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P75. Purification of α-2HS-glycoprotein from a human plasma fraction and development of an enzyme linked immunoabsorvent assay
134 free calcium (IFC) by ratio imaging of FURAdye excitation showed the release of IFC within 160 milliseconds released from intracellular stores. Ratiometric imaging of membrane potential showed a complex response where initially the membrane potential hyperpolarised and then returned to normal values (about 7OmV) within 5OOmsec. Similar results were gained in the absence of extracellular ions, thus reducing any zeta potential (streaming potential) on the cell surface. The role of strain related potentials does not thus appear to be involved in this pathway. These results are consistent with the presence of a strain activated phospholipase C which activates the same classical sequence of events that follows exposure to certain hormones and factors.
P74. Stiffening of cancellous bone by surrounding it with cortical bone R Bryce and RM Aspden Department of Orthopaedics, Unioersily of Aberdeen, Scotland When materials are compressed along one direction they tend to expand in perpendicular directions. A theoretical analysis shows that a shell of cortical bone will constrain the lateral expansion of loaded cancellous bone and increase its apparent stiffness and strength. Experimental results show that cancellous bone from vertebral bodies appears stiffer by a factor of about four when tested in situ in an intact vertebra than when removed as a cylindrical plug. These results are in good agreement with the theoretical analysis. Isolated cancellous bone is light but not very stiff or strong whereas cortical bone is stiffer and stronger but more dense. These results show that combining these two materials leads to struchues which are light but stronger than would be predicted from laboratory tests on the isolated components. It was also found that water content increased and organic content decreased as the density of the tissue increased. The composition, described in terms of volume fractions of water, organic and mineral phases, entirely explained the measured density in that order of strength of correlation. We conclude that (i) the stiffness of cancellous bone in zpivo is considerably greater than that found in conventional laboratory testing, (ii) although the density of cancellous bone may be predicted from its composition this is not the principal determinant of stiffness, and (iii) mineral content is a poor predictor of cancellous bone stiffness.
P75. Purification of a-2HS-glycoprotein from a human plasma fraction and development of an enzyme linked inununorbeorvent assay L Kalabay, R Gwilliam, P Matejtschuk’ and I Dickson Department of Biology and Biochemistry, Brunei University, Uxbridge UB8 3PH and *Research & Development Department Bioproducls Laboratory, EIstree Herts WD6 3BX Alpha-2HSglycoprotein (AH%) is a 50 kDa plasma glycoprotein which becomes incorporated into bone during mineralization. It is enriched there relative to other plasma proteins and constitutes a major non-collagenous protein of bone matrix. The objective of the research was to purify the glycoprotein for studies of ib biological properties and to devise a sensitive assay. Fraction V supernatant, obtained during the industrial scale fractionation of human plasma by Ihe procedure of Kiestler and Nitschmann, was used as starting material; preparations from fraction IV residue, which also contains AHSG, were found to contain some degraded material. Much of the AHSG present in
Abstracts from the Joint Meeting, September 1992
traction supernatant could be recovered by cryoprecipitation an was purified by column chromatography on DEAE cellulose, Affi-Gel Blue gel, and hydroxylapatite. An indirect enzymelinked immunoabsorbent assay (ELlSA) was developed with rabbit antibody to AHSC as first antibody and peroxidaseconjugated goat antibody to rabbit 1gG as second antibody, ophenylyene diamine being used a substrate. With this assay AHSG could be quantitated in the range 15 - 150 ng/ml. The assay is more convenient, rapid and sensitive than onedimensional immunoelectrophoresis, which is usually used to determine AHSG in serum or urine specimens
P76. A histochomical assessment osteosarcoma SD Shnyder, J I’ringle, ID Bowen’ and Institute of Orthopaedics, University ‘Departments of Pure Middlesex, “Anatomy, University Wales College
of cell death in human CW Arche?’ College London, StanmorE, b Applied Biology, and of Cardiff, Cardiff
Very little is known about the precise status of a tumour which is histologically “altered” as a result of chemotherapy given preresection as part of the treatment for osteosarcoma. Alkaline phosphatase positive cells are present in variable numbers, indicating that the tumour is metabolically active, but few mitotic figures are seen. In planning further chemotherapy, it would be useful to know if these cells are in the initial stages of cell death or are merely mitotically quiescent. The presence of acid phosphatase has been associated with cell death. Using a methacrylate embedding technique (Lewis & Bowen, 1985) to enhance tissue resolution, a histochemical method for the detection of acid phosphatase was utilised on viable, “altered” taken from osteosarcomas on samples necrotic and resection/amputation. A variety of patterns of positive staining were seen from specimen to specimen. However, preliminary data suggest that “altered” areas of tumour show elevated levels of acid phosphatase in non-reactive cells when compared with viable areas.
P77. The effects of alterations in oestrogen treatment on the development of osteopenia in female mice SR Milligan, MW Robins and DMJ Tissera Physiology Group, King’s College, London WC2 48 adult (80 days old) female mice were divided into 3 groups ovariectomized (OVEX), mock-operated (CONT) and ovariectomized with oestrogen-replacement therapy by a subcutaneous, silastic implant (OEST). After 60 days half of each group was killed for analysis. Then the remaining OVEX animals then were given oestrogen replacement (OVEX/OEST), and the OEST group were withdrawn from oestrogen (OEST/OVEX). After a h.uurther 60 days remaining animals were killed. Analysis of material was as follows - linear dimension and dry weights of parietal, femur and tibio-fibula, extent of parietal diploe, wet weight of uterus, gastrocnemius, plantaris and soleus muscles. At day 60 osteopenia was found in OVEX (dry weights). Additionally, the extent of the diploe in the parietals was much greater in OVEX than in OEST and CONT. At day 120, following sixty days reversal of therapy there were the expected changes in bone weights (relative loss in OEST/OVEX, relative gain in OVEX/OEST), but these were not significant. Probably a longer period is required for the the reversal of therapies to produce significant changes.
P78. The effects of castration and testosterone therapy bones of male mice SR Milligan, MW Robins and S Yogorajah Physiology Group, King’s College, Londotr WC2
on the
P74. Stiffening of cancellous bone by surrounding it with cortical bone R Bryce and RM Aspden Department of Orthopaedics, Unioersily of Aberdeen, Scotland When materials are compressed along one direction they tend to expand in perpendicular directions. A theoretical analysis shows that a shell of cortical bone will constrain the lateral expansion of loaded cancellous bone and increase its apparent stiffness and strength. Experimental results show that cancellous bone from vertebral bodies appears stiffer by a factor of about four when tested in situ in an intact vertebra than when removed as a cylindrical plug. These results are in good agreement with the theoretical analysis. Isolated cancellous bone is light but not very stiff or strong whereas cortical bone is stiffer and stronger but more dense. These results show that combining these two materials leads to struchues which are light but stronger than would be predicted from laboratory tests on the isolated components. It was also found that water content increased and organic content decreased as the density of the tissue increased. The composition, described in terms of volume fractions of water, organic and mineral phases, entirely explained the measured density in that order of strength of correlation. We conclude that (i) the stiffness of cancellous bone in zpivo is considerably greater than that found in conventional laboratory testing, (ii) although the density of cancellous bone may be predicted from its composition this is not the principal determinant of stiffness, and (iii) mineral content is a poor predictor of cancellous bone stiffness.
P75. Purification of a-2HS-glycoprotein from a human plasma fraction and development of an enzyme linked inununorbeorvent assay L Kalabay, R Gwilliam, P Matejtschuk’ and I Dickson Department of Biology and Biochemistry, Brunei University, Uxbridge UB8 3PH and *Research & Development Department Bioproducls Laboratory, EIstree Herts WD6 3BX Alpha-2HSglycoprotein (AH%) is a 50 kDa plasma glycoprotein which becomes incorporated into bone during mineralization. It is enriched there relative to other plasma proteins and constitutes a major non-collagenous protein of bone matrix. The objective of the research was to purify the glycoprotein for studies of ib biological properties and to devise a sensitive assay. Fraction V supernatant, obtained during the industrial scale fractionation of human plasma by Ihe procedure of Kiestler and Nitschmann, was used as starting material; preparations from fraction IV residue, which also contains AHSG, were found to contain some degraded material. Much of the AHSG present in
Abstracts from the Joint Meeting, September 1992
traction supernatant could be recovered by cryoprecipitation an was purified by column chromatography on DEAE cellulose, Affi-Gel Blue gel, and hydroxylapatite. An indirect enzymelinked immunoabsorbent assay (ELlSA) was developed with rabbit antibody to AHSC as first antibody and peroxidaseconjugated goat antibody to rabbit 1gG as second antibody, ophenylyene diamine being used a substrate. With this assay AHSG could be quantitated in the range 15 - 150 ng/ml. The assay is more convenient, rapid and sensitive than onedimensional immunoelectrophoresis, which is usually used to determine AHSG in serum or urine specimens
P76. A histochomical assessment osteosarcoma SD Shnyder, J I’ringle, ID Bowen’ and Institute of Orthopaedics, University ‘Departments of Pure Middlesex, “Anatomy, University Wales College
of cell death in human CW Arche?’ College London, StanmorE, b Applied Biology, and of Cardiff, Cardiff
Very little is known about the precise status of a tumour which is histologically “altered” as a result of chemotherapy given preresection as part of the treatment for osteosarcoma. Alkaline phosphatase positive cells are present in variable numbers, indicating that the tumour is metabolically active, but few mitotic figures are seen. In planning further chemotherapy, it would be useful to know if these cells are in the initial stages of cell death or are merely mitotically quiescent. The presence of acid phosphatase has been associated with cell death. Using a methacrylate embedding technique (Lewis & Bowen, 1985) to enhance tissue resolution, a histochemical method for the detection of acid phosphatase was utilised on viable, “altered” taken from osteosarcomas on samples necrotic and resection/amputation. A variety of patterns of positive staining were seen from specimen to specimen. However, preliminary data suggest that “altered” areas of tumour show elevated levels of acid phosphatase in non-reactive cells when compared with viable areas.
P77. The effects of alterations in oestrogen treatment on the development of osteopenia in female mice SR Milligan, MW Robins and DMJ Tissera Physiology Group, King’s College, London WC2 48 adult (80 days old) female mice were divided into 3 groups ovariectomized (OVEX), mock-operated (CONT) and ovariectomized with oestrogen-replacement therapy by a subcutaneous, silastic implant (OEST). After 60 days half of each group was killed for analysis. Then the remaining OVEX animals then were given oestrogen replacement (OVEX/OEST), and the OEST group were withdrawn from oestrogen (OEST/OVEX). After a h.uurther 60 days remaining animals were killed. Analysis of material was as follows - linear dimension and dry weights of parietal, femur and tibio-fibula, extent of parietal diploe, wet weight of uterus, gastrocnemius, plantaris and soleus muscles. At day 60 osteopenia was found in OVEX (dry weights). Additionally, the extent of the diploe in the parietals was much greater in OVEX than in OEST and CONT. At day 120, following sixty days reversal of therapy there were the expected changes in bone weights (relative loss in OEST/OVEX, relative gain in OVEX/OEST), but these were not significant. Probably a longer period is required for the the reversal of therapies to produce significant changes.
P78. The effects of castration and testosterone therapy bones of male mice SR Milligan, MW Robins and S Yogorajah Physiology Group, King’s College, Londotr WC2
on the