Biomedical subjects

T C Spelsberg

Publications and source records attributed to T C Spelsberg.

At least 127 records · Page 7Linked to original sources

PubMed1987

Characterization of the chromatin acceptor sites for the avian oviduct progesterone receptor using monoclonal antibodies.

Monoclonal antibodies (MAb) against the chromatin acceptor sites for the avian oviduct progesterone receptor were prepared with highly purified hen oviduct acceptor proteins reconstituted to hen DNA. Addition of the MAbs to a cell-free assay blocked progesterone receptor from chick oviduct (PRov) binding to native-like acceptor sites on nucleoacidic protein (NAP) representing a partially deproteinized chromatin, which has been shown to be enriched in these binding sites. However, the antibodies do not block PRov binding to pure DNA, nor do they affect the receptor itself. Estrogen receptor binding to NAP was not inhibited, supporting a receptor specificity of the PRov acceptor sites as reported previously from direct competition studies. These data support earlier studies showing that (1) the reconstituted PRov acceptor sites resemble the native sites, (2) the acceptor sites are receptor specific, and (3) the PRov binding sites of NAP are different from those of pure DNA. While some animal-species specificity in the PRov binding inhibition was observed, no tissue specificity was seen. Direct binding of the antibodies to native acceptor sites was demonstrated in an enzyme-linked immunosorbent assay (ELISA) system. The antibodies showed little recognition of free acceptor protein or DNA alone, indicating specificity for the protein-DNA complex. A partial evolutionary conservation of the nuclear acceptor sites for PRov was shown by the fact that about 50% of the inhibition seen with hen NAP was obtained with NAPs from several other species, and this partial cross-reactivity of the MAbs with the same NAPs from other animal species was also seen in the ELISA.

Animals↗

PubMed1987

Monoclonal antibodies against putative nuclear acceptor sites of the avian oviduct progesterone receptor.

Evidence from this and other laboratories has suggested that the nuclear binding sites (acceptor sites) for steroid receptors on chromatin involves chromatin protein-DNA complexes. A saturable high affinity receptor-dependent nuclear binding to these sites by isolated steroid receptor complexes has been reported. Addition of nonradiolabelled progesterone receptor from the chicken oviduct (PRov) successfully competes for the [3H]PRov binding to these acceptor sites in isolated chromatin or in nucleoacidic protein (NAP), a partially deproteinized chromatin enriched in these binding sites. This competition does not occur with pure DNA. This laboratory has isolated and enriched the chromatin proteins (acceptor proteins) involved in the nuclear acceptor sites for the avian oviduct PRov. Monoclonal antibodies against the nuclear acceptor sites for the PRov have been prepared using highly purified hen oviduct acceptor proteins reconstituted to hen DNA. Addition of the MAbs to a cell-free assay blocks PR binding to native oviduct chromatin as well as to NAP. However, the antibodies do not block PR binding to pure DNA nor do they affect the receptor itself. A partial animal species specifically was observed with the Ab inhibition of the PR binding, whereas no tissue specificity was seen. Direct binding of the antibodies to native acceptor sites was demonstrated using an ELISA system. The antibodies showed little recognition of free acceptor protein or DNA alone, indicating specificity for the protein-DNA complex. The partial evolutionary conservation of the nuclear acceptor sites for PR, as shown by the inhibition of PRov binding, was further supported by the partial crossreactivity of the MAbs with the NAPs from the same animal species using the ELISA. These data support earlier studies using PR binding assays showing that: 1) the reconstituted PR acceptor sites resemble the native sites; 2) the sites on whole chromatin and on NAP are similar; 3) the PR binding sites of chromatin and NAP are different from those of pure DNA; and 4) the nuclear acceptor sites for PR are different from those of the estrogen receptor. These results support a receptor specificity of the PR acceptor sites as reported previously using direct receptor competition studies.

Animals↗

PubMed1987

Nuclear acceptor sites for sex steroid hormone receptors in chromatin.

The steroid receptor interactions in vitro with specific acceptor sites composed of acceptor protein-DNA complexes fulfill many of the criteria of a physiologically significant binding system. Chromatin acceptor sites for many steroid receptors (especially for the progesterone and estrogen receptor) are specific since they are saturable and competitive with unlabelled receptors, have high affinity for the receptor, distinguish between functional and nonfunctional receptors and demonstrate target tissue specificity. Pure DNA as acceptor sites does not display many of these properties. Therefore, it is clear that certain chromatin proteins provide the necessary specificity for the acceptor sites for the steroid receptors. For the progesterone receptor in the chick oviduct, these nuclear sites appear to contain specific chromosomal proteins as well as specific DNA sequences. The substitution of other chromosomal proteins or the genomic DNAs from evolutionarily distant organisms results in a loss of the specific nuclear binding. The nuclear acceptor sites appear to be resistant to the DNase activity which is not characteristic of transcriptionally active domains of the genome. Further studies using the ovalbumin gene sequences from genomic clones also indicate that none of the sequences within this domain and the 3-k flanking regions appear to contain the specific acceptor sequences. These observations have led to development of a model suggesting that the steroid receptors bind to acceptor sites distant from the structural genes the steroids ultimately regulate. Neighboring these acceptor sites are regulatory genes which code for regulatory substances which in turn (as secondary messengers) regulate at great distances the expression of the structural gene. This model might better fit the sex steroids which require 1-2 h to measurably alter gene transcription, as opposed to the glucocorticoids which more rapidly alter gene expression.

Animals↗

PubMed1987

A nuclear binding assay to assess the biological activity of steroid receptors in isolated animal and human tissues.

This paper describes a nuclear binding assay (NB assay) which measures not only the presence of a steroid receptor in a tissue, but also the quantity of that receptor which is biologically active or functional, i.e. able to bind to nuclear acceptor sites. The assay involves the isolation viable cells from tissues and their incubation with an excess of radiolabeled steroid to encourage the activation and nuclear binding of all cellular receptors. The nuclei are isolated under conditions that remove unactivated (unbound) steroid-receptor complexes. This NB assay demonstrates, in both animal and human steroid target tissues, a saturable, tissue- and steroid-specific, and temperature- and time-dependent nuclear binding of radiolabeled steroids. These properties support a receptor-dependent nuclear binding of steroids. This assay is reproducible and requires relatively small amounts of tissue. The patterns of nuclear binding of the progesterone receptor, achieved with the assay in the avian oviduct model system, are shown to correlate with the nuclear binding of progesterone in vivo, the ability of the steroid to alter transcription, and the expression of a specific gene product, the protein avidin. The assay has been used to identify the existence of nonfunctional steroid receptors in endometrial and breast carcinomas. Therefore, this NB assay combined with the standard charcoal/hydroxylapatite methods of quantitating total cellular receptors should provide a means of assessing changes in the regulation of the biological activity of steroid receptors. Further, the assay should be useful to assess the ability of steroid analogs to properly activate their respective receptors for subsequent nuclear binding.

Animals↗

PubMed1987

A new assay to assess steroid-hormone responsiveness in head and neck cancer.

A new assay has been developed to predict the effectiveness of steroid-hormone therapy in various tumors. The Biopsy Nuclear Binding assay measures the amount of biologically active receptor that binds both steroid hormone and acceptor sites in the nucleus. This assay does not measure the entire receptor population, only those that are biologically active; therefore, it should more accurately predict the response to steroid-hormone therapy. We applied this assay effectively in breast and endometrial carcinoma. Preliminary studies have shown that 40% of patients with head and neck squamous-cell carcinomas have biologically active (nuclear bound) progesterone receptors. If nuclear binding predicts a remission with hormonal therapy, then the quality of life of appropriately selected patients could greatly improve.

Androgen Antagonists↗

PubMed1986

Nuclease resistance and the enrichment of native nuclear acceptor sites for the avian oviduct progesterone receptor.

High-affinity nucleoprotein acceptor sites for the avian oviduct progesterone receptor (PR) have been enriched by a combination of nuclease digestion and centrifugation. These enriched binding elements exhibited markedly enhanced PR binding on a per mass DNA basis compared to chromatin (20- to 25-fold) or dehistonized chromatin (4- to 5-fold). Electrophoretic analysis of the nuclease-resistant DNA showed that there is a set of DNA fragments of 100-150 base pairs that are protected from digestion. Excessive digestion resulted in smaller DNA fragments and a loss of PR binding activity. The PR binding was saturable using a crude receptor preparation and displayed a competition with the same receptor preparation that was labeled with nonradioactive progesterone. The enhanced binding was also demonstrable using highly purified receptor preparations that exhibit two classes of binding sites both of which are of high affinity and saturable as assessed by Scatchard analyses. These two high-affinity classes of binding sites are shown to be competed by unlabeled purified PR. The nuclease resistance of these nucleoprotein acceptor sites from chromatin is a property similar to the nuclear matrix binding sites suggesting a relationship between these two classes of nuclear acceptor sites.

Animals↗

PubMed1986

Monoclonal antibodies recognizing the nuclear binding sites of the avian oviduct progesterone receptor.

Monoclonal antibodies which block the binding of the avian oviduct progesterone receptor (PR) to nuclear acceptor sites have been prepared. We have previously shown that such acceptor sites consist of complexes of specific nonhistone proteins and DNA. The antigen was a reconstituted, enriched nuclear site for avian oviduct PR formed by reannealing a partially purified acceptor protein to pure hen DNA to reconstitute native-like acceptor sites. These reconstituted acceptor sites were then partially digested with deoxyribonuclease I and injected into BALB/c mice. The spleen cells were fused with NS-1 mouse myeloma cells. Hybridomas were then grown and tested for the ability of their culture media to 1) inhibit PR binding to avian oviduct nucleoacidic protein (NAP) which contains the native-like acceptor sites, but 2) to not inhibit PR binding to pure hen DNA. Three hybridoma clones produced ascites fluids which inhibited PR binding to intact oviduct chromatin and NAP but not to pure hen DNA. Control ascites fluids, prepared against other protein antigens, showed no inhibition of PR binding. The three positive ascites fluids contained low concentrations of immunoglobulin (0.3-0.5 mg/ml). The antibodies did not affect the stability of the receptor and did not interact with PR when analyzed by sucrose density gradient sedimentation. Direct binding of the antibodies to the NAP is shown by an enzyme-linked immunosorbent assay. The monoclonal antibodies display a partial species specificity with regard to the source of NAP in that PR binding to hen oviduct NAP was inhibited by greater than 90%, while PR binding to human uterine NAP was inhibited less than 40% by the antibodies. Further characterization of these candidate antiacceptor site monoclonal antibodies with regard to tissue, species, and steroid receptor specificities are underway.

Animals↗

PubMed1986

Estrogen regulation of the biological activity of the avian oviduct progesterone receptor and its ability to induce avidin.

During primary estrogen stimulation of chick oviduct development, estrogen withdrawal, or secondary estrogen treatment, changes in the oviduct progesterone receptor (PR) occur. The presence of estrogen appears to regulate not only PR concentration but also its biochemical activity, i.e. its capacity to bind to nuclear acceptor sites and alter RNA synthesis. This study reports that estrogen regulates the nuclear binding capacity of the PR even more rapidly than previously reported in fully developed oviducts of chicks that have been injected daily for 4 weeks with diethylstilbestrol (DES). Further, the nuclear binding capacity of the PR correlates with the ability of progesterone (P) to induce avidin protein concentrations in the oviducts in vivo. The PR concentration in the oviducts increases 2-fold within 8 h of the last injection and the decreases to a minimal value by 24 h. Injection of [3H]P into the chicks shows that the in vivo nuclear localization of the steroid increases almost 4-fold at 8 h, followed by a similar decrease to minimal values by 24 h. Cell-free nuclear binding assays, using PR isolated at various times after the last DES injection and oviduct nucleoprotein complexes, indicate that the capacity of the receptors to bind to nuclear acceptor sites is regulated by the estrogen. The enhanced nuclear binding capacity of the isolated PR increases to maximal values by 12-14 h after the last estrogen treatment and then begins to decrease to minimal values by 24 h. Similarly, the ability of P to induce in vivo avidin protein concentrations and to alter general RNA synthesis in the oviducts is reduced by 70% (of the estrogen non-withdrawn chick levels) by 24 h after the last estrogen injection. These changes over the 24-h period after the last DES treatment are not due to changes in the serum DES concentrations. The following 10-day period of estrogen withdrawal reveals a cyclic decaying pattern in the capacity of the PR for nuclear binding. The P induction of avidin and alteration of RNA polymerase II activity, using nuclear run-off experiments, also show a similar cyclic decaying pattern. By 6 days of estrogen withdrawal, the PR is incapable of any nuclear binding, and P cannot induce avidin protein concentrations in the oviducts. Serum DES concentrations over this 10-day period display only a gradual decay.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1985

Proteins that mask the nuclear binding sites of the avian oviduct progesterone receptor.

The binding of a steroid receptor to specific nuclear sites (i.e., nuclear acceptor sites) represents the immediate event preceding the steroid regulation of gene transcription. How the same steroid receptor regulates different genes in different tissues is unknown. Since a major fraction of the nuclear acceptor sites for a variety of steroid receptors has been reported to be masked in the chromatins of a variety of tissues, the differential expression of the nuclear acceptor sites may explain this regulation of different genes. In the avian oviduct, the removal of a subfraction of chromosomal non-histone proteins, termed CP-2, results in the unmasking of the nuclear acceptor sites for the progesterone receptor (PR). Further, the extent of masking of these nuclear acceptor sites for PR has been reported to vary during cytodifferentiation of the avian oviduct. This paper describes a method for the reconstitution of the masking of PR nuclear acceptor sites in the avian oviduct chromatin using a partially purified chromosomal protein fraction (CP-2b). The reannealling of the CP-2b fraction to unmasked avian oviduct chromatin (termed nucleoacidic protein or NAP) results in the "remasking" of about the same number of nuclear acceptor sites for PR as found in intact chromatin. Because some of the PR acceptor sites on the NAP cannot be remasked, these sites either must be protected from masking or not be recognized by the masking proteins. The masking activity apparently involves only protein(s) because the unmasking of acceptor sites can be achieved with protease but not ribonuclease activities and because the dissociated masking activity is destroyed only by proteases. The masking appears to be reversible because the reconstituted masked sites can again be unmasked. Preliminary purification and characterization of the masking activity in fraction CP-2b by molecular sieve chromatography indicate a heterogeneity of size with the activity eluting in a molecular weight range of from 60 000 to greater than 150 000. Whether the masking proteins prevent the binding of the progesterone receptor by directly binding the acceptor sites or by binding neighboring domains to condense the chromatin is unknown. It is speculated that the masking of acceptor sites may be responsible in part for determining the tissue-specific gene expression induced by steroids and/or may play a role in the unresponsiveness of certain human tumors containing steroid receptors.

Animals↗

PubMed1985

The androgen receptor in normal human foreskin. I. Stabilization and identification of two receptor subunits.

Use of glycerol and the protease inhibitor phenylmethylsulfonyl fluoride resulted in a fourfold increase in the yield of androgen receptor from human neonatal foreskin and a tenfold increase in stability of this receptor. The general physical properties of the androgen-binding component isolated from the cytosol of human neonatal foreskin were found to be consistent with those previously reported for androgen receptors. The synthetic androgen methyltrienolone (R1881) was bound with high affinity (a mean dissociation constant of 0.51 nmol/L) and low capacity (5.6 fmol/mg of protein); maximum binding required 4 hours. Affinity of the receptor was highest for R1881 followed by dihydrotestosterone, testosterone, and progesterone. 17 beta-Estradiol showed little or no competition. The receptor exhibited a change of sedimentation coefficient from 8S to 4S upon treatment with 0.1 mol/L potassium chloride. Isoelectric focusing demonstrated the existence of two molecular species with apparent isoelectric points of 7.2 and 5.7.

Binding, Competitive↗

PubMed1985

A rapid, sensitive assay for analyzing defects in metabolism of steroids in normal and diseased human tissues.

In this report, we describe a simple, rapid biopsy-steroid metabolism assay that is applicable to any steroid tissue system. It consists of mincing the sample, tissue culture incubation, extraction of the steroids and their metabolites from the tissue, and fractionation of the metabolites by high-pressure liquid chromatography (HPLC). Radioimmunoassay is used to verify the elution patterns of certain steroids. Studies of the metabolism of [3H]progesterone in the avian oviduct showed the generation of metabolites that eluted from the HPLC system in a pattern similar to androgens, estrogens, and glucocorticoids. Studies of the metabolism of [3H]testosterone in the human foreskin showed the production of metabolites that eluted from the HPLC system similar to 5 alpha-dihydrotestosterone and 5 alpha-androstane-3,17-dione (androstanedione) from the parent [3H]testosterone. In the production of the metabolite that eluted as androstanedione in samples of foreskin from normal subjects, a significant (P less than 0.001) correlation was found with the age of the donor. Preliminary studies of patients with hypospadias showed a significant (P less than 0.005) decrease in the production of "androstanedione" compared with that in normal subjects. Because of the wide range in rates of metabolism of testosterone in the patients with hypospadias, the effect of age does not seem to be the sole determinant of a low rate of metabolism in these patients. Some samples of hypospadias foreskin had a decreased rate of production of a metabolite that eluted as dihydrotestosterone in comparison with normal foreskin, even when the age of the donor was considered. The assay described herein should be applicable to any surgical biopsy specimen and to all steroids.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Evidence for specific DNA sequences in the nuclear acceptor sites of the avian oviduct progesterone receptor.

Recent studies have shown that saturable high-capacity nuclear binding sites (termed acceptor sites) for the avian oviduct progesterone receptor can be reconstituted by rehybridizing a specific oviduct chromatin protein fraction (CP-3) to pure hen DNA to generate a reconstituted nucleoacidic protein (NAP). Only a limited number of acceptor sites can be generated on hen DNA even at high protein/DNA ratios. This suggests the existence of a limited number of specific sequences in the avian genome that can participate in the acceptor sites. The studies presented in this paper show a specificity as to the source of DNA that can generate acceptor sites using hen oviduct CP-3 protein. The acceptor protein binds to all DNAs but generates acceptor sites only on DNAs from certain animals. The acceptor sites for the progesterone receptor, generated with heterologous mammalian DNAs and the avian oviduct CP-3 fraction, show saturation not only in number of acceptor sites generated on the DNAs but also in progesterone receptor binding. Binding to these sites is also receptor dependent. Using oviduct receptors from particular physiological states of the birds wherein the receptors do not bind to nuclear sites in vivo, it was found that the cell-free binding to these heterologous complexes of hen CP-3 protein and DNA from another species, termed heterologous NAP, is similarly absent. Thus, the cell-free binding to the native oviduct NAP and the heterologous NAP markedly resembles the nuclear binding in vivo. Interestingly, synthetic DNAs rich in adenine and thymine, but not those rich in guanine and cytosine, are capable of generating acceptor sites. Species-specific DNA sequences, as well as specific chromatin proteins, therefore, appear to be involved in the nuclear acceptor sites for the avian oviduct progesterone receptor. The DNA sequences appear to be conserved throughout most of the vertebrates but not among nonvertebrates as are the steroid hormones and their receptors. The exact numbers and distributions of these sequences in the avian genome are not known.

Animals↗

PubMed1985

Problems and artifacts in the identification of nuclear acceptor sites for the avian oviduct progesterone receptor.

Steroid receptor complexes bind to specific nuclear acceptor sites on chromatin to induce alterations in gene expression. This paper describes many factors regulating the nuclear binding of the avian oviduct progesterone receptor. Some of these factors occur in vivo and appear to be involved in natural regulatory processes, whereas other factors are detected only in cell-free in vitro binding assays and affect the amount of nonspecific binding. Such variations in nonspecific binding in vitro could cause misinterpretation of cell-free nuclear binding data and could lead to erroneous identification of nuclear acceptor sites for other steroid receptors in other animal tissues.

Animals↗

PubMed1984

Androgen insensitivity syndrome: gonadal androgen receptor activity.

To determine whether abnormalities of the androgen receptor previously observed in skin fibroblasts from patients with androgen insensitivity syndrome also occur in the gonads of affected individuals, androgen receptor activity in the gonads of a patient with testicular feminization syndrome was investigated. Using conditions for optimal recovery of androgen receptor from human testes established by previous studies, we detected the presence of a high-affinity (dissociation constant = 3.2 X 10(-10) mol/L), low-capacity (4.2 X 10(-12) mol/mg DNA), androgen-binding protein when tritium-labeled R1881 was incubated at 4 degrees C with nuclear extracts from the gonads of control patients or from a patient with testicular feminization syndrome but not when incubated at 37 degrees C. Thus this patient has an androgen receptor with a temperature lability similar to that of receptors from normal persons.

Adolescent↗

PubMed1984

Evidence for an androgen receptor in human testis.

The present study identified and characterized an androgen-binding protein in human testicular tissue. Human testes were homogenized in dilute Tris buffer containing thioglycerol, phenylmethylsulfonylfluoride, and molybdate. The supernatant (termed cytosol) was incubated with radiolabeled androgen methyltrienolone (tritium-labeled R1881), and nonspecific binding was determined by adding 100-fold excess of unlabeled R1881 together with [3H]R1881 to cytosol. Specific binding with saturation at 24 hours was observed. Scatchard analysis of the specific binding with the use of increasing concentrations of [3H]R1881 alone and [3H]R1881 plus 200-fold excess unlabeled R1881 demonstrated a high-affinity (dissociation constant = 2.18 X 10(-10) mol/L), low-capacity (2924 molecules per cell) class of binding sites. A second class of lower-affinity sites was identified with a dissociation constant equaling 1.2 X 10(-8) and 26,300 molecules per cell. The bulk of the higher-affinity class of sites was precipitated at 35% ammonium sulfate. In competitive binding assays, dihydrotestosterone and testosterone greatly diminished binding to this high-affinity class of sites. Progesterone also diminished binding but to a lesser degree. Estradiol, estriol, and estrone failed to compete for these sites. Analysis of the receptor, using sucrose gradients, revealed a major peak in the 4S region and a small peak at 8S. A similar high-affinity (dissociation constant = 4.28 X 10(-9), low-capacity (4860 molecules per cell) binding protein was identified in purified nuclei. Binding to nuclear chromatin was demonstrated in the cell-free binding assay, and nuclear binding was further illustrated in the biopsy assay of intact tissue, suggesting translocation in vivo. These properties are characteristic of the androgen receptor and suggest that human testis is a target tissue for androgen, as has been found in animal tissue.

Aged↗

PubMed1984

Reconstitution of nativelike nuclear acceptor sites of the avian oviduct progesterone receptor: evidence for involvement of specific chromatin proteins and specific DNA sequences.

A specific fraction of avian oviduct chromosomal proteins can be reannealed to pure avian DNA to reconstitute nativelike specific nuclear binding sites (acceptor sites) for the oviduct progesterone receptor (PR). These specific nuclear binding sites represent the difference between the binding to the reconstituted NAP and that to pure DNA. The specific fraction of chromatin protein which contains the acceptor activity, fraction CP-3, is very tightly bound to hen DNA in a complex termed nucleoacidic protein (NAP). Removal of the CP-3 fraction from NAP results in a loss of specific PR binding sites. Resins containing chromatin adsorbed to hydroxylapatite are used as a rapid method to isolate the CP-3 fraction. Reconstitution of the CP-3 fraction to DNA by the described method involving a regressing gradient of 6-0 M guanidine hydrochloride (Gdn-HCl) results in a reconstituted NAP which displays specific PR binding sites identical with those in native (undissociated) NAP and whole chromatin. Optimal conditions and potential problems for reconstituting these nucleoproteins are described. Only partially purified receptor preparations were used in these cell-free binding analyses since they have been shown to bind with similar properties and patterns as the nuclear binding in vivo. Therefore, the binding of PR to the reconstituted NAPs was demonstrated to be receptor dependent, saturable, and of high affinity. Further, the pattern of binding to the reconstituted sites mimics those which are observed in vivo. Thus, nonfunctional receptors that cannot translocate and bind to the nuclear acceptor sites in vivo also failed to bind to the acceptor sites on the reconstituted NAPs generated by the acceptor proteins. In contrast, the binding to pure DNA does not reflect these receptor differences in receptor bindings. Specific binding of PR to reconstituted NAP can be reversed by again removing the protein fraction. Moreover, the specific binding can be destroyed by proteases and protected by protease inhibitors, indicating that acceptor activity is proteinaceous in nature. The reconstitution of the activity is both a concentration-dependent and time-dependent process. During the reconstitution, acceptor activity appears to reconstitute on the DNA when the Gdn-HCl concentration reaches 2.0 M. By use of the reconstitution method as an assay for acceptor activity, the activity in the CP-3 fraction was shown by molecular sieve chromatography to elute in a relatively broad molecular weight range between 13 000 and 25 000. The activity also focuses in isoelectric focusing resins with apparent pI's of 5.2 and 6.4.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1984

Regulation of nuclear binding of the avian oviduct progesterone receptor. Changes during estrogen-induced oviduct development, withdrawal, and secondary stimulation.

The progesterone receptors from various stages of estrogen induced oviduct development, estrogen withdrawal, and secondary stimulation with estrogen were examined. The progesterone receptors were characterized for their biological function (i.e. capacity for nuclear translocation, nuclear binding, and effects on RNA polymerase II activity) as well as certain physical properties. The progesterone receptors from the undeveloped or partially developed oviducts (0 to 8 days of estrogen treatment) displayed little or no nuclear translocation and binding in vivo or in vitro. Similarly, progesterone showed little or no effect in vivo on RNA polymerase II activity at the early stages of development. As development progressed from 8 to 12 days of estrogen treatment, the above parameters rapidly increased to maximal levels and plateaued through day 23 of estrogen treatment. A marked decrease in these parameters occurred within 1 day of estrogen withdrawal. The reverse series of events occurred during secondary estrogen stimulation of 10-day-old withdrawn chicks. While the receptor concentrations increased rapidly to maximum values by 2 days of restimulation, receptor function did not return until day 4. Similarly, the effects of progesterone on RNA polymerase II activity reached maximal values by day 4. The progesterone receptor isolated from oviducts during development, estrogen withdrawal, and restimulation, displayed similar patterns of cell-free binding to chromatin and nucleoacidic protein as that observed in vivo supporting the nativeness of the in vitro binding assay. In contrast, the cell-free binding of these same progesterone receptor to pure DNA were not similar to the in vivo binding, i.e. no patterns (differences) in progesterone receptor binding were observed. These data support that protein DNA complexes and not pure DNA represent the native acceptor sites for oviduct progesterone receptor. Comparison of the progesterone receptor between the functional and nonfunctional states revealed no differences in the steroid affinity for the receptor, in the apparent pI of the species, or in the sedimentation of the receptor under high salt conditions. However, the nonfunctional receptors consistently displayed a deficiency in one of the two monomer molecular species (the B species) as determined by isoelectric focusing. These results suggest that both monomer species of progesterone receptor are required for biological activity. Interestingly, the 7S "aggregate" species of the progesterone receptor was constantly detected even when only one of the monomer species was present.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗

PubMed1984

The isolation of large quantities of undamaged cellular organelles and cytosolic enzymes using a low-shear continuous tissue homogenizer.

There is often a need to isolate large quantities of subcellular components such as membrane-coated organelles (e.g., nuclei, lysosomes, and mitochondria), cell membranes, and soluble (cytosolic) proteins. Instruments which can homogenize relatively large masses of tissue, primarily those with rapidly rotating blades and cylinders, are excessively vigorous, often resulting in damaged and/or low yields of the subcellular components. This paper describes procedures for obtaining high yields of undamaged subcellular components using a continuous bulk tissue homogenizer which performs with low shear (the low-shear continuous homogenizer or LSC). This homogenizer is simple in operation, durable and can be used with a variety of tissues. Fibrous tissues are more difficult to homogenize using this instrumentation and require a premincing to small pieces (0.2 to 1.0-cm diam) followed by filtration through 2-4 mesh (two to four apertures per inch). Methods for bulk preparations with enhanced recoveries of undamaged nuclei, and a typical soluble multimeric enzyme, phosphofructokinase, are presented. Electron microscope views of the homogenates show the preserved state of the other subcellular components. The LSC homogenizer requires less physical effort with no "hands on" operation and thus is safer. This homogenizer requires less homogenization time compared to the smaller, hand-held Potter-Elvehjem-type homogenizers. Operations requiring low temperature can be performed at room temperature as long as the continuously passing homogenate solutions are kept chilled.

Animals↗