130 Abstracts from the Joint Meeting, September 1992

sequences must undergo a demethylation event in order for gene expression to occur. To elucidate the possible role of DNA methylation in the transcriptional regulation of the collagen type X gene, the effect of the hypomethylating agent, 5- azacytidine, was explored. Chondrocytes treated with 15pg/mI 5- azacytidine switched to type X collagen synthesis after 16 days in culture.

persists even following prolonged periods of heating or reduction with f&mercaptoethanol indicating the presence of non-reducible covalent cross-links. Such cross-links may have a role in the maintenance and stabilisation of an extracellular matrix special&d for endochondral ossification.

P57. Cultured cells from the growing tip of deer antler express alkaline phosphatase activity and synthesise type I collagen JS Price, A Frazer, 80 Oyajobi and RGG Russell Department of Human Metabolism and Clinical Biochemistry, Uniuersily of Sheffield Medical School, Sheffield SIU 2RX

P59. The effect of 1,25-dihydmxyvitamin Ds on the proliferation and differentiaiton of l vian chondrocytes maintained in oioo C Farquharson, CC Whitehead, JS Rennie and N Loveridge InstiMe of Animal Physiology and Genetics Research, Roslin and Rowett Research Institute, Aberdeen

The annual regrowth of antlers in male deer is by a process of rapid bone formation. However, controversy exists as to whether this growth is “true” endochondral ossification. To further investigate the mechanism controlling this accelerated cellular differentiation, we have set up a system for culturing cells from tissue digests of three different zones of the growing tip; namely the perichondrium (zone l), the chondroblastic zone (zone II), and the vascular hypertrophic cartilage (zone 111). The aim of this study was to characterise, in part, the phenotype of these cells and to compare this with that seen in corresponding regions of tissue sections. Alkaline phosphatase (ALP) activity was detected in fixed cryostat sections of the growing tip by enzyme histochemistry. As has been previously described, ALP activity was strongly expressed with a distinct regional distribution. Type I collagen was immunolocalised to all three regions. We provide the first evidence that, in primary culture,

cells from all three zones also express ALP activity as detected by enzyme histochemistry. This expression was dependent on factors such as the method of tissue digestion, plating density and fixation techniques. The carboxyterminal propeptide of type I collagen (PICP), measured by radioimmunoassay in media conditioned by cells, was used as a marker of type I collagen synthesis. Interestingly, cells from all three zones secrete significant amounts of PICI? In conclusion, cells cultured from the growing region of the deer antler maintain phenotypic characteristics seen in nivo. The pattern of collagen synthesis suggests differences from epiphyseal growth plate.

P5.9. Partial characterisation of the C-terminal domain of type X collagen RE Barber and APL Kwan Department of Biochemistry and Molecular Biolog, Unwersity

of Manchester, Manchester

Type X collagen is a short-chain collagen expressed exclusively in the hypertrophic region of growth plate cartilage undergoing endochondral ossification. Chicken type X collagen consists of three identical a-chains of Mr59,000, each containing a triple helical region of Mr45,000, flanked by a short N-terminal sequence and a larger non-collagenous C-terminal domain of approx. Mr15,OOO. The amino acid sequence of the C-terminal region particular, shows a high degree of interspecies homology. Interesting features include 13 conserved tyrosine residues and a potential N-glycosylation site. Furthermore, studies have shown that type X molecules associate via their C-termini to form a regular hexagonal lattice, which in V&J may provide a modified matrix for the subsequent events of endochondral ossification. The present study has investigated the structure and aggregation properties of the C-terminal globular domain independently of the triple helix. A polypeptide of approx Mr58,OOO derived following bacterial collagenase digestion of cartilage collagens, has been identified as a trimer of al(X) C-terminal domains. When heated to 90-100°C in Laemmli sample buffer, this species yields a dimer (Mr38,OOO) and a monomer (Mr21,OOO). The dimer

Chondrocyte culture and the rachitic growth plate model have indicated that 1,25(OH)zD3 is involved in chondrocyte metabolism. The effects of this hormone on normal chondrocytes in viva are however unknown. Chicks were fed a diet supplemented with 1,25(OHhD3 for 3 weeks and measurements were made in sections of growth plate of chondrocyte proliferation and rate of maturation through the growth plate (using BrdUrd labeling) and also chondrocyte differentiation (ALP activity). ALP activity was quantified by microdensitometry. The labelling index of th control and supplemented chicks were similar (23.1 f 1.3% vs 23.2 f 1.6%) however the BrdUrd positive cells of the supplemented chicks had moved 12.0% (P-zO.05) further down the growth plate than in the control chicks within a 21 h period. Greater ALP activity higher up the growth plate of the supplemented chicks indicated a more differentiated phenotype in cells closer to the top of the growth plate (epiphyseal junction). Within individual transitional chondrocytes ALP activity was 38.5% (P-zO.001) higher in the supplemented chicks. These results suggest that 1,25(OH)sD3 in vivo does not increase the rate of chondrocyte proliferation but does accelerate the onset of maturation.

P60. Parathyroid hormone action on human placenta

K Page, D Abramovich, S Clarke, C Dacke*, T Mayhew” and J Williams Departments of Biomedical Sciences Obstetrics and Gynaecology. Aberdeen University, ‘School of Pharmacy, Portsmouth

UnirTersity, “Dept of Human Morphology, Nottingham University

The actions of bovine parathyroid hormone 1-34 [bPTH(l-3411 on term human placenta were investigated. Compared with control perfused lobules in separate regions of the placenta, exposure to bPTH(l-34) enhanced tissue CAMP content (79ti2%), 3OnM dose in maternal perfusate, n=8, 60+20%, 120 nM dose in fetal perfusate, n=7). Localisation of the peptide using the streptavidin-biotin peroxidase staining technique with a polyclonal hPTH antibody, indicated bI’TH(l-34) crosses from maternal to fetal sides of the syncytiotrophoblast (n=6). Thus bPTH(l-34) probably interacts with basal border (fetal facing) receptors in the syncytiotrophoblast, as adenylate cyclase is localised in these membranes. *5Ca uptakes by microvillous border (maternal facing) membrane vesicles extracted from the trophoblast were measured using tissue obtained either from lobules perfused with 34 nM bPTH(l-34) on maternal side (n=6), or from unperfused placentae, these vesicles being incubated in solutions containing 34 nM bPTH (l-34) (n=5). In neither case did PTH exposure significantly affect 45Ca uptake. It is therefore unlikely that the I’TH induced enhancement of CAMP alters the Ca transport properties of the microvillous membranes. We have not so far studied its effects on *sCa uptake by basal membranes. Support by Action Research is gratefully acknowledged.