Comparison of urinary bone resorption markers in women of 40-70 years; day-to-day and long-term variation in individual subjects.

OBJECTIVES: Bone resorption can be judged using biochemical markers in urine and blood. Our aim was to study the patterns of markers in the postmenopausal period. METHODS: The urinary excretion of bone resorption markers was tested using different assays. The study was undertaken to determine the day-to-day and the long-term variation, over 8 years, of these markers in individual women. RESULTS: Over a period of 2 weeks, the median of the day-to-day variation of the pyridinium crosslink markers varied between 12 and 23%, the median value of the long-term variation over 8 years between 10 and 21%, for the telopeptide markers median day-to-day variation was 18 and 20% and the long-term variation was 17 and 19%. The correlations between the different crosslink markers varied between 0.63 and 0.92, depending on the kind of the crosslink and on the method of determination. The two telopeptide markers showed an excellent correlation with r of 0.95. The excretion of all bone resorption markers varied with postmenopausal age, some differences were found between the crosslink and the telopeptide excretions with age, in women more than 20 years postmenopausal the telopeptides decrease whereas the crosslinks show an increase. CONCLUSIONS: This study shows that crosslinks and telopeptides give similar information on the rate of bone resorption: an increase during the first 5 years and a slight decrease in the next 5 years after menopause, discrepancies were found after 10 or more postmenopausal years.

Human steroidogenic acute regulatory protein: functional activity in COS-1 cells, tissue-specific expression, and mapping of the structural gene to 8p11.2 and a pseudogene to chromosome 13.

Steroidogenic acute regulatory protein (StAR) appears to mediate the rapid increase in pregnenolone synthesis stimulated by tropic hormones. cDNAs encoding StAR were isolated from a human adrenal cortex library. Human StAR, coexpressed in COS-1 cells with cytochrome P450scc and adrenodoxin, increased pregnenolone synthesis > 4-fold. A major StAR transcript of 1.6 kb and less abundant transcripts of 4.4 and 7.5 kb were detected in ovary and testis. Kidney had a lower amount of the 1.6-kb message. StAR mRNA was not detected in other tissues including placenta. Treatment of granulosa cells with 8-bromo-adenosine 3',5'-cyclic monophosphate for 24 hr increased StAR mRNA 3-fold or more. The structural gene encoding StAR was mapped using somatic cell hybrid mapping panels to chromosome 8p. Fluorescence in situ hybridization placed the StAR locus in the region 8p11.2. A StAR pseudogene was mapped to chromosome 13. We conclude that StAR expression is restricted to tissues that carry out mitochondrial sterol oxidations subject to acute regulation by cAMP and that StAR mRNA levels are regulated by cAMP.

Purification of, generation of monoclonal antibodies to, and mapping of phosphoribosyl N-formylglycinamide amidotransferase.

5'-Phosphoribosyl N-formylglycinamide (FGAR) amidotransferase (EC 6.3.5.3) catalyzes the fourth reaction in the de novo synthesis of purines, that is, the conversion of FGAR to 5'-phosphoribosyl N-formylglycinamidine (FGAM). This is the only step of the pathway for which a vertebrate gene has not been cloned. FGAR amidotransferase has been highly purified from Chinese hamster ovary (CHO) cells, and this preparation has been used to generate monoclonal antibodies in mice. Two of these antibodies, designated BD4 and DD2, have been shown to recognize a 150-kDa protein in CHO-K1 cells that is of very low abundance in Ade-B cells, a CHO line in which FGAR amidotransferase activity is undetectable. Furthermore, the protein recognized by these antibodies is 5-10-fold more abundant in Azr cells. The CHO Azr cell line was made resistant to azaserine, a potent inhibitor of FGAR amidotransferase, and displays a 5-10-fold increase in FGAR amidotransferase activity over the parental K1 line. FGAR amidotransferase activity and the 150-kDa protein recognized by both monoclonal antibodies were found to immunoprecipitate concomitantly using antibody BD4. Monoclonal antibody DD2 cross-reacted with a human protein of identical molecular mass. A number of Ade-B/human hybrid cells were generated by somatic cell fusion and subsequent 5-bromo-2-deoxyuridine segregation. Analysis of these lines, together with two independently generated human/mouse hybrid cell lines, by both cytogenetics and immunoblotting with antibody DD2 revealed that the human FGAR amidotransferase gene is located on chromosome 17p.

Mapping of a locus correcting lack of phosphoribosylaminoimidazole carboxylase activity in Chinese hamster ovary cell Ade-D mutants to human chromosome 4.

The human phosphoribosylaminoimidazole (AIR) carboxylase locus has been until this report one of the genes encoding purine biosynthetic enzymes that had not been assigned to an individual human chromosome. Characterization of Chinese hamster ovary (CHO) cell mutant Ade-D showed that the cell line was unable to produce IMP and accumulated AIR. CHO Ade-D cells were fused with normal human lymphocytes utilizing inactivated Sendai virus and the resulting hybrid cell lines were selected for purine prototrophy. Cytogenetic analysis showed a 100% concordance value for chromosome 4. Two of the isolated subclones contained only the long arm of chromosome 4 translocated onto a CHO chromosome, providing evidence for a regional assignment of the Ade-D gene to the long arm of chromosome 4. Two of the subclones containing chromosome 4 were subjected to the BrdU visible light segregation. All of the isolated purine auxotrophic cell lines showed a loss of the q arm of chromosome 4. The localization of the Ade-D locus to the long arm of chromosome 4 may reveal further clustering of the mammalian purine genes since the Ade-A locus has previously been regionally assigned to 4pter-q21.

A gene correcting the defect in the CHO mutant Ade -H, deficient in a branch point enzyme (adenylosuccinate synthetase) of de novo purine biosynthesis, is located on the long arm of chromosome 1.

Somatic hybrids between human cells and the Chinese hamster ovary (CHO) K1 mutant, Ade -H cells, were selected for purine prototrophy by growth in adenine-free medium. The Ade -H mutant is defective in the enzyme adenylosuccinate (AMPS) synthetase (ADSS; EC 6.3.4.4), which carries out the first of a two-step sequence in the biosynthesis of AMP from IMP, and therefore requires exogenous adenine for growth. The presence of the long arm of human chromosome 1 in the hybrids is 100% concordant for the ability to grow in adenine-free medium and restoration of the enzyme activity. Hybrid segregants that lose the ability to grow in adenine-free medium lose all or a portion of chromosome 1 and enzyme activity. Southern blot hybridization with a chromosome 1-specific probe, BCMI, confirms the existence of human chromosome 1 in these hybrids. Analysis of a human/CHO translocation chromosome that arose in one of the hybrids suggests that the gene correcting the defect lies in the region 1 cen-1q12. In summary, we have shown by cytogenetics, segregant analysis, biochemical assay, and Southern blot analysis that human chromosome 1, most likely in the region 1cen-1q12, corrects the defect in ADSS-deficient mutant Ade-H cells.

Chromosomal location of human genes encoding major heat-shock protein HSP70.

The HSP70 family of heat-shock proteins constitutes the major proteins synthesized in response to elevated temperatures and other forms of stress. In eukaryotes members of the HSP70 family also include a protein similar if not identical to bovine brain uncoating ATPase and glucose-regulated proteins. An intriguing relation has been established between expression of heat-shock proteins and transformation in mammalian cells. Elevated levels of HSP70 are found in some transformed cell lines, and viral and cellular gene products that are capable of transforming cells in vitro can also stimulate transcription of HSP70 genes. To determine the organization of this complex multigene family in the human genome, we used complementary approaches: Southern analysis and protein gels of Chinese hamster-human somatic cell hybrids, and in situ hybridization to human chromosomes. We demonstrate that functional genes encoding HSP70 proteins map to human chromosomes 6, 14, 21, and at least one other chromosome.

A somatic cell hybrid with a single human chromosome 22 corrects the defect in the CHO mutant (Ade-I) lacking adenylosuccinase activity.

Adenine-requiring Chinese hamster ovary (CHO-K1) auxotrophs of the complementation group Ade-I were hybridized with various human cells, and hybrids were isolated under selective conditions in which retention of the complementing gene on the human chromosome is necessary for survival. Ade-I cells are deficient in adenylosuccinase activity. This enzyme carries out two independent, but similar, steps of purine biosynthesis: the removal of a fumarate from succinylaminoimidazole carboxamide ribotide to produce aminoimidazole carboxamide ribotide and the removal of fumarate from adenylosuccinate to produce AMP. These are the 9th and 13th steps of adenylate biosynthesis, respectively. Analysis of hybrids by cytogenetics and by Southern blot techniques using chromosome 22-specific DNA probes, one of which encodes an antigen expressed in human fetal brain, indicated that human chromosome 22 was 100% concordant for growth without adenine. One hybrid subclone, isolated after two successive rounds of subcloning, was found to be capable of growth without adenine; the only human chromosome present was 22. In addition, segregants that had lost the ability to grow in adenine-free media had also lost human chromosome 22. These results suggest that the human gene for adenylosuccinase resides on chromosome 22.