Search

The structural features of 1,25-dihydroxyvitamin D3 that permit its high affinity binding to a 3.7 S protein from chick intestinal cytosol were determined in a series of binding and competition experiments analyzed by sucrose density gradient centrifugation. Optimal binding to the 3.7 S protein was achieved when both 1α- and 25-hydroxyls were present in the vitamin D3 molecule. Modification of the side chain by the introduction of a methyl on C-24 and a double bond on C-22,23 (1,25-dihydroxyvitamin D2) did not alter the binding of 1,25-dihydroxyvitamin D3, but significantly diminished the binding of 25-hydroxyvitamin D3. However, introduction of a hydroxyl on C-24 decreased the ability of either 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 to compete, especially when the 24-hydroxyl was in the S configuration. These results reveal that the 3.7 S protein requires specific ligand structural features for binding and suggest that metabolite discrimination by the chick intestinal receptor system is likely located in the 3.7 S cytosol protein.

In situ hybridization with 32 P- or 31 S-labeled double-stranded DNA or single-strandedRNA probes was used to investigate the temporal and spatial distribution of cartilage-characteristic type II collagen mRNA during embryonic chick limb development and cartilage differentiation in vivo . When the type II collagen probes were hybridized to sections through embryonic limb buds at the earliest stages of their development (stages 18–25), an accumulation of silver grains representing type II collagen mRNA first became detectable in the proximal central core of the limb coincident with the prechondrogenic condensation of mesenchymal cells that characterizes the onset of cartilage differentiation. At later stages of development (stage 32; 7 days) intense hybridization signals with the type II collagen probes were localized over the well differentiated cartilage rudiments, whereas few or no silver grains above background were observed over the non-chondrogenic tissues. In contrast, sections hybridized with a probe complementary to mRNA for the α1 chain of type I collagen exhibited an intense hybridization signal over the perichondrium and little or no signal over the cartilage primordia. At all stages of development examined, 32 P-labeled double-stranded DNA probes or single-stranded RNA probes labeled with either 32 P or 35 S provided adequate hybridization signals. Several experimental protocols were employed to control for the potential cross-hybridization and non-specific hybridization of the type II collagen probes. These included the utilization of labeled noncomplementary “sensestrand” type II collagen RNA as a control probe for nonspecific background, and prehybridization with a large excess of appropriate unlabeled RNA to block sequences in heterologous collagen RNAs that might cross-hybridize to the specific labeled probe.

A competitive binding assay for 1,25-dihydroxyvitamin D [1,25-(OH2D] in plasma has been developed in which intestinal cytosol preparations from rachitic chicks are used as the binding protein. A new method of extraction and two new chromatographic procedures are used for this assay. The method is sensitive to as little as 10 picograms of 1,25-(OH)2D, and triplicate assays can be done on 5 milliliters of plasma. This assay shows that in the plasma of normal adult subjects there is a 1,25-(OH)2D concentration of 29 +/- 2 picograms per milliliter, while none can be detected in the plasma of nephrectomized subjects and end-stage renal failure patients.

Activation of rabbit serum complement caused a marked reduction in collagen synthesis but a much smaller change in noncollagen protein synthesis in fetal rat calvaria maintained in organ culture. In the periosteum of the fetal rat calvarium, both collagen and noncollagen protein synthesis were reduced, whereas in the central bone, presumably enriched in osteoblasts, only collagen synthesis was inhibited. This large decrease in bone collagen synthesis could not be attributed to enhanced degradation of newly synthesized collagen or its release into the culture medium. Activation of complement also stimulated the production of PGE in fetal rat calvaria. Antagonists of prostaglandin cyclooxygenase decreased prostaglandin synthesis but did not restore collagen synthesis in complement-treated bones, suggesting that complement decreases osteoblast collagen synthesis by a mechanism largely independent of prostaglandin production.