Biomedical subjects

G Shyamala

Publications and source records attributed to G Shyamala.

At least 55 records · Page 3Linked to original sources

PubMed1988

Distribution of estrogen receptors in various cell types of normal, hyperplastic, and neoplastic human endometrial tissues.

To facilitate our understanding of estrogen participating in the genesis of human endometrial hyperplasia and carcinoma and the influence of the surrounding mesenchyme in their development, we assayed estrogen receptors in the epithelium and stroma of various human endometrial tissues by immunocytochemistry. The receptors were localized in fresh frozen sections using the ER-ICA kits (Abbott Laboratories, North Chicago, Illinois). The receptor content was highest in the proliferative endometrium and decreased gradually throughout the postovulatory phase of the menstrual cycle in both the epithelium and the stroma and this occurred more rapidly in the stroma. Estrogen receptor content was high in both the epithelium and the stroma of hyperplasia and was thus similar to that of the proliferative endometrium, the receptor content was low in the epithelium of endometrial intraepithelial neoplasia (hyperplasia with cytologic atypia) closely resembling that of carcinoma. In carcinomas there was a heterogenous distribution of estrogen receptors both in the epithelium and the stroma, but in general, the receptor content was low as compared with normal proliferative or hyperplastic endometrium; however, in selected cases the content of estrogen receptor in the stroma was high, despite the lack of receptors in the epithelium.

Adult↗

PubMed1987

Binding of glucocorticoid receptors to mammary chromatin acceptor sites.

We have recently characterized the interaction of mouse mammary estrogen receptors (ER) with mammary chromatin acceptor sites and demonstrated that ER from estrogen resistant lactating mammary glands do not bind to chromatin. In this study we have characterized the chromatin binding of the glucocorticoid receptor from mouse mammary glands isolated from nulliparous and lactating mice in order to better understand the relationship between receptor binding to chromatin and steroidogenic sensitivity of the tissue. Mammary chromatin was linked covalently to cellulose and deproteinized sequentially by 0-8 M Gdn-HCl. Binding to intact chromatin as well as to chromatin deproteinized by Gdn-HCl was determined using partially purified [3H]dexamethasone labelled glucocorticoid-receptor complexes (GR) obtained by fractionation on DEAE-cellulose columns. The binding of [3H]GR from mammary glands of nulliparous mice to chromatin fractions from the same tissue revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 5-6 M Gdn-HCl. Binding of [3H]GR was of high affinity (Kd = 0.2 nM) and saturable. A simultaneous comparison of the chromatin binding patterns for [3H]ER and [3H]GR isolated from mammary glands of nulliparous mice revealed that the chromatin subfractions obtained with 4-6 M Gdn-HCl extraction contained acceptor sites for both [3H]ER and [3H]GR; however, while the [3H]ER bound to a 4.5 M and a 5.5 M site, the [3]GR bound a 5 M and a 6 M site. Competition experiments supported the steroid receptor specificity of the chromatin acceptor sites. Thus, the 4-6 M chromatin fractions contain distinct acceptor sites for the glucocorticoid receptor and for the estrogen receptor. In addition our studies reveal that the binding patterns of [3H]GR isolated from mammary glands of nulliparous and lactating mice to their homologous chromatin is essentially similar. Thus, in contrast to estrogen receptors, glucocorticoid receptors from lactating mammary glands are able to effectively bind to chromatin acceptor sites which supports our previous suggestion that the estrogenic insensitivity of lactating mouse mammary glands may at least be in part due to the impeded interaction of ER with chromatin acceptor sites.

Animals↗

PubMed1986

The effect of dithiothreitol on the kinetics of dissociation of dexamethasone from the non-transformed mammary cytosolic glucocorticoid receptor.

Sulfhydryl reducing agents such as dithiothreitol are required for maximum binding of dexamethasone to the mammary cytosolic glucocorticoid receptor, but little is known concerning the effects of dithiothreitol on the kinetics of the binding reaction. In this report we have examined the influence of dithiothreitol on the dissociation kinetics of dexamethasone from the non-transformed glucocorticoid-receptor complex at 0-4 degrees C under various experimental conditions. Without dithiothreitol, the rate of dissociation of dexamethasone remains essentially the same (t1/2 approximately 17 h) regardless of the method chosen to monitor dissociation. With dithiothreitol, however, there is a marked acceleration in the rate of dissociation of receptor-bound dexamethasone when an excess of unlabeled dexamethasone is used to study dissociation (t1/2 approximately 5 h) but not when dissociation is investigated by removal of free labeled dexamethasone by charcoal adsorption (t1/2 approximately 21 h); dithiothreitol also accelerates the observed rate of dissociation when a combination of these methods is used. An acceleration in the rate of receptor-bound dexamethasone is also observed when an excess of the synthetic progestin, R5020, is used in the dissociation assay. The possible reasons and importance underlying these findings have been discussed.

Animals↗

PubMed1986

Relationships between mammary estrogen receptor and estrogenic sensitivity. Molecular properties of cytoplasmic receptor and its binding to deoxyribonucleic acid.

The cytoplasmic estrogen receptors (ER) from mammary glands of estrogen-responsive nulliparous and estrogen-resistant lactating mice have been studied to delineate various relationships between the molecular properties of ER and estrogenic sensitivity. These studies indicate that there are essentially no differences in the hydrodynamic parameters of native ER isolated in hypotonic buffer; the ER from both tissues have a stokes radius of 80-85 A, sedimentation coefficient of 9-10S, and mol wt of 300,000-340,000. However, while 60-80% of the total ER in mammary glands of nulliparous mice, upon exposure to 400 mM KC1 is able to bind to DNA, under identical experimental conditions only approximately 20% of total ER from lactating mammary glands binds to DNA. Analyses of ER in buffers containing 400 mM KC1 reveal that the ER in lactating mammary glands have a larger mol wt (100,000-130,000) as compared to ER in mammary glands of nulliparous mice (70,000). The ER in lactating mammary glands also appear to be more acidic when analyzed by diethylaminoethyl cellulose chromatography. Experiments performed with mixed cytosol reveal that lactating mammary cytosol contains factors which can impede the ability of ER to bind to DNA subsequent to exposure to KC1. The possible significance of the observed differences in the properties of ER from estrogen-responsive and unresponsive mammary glands has been discussed.

Animals↗

PubMed1986

Relationships between mammary estrogen receptor and estrogenic sensitivity. II. Binding of cytoplasmic receptor to chromatin.

Mammary glands from nulliparous mice are responsive to estradiol, whereas mammary glands from lactating mice are unresponsive, despite the presence of high concentrations of estrogen receptors. This study examined the relation between mammary estrogenic sensitivity and ability of mammary estrogen receptors to bind to intact chromatin as well as to partially deproteinized chromatin. Mammary chromatin was prepared from nulliparous and lactating mice, linked covalently to cellulose and deproteinized sequentially by 0-8 M guanidine chloride (Gdn X HCl). The binding of receptors to these various chromatin preparations was determined using partially purified [3H]estradiol-receptor complexes ([3H]ER) obtained by fractionation on diethylaminoethyl cellulose columns. The binding pattern of [3H]ER from nulliparous mice to chromatin fractions from either nulliparous or lactating mice revealed maximal binding activity with chromatin previously extracted with 4-6 M Gdn X HCl. Binding was of high affinity [dissociation constant (Kd) 3.6 X 10(-10) M], saturable and steroid receptor and species specific. However, mammary [3H]ER preparations from lactating mice bound poorly to intact chromatin as well as to the Gdn X HCl extracted chromatin fractions isolated from either mammary gland of nulliparous or lactating mice. In mixing experiments the estrogen receptor preparation from lactating mice decreased substantially the binding activity of [3H]ER from nulliparous mice to chromatin suggesting the presence of an inhibiting factor. Thus, these studies reveal that the unresponsiveness of lactating mammary glands to estradiol coexists with the inability of estrogen receptors from lactating mice to interact with specific high affinity sites on mammary chromatin and also that this impeded interaction of estrogen receptors with chromatin may be due to some inhibitor(s) present in the cytosol of lactating mammary glands.

Animals↗

PubMed1985

Glucocorticoid receptors in primary cultures of mouse mammary epithelial cells: characterization and modulation by prolactin and cortisol.

Mammary epithelial cells isolated from midpregnant mice and cultured on collagen gels contain soluble glucocorticoid receptors. The kinetics of binding of dexamethasone reveal a saturable binding site [dissociation constant (Kd), approximately 1 nM], and the binding site obeys a steroid specificity characteristic of a glucocorticoid receptor. As with the receptor isolated from intact glands, the receptor from the cultured cells also requires the addition of dithiothreitol for maximal binding of dexamethasone. The receptors are maintained at in vivo levels (approximately 1.3 pmol/mg DNA) for at least a period of 10 days in culture. However, the presence of both cortisol and PRL is required for the maintenance of the receptors, and the effect of both these hormones is dose dependent.

Animals↗

PubMed1984

Phosphorylation of high-mobility-group proteins by the calcium-phospholipid-dependent protein kinase and the cyclic AMP-dependent protein kinase.

Purified lamb thymus high-mobility-group (HMG) proteins 1, 2, and 17 have been investigated as potential substrates for the Ca2+-phospholipid-dependent protein kinase and the cAMP-dependent protein kinase. HMG proteins 1, 2, and 17 are phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the reactions are totally Ca2+ and lipid dependent and are not inhibited by the inhibitor protein of the cAMP-dependent protein kinase. HMG 17 is phosphorylated predominantly in a single seryl residue, Ser 24 in the sequence Gln-Arg-Arg-Ser 24-Ala-Arg-Leu-Ser 28-Ala-Lys, with the second seryl moiety, Ser 28, modified to a markedly lesser degree. HMGs 1 and 2 are also phosphorylated in only seryl residues but with each there are multiple phosphorylation sites. HMG 17, but not HMG 1 or 2, is also phosphorylated by the cAMP-dependent protein kinase with the site phosphorylated being the minor of the two phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the Km for phosphorylation by the cAMP-dependent enzyme is 50-fold higher than that by the Ca2+-phospholipid-dependent enzyme. HMG 17 is an equally effective substrate for the Ca2+-phospholipid-dependent protein kinase either as the pure protein or bound to nucleosomes. Preliminary evidence has indicated that lamb thymus HMG 14 is also a substrate for the Ca2+-phospholipid-dependent enzyme. It is phosphorylated with a Km similar to that of HMG 17 (4-6 microM), and a comparison of tryptic peptides suggests that it is phosphorylated in a site that is homologous with Ser 24 of HMG 17 and distinct from the sites phosphorylated by the cAMP-dependent protein kinase.

Amino Acid Sequence↗

PubMed1984

Heparin-mediated inactivation and transformation of mammary cytoplasmic glucocorticoid receptor.

Glucocorticoid receptor of lactating mouse mammary gland cytosol was exposed to heparin when the receptor was either steroid-free or steroid-bound. Heparin caused a dose-dependent and time-dependent loss of steroid binding activity (inactivation) of the steroid-free receptor; this heparin-induced inactivation was inhibited by molybdate. In contrast, steroid-bound receptor maintained its steroid binding capacity in the presence of heparin but the heparin caused transformation of receptor as detected by increased binding to DNA-cellulose and ATP-Sepharose. Heparin also converted steroid-bound receptor from the 7-8S form to the 4S form. Molybdate inhibited both the heparin-induced transformation and associated conversion to the 4S form.

Animals↗

PubMed1984

Mammary fat pad may be a potential site for initiation of estrogen action in normal mouse mammary glands.

The effect of estradiol (E2) on mammary and uterine DNA synthesis has been examined in castrated virgin mice. In both these target tissues for E2, in vitro incorporation of thymidine into DNA begins 12 h after E2 treatment, and by 24 h there is approximately a 3-fold increase in the mammary glands and a 9-fold increase in the uterus. Whereas in the uterus by 48 h after E2 the rate of DNA synthesis has basically returned to control values, in the mammary gland a maximal stimulation of DNA synthesis is observed at this time, and even at 72 h after E2 there is still approximately a 2-fold increase in DNA synthesis. In vivo histoautoradiography reveals that, unlike in the uterus, 24 h after E2 there is virtually no uptake of labeled thymidine in the mammary epithelium; the increase in the uptake of thymidine is now mostly confined to the mammary adipose and connective tissues. However, 48 and 72 h after E2 there is extensive labeling of the mammary epithelium in addition to the adipose and connective tissues. These data lead us to suggest that both the mammary fat pad and epithelium are responsive to E2, and that, at least with respect to mammary cell proliferation, the mammary fat pad may be the site of initiation of estrogen action.

Adipose Tissue↗

PubMed1983

Similarities and differences of the binding of estradiol and 4-hydroxytamoxifen (an antiestrogen) in the chick oviduct cytosol.

The binding characteristics of [3H]estradiol and 4-[3H] hydroxytamoxifen (a powerful estradiol antagonist) in the chick oviduct cytosol was analyzed by sucrose gradient centrifugation and dissociation kinetics experiments at 28 degrees C. Heating the cytoplasmic estradiol-estrogen receptor complexes led to the 'transformation' of the receptor; as with the estrogen receptor in other target tissues and species, the transformed receptor sedimented in the 5 S region of sucrose gradients containing 0.4 M KCl and had a slower rate of dissociation of bound estradiol. Upon heating, the cytoplasmic 4-hydroxytamoxifen complexes also appeared to undergo similar changes in their physical states as analyzed by sedimentation rates and dissociation kinetics, and we conclude that antiestrogen can transform the receptor. Sodium molybdate inhibited the temperature mediated changes with both estrogen and antiestrogen complexes. Slight but consistent differences in the sedimentation coefficient and rate of ligand dissociation were observed between the complexes formed by estradiol and 4-hydroxytamoxifen but the relevance to opposite biological activities remains unknown.

Animals↗

PubMed1983

An evaluation of peroxidase as a marker for estrogen action in normal mammary glands of mice.

The present studies were initiated to study peroxidase and its possible regulation by estrogen in normal mammary glands. The activity of peroxidase was measured biochemically using guaiacol as the substrate for oxidation. Significant levels of peroxidase activity were associated with the particulate fraction of mammary glands from virgin mice, pregnant mice, and mice undergoing lactational involution. However, during lactation there was no detectable level of peroxidase activity in the mammary glands. Although ovariectomy led to a decrease in mammary peroxidase, detailed studies using various hormonal manipulations revealed that mammary peroxidase was perhaps not a product of estrogen action alone, but might be the result of a complex hormonal control related to growth. Alternatively, a critical evaluation of all of the data obtained with mammary glands and a comparison of these data obtained with the uterus also suggest that the presence of peroxidase in mammary glands may be due to infiltration of eosinophils and macrophages in these tissues resulting from mast cell degranulation.

Animals↗

PubMed1982

Inhibition of the binding of dexamethasone to mammary cytoplasmic glucocorticoid receptor by phenylglyoxal.

We have studied the effects of phenylglyoxal and other related arginine-specific reagents on the mammary cytoplasmic glucocorticoid receptor. Our studies show that phenylglyoxal can inhibit the binding of [3H]dexamethasone to the steroid-free receptor and also displace the bound [3H]dexamethasone from the steroid-receptor complex. The kinetics of the binding reaction reveals that the inhibition of phenylglyoxal may be due to its ability to interact with the steroid binding site of the receptor. Similar data are also obtained with other arginine-modifying agents such as 1,2-cyclohexanedione. Thus, these studies suggest that arginine residues may be involved in the binding of glucocorticoids to mammary cytoplasmic glucocorticoid receptor.

Aldehydes↗

PubMed1982

A comparison of the cytoplasmic estrogen receptors of mammary gland from virgin and lactating mice.

Recent studies suggest that the mammary cytoplasmic estrogen receptor of virgin mice sediment predominantly as 45 species on low ionic strength sucrose gradients. In the present studies we show that the mammary cytoplasmic estrogen receptor from both the intact and ovariectomized virgin and lactating mice sediment at 9S on sucrose gradients prepared in low ionic strength buffers. However, the receptor from the virgin mice is susceptible to cleavage, resulting in the appearance of 3-4S forms of the receptor. If, however, phosphate buffers or buffers containing sodium molybdate are used in the preparation of cytosol and its analysis, it preserves the integrity of the native 9S estrogen receptor.

Animals↗

PubMed1982

The nonresponsiveness of lactating mammary gland to estradiol.

The effect of estradiol on mammary glucose metabolism and DNA synthesis has been studied. Estradiol causes a significant increase in both the rate of glucose oxidation and DNA synthesis in the mammary glands of virgin mice, but is without any stimulatory effect on the glands of lactating mice. The reason for the inability of lactating mammary gland to respond to estradiol appears to reside in the lactating tissue itself and does not seem to be related to the hormonal milieu of lactation. Previously, we have shown that, in contrast to mammary gland of virgin mice, lactating mammary glands do not respond to estradiol with an increase in progesterone receptor levels. Therefore, all of these studies taken together reveal that during lactation, mammary glands may be nonresponsive with respect to most known parameters of estrogen action.

Animals↗

PubMed1981

Transformation of mammary cytoplasmic glucocorticoid receptor under cell-free conditions.

The transformation of glucocorticoid--receptor complex in the cytosol from lactating mouse mammary tissue was studied by using elevated temperature and KCl as promoters of the transformation reaction. The transformed receptor was identified from the nontransformed receptor by the following criteria: (a) increased binding to DNA--cellulose, (b) increased binding to ATP--Sepharose, (c) higher affinity for the steroid as determined by steroid dissociation kinetics, and (d) different sedimentation profiles on sucrose gradients containing KCl and sodium molybdate. A greater percentage of the nontransformed receptor was converted to the transformed state by an increased KCl concentration as opposed to increased temperature. Pretreatment of cytosol with 10 mM sodium molybdate prevented both the temperature- and salt-mediated transformation of the receptor.

Animals↗

PubMed1981

Relative distribution of estrogen and progesterone receptors among the epithelial, adipose, and connective tissue components of the normal mammary gland.

The present studies were undertaken to determine the relative distribution of estrogen and progesterone receptors (PgR) in the epithelial, adipose, and connective tissues of normal mouse mammary gland and to determine in which tissue(s) the modulations in PgR concentration and mammary gland sensitivity to estrogenic stimulation of PgR occur. Mammary epithelium was separated from adipose and connective tissues surgically in vivo according to the cleared fat pad technique. Binding studies using [3H]R5020 or [3H]estradiol were carried out on cytoplasmic extracts of intact mammary gland or cleared fat pads which were devoid of epithelium and contained only mammary adipose and connective tissues. We found that mammary adipose and connective tissues contain separate, high affinity, steroid-specific, macromolecular binding sites for estrogen and progesterone, and the majority of PgR are present only in the glandular epithelium. Furthermore, the modulation in PgR concentration relative to mammary gland development as well as the estrogenic regulation of PgR concentration appear to be restricted to the epithelial component of mammary gland. Since the major mammary gland developmental changes that occur during pregnancy and lactation also occur in the epithelium, it is proposed that the receptor modulations may serve a regulatory function in mammary gland.

Adipose Tissue↗

PubMed1981

The effect of sodium molybdate on the cytoplasmic estrogen and progesterone receptors in human endometrial tissues.

We have investigated the effects of including 10 mM sodium molybdate in the buffers used for analysis of cytoplasmic estrogen and progesterone receptors of normal human proliferative endometrium and endometrial carcinoma. Sodium molybdate does not increase the steroid binding capacity of the receptors, but imparts stability to the steroid bound receptor; the steroid-bound receptors in molybdate treated cytosol sediment as distinct 8-9S moieties in sucrose gradients of low ionic strength in contrast to those in untreated cytosol which sediment mainly as 4S. We conclude that sodium molybdate is a useful reagent for the assay and isolation of estrogen and progesterone receptors of human endometrial tissues.

Adenocarcinoma↗

PubMed1980

Inhibition of uterine estrogen receptor transformation by sodium molybdate.

The effects of sodium molybdate on the transformation of mouse uterine cytoplasmic estradiol . receptor complex has been studied. If molybdate is added to cytosol containing the nontransformed estradiol . receptor complex, it blocks the receptor transformation which otherwise occurs during incubation at 28 degrees C. The inhibition by molybdate is apparent both in sucrose gradient centrifugation analyses of estradiol . receptor complexes and [3H]estradiol dissociation kinetic studies. A molybdate-sensitive change of the nontransformed estradiol . receptor complex can also be inferred from behavior of the estradiol . receptor complex on low ionic strength sucrose gradients.

Animals↗