least an additional 20 h. Progesterone administration at the peak 12-h interval induces maximal nuclear accumulation of its own receptor within 1-2 h, with apparent extensive processing occurring thereafter. To this point, no differences were seen between anterior pituitary and hypothalamic responses. If animals were administered progesterone (0.8 mg/kg BW) at the 12-h peak interval, subsequent nuclear accumulation of anterior pituitary estrogen receptor by an injection of estradiol was suppressed if, and only if, the interval between progesterone and estradiol injection did not exceed 2 h; at no time interval did progesterone have an effect in the hypothalamus. In both tissues, estradiol readministration at 12 h after an initial injection stimulates a second wave of progesterone receptor activity, again peaking 12 h later. A single injection of progesterone 1 h before the second estradiol administration blocks the second peak of progesterone receptor in the anterior pituitary, but not in the hypothalamus. If the interval between the progesterone and second estradiol injections is extended to 4 h, the second progesterone receptor peak appears as though no progesterone had been introduced. The results indicate a critical temporal reliance of the inhibitory effects of progesterone on estrogen receptor activity and estrogen function in a well defined animal model. The effect is progesterone receptor-mediated and is manifested in the anterior pituitary, but not in the hypothalamus, even though the kinetics of estrogen-induced progesterone receptor activity are indistinguishable between the two tissues.

Animals↗
PubMed1987

Prolactin mediation of estrogen-induced changes in mammary tissue estrogen and progesterone receptors.

Selective regulation of estrogen receptor (ER) content, distribution, and function has been studied in mouse mammary gland, as influenced by PRL and estradiol administration in vivo. In virgin female C3H+ mice, short-term treatment with PRL causes an apparent increase in the affinity with which activated ER binds to DNA. However, a contrasting effect of PRL to diminish nuclear accumulation of ER and progesterone receptors is consistently observed in PRL-treated mice. The ER-enhancing activity of PRL and the PRL mediation of estrogen stimulation of ER are more pronounced in C3H than in BALB/c mammary tissue, evidenced by the ability of bromocryptine (CB-154) to eradicate estrogen-stimulated appearance of ER in C3H, while having no effect on this parameter in the BALB/c animal. Uterine ER in either strain is independent of PRL and is stimulated with equivalent efficiency by estradiol. Stimulation of mammary gland ER activity by estradiol is dose responsive, being linear within the range of 0.25-5.0 micrograms/kg body weight. The simultaneous rise in cytosolic progesterone receptor binding activity is also linear over the same range. With respect to PRL responsiveness, ovariectomized adult (but not immature) mouse tissue shows a doubling of ER content with 10(3) or 10(4) micrograms/kg body weight, but no elevation with 10(2) or lower levels. The upper level is approximately equivalent to that seen in the intact or estrogen-replaced castrate mouse. High levels of PRL administered to these latter animal groups cause a secondary increase in ER activity. A PRL-independent component of estradiol-stimulated ER activity is demonstrable from experiments using various doses of CB-154 in animals treated with different levels of estradiol. High dosages of CB-154 superimposed on a highly inducing level of estradiol have a side effect of redistributing nuclear ER to the cytosol; sucrose gradient analyses of nuclear receptor levels demonstrate that this pharmacological action of CB-154 is not mediated through its PRL-suppressive effect. The suppression of nuclear ER by CB-154 is reflected in a loss of estrogen-induced progesterone receptor activity. The ability of tamoxifen to inhibit estrogen-stimulated progesterone receptor activity is appreciably curtailed by concomitant treatment with PRL. These results demonstrate that, of PRL and estradiol, PRL is the dominant factor in eliciting ER activity in mouse mammary gland. Basal conditions are established for quantifying the relative contributions of these and other modifiers of mammary tissue function at the molecular level, and for uncovering otherwise-masked differential dose-related effects of such agents.

Animals↗
PubMed1987

Initial studies of a phytoestrogen-deoxyribonucleic acid interaction.

Molecular modeling studies show that estrogens such as estradiol complement the topography of spaces between base pairs in unwound DNA and simultaneously hydrogen bond phosphate moieties on opposite strands. We demonstrate here that the phytoestrogen coumestrol has this capability, in addition to its documented properties of UV absorbance at lambda greater than 300 nm and fluorescence. The latter properties enable spectroscopic examination of interactions with DNA by methods not possible with estrogenic steroids. On exposure to calf thymus DNA, the UV spectrum of coumestrol displays a bathochromic shift and simultaneous hypochromic effect with an isosbestic point at 370 nm, suggesting a shift between coexisting free and bound states. Similar results are observed with the intercalating agents adriamycin, ethidium bromide, and acridine. The fluorescence spectrum of coumestrol is quenched on exposure to DNA as are those of adriamycin and acridine. Coumestrol differs from the intercalators in that denatured DNA does not affect its UV spectrum or alter its relative fluorescence yield. Unlike classical intercalators, coumestrol has no influence on the thermal stability of calf thymus DNA. Preliminary electrophoretic analysis of DNA plasmid conformers indicates that coumestrol is incapable of significantly altering DNA superhelical density, in contrast to ethidium bromide. These initial physicochemical data provide evidence for the DNA base-estrogen electronic and/or hydrophobic interactions suggested by modeling studies, yet tend to rule out classical intercalation as an explanation for these phenomena.

Animals↗
PubMed1987

Stereochemical modelling studies of the interaction of antineoplaston A10 with DNA.

Antineoplaston A10 (3-phenylacetylamino-2,6-piperidinedione), a peptide analogue originally isolated from human urine, has been demonstrated to fit between base pairs in DNA. Examination of the fit of A10 into the 10 possible sites in unwound DNA using published criteria revealed a preference, but not absolute specificity, for the sequence 5'-dTdT-3' X 5'-dAdA-3'. Good fits were also observed for the sequences 5'-dTdC-3' X 5'-dGdA-3' and 5'-dCdT-3' X 5'-dAdG-3'. In each case at least one stereospecific hydrogen bond was possible between the imino proton of the piperidinedione ring stacked between the two pyrimidines and a neighbouring phosphate oxygen of the DNA backbone. These findings support the prediction that A10 may interact reversibly with DNA and thereby compete with carcinogens that form covalent linkages with DNA (e.g., arene oxides). It follows that such interactions should prevent the growth of tumours induced by various carcinogens.

Antineoplastic Agents↗
PubMed1987

Inhibition of spontaneous mouse mammary tumour development by antineoplaston A10.

Antineoplaston A10, a human urinary product of minimal toxicity and demonstrable antineoplastic activity, was examined as a modulator of spontaneous mammary tumour development in C3H+ mice. These animals normally express the viral response with appearance of tumours in greater than 95% of the mice by 9-10 months of age. Inclusion of A10 as a 1% dietary supplement from the age of 3 months dramatically increased the disease-free interval. At 10-11 months of age, none of the animals had developed tumours, and the incidence reached 95% only at 21 months of age. In spite of this effect, the survival interval following tumour detection could not be prolonged by treatment with A10. Male C3H+ mice, normally fully protected against spontaneous mammary tumour by their testicular androgens, can be made susceptible to such tumorigenesis by castration. The age at which castration is performed influences the age of onset of the disease: castration at one month leads to initial appearance at 5 months of age; castration at 2 months to appearance at 7-8 months. When such animals are maintained on A10-containing diets following castration, the onset of tumour appearance is delayed until 7 months and 10 months for the 1- and 2-month castrated animals respectively. The results indicate a clear inhibitory action of A10 on MMTV-induced spontaneous mammary tumour occurrence in a manner similar to the protective effect of androgens.

Animals↗
PubMed1986

Cytosol estrogen receptor content of female parietal peritoneum.

The ovarian and peritoneal mesothelium are believed to derive from the same embryonal coelomic epithelium. Neoplastic proliferation of these epithelia is sometimes grossly and histologically similar. Recently estrogen receptors have been identified in gynecologic neoplasms. With the use of standard techniques, the parietal peritoneum was assessed for estrogen receptor content in 17 women undergoing celiotomy for various reasons. All peritoneal specimens were negative for estrogen receptor content. This suggests a heterogeneous origin of the peritoneal surface epithelium and that peritoneum may not be müllerian in origin.

Chromatography, Paper↗
PubMed1986

Correlation between LH and estrogen receptor turnover in pituitary and hypothalamus of castrate rats following estrogen agonists and antagonists.

A series of studies was undertaken to correlate the short-term dynamics of LH secretion and depletion-replenishment patterns of estrogen receptors (ER) in hypothalamic and pituitary cytosols of ovariectomized rats. Animals castrated for 2 weeks were administered various test compounds and analyzed at 1, 3, 5, 10 and 15 h post-treatment. A single injection of 10 micrograms 17 beta-estradiol (E2) to ovariectomized rats elicited a rapid depletion of ER in both pituitary and hypothalamus and a dramatic, though delayed, fall in serum LH. ER replenishment occurred in both tissues through 15 h and LH recovered in a similar manner. When cycloheximide was administered along with E2, ER replenishment was completely inhibited in both tissues; serum LH fell and failed to recover. Actinomycin D injected with E2 blocked replenishment in pituitary but not hypothalamus; serum LH recovered in parallel with the hypothalamic ER pattern. 17 alpha-E2 elicited only slight changes in ER and LH was suppressed 10-20% through 15 h. CI-628 caused a near total depletion of pituitary ER with no subsequent replenishment, whereas hypothalamic ER content was virtually unaltered; serum LH was suppressed and later recovered. Orchidectomized rats given 5 micrograms E2 demonstrated a less complete ER depletion in hypothalamus, and an earlier replenishment than that seen in pituitary or hypothalamus of similarly treated ovariectomized females. Serum LH rebounded to 157% of control levels at 15 h. The results indicate that the acute feedback suppression of LH by exposure to estrogens correlates with binding to ER and nuclear translocation. Replenishment and/or retention of cytoplasmic ER in hypothalamus appears to be required for full resumption of LH secretion, following acute suppression.

Animals↗
PubMed1986

The stereochemical complementarity of DNA and reproductive steroid hormones correlates with biological activity.

Modeling studies revealed that progesterone, testosterone, and estradiol are stereochemically complementary to the cavity formed between base pairs in the DNA sequence, 5'-dTdG-3' X 5'-dCdA-3'. Each steroid aligned precisely with the topography of the cavity and formed 2 stereospecific hydrogen bonds linking phosphate oxygens on adjacent DNA strands. Hydrogen bonding donor-acceptor relationships were different for each hormone. The remarkable stereochemical specificity of the hormone-DNA complexes was demonstrated by the lack of complementarity of steroid enantiomers and steroid analogs having alternate ring systems and/or changes in the position of functional groups. Fit of molecules into DNA in the manner of the parent hormone correlated with biological activity. Antagonists also fit into the cavity but differed from agonists in their hydrogen bonding linkages to DNA and/or extended out of the cavity beyond the helix. Unlike flat intercalating agents which form stable complexes with DNA, wedge shaped steroids may thus be capable of forming reversible sequence-specific complexes with DNA. We conclude that the stereochemistry of DNA can be used to predict hormonal activity.

Animals↗
PubMed1986

Steroid hormone receptor regulation by various hormonal factors during mammary development and growth in the normal mouse.

The studies described herein are focused on the nature and regulation of estrogen receptors in normal mammary tissue, with the rationale that manipulation of these receptors is the sole basis for endocrine therapy of human breast cancer. Various features of this complex system have been uncovered by our studies. The presence of different forms of cytosol estrogen receptor, fluctuating in unison with glandular stimulation, results in differential responsiveness of the cells. Tissue that is relatively estrogen-starved presents its receptors in a form that avidly attract the limited available ligands and can hastily put them to use in the nucleus. In contrast, the receptor system in the highly stimulated state displays a sort of refractoriness to estrogen, being relatively sluggish in its responsiveness. Once formed, however, these latter complexes are probably far more effective in terms of eliciting estrogenic responses, since they have an enhanced affinity for DNA and a prolonged half-life. (Table; see text) Prolactin is clearly a very important mediator of the action of estrogen on the mammary gland estrogen receptors, presenting a tissue-specific difference in comparison with the regulation of the uterine estrogen-receptor system. An odd finding was that prolactin inhibits nuclear retention of the estrogen receptor (and probably the progesterone receptor), an effect that is counterproductive to its very strong positive action on the level of intracellular receptor. Perhaps prolactin is the gross effector of receptor fluctuation, allowing estrogen the privilege of dictating the degree of receptor function; indeed our data on the dose-responsiveness of estrogen action and the effects of bromocriptine indicate that a portion of the estrogenic stimulation is not mediated by prolactin. An interesting sidelight of these studies was the finding that high levels of bromocriptine, pharmacologic in terms of prolactin suppression, exhibited an inhibitory effect on nuclear retention of estrogen receptors that was independent of, and did not prevent subsequent elicitation of, the action of prolactin on the receptors. The role of prolactin in tumorigenesis is well established in experimental animals, but its role in the human disease is not clear. Although prolactin receptors have been measured in human breast cancer, there does not appear to be any distinguishable correlation with the presence of estrogen or progesterone receptors. While it is a bit premature to draw conclusions, initial trials of bromocriptine usage have not been supportive of beneficial effects of such treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Baseline and luteinizing hormone-releasing hormone-perturbed patterns of estrogen receptor distribution in anterior pituitary cell nuclei.

We have previously demonstrated that LHRH elicits a direct and dramatic elevation of nuclear estradiol receptor (ERn) levels in the anterior pituitary of young adult female rats. We now describe the effect of LHRH on subpopulations of ERn in the anterior pituitary. Intact purified pituitary nuclei were prepared from adult ovariectomized rats primed with estradiol (E2) and incubated with or without (control) 100 pmol LHRH/pituitary equivalent for 30 min at 37 C. The nuclei were subjected to salt extraction, and the number of occupied and unoccupied specific E2-binding sites in the salt-soluble and salt-resistant fractions of nuclei were measured. In the control pituitary nuclei, 70% of ERn were in the salt-soluble fraction, of which the great majority were occupied by endogenous steroid. The remaining ERn in the salt-resistant fraction consisted of an almost equal distribution of free and occupied sites. On preincubation of the nuclei in the absence of LHRH at 37 C for 30 min, a 40% decrease in the total number of ERn was observed, which reflected primarily a significant decrease in the number of salt-soluble ERn. Incubation of the nuclei in the presence of LHRH led to the expected increase in total ERn levels, and this was traced to a dramatic and significant increase in the salt-resistant forms of ERn, while number of salt-soluble ERn was not significantly changed from the control level. Triton X-100 treatment of nonextracted nuclei had no effect on control or LHRH-induced levels of ERn. Salt-resistant ERn were subjected to DNase and RNase treatment, and the majority of the specific binding sites (70%) remained resistant to the digestion. The enzyme-resistant forms increased significantly in the presence of LHRH, while the enzyme-soluble forms did not change significantly. It is clear from these studies that LHRH affects a specific subpopulation of ERn, which appears to be an integral part of the protein matrix of the nuclei. These observations pose new questions about the mechanism of peptide hormone action and advance our understanding of the molecular basis for LHRH-E2 interactions in regulation of reproductive functions.

Animals↗
PubMed1985

Androgen receptor dynamics in the rat ventral prostate.

Upon testosterone administration, a dose-dependent cytosolic depletion and nuclear accumulation of androgen receptors in the ventral prostate of 1-day-castrated male rats is observed. Replenishment in the cytosol is rapid with a return to control levels 3 h after testosterone stimulation. The process of nuclear retention (as measured 4-6 h post-injection) is both dose-dependent and time-dependent (there is no retention of the androgen receptor 15 h after testosterone). When assayed 1 h after testosterone, the increase in nuclear binding sites was not sufficient to conclude that the disappearance of cytosolic binding sites could be accounted for by translocation of cytosolic receptors to the nucleus. The cytosolic compartment contained more than 70% of the total cellular receptors whether testosterone was present (in the range 50-400 micrograms/100 g body wt.) or not. Nuclear processing of androgen receptors is extensive and it is dose-dependent. Turnover of prostatic androgen receptors was studied simultaneously in the cytosolic, microsomal and nuclear compartments 1, 2, 3, 4, 5 and 6 h after testosterone administration. Cytosolic and microsomal depletion-replenishment patterns are similar, displaying a nadir after 1 h and a full replenishment 3-4 h post-testosterone. Cycloheximide, but not actinomycin D, inhibits cytosolic and microsomal replenishment. Nuclear accumulation and retention of androgen receptors is insensitive to both drugs. The very rapid and RNA synthesis-independent turnover of androgen receptors in the ventral prostate suggests that testosterone regulates the receptor levels acutely by both a rapid post-transcriptional positive action and a similarly rapid negative effect on nuclear receptor half-life.

Animals↗
PubMed1985

Apparent stereochemical complementarity of estrogens and helical cavities between DNA base pairs: implications for the mechanism of action of steroids.

The shape of the space occupied by a model of the estrogenic steroid hormone estradiol-17 beta conforms closely to a helical cavity between neighboring base pairs in partially coiled B-DNA. The orientation of estradiol-17 beta when fitted into DNA allows stereochemically complementary hydrogen bonding of both the 3- and 17 beta-hydroxyl groups to phosphate oxygens of the deoxyribose-phosphate backbone on adjacent strands. Changes in the chirality (handedness) of the steroid skeleton or in the absolute stereochemistry of hydrogen bonding groups prevent formation of complementary fits in the DNA. Synthetic estrogens can also adopt conformations which are stereochemically complementary to the cavities between base pairs. The complementary relationships between active estrogens and nucleic acids may be related to constraints on the evolution of the structure and the biological function of steroids.

Base Composition↗
PubMed1985

Apparent lack of involvement of cAMP as a mediator of LHRH stimulation of nuclear estrogen receptor activity in the rat anterior pituitary.

We have previously shown that incubation of rat pituitary cells in the presence of LHRH results in specific enhancement of nuclear estrogen receptor (ER) binding which cannot be accounted for by simple intracellular translocation of cytoplasmic receptor. In the present study, the role of cAMP in this response has been examined. Suspended pituitary cells from adult ovariectomized rats primed with estradiol were incubated with varying concentrations of LHRH or a highly active LHRH analog (LHRH-A) for 30 min at 37 degrees C, and levels of cAMP were determined. Total cAMP levels changed only in response to a concentration of 100 pmol/pituitary of either peptide; the stimulation by LHRH was twice that by LHRH-A. When the priming dose level of estradiol was reduced from 1.0 to 0.5 micrograms/day, stimulation of cells by 100 pmol of LHRH caused a much greater increase in cAMP levels. Separate incubation of subcellular fractions with a wide dose range of dibutyryl-cAMP (DBcAMP) resulted in a progressive loss of cytosol receptor binding capacity (which was also observed in whole cultured or suspended cells), but no significant concomitant change in nuclear receptor binding; however, whole cells in suspension or culture did show an increase in nuclear ER activity in the presence of 100 nM DBcAMP. This response was qualitatively, but not quantitatively, similar to that elicited by LHRH. When the effects of whole cell incubation with LHRH and LHRH-A on nuclear ER were compared with their effects on total cAMP levels, no correlation was observed; rising levels of cAMP did, however, coincide with falling levels of cytosol ER.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Specific binding of estrogen and estrogen-receptor complex by microsomes from estrogen-responsive tissues of the rat.

Rat uterine and anterior pituitary microsomes each contain a population of specific estrogen-binding sites. Saturation binding of estradiol is demonstrable, with an affinity similar to that of the cytosol estrogen receptor (Ka = 1-2 X 10(10) M-1). Dissociation rate kinetic determinations, however, revealed that estrogen-microsomal complexes are 4 times as stable as cytosol estrogen-receptor complexes. Sedimentation properties in sucrose gradients were salt-dependent, yielding values of 10S in KCl-free buffer and 5.5S in the presence of 0.4 M KCl. The concentration of microsomal sites varies in proportion to the level of cytosol estrogen receptor, such that microsomal binding constitutes a consistent 20% of the total extranuclear binding capacity. Binding is sensitive to pronase, but not to ribonuclease or deoxyribonuclease; steroidal specificity differs from cytosol receptor only with respect to a greater extent of competition by progesterone. Microsomal binding sites are readily extractable with KCl-free hypotonic buffer or with 0.4 M KCl, but are resistant to extraction by 0.15 M KCl. The presence of estradiol lends stability to the microsomal binding sites, while high salt has a deleterious effect on their longevity. After exhaustive extraction of binding sites, microsomes are capable of accepting cytosol estradiol-receptor complexes to a level corresponding to the concentration of depleted binding sites; microsomes from nontarget tissue do not manifest such capability. However, the original microsomal estrogen-binding sites are not simply cytosol receptor contaminants, as evidenced by the observations that the microsomal binding site concentration is independent of the volume of tissue homogenate (indicating that a trapping phenomenon is not operative) and that nonextracted microsomes are not potential acceptor sites for cytosol estradiol-receptor complexes. In considering total cellular dynamics of estrogen and estrogen receptor turnover, it thus becomes important to explore the role of the microsomal compartment, since it functions as a repository of specific estrogen-binding sites and may have significant acceptor capability for the cytosol estrogen-receptor complex.

Animals↗
PubMed1984

Cryptic estrogen binding protein complicates analysis of estrogen receptor distribution.

The response of rat uterine estrogen receptor sub-species to injection of 5 micrograms estradiol has been investigated in intact and 4-weeks' ovariectomized adult animals. Determinations of occupied and unoccupied receptor subcellular fluctuations reveal significant differences not detectable under standard assays which measure only total nuclear and unoccupied cytosolic receptors. Both animal models manifest a high level of unoccupied nuclear receptors which are inaccessible to estrogen. In contrast to the intact animal, uteri from castrate animals have a high level of occupied receptors in the cytosol, which remains high following estrogen exposure. Receptor processing occurs in the castrate, but not the intact, animal. The results demonstrate that traditional assays are complicated by the presence and simultaneous measurement of non-responsive receptor species which quantitatively differ widely among animal models and will give rise to an erroneous interpretation of the pattern of estrogen-induced turnover of its receptor.

Animals↗