E. Travis Littledike

1,25-Dihydroxyvitamin D3 receptor in bovine thymus gland

Abstract 1,25-Dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ] receptor was characterized after partial purification of thymus cytosol by ammonium sulfate fractionation. The 1,25-(OH) 2 D 3 receptor sediments at 3.7S in 5–20% sucrose gradients. The binding of 1,25-(OH) 2 D 3 in thymic cytosol was a saturable process with high affinity (Kd = 0.12−0.48 nM) at 4°C. Competition for 1,25-(OH) 2 [ 3 H]D 3 receptor by nonradioactive analogs demonstrated the affinities of these analogs to be in order; 1,25-(OH) 2 D 3 = 1,24R,25-(OH) 3 D 3 = 1,25S,26-(OH) 3 D 3 = 1,25R,26-(OH) 3 D 3 > 1,25-(OH) 2 D 3 -26,23 lactone > 25-OHD 3 > 23R,25-(OH) 2 D 3 > 24R,25-(OH) 2 D 3 > 23S,25-(OH) 2 D 3 ⋙ 25-OHD 3 -26,23 lactone. The receptor bound to DNA cellulose columns in low salt buffer and eluted as a single peak at 0.21 M KCl. These findings provide evidence that the thymus possesses a 1,25-(OH) 2 D 3 receptor with properties indistinguishable from 1,25-(OH) 2 D 3 receptors in other tissues.

Impaired 24,25-Dihydroxyvitamin D Production in Anephric Human and Pig

Plasma 25-hydroxyvitamin D and 24, 25-dihydroxy-vitamin D [24,25-(OH)(2)D] concentrations were measured in normal and chronically dialyzed anephric humans and pigs. Measurement of the 24, 25-(OH)(2)D was preceded by three purification steps involving one Sephadex LH-20 column and two high-pressure liquid chromatographic columns. The final high-pressure liquid chromatography step involved resolution of 25-hydroxy-vitamin D(3)-26,23 lactone and 25,26-dihydroxy-vitamin D(2) from 24,25-dihydroxyvitamin D(2) and 24,25-dihydroxyvitamin D(3) [24,25-(OH)(2)D(3)]. The total 25-hydroxyvitamin D [25-hydroxyvitamin D(2) plus 25-hydroxyvitamin D(3) (25-OHD(3))] was 31.7+/-3.6 ng/ml in the plasma of eight anephric human subjects and 40.1+/-3.7 ng/ml in five normal human subjects. Six of the eight anephric patients had undetectable (<0.2 ng/ml) 24,25-(OH)(2)D concentrations. Two of the eight patients had very low (0.51 and 0.41 ng/ml), but detectable, 24,25-dihydroxyvitamin D(2). The normal human volunteers had plasma 24,25-(OH)(2)D concentrations of 2.8+/-0.7 ng/ml. Chronically dialyzed anephric and normal pigs were given intramuscular injections of massive amounts (5 x 10(6) IU) of vitamin D(3) immediately after surgery (day 0) and again on day 7. In anephric pigs, plasma 25-OHD(3) progressively rose from 12+/-4 ng/ml on day 0 to 705+/-62 ng/ml on day 10. The 25-OHD(3) concentrations in normal pigs rose from 8+/-2 ng/ml on day 0 to 439+/-64 ng/ml on day 10. Plasma 25-OHD(3) was higher in anephrics throughout the experiment, and concentrations were significantly higher (P < 0.05) on days 9 and 10. Plasma 24,25-(OH)(2)D(3) concentrations declined progressively in anephric pigs from 3.6+/-0.6 ng/ml on day 0 to 3.2+/-0.7 ng/ml on day 2. During days 4-10, plasma 24,25-(OH)(2)D(3) was not apparent until plasma 25-OHD(3) was >400 ng/ml. In control pigs, plasma 24,25-(OH)(2)D(3) was elevated from 4.3+/-0.6 ng/ml on day 0 to 178+/-2.7 ng/ml on day 3. Plasma 24,25-(OH)(2)D(3) was significantly higher (P < 0.05) in controls on days 1-8. At the end of the experiment (day 10), 24,25-(OH)(2)D(3) concentrations were similar and not significantly different in both groups (87.0+/-18.4 ng/ml in anephric and 110.3+/-32.1 ng/ml in normal pigs). The identity of the 24,25-(OH)(2)D(3) isolated from anephric pig plasma was confirmed by mass spectroscopy. Our data suggest that anephric humans receiving normal dietary levels of vitamin D(3) have little or no ability to produce 24,25-(OH)(2)D. However, we have shown that pigs produce 24,25-(OH)(2)D(3) when plasma 25-OHD(3) is extremely high (>400 ng/ml).

A sensitive competitive protein binding assay for vitamin D in plasma.

A sensitive protein binding assay for vitamin D is described. The vitamin D3 was extracted from plasma with diethyl ether and methylene chloride. The lipid extract was purified in Sephadex LH-20 followed by Lipidex 5000 and finally by high pressure liquid chromatography on a Zorbax Sil column (0.79 x 25 cm) developed in 0.25:99.75 isopropanol: methylene chloride. The vitamin D fraction was collected and quantitated by competitive protein binding assay with a 1/50,000 dilution of sheep plasma in 0.05 M potassium phosphate buffer (pH 7.5) containing 0.01% gelatin. [H3]-25-Hydroxyvitamin D3 was used as a radioactive tracer in the assay. We found that under these conditions, sheep plasma had equal affinity for vitamin D2 and vitamin D3 and could detect as little as 0.1 ng of vitamin D. When rat, cow, or human plasma was substituted for the sheep plasma, the decline in sensitivity to vitamin D2 was fivefold to tenfold. With this assay, we found excellent agreement (r = 0.98) between the results obtained by competitive protein binding analysis and direct U.V. absorbance analysis by high pressure liquid chromatography.

25-OHD3-26,23 lactone: demonstration of kidney-dependent synthesis in the pig and rat.

Summary Plasma concentrations of polar metabolites of vitamin D 3 were measured in intact and bilaterally nephrectomized pigs and rats receiving large doses of vitamin D 3 and undergoing peritoneal dialysis. Plasma concentrations of 25-OHD 3 were increased one day after the vitamin D 3 injections. Bilaterally nephrectomized pigs had higher plasma concentrations of 25-OHD 3 and 25,26-(OH) 2 D 3 throughout the experiment than control pigs had. Bilaterally nephrectomized rats, however, had lower concentrations of 25-OHD 3 and 25,26-(OH) 2 D 3 than the control rats had. Both bilaterally nephrectomized pigs and rats were unable to produce 25-OHD 3 -26,23 lactone (lactone) after a challenge of massive I.M. injections of vitamin D 3 . Intact animals of both species had elevated concentrations of lactone (30–50 ng/ml) at the end of the experiment. Our results show that lactone, like 1,25-(OH) 2 D 3 , requires the kidney for its production.

1,24,25-Trihydroxyvitamin D3: A circulating metabolite in vitamin D3-treated bovine☆☆☆

Abstract 1,24,25-Trihydroxyvitamin D3 was detected using a modified receptor-binding assay in plasma of cows injected intramuscularly with 375 mg of vitamin D3 once a week for 4 weeks. The metabolite was purified to homogeneity and identified by its comigration with synthetic 1,24,25-trihydroxyvitamin D3 on three different high-pressure liquid chromatography systems, mass spectrometry, and by comigration of sodium metaperiodate cleavage products obtained from isolated and synthetic 1,24,25-trihydroxyvitamin D3. Plasma concentrations of 1,24,25-trihydroxyvitamin D3 and 1,25,26-trihydroxyvitamin D3 were approximately 30 and 20 pg/ml, respectively (uncorrected for recovery), in vitamin D3-treated cows. Neither of these metabolites were detected in plasma of untreated cows.

Intracellular location of unoccupied 1,25-dihydroxyvitamin D receptors: A nuclear-cytoplasmic equilibrium☆☆☆

In order to investigate the subcellular distribution of unoccupied 1,25-dihydroxyvitamin D3 receptors, highly purified cytoplasts and nucleoplasts were prepared from two kidney cell lines (PK1 and MDBK). This was accomplished utilizing the technique of enucleation by cytochalasin B and density gradient centrifugation. Unoccupied 1,25-dihydroxyvitamin D3 receptors were found in both the nuclear and cytosolic compartments, with approximately 70% of the receptors localized in the cytoplasm. When cells were pretreated with 1,25-[3H]dihydroxyvitamin D, prior to enucleation, it was found that 90% of the receptor-hormone complex was associated with nucleoplasts, thus demonstrating that cytochalasin B treatment does not alter the high-affinity association of the receptor-hormone complex with the nucleus. The ratio of unoccupied receptor/protein was found to be the same in whole cells, cytoplasts, and nucleoplasts for both cell types. The ratio of unoccupied receptor/DNA was highest in cytoplasts and lowest in nucleoplasts. Taken together, these data indicate that the unoccupied 1,25-dihydroxyvitamin D receptor is generally associated with cell proteins and not specifically associated with cell DNA. We therefore propose, at least for these cells, that the unoccupied 1,25-dihydroxyvitamin D receptor exists in equilibrium between the nuclear and cytosolic compartments of the whole cell, and receptor-hormone binding shifts this equilibrium to favor nuclear localization.

Bovine Viral Diarrhea Serologic Diagnostic Reagents Prepared from Bacterially Expressed Recombinant Proteins

Bovine viral diarrhea virus (BVDV) is a positive-stranded RNA virus and is classified in the Pestivirus genus of the family Flaviviridae. The BVDV genome has been cloned and sequenced, and a map of protein-encoding genes has also been established. According to this map, the BVDV genome encodes at least 4 primary gene products: p20, gp116, p125, and p133. There is evidence that the glycoprotein precursor gp116 gives rise to gp48, gp25, and gp53 through proteolytic processes, and p125 gives rise to p80 by proteolysis when cells are infected with cytopathic BVDV. The p125 is produced only in cells infected with noncytopathic BVDV. When an antibody against BVDV is analyzed by using radioimmunoprecipitation or western blots, gp48, gp25, gp53, and p80 (or p125) are detected. 1,2,8,9 Thus, these proteins are good candidates for specific enzyme-linked immunosorbent assays (ELISAs). Currently, virus neutralization (VN) is the standard serologic test for BVDV, and virus-neutralizing antibody binds primarily to gp53. 5,11 Other diagnostic tests, such as wholevirus ELISA, fluorescent antibody, and immunoperoxidase staining, also are used and likely detect additional viral proteins. All of these tests require specialized tissue culture equipment. Consequently, these procedures are often impractical for screening a large number of samples. Recombinant ELISA is now widely accepted for serodiagnosis of many important human and veterinary diseases. This test is a simple, rapid, highly sensitive, and inexpensive tool for screening for an antibody. In this report, we describe the production of large amounts of gp48, gp25, and p80 recombinant proteins, use of these recombinant proteins in the ELISA, and comparison of the ELISA with the standard VN test for the serodiagnosis of bovine viral diarrhea (BVD) in cattle. To express gp48, gp25, and p80, each gene fragment was cloned into the expression vector pGex2T or pGex3X. In each construct, the correct translational frame required for expression of glutathione-S-transferase (GST) fusion protein was created. The gp48 and p80 restriction fragments were isolated from pBV4-gp62 and pBV4-p80 clones. The construction and production of these recombinant proteins have been previously described. A 359-base pair (bp) fragment of the gp48 region (nucleotides 1295-1653) and a 918-bp