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W S Mellon

Publications and source records attributed to W S Mellon.

At least 19 recordsLinked to original sources
PubMed1992

Identification of actin and HSP 70 as cyclosporin A binding proteins by photoaffinity labeling and fluorescence displacement assays.

A novel family of cyclosporin A (CsA) binding proteins was identified by using the biologically active, radioiodinated photoaffinity probe [D-Lys-N epsilon-(4-azido-3-[125I]iodophenyl)propionyl)]8-CsA. In addition to cyclophilin, proteins with molecular masses of 43 kDa and approximately 50-55 kDa were labeled in Jurkat extracts and bovine calf thymus. Sequence analysis of the 43-kDa protein purified from calf thymus and subsequent Western analysis of CsA affinity-purified material from Jurkat extracts identified the 43-kDa component as actin. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA, a fluorescent analogue of CsA, was prepared and used to measure the binding constants of cyclosporin derivatives to actin by means of a new fluorescence displacement assay. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA and [N delta-t-butoxycarbonyl diaminobutyryl)]8-CsA bind to bovine actin at physiologically relevant concentrations, with dissociation constants of 60 +/- 33 and 570 +/- 380 nM, respectively. Because the ATPase fragment of heat shock cognate 70 (HSC 70) is structurally related to actin, the yeast homologue SSA1 was tested and found to be radiolabeled by the cyclosporin A photoaffinity reagent. The binding constant for [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA to SSA1 was determined and is 53 +/- 48 nM. These results indicate that actin and the 70-kDa heat shock protein family contain a structurally related domain for binding of cyclosporin A-related peptides.

Actins↗
PubMed1989

Immunological identification of 1,25-dihydroxyvitamin D3 receptors in human promyelocytic leukemic cells (HL-60) during homologous regulation.

Immunological techniques were utilized to detect 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) receptor levels and to characterize physical/chemical changes in receptors in human promyelocytic leukemic cells (HL-60) during continuous exposure to hormone. The monoclonal antibody (IVG8C11) raised against the porcine intestinal 1,25-(OH)2D3 receptor immunoprecipitated quantitatively 1,25-(OH)2D3 receptors in nuclear extracts from HL-60 cells. The highly enriched immunoprecipitated receptors were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes, which were probed with 125I-labeled IVG8C11. The basal receptor from the cells treated with 1,25-(OH)2D3 for 2 h was detected as a single form at 53 kDa. Moreover, receptors were shown to be up-regulated at 12 h and down-regulated at 48 and 72 h in the continuous presence of hormone as evidenced by the ratio of density of the bands, 1.0 (2 h):4.2 (12 h):1.2 (48 h):0.9 (72 h), as measured by laser scanning densitometry. The up- and down-regulated receptors were also detected as single forms and had the same molecular mass as the basal receptor. Therefore, the data presented here strongly support the hypothesis of homologous regulation of 1,25-(OH)2D3 receptors in intact human target cells.

Antibodies, Monoclonal↗
PubMed1987

Induction of monocytic differentiation by calcitriol (1,25-dihydroxyvitamin D3) in the human promyelocytic leukemic cell line (HL-60) in serum-free medium.

The effect of calcitriol on the induction of differentiation in human promyelocytic leukemic cell line (HL-60) cultured in serum-free chemically defined medium (SFM) was investigated. The utilization of SFM containing RPMI-1640 basal medium supplemented with insulin (5 micrograms/ml), transferrin (5 micrograms/ml), sodium selenite (5 ng/ml), and bovine serum albumin (0.5 micrograms/ml), transferrin examination of the cellular/molecular mechanism of calcitriol's action in HL-60 cell differentiation without interference of components present in serum. HL-60 cells grown in SFM were induced to differentiate into monocytes/macrophages by calcitriol as indicated by induction of differentiation-associated biological and biochemical parameters: chemiluminescent (CL) responsiveness, lysozyme activity, nonspecific esterase, expression of cell surface antigens, and reduced proliferation. The exposure of HL-60 cells in SFM to calcitriol (from 10(-10) to 10(-8)M) resulted in dose-dependent induction of these parameters, which was similar to those obtained with cells grown in 10% fetal calf serum containing medium (10% SCM). However, calcitriol was 5-fold more potent for HL-60 cells cultured in SFM than those cultured in 10% SCM as indicated by shifts in dose-response curves for induction of CL responsiveness and lysozyme activity. The effect of calcitriol on the proliferation and acquisition of several monocyte-associated cell surface antigens was also more sensitive for HL-60 cells cultured in SFM than for cells grown in 10% SCM. We characterized and quantitated calcitriol receptors in HL-60 cells cultured in SFM in comparison to those in 10% SCM after exposing intact cells to radiolabeled calcitriol. Cells cultured in either SFM or 10% SCM exhibited calcitriol receptors that migrated at 3.4S as a single peak on sucrose gradients and elicited inherent DNA binding ability. There was essentially no difference in the apparent dissociation constants (Kd) nor in the number of calcitriol binding sites per HL-60 cell, that is approximately 6.0 X 10(-11) M and approximately 3000 binding sites/cell respectively. It is concluded that culturing HL-60 cells in SFM results in full expression of calcitriol-induced phenotypic changes excluding the possibility that such changes result from the indirect effect of calcitriol mediated by identified and/or unidentified components present in serum.

Calcitriol↗
PubMed1986

1,25-Dihydroxyvitamin D3 receptor replenishment in the chicken intestine.

1,25-Dihydroxyvitamin D3 intestinal receptor replenishment was examined in rachitic chickens after hormone administration. A single injection of 1,25-dihydroxyvitamin D3 caused an increase in the level of occupied receptors with a concomitant decrease in the amount of unoccupied receptors. Maximum occupancy occurred 1 h after hormone injection. The metabolic inhibitor of protein synthesis, cycloheximide, was employed to obtain additional information concerning the fate of 1,25-dihydroxyvitamin D3 receptor complexes. Cycloheximide, at a dose that effectively blocked protein synthesis, had no effect on the time-course or the magnitude of replenishment of nuclear receptors. Additionally, repletion with vitamin D3 or administration of several injections of 1,25-dihydroxyvitamin D3 did not lead to a lag in replenishment time or a significant decrease in total receptor levels. These findings demonstrate that recycling of receptors plays an important functional role for the replenishment of unoccupied 1,25-dihydroxyvitamin D3 intestinal receptors.

Animals↗
PubMed1985

Analysis of hormone- and polynucleotide/histone-binding sites of the chicken intestinal 1,25-dihydroxyvitamin D3 receptor by means of proteolysis.

Several discrete forms of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor from chicken intestinal cytosol were characterized by ultracentrifugation; gel filtration; DNA-, histone-, dye-ligand-binding affinity; and several other chromatographic media. Formation of altered receptor complexes was carried out through partial proteolysis using trypsin, alpha-chymotrypsin, and papain. The holoreceptor complex was found to be asymmetric and have a Stoke's radius of 37 A. Depending on the concentration of protease, several sterol-binding fragments of 33 and 27 A were produced which tended to be more globular in shape. The 27 A complex was incapable of binding DNA, histones, or phosphocellulose, but still retained affinity for DEAE-cellulose. Resolution of three forms of 1,25-(OH)2D3 receptors was demonstrated by cibacron blue F3GA-agarose chromatography. The 37 A complex eluted at 1.1 M KCl, and the 33 A form eluted at 0.6 M KCl, whereas the 27 A complex was not retained by the column and eluted in the 0.15 M KCl wash. The specificity of the 37 A complex for histone binding was assessed. The order of binding preference was: histone f3 greater than histone f2a approximately equal to histone f2b much greater than histone f1. Results also indicated that DNA could inhibit receptor binding to histone and that this was competitive with respect to histone-agarose binding, suggesting that the interaction of histones and DNA is at a domain common to polynucleotides. It is concluded the 1,25-(OH)2D3 receptor has separate and distinct binding sites for hormone and polynucleotides/histones. These in vitro findings of histone binding suggest that the polynucleotide domain of this receptor is capable of recognizing several nuclear derived components that may be important for the alteration of gene expression.

Animals↗
PubMed1984

Inhibitory action of aurintricarboxylic acid and rifamycin AF/013 at the polynucleotide domain of 1,25-dihydroxyvitamin D3-receptor complexes.

The binding of 1,25-dihydroxyvitamin D3-receptor complexes from chicken intestine to DNA-cellulose and isolated intestinal nuclei is inhibited in a dose-dependent manner by aurintricarboxylic acid and rifamycin AF/013. Since both nuclear- and cytoplasmic-associated receptors have been identified, some experiments were carried out on both populations of receptors. Concentrations resulting in 50% displacement of cytoplasmic receptor complexes were 3.2 X 10(-6) M and 1.2 X 10(-4) M for aurintricarboxylic acid and rifamycin AF/013 respectively. Moreover, rifamycin AF/013 was approximately nine times more potent at inhibiting nuclear receptor binding to DNA-cellulose compared to cytoplasmic receptors. Contrary to these findings, rifampicin, which does not inhibit eukaryotic RNA or DNA polymerases, did not cause a loss of receptor complex binding to DNA-cellulose at the doses tested. Neither aurintricarboxylic acid, rifampicin, nor rifamycin AF/013 resulted in any significant loss of sterol binding. Inhibition of receptor binding to DNA-cellulose by these polymerase inhibitors was not due to alteration of the DNA and was reversed by dialysis. Incubation of receptor complexes with aurintricarboxylic acid or rifamycin AF/013 inhibited binding to Cibacron blue-agarose and phosphocellulose. Furthermore, these polymerase inhibitors were utilized specifically to desorb receptor complexes from Cibacron blue-agarose columns. Sucrose density gradient analysis of inhibitor treated and untreated receptor revealed that rifamycin AF/013 treatment resulted in the appearance of a broadened 3.7 S sedimenting receptor in addition to specific bound 1,25-dihydroxyvitamin D3 in the 6.0 S region and in the pellet of the gradient.

Animals↗
PubMed1984

An exchange assay for quantitating 1,25-dihydroxyvitamin D3 receptors.

Steroid hormone receptors, including those for vitamin D3, contain reactive sulfhydryl group(s) essential for hormone binding. On this basis, several compounds that are capable of interacting with sulfhydryl groups were tested for their ability to dissociate 1,25-dihydroxyvitamin D3 from chicken intestinal receptors. At concentrations resulting in 50% displacement of specifically bound cytoplasmic 1,25-dihydroxyvitamin D3, the order of potency for these displacing reagents is mersalyl acid greater than sodium thiocyanate approximately equal to p-hydroxymercuribenzoate approximately equal to mercuric chloride much greater than 5,5'-dithiobis-(2-nitrobenzoic acid). Hormone displacement by mersalyl acid and p-hydroxymercuribenzoate was completely reversible upon dithiothreitol addition. In contrast limited rebinding of 1,25-dihydroxyvitamin D3 occurred after, 5,5-dithiobis-(2-nitrobenzoic acid) and mercuric chloride treatment. Furthermore, at least for mersalyl acid treatment, hormone displacement and subsequent regeneration of sterol binding did not seem to alter the integrity of the receptor as evidenced by sucrose gradient analysis, DNA-cellulose and phosphocellulose chromatography. Additionally, treatment of the nuclear 1,25-dihydroxyvitamin D3 receptor did not significantly affect the apparent equilibrium dissociation constant for hormone binding (Kd = 2.7 X 10(-10) M; Kd = 1.7 X 10(-10) M, for control and mersalyl acid treated receptor, respectively). Finally, a method has been developed for measurement of occupied 1,25-dihydroxyvitamin D3 receptors. The unfilled receptors were quantitated in the cytoplasm or chromatin extracted fraction by incubation with radioactive 1,25-dihydroxyvitamin D3 at 0-4 degrees C for 3-6 h without interference from previously filled sites. The total receptor measurement is carried out by incubation of cytosol or nuclear extract with 0.5-1.0 mM mersalyl acid for 1.0 h. Exchange of radioactive sterol for the bound nonradioactive sterol is accomplished by incubation with 1-2 mM dithiothreitol and 1.0 nM radioactive 1,25-dihydroxyvitamin D3 at 0-4 degrees C for 16 h. Subtracting the unfilled sites from total sites results in a measurement of filled sites. With this exchange assay, estimates of total receptor in untreated and 1,25-dihydroxyvitamin D3 treated chicks (estimated 2 h after injection of doses 0.3-300 nmol) were not significantly different (66.2 +/- 4.5 versus 64.4 +/- 10.4 to 69.0 +/- 5.9 pg/mg protein, respectively). Furthermore, quantitation of total receptor by direct or exchange assay obtained from in vitro incubations of intestinal slices with radioactive and nonradioactive 1,25-dihydroxyvitamin D3 were not

Animals↗
PubMed1984

Dye-ligand interactions with 1,25-dihydroxyvitamin D3-receptor complexes from chicken intestine.

A number of dye-ligand adsorbents have been examined for purifying and characterizing 1,25-dihydroxyvitamin D3-receptor complexes from intestines of vitamin D3-deficient chickens. In particular, several triazinyl dyes--Cibacron blue F3GA, Procion red HE3B, and Green A dye, immobilized to agarose via an ether linkage--retain specifically bound 1,25-dihydroxyvitamin D3-receptor complexes formed at 0-4 degrees which are eluted at high salt concentrations. Moreover, receptor binding to these dye-ligand matrices occurs in the presence and absence of sterol. At least for Cibacron blue, the strength of receptor binding depends critically on the method of dye coupling to matrix. The concentration of KCl required for elution of receptor from the triazine ether-linked matrix is greater than coupling through the amine of the anthraquinone via a 10-atom spacer arm approximately equal to coupling through the amine of the anthraquinone via an isourea bond greater than Cibacron blue dextran. Data are presented which demonstrate that sterol-receptor complexes formed at 25 degrees have reduced affinity for dye-ligands when compared with sterol-receptor complexes formed at 0-4 degrees. It is suggested that this finding is related to proteolytic alterations of the receptor, since limited digestion with trypsin can mimic this phenomenon and several protease inhibitors can reduce the thermal-induced alterations. Biospecific elution of receptor is demonstrated using synthetic polyribonucleotides. Preference for polyguanylic and polyinosinic acid is observed over several other polyribonucleotides and mononucleotides. The data in this study, viewed collectively, suggest that there is a specific interaction between the polynucleotide domain of the 1,25-dihydroxyvitamin D3-receptor and several triazinyl dye-ligands. It is concluded that these dye-ligands should prove to be of considerable interest for facile chromatography to purify and characterize this receptor.

Animals↗
PubMed1984

Specificity of dye-ligand interaction with the polynucleotide binding domain of 1,25-dihydroxyvitamin D3-receptor complexes of chicken intestinal cytosol.

The binding of 1,25-dihydroxyvitamin D3-receptor complexes from chick intestinal cytosol to DNA-cellulose and isolated intestinal nuclei is inhibited by several dye-ligands in a dose-dependent manner. Concentrations of Cibacron blue F3GA, blue dextran, Procion red HE3B, and Green A dye causing 50% competition for receptor binding to DNA-cellulose ranged from 2.8 to 3.6 microM. A structural analogue of the anthraquinone moiety of Cibacron blue F3GA, bromaminic acid, was 111-fold less potent in inhibiting DNA-cellulose binding. Moreover, the inhibitory effects of these dye-ligands is not due to a simple electrostatic effect, since two other polyanions, heparin and poly-L-glutamate, are much less effective. Whereas dye-ligands can cause the release of receptors bound to DNA-cellulose, they do not alter the dissociation of 1,25-dihydroxyvitamin D3 from its receptor nor do they affect the apparent equilibrium binding constant of the receptor or the concentration of available sterol-binding sites. The inhibition of binding by dye-ligands is competitive with respect to DNA-cellulose binding, indicating that the effect of these dyes is at a domain common to polynucleotides.

Animals↗
PubMed1982

Computerized tomography and adenocarcinoma of the esophagus.

A case of adenocarcinoma of the stomach spreading to the esophagus is described. It was well seen on computerized tomography and appeared more ominous than the esophagram. The latter illustrated an appearance simulating narrowing secondary to peptic esophagitis; the former demonstrated a relatively large encompassing mass. The definitive diagnosis was made during endoscopy and confirmed by biopsy. Although computerized tomography is not considered the method of choice for establishing an abnormality in this organ, it can occasionally help. It can usually show the total extent of the tumor better. Additional cases are presented.

Adenocarcinoma↗
PubMed1980

Direct chemical synthesis of 1 alpha,25-dihydroxy[26,27-3H]vitamin D3 with high specific activity: its use in receptor studies.

The first direct chemical synthesis of radiolabeled 1 alpha,25-dihydroxyvitamin D3 is reported. Unlike all previous syntheses, the new approach does not rely on enzymatic 1 alpha-hydroxylation of radiolabeled precursors. Rather, isotope is introduced in the last synthetic step by reaction of [3H]-methylmagnesium bromide with methyl 1 alpha-hydroxy-26,27-dinorvitamin D3-25-carboxylate to give 1 alpha,25-dihydroxy-[26,27-3H]vitamin D3 with a specific activity of 160 Ci/mmol. Mass spectroscopy confirmed that the radiohormone consists of a single isomer with six tritium atoms bound to carbons 26 and 27. Synthetically produced 1 alpha,25-dihydroxy[26,27-3H]vitamin D3 is indistinguishable from 1 alpha,25-dihydroxy-[26,27-3H]vitamin D3 obtained from the enzymatic 1 alpha-hydroxylation of 25-hydroxy[26,27-3H]vitamin D3 (160 Ci/mmol) by high-pressure liquid chromatography analysis and in the competitive binding assay using chick intestinal cytosol as the receptor source. Equilibrium dissociation constant measurements with the high specific activity radiohormone indicate a Kd of 8.2 x 10(-11) M for the chick intestinal cytosol 1 alpha,25-dihydroxyvitamin D3 receptor--a value considerably lower than the constants in the range of (1-5) x 10(-9) M previously reported.

Animals↗
PubMed1980

A specific 1,25-dihydroxyvitamin D3 binding macromolecule in chicken bone.

Cytosol prepared from homogenates of bone from vitamin D3-deficient chicks contains a 3.7 S macromolecule having high affinity and low capacity for 1,25-dihydroxyvitamin D3. Employing 1,25-dihydroxy-[26,27-3H]vitamin D3 (160 Ci/mmol) an apparent Kd has been calculated to be 7.6 x 10(-11) M while the association and dissociation rate constants for the binding process at 25 degrees C were determined to be 9.5 x 10(8) M-1 min-1 and 2.3 x 10(-2) min-1, respectively. A 5.5 S molecule is also present which binds 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 but appears to prefer 25-hydroxyvitamin D3 and is increased by the addition of chick serum to cytosol. The 3.7 S material is neither a serum contaminant nor a component of the 5.5 S molecular species and is likely of intracellular origin. Under low salt conditions the 3.7 S macromolecule migrates to 4.3 S and 5.5 S regions on sucrose gradients suggesting aggregation of the protein. Several vitamin D3 metabolites are capable of specifically binding to the 3.7 S macromolecule. The relative order of potency for several analogs causing displacement of specifically bound 1,25-dihydroxy-[26,27-3H]vitamin D3 is: 1,25-dihydroxyvitamin D3 greater than 1 alpha-hydroxyvitamin D3 greater than or equal to 25-hydroxyvitamin D3 greater 24(R),25-dihydroxyvitamin D3. It is concluded that chick bone cytosol contains a macromolecule of high affinity and low capacity for 1,25-dihydroxyvitamin D3 which may function as a receptor for some physiological events in bone.

Animals↗