No abstract

To examine whether either of the two known active vitamin D metabolites 1,25(OH)2D3 or 24,25(OH)2D3 could reverse the mineralization defect induced by 1-hydroxyethylidene-1,1-bis phosphonate (EHDP), a model of EHDP-induced rickets was used. Rats at the age of 31 days were injected for 10 consecutive days with EHDP (10 mg/kg). Other littermates were treated with a combination of EHDP and either 1,25(OH)2D3 or 24,25(OH)2D3 or were treated following 10 days of EHDP, with either of the vitamin D metabolites for an additional 72 hr. Samples of cartilage fluid (Cfl) and of blood were removed prior to sacrifice for biochemical studies of some parameters of calcification. These parameters were correlated with the results of light and electron microscope studies of growth plate cartilage and bone. EHDP-treated rats revealed signs of typical rickets, manifested by widened growth plates and impaired bone mineralization. Transmission electron microscope (TEM) examination revealed matrix vesicles distributed throughout the growth plate; however, there appeared to be an arrest of the spread of the crystals at the provisional zone of calcification. Treatment with either 1,25(OH)2D3 or 24,25(OH)2D3 failed to reverse the rachitic condition of the animals. Serum calcium blood levels were elevated in the 1,25(OH)2D3 and EHDP-treated group. 1,25(OH)2D3 and 24,25(OH)2/D3 further increased the already elevated serum alkaline phosphatase levels observed in EHDP rats, although the increase observed with 1,25(OH)2D3 was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Diaphyseal bone from normal Sprague-Dawley rats was delipidated in chloroform-methanol and demineralized in 0.6 N HCl at 4 degrees C. The bones were then implanted for 7-28 days into rats made rachitic by a low-phosphate, vitamin D-deficient diet (VDP-) for 3 weeks. Bones from VDP- and normal rats were also implanted into normal hosts. When normal rats were used as the host environment, a consistent sequence of cartilage induction and bone formation was observed. Demineralized rachitic bone (RB) implanted into normal host rats resulted in cartilage and bone induction similar to that seen for normal bone (NB) implants. Transmission electron microscopy of RB in normal hosts revealed morphologically normal chondrocytes and cartilage matrix with normal mineralization. In contrast, implantation of NB in VDP- hosts resulted in delayed chondrogenesis and lack of calcification. Furthermore, similar results were observed when RB was implanted into VDP- hosts. Treatment of VDP- hosts with either 1 alpha-hydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 did not accelerate the sequential appearance of precartilage or cartilage. However, 24,25-(OH)2D3 administered alone or in combination with 1 alpha-OHD3 significantly increased the amount of calcified cartilage observed at 2 weeks postimplantation compared to implants from either untreated VDP-hosts or those treated only with 1 alpha-OHD3. New bone formation was observed at 4 weeks postimplantation in all vitamin D-treated groups as determined by von Kossa staining or direct electron microscope examination. There was no apparent difference in the quantitative or qualitative bone formed within the various vitamin D-treated groups. Serum calcium and phosphorus levels were lower and alkaline phosphatase levels were higher in VDP- hosts compared with normal animals or those treated with vitamin D metabolites. The results of this study show a reduction in the capacity of progenitor cells in VDP- rat hosts to respond to osteoinductive factor(s). This impaired response appears to be corrected by vitamin D metabolites.

Extrachromosomal DNA in the form of covalently closed circular DNA molecules was isolated from killer and nonkiller xenosomes, bacterial endosymbionts of the marine protozoan Parauronema acutum. Restriction endonuclease digests of these molecules derived from 12 isolates revealed consistent, readily identifiable, differences in the pattern of fragments of the killer as compared with those present in the nonkiller. Transformation of the nonkiller to killer by infection is also accompanied by a change from the nonkiller to killer pattern. Based on analysis of fragments resulting from restriction endonuclease digests, two circular duplex DNA molecules, each 63 kilobase pairs (kbp) in length, were identified in the 263-20 nonkiller stock and mapped. The maps revealed that each possesses a single BamHI site and multiple Bgll, BstIIE, PstI, and Sall sites. A distinguishing feature of these maps is that the two molecules share a region about 17 kbp in length in which multiple restriction sites are in register with each other. Allowing for a 0.5-kbp insertion or deletion and the introduction or removal of only a few restriction sites, an additional stretch extending approximately 31 kbp beyond this sequence could also be considered to be homologous. The structure of the killer plasmid appears to be more complex, and we have been unable, as yet, to construct physical maps for this DNA. We postulate that the killer plasmid DNA is composed of three, perhaps four, circular 63-kbp duplexes, at least one which contains a single BamHI site and another which contains two BamHI sites. The remaining molecules may represent copies of either or both of the other two, modified to contain additional restriction sites. Transformation from the nonkiller to the killer is visualized as the insertion of restriction sites at various points along parent nonkiller plasmid DNA molecules. The mechanism by which these sites are introduced is unknown.Several stocks of the marine protozoan Paraiuronema acutum contain intracytoplasmic symbiotic bacteria which grow and divide in synchronism with the host (13). The symbionts, which we have termed xenosomes, sensu strictu, are obligate parasites and cannot be cultured outside the protozoan. Xenosomes released from the cytoplasm by mechanical rupture of the host cell are capable of infecting homologous and heterologous strains of P. acutuem as well as one other marine protozoan, Miamiensis aliidus Ma. Xenosomes from some stocks (killers) are capable of killing certain marine protozoa of the genus Uronema as evidenced by exposure of these organisms to breis of symbiont-bearing P. acutum. We show here that xenosomes from other stocks of P. acutum (nonkillers) do not have this capacity. Significantly, P. acutum is insensitive to the toxin produced by the killer xenosomes. In an effort to determine the mechanism responsible for the killer effect and to establish its molecular basis, we previously examined chromosomal DNA derived from killer and nonkiller xenosomes by DNA-DNA hybridization (9). Whereas a h...

Pregnant mice were injected with pharmacological doses of vitamin A during days 11-19 of gestation with the purpose of studying the long bones of offspring up to the age of 1 week. Tibiae were collected for routine light microscopic examination and tranmission electron microscopic examination. In addition, biochemical studies were conducted to determine the calcium, phosphorus, and magnesium content as well as the hydroxyproline and protein content of the bones. Treatment with vitamin A resulted in reduced weight and length of the long bones, as well as the presence of excessive calcification throughout the hypertrophic zone of the cartilaginous epiphyses. Matrix vesicles, many of them containing hydroxyapatite crystals, were observed and found to be distributed within the cartilaginous epiphyses in a similar pattern as in untreated control mice offspring, but mineral crystals were also observed unassociated with the matrix vesicles. The calcium, phosphate, magnesium, and hydroxyproline content was reduced in the vitamin A offspring. However, the percentage of these minerals expressed per dry weight bone was higher than in controls, verifying the morphological findings that although vitamin A inhibits bone growth, it enhances calcification in the growth plate.