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T C Spelsberg

Publications and source records attributed to T C Spelsberg.

At least 145 records · Page 8Linked to original sources
PubMed1984

Lack of an effect of estrogen on endogenous RNA polymerase II in guinea pig brain.

An early action of estrogen in peripheral target tissues is to increase endogenous RNA polymerase II activity which precedes changes in protein synthesis. Previous reports have indicated that estrogen causes a similar increase in polymerase activity in rat brain regions containing high affinity receptors for this hormone. The present series of experiments was designed to test whether a similar action of estrogen on polymerase activity occurs in guinea pig brain, with the expectation being that this enzyme might prove to be a useful marker for the early actions of estrogen. Injections of 25 or 100 micrograms estradiol-17 beta in an ethanol-saline vehicle failed to induce significant changes in polymerase II activity in the basal hypothalamus, preoptic area-septum or cortex 1, 2, 6, 13 or 24 h after hormone administration. It is concluded that the modification of RNA polymerase II activity by estrogen is not a good marker for estrogen action in the brain. This may be due to the intrinsic heterogeneity of the tissue resulting possibly in (1) increased activity in some cells and decreased activity in other cells with no net change in overall polymerase activity after estrogen treatment (2) only a small percentage of the cells responding to the steroid with altered polymerase activity with this change not detectable when whole tissue is measured or (3) only a few genes within the cells being altered in transcription by the steroid.

Animals↗
PubMed1984

Pulmonary lymphangiomyomatosis: with particular reference to steroid-receptor assay studies and pathologic correlation.

The symptoms of progressive dyspnea, hemoptysis, spontaneous pneumothorax, chylous effusions, and cough in conjunction with ventilatory obstruction and abnormal gas exchange in a young woman should prompt the diagnosis of pulmonary lymphangiomyomatosis. Cytosol steroid-receptor assays and postmortem studies were conducted in an extensive investigation of a case of this disease. A biopsy specimen of the lung disclosed evidence of nuclear translocation of [3H]progesterone and the presence of a cytosolic receptor for progesterone, an indication that this disease could be treated effectively with progestin.

Adult↗
PubMed1984

Characterization of Epstein-Barr virus nuclear antigen(s) in different cell lines by radioimmunoelectrophoresis.

Utilizing radioimmunoelectrophoresis, Epstein-Barr virus nuclear antigens (EBNA) with molecular weights of 67,000 and 70,000 were detected in NC37 cells, while the Raji cell line contained an antigen with a molecular weight of 70,000. Cell lines containing the B-95 strain of Epstein-Barr virus (EBV) expressed antigens with a molecular weight of 73,000. Three different B-lymphocyte cell lines, established by infection with the B-95 virus, also exhibited 73,000 molecular weight EBNA proteins. Cell lines containing the P3HR-1 strain of EBV demonstrated a single antigen with a molecular weight of 72,000. The results indicate that the molecular weight of EBNA is determined by the strain of infecting virus.

Antigens, Viral↗
PubMed1983

Androgen receptor in human foreskin. II. Characterization of the receptor from hypospadiac tissue.

Hypospadias represents a spectrum of disorders known as androgen insensitivity syndromes, which are characterized by a nonresponsiveness of an androgen target tissue to the steroid. The basic defect in this disorder is unknown. This article presents results of studies of the androgen receptor with use of human foreskin from normal neonates undergoing circumcision and from patients undergoing urethroplasty necessitated by hypospadias. It is speculated that a defect in receptor level or function might explain this disorder. The androgen receptors from both normal and hypospadiac tissue were stabilized with a buffer containing 50 mM Tris hydrochloride, 1 mM ethylenediaminetetraacetic acid, 12 mM monothioglycerol, 10% (v/v) glycerol, and 10 mM sodium molybdate plus 1 mM phenylmethylsulfonylfluoride (pH 7.5). The receptors were characterized by Scatchard analysis, competitive binding assays, sedimentation analysis, molecular sieve chromatography, and isoelectric focusing techniques. Specific R 1881 binding to soluble proteins from normal human foreskin was shown to be high affinity (dissociation constant = 0.5 nM), low capacity (6 fmol/mg of protein), and steroid specific. Sedimentation analysis by sucrose density gradients demonstrated the 8S-to-4S shift from low- to high-salt treatments. The receptor from the foreskins of hypospadiac patients also contained androgen receptor in a concentration similar to that in the foreskins of normal persons. The affinity for steroid binding and sedimentation profiles or sucrose gradients of the androgen receptor between normal persons and hypospadiac patients were also similar. However, the androgen receptor from hypospadiac patients displayed different patterns of molecular sieve chromatography from those displayed by the receptor of normal persons. The reason for this difference is not known but may reflect a defective receptor and explain the basic mechanism of this disorder.

Androgen-Binding Protein↗
PubMed1983

Partial purification of the Epstein-Barr virus nuclear antigen(s).

The Epstein-Barr virus nuclear antigen (EBNA) is speculated to be involved in cell transformation by the virus. Studies on the molecular properties of EBNA, however, have yielded conflicting results. In this study, three Epstein-Barr virus(EBV)-induced antigens were isolated and purified from extracts prepared from Raji cells. These antigens were able to block the anticomplement immunofluorescence reaction, indicating that all three were related to EBNA. The soluble antigen was found wholly in the cytosol fraction. An EBV-induced nuclear antigen I was found both in the cytosol and the nucleus. The EBV-induced nuclear antigen II was found associated with the chromatin. The soluble antigen and the nuclear antigen I were separated and partially purified using phosphocellulose chromatography. Each was further purified 1,400-fold with respect to the whole cell state by chromatography on CL-Sepharose 6B followed by blue dextran-Sepharose. subunit molecular weights of 70,000 were determined for each of these antigens, both in the crude and purified state, by radioimmunoelectrophoresis and gel filtration. The nuclear antigen II was purified 2,500-fold using hydroxylapatite, CL-Sepharose 6B, and blue dextran-Sepharose chromatographies. This antigen displayed two subunits by radioimmunoelectrophoresis with molecular weights of 65,000 and 70,000. Although all antigens shared similar molecular weights, the extent of their homology remains to be determined.

Antigens, Viral↗
PubMed1983

Acceptor sites for the oestrogen receptor in hen oviduct chromatin.

Partially purified hen oviduct oestrogen receptors, charged with [3H]oestradiol, were shown to specifically bind in vitro to purified hen oviduct chromatin. Maximal binding occurred within 60min at 0 degrees C in a Tris buffer containing 0.1 M-KCl and 0.5 mM-phenylmethanesulphonyl fluoride. The binding of the [3H]oestradiol-receptor complexes to intact purified chromatin was saturable, whereas the receptor binding to hen DNA remained linear. Saturation was further demonstrated by the minimal acceptor binding of receptor charged with [3H]oestradiol plus 200-fold oestradiol compared with [3H]oestradiol receptors at equal [3H]oestradiol concentrations. Scatchard analysis of [3H]oestradiol-receptor binding to chromatin above DNA levels gave indications of high-affinity binding with a low capacity. Further, the nuclear binding was tissue-specific since the binding to hen spleen chromatin was negligible. To further uncover the specific acceptor sites, proteins were removed from hen oviduct chromatin by increasing concentrations of guanidine hydrochloride (1-7M). Those residual fractions extracted with 3-7 M-guanidine hydrochloride had the highest acceptor activity (above DNA levels) with the peak activity uncovered by 5 M-guanidine hydrochloride. To further characterize the oestrogen-receptor acceptor sites, oviduct chromatin was bound to hydroxyapatite in the presence of 3 M-NaCl and then protein fractions were extracted sequentially with 1-7 M-guanidine hydrochloride. Each fraction was then reconstituted to pure hen DNA by reverse gradient dialysis. [3H]Oestradiol receptors were found to bind to the greatest degree to the fraction reconstituted from the 5 M-guanidine hydrochloride protein extract. Reconstituted nucleoacidic proteins (NAP) from combined 4-7 M-guanidine hydrochloride protein extracts showed saturable binding by [3H]-oestradiol receptors, whereas binding to hen DNA did not saturate. The high affinity, low capacity, and specificity of binding of oestrogen receptors to NAP was similar to that found in intact chromatin. Thus, chromatin acceptor proteins for the oestrogen receptor have been partially isolated and characterized in the hen oviduct and display properties similar to that reported for the acceptor proteins of the progesterone receptor.

Animals↗
PubMed1983

A rapid method for analysis of ligand binding to deoxyribonucleic acid and soluble nucleoproteins using streptomycin: application to steroid receptor ligands.

A method is described which allows the rapid analysis of the binding of practically any molecules to DNA or to protein--DNA complexes (termed nucleoacidic protein or NAP). The antibiotic streptomycin sulfate, a soluble aminoglycoside, is used to precipitate the DNA after the ligand binding. Comparison of different sources and commercial batches of the antibiotic is described. Optimal conditions for precipitating DNA or NAP and the application of this method to the binding of the chick oviduct progesterone receptor to soluble NAP are described. The streptomycin method can be used with DNA molecules whose size ranges from 750 base pairs to greater than 50 000 base pairs. The method works with a DNA or NAP from a variety of sources, including synthetic homo- or heteropolymers. The precipitation of DNA or NAP by streptomycin occurs rapidly and has minimal effects on the steroid receptor complex or binding of the steroid receptor to DNA or NAP. The requirements and limitations of the method as well as the optimal conditions for binding of the progesterone receptor to DNA or NAP are described.

Animals↗
PubMed1983

Characterization of the glucocorticoid receptor in human skin.

Because of the profound importance glucocorticoids have in dermatologic therapy, we studied the glucocorticoid receptor in human skin. A cytosol fraction was prepared from frozen skin by homogenization and centrifugation. When reacted with [3H]dexamethasone, this cytosol contained saturable, low-capacity binding. The glucocorticoid binding was stabilized by a protease inhibitor, phenylmethylsulfonylfluoride, and by sodium molybdate and was destroyed by trypsin. Sedimentation analysis of the glucocorticoid binding protein showed an 8S to 4S transition in high salt, a property of many known steroid hormone receptors. The binding was steroid specific, supporting the conclusion that this binding protein was a glucocorticoid receptor. The receptor molecule had a frictional ratio of 1.60 and a Mr of about 226,000 under low-salt conditions (0.05 M KCl) and a frictional ratio of 1.86 and a Mr of about 100,000 under high-salt conditions (0.3 M KCl) consistent with a nonglobular, elongated molecule. Isoelectric focusing showed that the receptor had 2 molecular species with isoelectric points of approximately 5.8 and 7.5. Quantitation of receptor in human skin showed 4-7 times more receptors in the epidermis and papillary dermis than in the lower dermis and nearly equal numbers in epidermis and papillary dermis. The concentration of receptors varied in different anatomic areas, with male foreskin showing the highest concentration, followed by female face, breast, and abdominal skin. Interestingly, the concentration of glucocorticoid receptors also varied with age; the highest levels were present at the extremes of life and a significantly lower level at midlife.

Aging↗
PubMed1982

Comparison of estrogen receptors in human premenopausal and postmenopausal uteri: indication of biologically inactive receptor in postmenopausal uteri.

Analysis of the estrogen receptor (ER) from uteri of premenopausal and postmenopausal patients has largely been limited to quantitation. The present study examined the biologic activity of the receptors with regard to their nuclear binding capacities. A Scatchard analysis of myometrial cytosol indicated high-affinity and low-capacity estradiol binding (Kd = 10(-10 M). A 35% ammonium sulfate fraction of cytosol was characterized for steroid specificity, receptor concentration, isoelectric focusing patterns, and capacity of the ER to bind to nuclear acceptor sites in a cell-free assay. The ER from premenopausal uteri displayed an extensive but saturable binding to the nuclear acceptor sites. In contrast, the postmenopausal ER displayed minimal binding. These cell-free binding differences were supported by tissue culture studies. This difference may be accentuated by the fact that much of the receptor is bound by endogenous estrogen in the premenopausal uterine preparation, which would decrease the subsequent binding of the [3H]ER to nuclear sites. This receptor in the postmenopausal uterus is largely unbound and thus would not compete for nuclear sites. These studies suggest that a significant fraction of the ER in postmenopausal uteri is biologically inactive; for example, it cannot translocate and bind to nuclear acceptor sites. This loss in nuclear binding may represent a change in the ER occurring at menopause as a result of changing endocrine states.

Adult↗
PubMed1982

Evidence for two classes of chromatin-associated Epstein-Barr virus-determined nuclear antigen.

A new class of Epstein-Barr virus nuclear antigen (EBNA) was identified by the complement fixation assay. This new species of EBNA is more tightly bound to chromatin and was termed class II EBNA, as opposed to the more weakly associated species, class I EBNA. Preparations of this new antigen(s) specifically reduced absorption with the titer of anti-EBNA antibodies as determined by the anticomplement immunofluorescence assay. Therefore, the complement fixation antigens (class II EBNA) appear to be related to the classical EBNA (class I EBNA). The class I EBNA was found to focus at the same pH (4.6) as the soluble antigen found in the cytosol. The class II EBNA differed from the class I EBNA with regard to its overall charge, molecular size, antigenicity, and affinity for chromatin. The class II EBNA appeared to be a basic protein, based on its apparent pI of 9.2 and its binding to cation-exchange resins. It differed from histones with regard to its molecular size (molecular weight between 60,000 and 70,000) and its elution from hydroxylapatite chromatography. Steps were taken to prevent proteolysis and artifacts in the immunological assays and in the overall charge estimation of the new antigen by nonspecific basic histone protein-acidic protein interactions. Both class I and class II EBNA were identified by radioimmunoelectrophoresis on two-dimensional polyacrylamide gels with pI values of 5.0 and 8.5, respectively, and a molecular weight range of 60,000 to 70,000 for both. A lower-molecular-weight antigen identified by molecular sieve chromatography appeared to be due to interference by histones in the immunoassays since it was not observed by the two-dimensional gel electrophoresis. Further characterization of this class II EBNA is in progress.

Antigens, Viral↗
PubMed1982

Nonfunctioning progesterone receptors in the developed oviducts from estrogen-withdrawn immature chicks and in aged nonlaying hens.

The progesterone receptor (PR) in the oviducts of aged, nonlaying hens was compared to that in laying hens. The PRs in the mature oviducts of chicks that were diethylstilbestrol (DES) primed and of chicks with DES withdrawn were similarly analyzed for comparative purposes. The oviducts from the DES-withdrawn chicks and from aged nonlaying hens showed marked atrophy. The PR concentrations in the oviducts of DES-withdrawn chicks and of aged nonlaying hens were reduced to one half and one fourth those of controls, respectively. The oviduct atrophy and reduced PR concentrations in the nonlaying hen were not caused by decreased plasma estrogen since estrogen concentrations were similar in aged nonlaying hen and in the laying hens. The stabilities of the PR from laying and nonlaying hen oviducts were identical. However, analysis in vivo and in vitro revealed that the PR from the oviducts of DES-withdrawn chicks and of aged, nonlaying hens had lost the capacity for nuclear translocation and binding. This was accompanied in the DES-withdrawn chick by the inability of progesterone to alter RNA synthesis in the oviduct in vivo. This loss in nuclear binding capacity of PR in both animal models was accompanied by a loss of one of the molecular species of the PR (the B species). The loss of the B species differs from the loss of the A species that occurs during the winter in the domestic fowl. Thus, two types of regulation of the receptor may exist. The results suggest that biologically inactive receptors may explain the cessation of reproductive function in these aged animals.

Aging↗
PubMed1981

Evidence for an androgen receptor in the human placenta.

A steroid-binding protein was examined in human placental and nuclei which had several characteristics of an androgen receptor. It binds R 1881 (methyltrienolone), a steroid specific for androgen receptors, with high affinity (dissociation constant equals 1.3 to 50 nM) and low capacity (n equals 1.2 to 21 moles/mg of protein). The steroid-binding protein is found in the nuclei as well as the cytosol of the placenta but is not found in either the cytosol or the nuclei of fetal membranes; thus, it is tissue-specific. Among the natural steroids, 5 alpha-dihydrotestosterone competes most efficiently with R1881 for binding to the protein. Testosterone is the next best competitor. When the ranges of dissociation constants and concentrations of binding sites for placentas of various gestational periods are compared, not significant differences are observed between period of gestation and magnitude of binding constants.

Binding, Competitive↗
PubMed1981

Covalent binding of benzo[a]pyrene metabolites to DNA, RNA and chromatin proteins in the AKR mouse embryo cell-line.

A specific fraction from the nuclei of the AKR mouse embryo cell-line (fraction I) displayed a much greater localization of radioactivity compared to fraction II and III when the chemical carcinogen, [3H]benzo[a]pyrene (B[a]P) was incubated with the cells for 24 h. The radioactivity in fraction I consisted of both covalently and non-covalently bound metabolites. Isolation of the DNA, RNA and protein of fraction I revealed that 94% of the covalently bound radioactivity was to protein, 5% to RNA and 1% to DNA. Analysis of the fraction I proteins by SDS gel electrophoresis revealed that there was more radioactivity covalently bound to the larger proteins than to smaller proteins. Isoelectric focusing (IEF) of the purified proteins displayed two peaks of radioactivity, one at a pH of 5 and the other at 11. The former proteins bound more radioactivity per mass of protein than the latter proteins. Analysis of fraction I histones on acid urea polyacrylamide gels showed that the radioactivity coincided with histones H3 and H2B and low levels of radioactivity associated with histones H1, H2A and H4. Two significant peaks of radioactivity closely migrated near but did not co-migrate with histone H1. The distribution of the bound radioactivity is probably a reflection of the availability of the proteins to the reactive carcinogen metabolites. The possible binding of B[a]P metabolites to phosphorylated histones and to the high mobility of group (HMG) proteins 1 and 2 is discussed.

Animals↗
PubMed1981

Binding of [3H]benzo(a)pyrene metabolites to the AKR mouse embryo cell line nuclear proteins.

Nuclear proteins from AKR-2B mouse embryo cells incubated 24 hr with [3H]benzo(a)pyrene (1 microgram/ml; 10 microCi/ml) were purified to remove nucleic acids and characterized by isoelectric focusing and sodium dodecyl sulfate gel electrophoresis on polyacrylamide gels. Analysis of the nuclear proteins by isoelectric focusing revealed a distribution of protein in the pH gradient with two prominent bands of proteins, one focusing at pH 5 and the other at pH 11, both of which bound significant amounts of the carcinogen. The acid-soluble proteins from the nuclear preparation were analyzed by isoelectric focusing, and the results also revealed two peaks of protein and radioactivity at pH's of 5 and 11. Further analysis of the acid-soluble fraction on acid urea gels revealed that significant radioactivity was associated with proteins that migrated near but slower than histone H1 which may be the high-mobility group proteins 1 and 2. Radioactivity also comigrated with histones H3 and H2B. Low levels of radioactivity were associated with histone H1 and H4.

Animals↗