An improved radioreceptor assay for 1,25-dihydroxyvitamin D in human plasma

Dokoh, Shigeharu; Pike, J. Wesley; Chandler, John S.; Mancini, Jennifer M.; Haussler, Mark R.

doi:10.1016/0003-2697(81)90346-8

Cited by 92 publications

(38 citation statements)

References 34 publications

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“…Retinoid binding assays were performed as described previously (22,37) with the following exceptions: the total protein concentration in each assay was 6 -12 g, and both 9-cis-RA and all-trans-retinol binding were determined exactly as described for all-trans-RA using either Western Blot Analysis-The levels of wild type and mutant RAR␣ protein were determined by Western blot analysis using CV-1 cells transfected with the indicated expression construct essentially as described previously (17,39).…”

Section: Methodsmentioning

confidence: 99%

Differential Role of Homologous Positively Charged Amino Acid Residues for Ligand Binding in Retinoic Acid Receptor α Compared with Retinoic Acid Receptor β

Scafonas

Wolfgang²,

Gabriel

et al. 1997

Journal of Biological Chemistry

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The diverse biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARs) and retinoid X receptors. Although it has been suggested that the ligand binding domains (LBDs) of RARs share the same novel folding pattern, many RAR subtype-specific agonists and antagonists have been synthesized demonstrating that the LBD of each RAR subtype has unique features. We have examined the role of several positively charged amino acid residues located in the LBD of RAR␣ in RA binding. These results are compared with previously published data for the homologous mutations in RAR␤.

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Section: Methodsmentioning

confidence: 99%

Differential Role of Homologous Positively Charged Amino Acid Residues for Ligand Binding in Retinoic Acid Receptor α Compared with Retinoic Acid Receptor β

Scafonas

Wolfgang²,

Gabriel

et al. 1997

Journal of Biological Chemistry

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“…The serum levels of bone gla protein (BGP) [18], parathyroid hormones (highly sensitive parathyroid hormone (HS-PTH) and intact parathyroid hormone (i-PTH) [19]) and 1, 25(OH)2D [20] were measured serially at 4-month intervals. And urinary hydroxyproline [13]), creatinine and calcium were determined serially.…”

Section: Serum and Urine Chemistrymentioning

confidence: 99%

Effects of 2 Years' Treatment of Osteoporosis with 1 a-Hydroxy Vitamin D3 on Bone Mineral Density and Incidence of Fracture: A Placebo-Controlled, Double-Blind Prospective Study.

et al. 1996

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Abstract.A two-year double-blind study monitored and evaluated the effects of 1a-hydroxy vitamin D3(1a(OH)D3) on the lumbar (L2.4BMD) and total body bone mineral densities (TBBMD) and occurrence of fracture in 113 female osteoporotic patients receiving 0.75 jig/day of 1a(OH)D3 (n=57) or a placebo (n=56) with calcium supplementation in both groups. L2~BMD increased 1.81% and 2.32% after one and 2 years in the 1a(OH)D3group, but decreased 1.89% (P<0.05) and 0.28% in the placebo group. A significant difference (P<0.01) existed between the two groups after one year. TBBMD decreased significantly in the placebo group by 3.34% (P<0.01) and 3.52% after one and 2 years. Six new fractures occurred in the control group, but only two in the 1a(OH)D3 group (Odd's ratio=0.343, 95% confidence range; 0.0648-1.815).There were no serious adverse effects of the 1a(OH)D3 treatment.It was concluded that two-year treatment with 1a(OH)D3 increased the lumbar BMD and inhibited the decrease in TBBMD. Altough it was not significant, new fracture occurrence in the 1a(OH)D3 group was around 1 /3 of that in the control group.

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“…Charcoal was activated according to ref. 18 and was then brought to 3% in 0.15 M NaCl/0.1 M Na2HPO4/0.039 M NaH2PO4/0.1% gelatin/0.015 M NaN3, pH 7.03. Charcoalabsorbed free RA was then separated from bound RA by centrifugation at 15,000 x g for 15 min.…”

mentioning

confidence: 99%

Characterization of DNA binding and retinoic acid binding properties of retinoic acid receptor.

Yang

Schüle

Mangelsdorf

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

102

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High-level expression of the full-length human retinoic acid receptor (RAR) a and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RARa protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a Kdof 2.1 x 10'10M.All-trans-retinoic acid (RA), a vitamin A derivative, is a biologically active vertebrate morphogen. It has profound effects on cellular differentiation, pattern formation, and embryonic development. RA also has a striking effect on regenerating limbs and has been implicated as a natural morphogen in chicken and frog embryogenesis (refs. 1-5 and references therein). Functions of RA are presumed to be mediated in part by its nuclear receptor proteins that are members of the steroid hormone receptor superfamily. Three species of RA receptor (RAR) cDNAs have been cloned and are referred to as a, ,, and y (refs. 6-10). However, despite the fact that certain genes regulated by RAR have been identified (refs. 11-13 and (refs. 11 and 13). However, the affinity of this binding is greatly enhanced by a heat-sensitive factor in eukaryotic cells. Upon addition of cellular extracts to the gel-mobility-shift assay, the RAR-DNA complex is further retarded. We conclude that in vivo, RAR protein requires another protein factor for high-affinity DNA binding. We show herein that at least one site of the interaction between RAR and the factor is localized in the DNA binding domain of RAR.Activation of RAR is known to be triggered by the association with its ligand RA. However, determination of the accurate dissociation constant of RA with RAR from mammalian sources has been hampered because of the interference of the cellular RA binding protein in mammalian tissues (15). In contrast, the RAR produced in E. coli binds RA with high affinity, displaying a Kd of 2.1 x 10-1o M. MATERIALS AND METHODSExpression of Human (h) RARa and RARDBD in E. coli. The cloning procedure offull-length hRARa cDNA was described in ref. 16. For expression of the DNA binding domain of RAR, the cDNA sequence of RARa corresponding to amino acids 87-156 (6) was PCR-amplified. An Nco I site and a BamHI site flanking the 5' and 3' ends, respectively, were put in for subsequent cloning into the pET-8c vector (14). The resulting plasmids pET-...

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An improved radioreceptor assay for 1,25-dihydroxyvitamin D in human plasma

Cited by 92 publications

References 34 publications

Differential Role of Homologous Positively Charged Amino Acid Residues for Ligand Binding in Retinoic Acid Receptor α Compared with Retinoic Acid Receptor β

Differential Role of Homologous Positively Charged Amino Acid Residues for Ligand Binding in Retinoic Acid Receptor α Compared with Retinoic Acid Receptor β

Effects of 2 Years' Treatment of Osteoporosis with 1 a-Hydroxy Vitamin D3 on Bone Mineral Density and Incidence of Fracture: A Placebo-Controlled, Double-Blind Prospective Study.

Characterization of DNA binding and retinoic acid binding properties of retinoic acid receptor.

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