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R W Harrison

Publications and source records attributed to R W Harrison.

At least 91 records · Page 5Linked to original sources
PubMed1987

Glucocorticoid receptor phosphorylation, transformation, and DNA binding.

Glucocorticoid receptors were isolated by immunoadsorption from cytosol of L cells that were cultured for 18 h in the presence of [32P]orthophosphate, and the phosphorylation state of the receptor was examined before and after transformation to the DNA-binding state. Temperature-mediated transformation of the glucocorticoid receptor under cell-free conditions results in no change in receptor size or degree of phosphorylation. When cytosol containing transformed receptors is incubated with DNA-cellulose, 30-50% of the receptors are able to bind to DNA and the remainder do not bind to DNA. Both the heated receptors that bind to DNA and the receptors that do not bind to DNA are phosphorylated to the same degree. When intact cells containing 32P-labeled receptors are incubated for 2 h at 0 degree C with triamcinolone acetonide and then for 20 min at 37 degrees C in the presence of the hormone, 80% of the receptor becomes tightly associated with the nucleus in a manner that is both temperature-dependent and ligand-dependent. Approximately 80% of the nuclear-bound receptor is extracted with 0.4 M NaCl. Both the cytosolic receptor from cells incubated at 0 degree C and the salt-extracted nuclear receptor from cells incubated at 37 degrees C have been resolved by immunoadsorption to protein A-Sepharose with the BuGR1 monoclonal antibody and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting and autoradiography of the immunoblots. In addition, direct measurements of the amounts of 32P contained per unit of receptor protein were performed for receptors transformed both in the intact cell and in cell-free lysates. The results demonstrate that the untransformed receptor and the nuclear-bound transformed receptor are labeled with 32P to the same extent.

Animals↗
PubMed1987

Ultrastructural localization of glucocorticoid receptor (GR) in hypothalamic paraventricular neurons synthesizing corticotropin releasing factor (CRF).

Corticotropin releasing factor (CRF) synthesizing neurons, located in the hypothalamic paraventricular nucleus (PVN), are the main central regulators of the pituitary-adrenal cortex endocrine axis. The hormone production and release of CRF-synthesizing neurons is regulated by neuronal messages and feedback action(s) of glucocorticoids secreted by the adrenal gland. In order to characterize the latter mechanism, glucocorticoid receptor (GR)-immunoreactive (IR) sites were studied in hypothalamic paraventricular neurons of intact, long-term adrenalectomized, and adrenalectomized plus glucocorticoid treated animals, by means of ultrastructural immunocytochemical labelling. In intact animals, glucocorticoid receptor immunoreactivity was found predominantly in the nuclei of parvocellular neurons. Following adrenalectomy GR-immunoreactivity was localized in the cytoplasm of the cells, and there was a concomitant disappearance of the label from the nuclei. After corticosterone administration to adrenalectomized animals, GR-IR sites were again concentrated within the cell nuclei. Immunocytochemical double labelling studies performed on adrenalectomized plus corticosterone-replaced animals demonstrated glucocorticoid receptor-IR sites in the cell nuclei of parvocellular paraventricular neurons that expressed CRF-immunoreactivity in their cytoplasm. These ultrastructural data indicate that the intracellular location of glucocorticoid receptor is dependent on the availability of glucocorticoids by the neurons. The simultaneous expression of GR- and CRF-immunoreactivity in parvocellular paraventricular neurons supports the concept of a direct feedback action of glucocorticoids upon CRF-synthesizing neurons.

Animals↗
PubMed1987

Analysis of the disruptive action of an epididymal protease and the stabilizing influence of molybdate on nondenatured and denatured glucocorticoid receptor.

The nucleomyofibrillar fraction of epididymides from sexually mature rabbits contains a novel leupeptin-sensitive protease that disrupts the oligomeric conformation of cytosolic estrogen and progesterone receptors, and molybdate inhibits this process. In this report we used the AtT-20 cell glucocorticoid receptor as substrate and performed analyses under nondenaturing vs. denaturing conditions to further investigate the effects of the epididymal protease and molybdate on steroid receptor structure. Analysis on low salt sucrose gradients indicated that the protease partially converted the oligomeric (9-10S) glucocorticoid receptor to several more slowly sedimenting forms (3-7S), and this effect was not observed in the presence of molybdate. Paradoxically, gradient analysis under high salt conditions revealed that the protease induced a discrete, quantitative and molybdate-insensitive conversion of the 4-5S steroid-binding subunit to a 3S form. Further studies were done using denaturing polyacrylamide gel electrophoretic analysis of receptor that had been labeled covalently with [3H]dexamethasone 21-mesylate and partially purified by DNA/cellulose chromatography. At 0-4 C, the protease cleaved the steroid-binding subunit (mol wt, 96,900) of the receptor to a single steroid-labeled fragment (mol wt, 42,600). Under these conditions, digestion was complete within 30-60 min and was inhibited by leupeptin, but was unaffected by thiol-reactive reagents or molybdate. The epididymal protease and alpha-chymotrypsin produced steroid-labeled receptor fragments that were indistinguishable in size, shared an epitope recognized by our BuGR-2 monoclonal antibody, and retained DNA-binding activity. Despite the apparent similarity of these two enzymes, they are distinct, since the chymotrypsin-dependent cleavage event was not inhibited by leupeptin. These studies show that the epididymal protease attacks a site on the steroid-binding subunit of glucocorticoid receptors as well as estrogen and progestin receptors. It also appears that the cleavage site is situated close to that most readily attacked by alpha-chymotrypsin. Finally, our data provide independent confirmation of a recent report indicating that molybdate ions interact directly with the cytosolic steroid receptor to stabilize its oligomeric structure even after proteolysis within the steroid-binding subunit.

Animals↗
PubMed1987

Benefit-risk considerations in preventive treatment for tuberculosis in elderly persons.

Of 2135 elderly residents of nursing homes in Arkansas (mean age, 79.4 years) who have been treated with isoniazid for prevention of tuberculosis, data from 1935 were suitable for analysis. About 12 months of therapy was successfully completed in 1600 persons. Therapy could not be completed in 84 persons (4.4%) because of incipient hepatic toxicity and in 116 (6.0%) because of other types of drug intolerance. Although 135 persons (7.0%) died during the course of therapy, no evidence was found that isoniazid contributed to any death. The ratio of benefit (reduction of risk for tuberculosis) to risk (for nonfatal isoniazid-related hepatitis) was clearly favorable in persons who had definite conversions (1.6 for women, 3.4 for men) but less so for persons who had tuberculin reactions of unknown duration and for persons with minor increases in size of tuberculin reaction (less than 12 mm increase from an initially negative reaction).

Aged↗
PubMed1986

Immunochemical characterization of the rat kidney glucocorticoid receptor. The role of proteolysis in the formation of corticosteroid binder IB.

Glucocorticoid receptors of rat kidney and liver were compared by physicochemical and immunochemical methods to investigate the role of proteolysis in the formation of corticosteroid binder IB. Kidney cytosol prepared in the presence of sodium molybdate contained receptor forms comparable to rat liver glucocorticoid receptor; [3H]triamcinolone acetonide-labeled receptors eluted from Sephacryl S-300 as a multimeric 6.1 nm component in the presence of molybdate and as a monomeric 5.7 nm component in the absence of molybdate. Both forms were recognized by the monoclonal antibody BUGR-1 which was raised against rat liver glucocorticoid receptor. When kidney cytosol was prepared in the absence of molybdate, labeled receptor complexes eluted from Sephacryl S-300 as a 5.8 nm component in the presence of molybdate. However, in the absence of molybdate, the receptor eluted as a smaller 3.4 nm component which was identical with the size of activated kidney glucocorticoid receptor chromatographed in either the presence or absence of molybdate. The 3.4 nm activated kidney glucocorticoid receptor did not bind to DEAE-cellulose under conditions where activated liver receptor was retained. These properties of the activated kidney receptor are characteristic of corticosteroid binder IB. Incubation of the activated kidney receptor complex with BUGR-1 resulted in a shift in apparent Stokes radius from 3.4 nm to 5.4 nm, indicating immunochemical similarity with rat liver receptor. Identification of the immunoreactive receptor subunit by Western blotting demonstrated that kidney cytosol prepared in the presence of molybdate contained a major 94-kDa immunoreactive component which co-migrated with rat liver glucocorticoid receptor, while cytosol prepared in the absence of molybdate contained principally a 44-kDa immunoreactive species. These results suggest that corticosteroid binder IB can be generated by in vitro proteolysis and does not represent a polymorphic form of the glucocorticoid receptor.

Animals↗
PubMed1986

Molybdate-stabilized nonactivated glucocorticoid-receptor complexes contain a 90-kDa non-steroid-binding phosphoprotein that is lost on activation.

We have used a monoclonal antibody to purify glucocorticoid-receptor complexes from WEHI-7 mouse thymoma cells. Molybdate-stabilized, nonactivated complexes were found to contain two distinct proteins which could be separated by polyacrylamide gel electrophoresis under denaturing and reducing conditions. One of the proteins, 100 kDa, was labeled when cytosol was incubated with the affinity ligand [3H]dexamethasone 21-mesylate. The second protein, 90 kDa, was not labeled. Several lines of evidence, including Western blot analysis of purified nonactivated complexes, indicate that only the 100-kDa protein is directly recognized by the antibody. The 90-kDa protein appears to be purified as a component of the nonactivated complex due to noncovalent association with the 100-kDa protein. Both the 100-kDa and 90-kDa components of the nonactivated complex become labeled with 35S when cells are grown in medium containing [35S]methionine. Using cells labeled in this manner, we have shown that activated (i.e. DNA-binding) cytosolic complexes, formed by warming either in intact cells or under cell-free conditions, contain only the 100-kDa protein. Complexes extracted from nuclei of warmed cells similarly contain only the 100-kDa protein. These results indicate that the 100-kDa and 90-kDa components of nonactivated complexes separate upon activation. Purification of nonactivated complexes from cells grown in medium containing [32P]orthophosphoric acid indicates that both the 100-kDa and 90-kDa components are phosphoproteins which can be labeled with 32P. Therefore, resolution of the two proteins will be essential in order to determine whether the receptor is dephosphorylated on activation.

Animals↗
PubMed1986

Staphylococcal alpha-toxin: a structure-function study using a monoclonal antibody.

A monoclonal antibody (A-Tox-653.1) selected for its reactivity in a dot immunoblot assay with denatured staphylococcal alpha-toxin has been isolated and its capacity to block the hemolytic and lethal activities of alpha-toxin measured. In addition, 'reactivity with monomer, hexamer, 125I-monoiodinated and CNBr peptides of alpha-toxin was studied. In all cases the reactions of the monoclonal antibody were compared to those obtained with anti-alpha-toxin rabbit hyperimmune serum. We find that while both the monoclonal antibody and the rabbit antiserum react with all forms of alpha-toxin, only the rabbit antiserum blocks hemolytic or lethal activity. Further, the rabbit antiserum reacts with CNBr fragments IV, V ad VII, whereas the monoclonal antibody reacts only with the carboxy terminal CNBr peptide VII. We conclude that, in solution, the carboxy terminal segment of alpha-toxin is relatively free and reaction with the monoclonal antibody neither impedes its binding to the specific receptor on the membrane nor interferes with formation of the hexamer complex.

Animals↗
PubMed1986

The use of positron emission tomography in pion radiotherapy.

The radioactive debris produced by pion radiotherapy can be imaged by the technique of Positron Emission Tomography (PET) as a method of non-invasive in situ verification of the pion treatment. This paper presents the first visualization of the pion stopping distribution within a tumor in a human brain using PET. Together with the tissue functional information provided by the standard PET scans using radiopharmaceuticals, the combination of pion with PET technique can provide a much better form of radiotherapy than the use of conventional radiation in both treatment planning and verification.

Brain Neoplasms↗
PubMed1985

Monoclonal antibody to the rat glucocorticoid receptor. Relationship between the immunoreactive and DNA-binding domain.

The region of the glucocorticoid receptor that reacted with a monoclonal antibody (BUGR-1) was identified. In order to identify the immunoreactive region, the rat liver glucocorticoid receptor was subjected to limited proteolysis; immunoreactive fragments were identified by Western blotting. The monoclonal antibody reacted with both the undigested Mr approximately 97,000 receptor subunit and a Mr approximately 45,000 fragment containing the steroid-binding and DNA-binding domains. Digestion by trypsin also produced two steroid-binding fragments of Mr approximately 27,000 and 31,000 which did not react with the antibody and an immunoreactive Mr approximately 16,000 fragment. This Mr approximately 16,000 fragment was shown to bind to DNA-cellulose, indicating that it contained a DNA-binding domain of the receptor. The undigested receptor must have steroid associated with it to undergo activation to a DNA-binding form. However, the Mr approximately 16,000 immunoreactive fragment binds to DNA-cellulose even if it is obtained by digestion of the steroid-free holoreceptor which does not itself bind to DNA.

Animals↗
PubMed1985

Characterization of wild type and mutant glucocorticoid receptors from rat hepatoma and mouse lymphoma cells.

Using a combination of immunological blotting techniques and hormone affinity labeling, we have characterized the glucocorticoid receptors present in wild type and mutant rat hepatoma (HTC) and mouse thymoma (S49 and WEHI7) cells. Mutant HTC and WEHI7 cells of the receptorless phenotype, which contain greatly reduced amounts of glucocorticoid hormone binding activity, show parallel decreases in immunoreactive material using a monoclonal antibody raised against the rat liver glucocorticoid receptor. This indicates that these receptorless mutant cells harbor defects in either the production or accumulation of receptor protein. Quantitation of immunoreactivity and hormone binding activity present in wild type and mutant S49 cells indicates that these cells contain significantly more immunoreactive material than hormone binding activity. We conclude that S49 cells produce, in addition to their well characterized wild type or mutant receptors, a mutant receptor from a second allele which is of wild type size, is immunologically reactive, but is unable to bind hormone. The S49 mutant cell line nti (nuclear transfer increase) contains a glucocorticoid receptor which has a molecular weight of 40,000, while the wild type receptor has a molecular weight of 94,000. Affinity labeling of glucocorticoid receptors in nti cells with [3H]dexamethasone mesylate indicates that nti cells do not contain wild type sized precursor molecules which bind hormone, nor do they contain immunoreactive fragments of a molecular mass smaller than 94 kDa. It is proposed that the 40-kDa nti receptor is produced as a truncated protein most likely resulting from a nonsense mutation or from a truncated messenger RNA.

Affinity Labels↗
PubMed1985

In vitro and in vivo studies of the TRIUMF pion therapy beam.

Patient treatments at TRIUMF (Tri-University Meson Facility, Vancouver, B. C.) use a moving spot raster scan technique where the pion range is modulated in depth for each position of the moving spot. The spot scans in a stepwise fashion and can produce any desired field shape. This approach provides very good dose uniformity across the treatment field and allows maximum flexibility in shaping the treatment volume. Survival of cultured cells has been used as a biological dosimeter to test the isoeffectiveness of the pion dose distributions, which must be shaped in depth to compensate for the depth-dependent LET distribution. Isoeffectiveness across the treatment field has also been verified using this system, which involves irradiating cells supported in a gelatin matrix. The response of pig skin to pion irradiation at TRIUMF has provided a check on the in vivo RBE for acute effects derived from our earlier studies with mouse foot. In addition, the pig skin reactions have been followed for several months to assess the later dermal response. The RBE of our pion beam relative to 270 kVp X rays is approximately 1.5 for both the acute epidermal and the later dermal responses.

Animals↗
PubMed1984

Relative potencies of histamine H1-agonists on guinea-pig tracheal smooth muscle.

The relative potencies of a series of histamine H1-agonists in causing contraction of spirally cut strips of guinea-pig trachea, measured in the presence of cimetidine, indomethacin, methylatropine and propranolol, were similar to those reported on other guinea-pig tissues. However, whereas 2-methylhistamine and N alpha,N alpha-dimethylhistamine are apparently partial agonists in potentiating the adenosine-stimulated accumulation of cyclic AMP in slices of guinea-pig cerebral cortex, on tracheal spirals they are full agonists producing the same maximum contraction as histamine.

Animals↗
PubMed1984

Characterization of a monoclonal antibody to the rat liver glucocorticoid receptor.

The rat liver glucocorticoid receptor was partially purified and used to immunize a BALB/c mouse whose splenic lymphocytes were fused with the nonsecreting myeloma cell line P3-AgX-653. The fusion products were selected in HAT (hypoxanthine, aminopterine, and thymidine) medium and a stable antibody-producing clone designated BuGR1 obtained from 1 of 81 positive wells. Immunological specificity for the receptor was confirmed by sucrose density gradient analysis when the sedimentation constant of the specifically labeled receptor was altered by reaction with the antibody and by Western blot analysis, which showed that the BuGR1 antibody detected a single band with a mobility (mol wt, approximately 95,000) identical to that of the [3H]dexamethasone 21-mesylate-labeled rat glucocorticoid receptor. Similar sucrose gradient and Western blot experiments showed that the BuGR1 antibody reacted with the mouse glucocorticoid receptor. BuGR1 is a stable hybridoma producing an antibody which detects loci common to both rat and mouse glucocorticoid receptors.

Animals↗
PubMed1983

Titratable effects of p-chloromercuriphenyl sulfonate, a thiol-attacking reagent, on glucocorticoid receptor binding.

Cytosol prepared from cultured AtT-20 mouse pituitary cells or mouse liver was treated with concentrations of p-chloromercuriphenyl sulfonate (PCMPS) which reduced but did not abolish receptor-binding activity. Scatchard analysis of triamcinolone acetonide binding to the treated cytosol showed that the PCMPS effect was caused by a reduction of binding affinity with little effect on the apparent binding site concentration. The effect on affinity was dose-dependent. Binding specificity appeared unaffected since the relative abilities of triamcinolone acetonide, dexamethasone, cortisol, progesterone, and corticosterone to compete with labeled triamcinolone were similar at various PCMPS concentrations which caused a progressive reduction of detectable cytosol binding. The PCMPS effect was reversible since cytosol treated with up to 200 microM PCMPS followed by dithiothreitol 15 min later showed nearly complete recovery of binding sites (62-100%). The possibility that several sulfhydryl groups were involved in this phenomenon was further explored in experiments using AtT-20 cytosol labeled with [3H]dexamethasone-mesylate, a glucocorticoid affinity label which binds covalently to sulfhydryl groups. Chromatography of dexamethasone-mesylate labeled receptor on a sulfhydryl affinity column resulted in binding, indicating that the receptor had at least two sulfhydryl groups, one bound to the mesylate moiety of the steroid and the other capable of binding to the affinity column.

4-Chloromercuribenzenesulfonate↗
PubMed1983

Evidence for carrier-mediated transport of glucocorticoids by human placental membrane vesicles.

Glucocorticoid uptake by isolated placental membrane vesicles has been studied in an attempt to identify a membrane-mediated carrier mechanism. A preliminary communication from this laboratory has reported that uptake of the glucocorticoid corticosterone by these vesicles was a time-dependent, saturable, osmotically sensitive process (Fant, M.E., Harbison, R.D. and Harrison, R.W. (1979) J. Biol. Chem. 254, 6218-6221), but did not conclusively demonstrate a carrier mechanism. Further studies of labeled corticosterone uptake by placental vesicles are described herein which indicate that steroid uptake by these vesicles is a carrier-mediated process. We found that corticosterone uptake was temperature-sensitive, and an apparent phase-transition effect on the rate of uptake was seen to occur at approximately 16 degrees C. Treatment of the vesicles with phospholipase A2 and the sulfhydryl group attacker, p-chloromercuriphenylsulfonate, inhibited corticosterone uptake. In contrast to our previous findings in intact cells, neuraminidase treatment of membranes did not inhibit steroid uptake, perhaps indicating a species variation. Lastly, it was possible to show that corticosterone movement across the membrane exhibited countertransport, a phenomenon common only to carrier-mediated transport mechanisms. These studies show that placental vesicles accumulate corticosterone by a carrier-mediated mechanism.

Aminoisobutyric Acids↗
PubMed1983

Cellular factors which modulate hormone responses: glucocorticoid action in perspective.

Virtually all glucocorticoid effects are mediated through changes in gene transcription. Steroid binding to the receptor and nuclear binding of the steroid-receptor complex are pivotal events in this process, but may be modified by the ability of a given steroid to traverse the cell membrane or cause receptor activation. The physical nature of the receptor, its precise subcellular location, and the process by which gene activation is accomplished are unknown. Preparation of purified receptor and further characterization of the nuclear binding sites will be crucial to a better understanding of this process.

Animals↗
PubMed1983

In vivo activation and nuclear binding of the AtT-20 mouse pituitary tumor cell glucocorticoid receptor.

AtT-20 mouse pituitary tumor cells were incubated at 25 C with the tritiated glucocorticoids triamcinolone acetonide (9 alpha-fluoro-11 beta, 16 alpha, 17,21-tetrahydroxy-pregna-1, 4-diene-3,20-dione 16,17-acetal with acetone), dexamethasone (9 alpha-fluoro-11 beta, 17,21-trihydroxy-16 alpha-methyl-pregna-1,4-diene-3, 20-dione), prednisolone (11 beta, 17,21-trihydroxypregna-1,4-diene-3,20-dione), and corticosterone (11 beta, 21-hydroxypregn-4-ene-3,20-dione) in order to examine the nuclear binding and glucocorticoid receptor activation produced in vivo. Although the total amounts of intracellular receptor labeled by each steroid were similar, each steroid caused different and characteristic percentages of occupied receptor to be translocated into the nucleus. DEAE chromatography of the nuclear receptor extracted in the presence of sodium molybdate to prevent spontaneous activation showed that, as expected, the nuclear receptor was in the activated form. Activated receptor in the cytosol was determined by DEAE chromatography of cytosol prepared from cells that had been incubated with the various labeled glucocorticoids mentioned above. Total intracellular activated receptor was determined as cytosolic activated receptor plus total nuclear receptor. The results showed that for the four agonists used, the extent of nuclear binding is proportional to the degree of activation and that both of these parameters correlate with steroid-receptor affinity. It was also found that the removal of steroid from the cell incubation medium caused the rapid return of nuclear receptor to the cytosolic compartment in an unactivated form. This reversal was not dependent on protein synthesis. These results are consistent with a model of nuclear binding in which the proportion of steroid-bound receptor that becomes activated is determined by steroid binding affinity. However, the activated receptor partitions between the cytoplasmic and nuclear compartments to the same extent regardless of the steroid used, suggesting that although the percentage of activated receptors is steroid dependent, the nuclear-binding ability of the activated receptor is an intrinsic and constant property of the protein itself.

Animals↗