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Birefringence and arrangement of collagen and cartilage
Sixth International Conference on the Molecular Biology and Pathology of Matrix
The Laminin 5 Synthesis and Extracellular Processing By Human Keratinocytes S. Amano, T. Nishiyama* and R. E. Burgeson MGH/Harvard Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA and Shiseido Research Ctr., Yokohama, Japan. Laminin 5 extracted from tissues is known to be composed of ¢z3 (165 or 145 kDa), 133 (140 kDa) and 72 (1 05 kDa) . Cultured keratinocytes synthesized an intracellular precursor composed of (z3 chain (200 kDa), J33 (140 kDa) and 72 (155 kDa). To further examine the observed proteolytic processing, the kinetics of the process were examined in cultured keratinocytes. Cells were pulse labeled and chased for various times. The results indicated that (1) synthesis of (~3 might be a ratelimiting step in iaminin 5 assembly; (2) processing of (~3 to 165 kDa began 10 min. after labeling, approximately corresponding to the time of secretion; (3) processing of 72 to 105 kDa occurred later, 3 hours after labeling; (4) processing of ~3 and 72 did not continue after release into the culture medium. Extracellular laminin 5 was shown to be concentrated in contacts with the plastic substrate by immunofluorescence, and was fully processed. EDTA and o-phenanthroline inhibited the processing. Unprocessed laminin 5, showed high affinity for heparin through the (z3 and 72 chains. These results suggest that both integrins and cell surface heparan sulfate proteoglycans can bind laminin 5, may participate in the extracellular processing or transport of the fully processed laminin 5 molecules to anchoring contacts. The processing of (z3 and 72 are temporally separate and therefore are likely to be due to the actions of different enzymes.
163
superficial split lines of articular cartilage. The split line pattern was demonstrated on the femoral and tibial condyles of canine knee. The optical path difference (= retardation, 7) and the cartilage zone thickness analysis of the sections was performed with a computerized analysis of the images of a polarized light microscope (Leitz Ortholux ® 2 POL) connected to a thermoelectrically cooled camera (Photometrics CH250/A). Fresnel's general equation was utilized to relate the intensity of incident light to the intensity of emergent light as altered by the retardation of the object and compensator. The mean birefrigence [= areaadjusted integrated retardation value (AIR) (nm/l~m2)] of collagen fibrils and thickness (mm or %) of articular cartilage zones were determined. The mean values of AIR were higher in the femoral than in the tibial cartilage. The mean thickness of the superficial zone, and the proportion of the superficial zone thickness to the whole thickness of the uncalcified cartilage, were higher at femoral than tibial test points. The specimen orientation related to the split lines affected both the AIR as well as the thickness of articular cartilage zones. Polarized light microscopy is a sensitive method and it gives a good overall insight into the general pattern of collagen fibril network of tissue. The orientation of the specimen relative to the superficial split lines is, however, critical since sections cut parallel to the artificial split lines show higher birefringence and thickness of the superficial zone than sections cut perpendicularly. Threfore, determination of split line direction is recommended prior to microscopy.
Running Activity of Mice Monitored with a Computer-Assisted System. Birefringence and Collagen in Cartilage.
Arrangement
of
J.P.A. Arokoski *t, M.M. Hyttinen*, M. EIo*, K. Kiraly, T. Lapvetel&inen, H.J. Helminen* * Department of Anatomy, University of Kuopio, P.O. Box 1627, FIN-70211, Kuopio, Finland; tDept, of Physical and Rehabilitation Medicine, Kuopio Univ. Hospital, Finland. We examined the collagen fibril network of articular cartilage from unstained histological sections cut parallel, perpendicular, or at 45° angles to the
T. Lapvetel,~inen, A. Tilhonen*, T. Nevalainen t, J. Linkblom, K. Kiraly, P. Halonen~, H.J. Helminen Department of Anatomy, University of Kuopio, Kuopio, Finland; * Technical Centre, t National Laboratory Animal Center, ~Computer Center, Univesity of Kuopio, Finland. We have taken into use a computer-based follow-up system of mice to record the running activity of the animals. It was anticipated that the running activity would be altered when a mutated gene of the musculoskeletal system affects the development and function of the Iocomotory system. Thus, running activity would be one
The Laminin 5 Synthesis and Extracellular Processing By Human Keratinocytes S. Amano, T. Nishiyama* and R. E. Burgeson MGH/Harvard Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA and Shiseido Research Ctr., Yokohama, Japan. Laminin 5 extracted from tissues is known to be composed of ¢z3 (165 or 145 kDa), 133 (140 kDa) and 72 (1 05 kDa) . Cultured keratinocytes synthesized an intracellular precursor composed of (z3 chain (200 kDa), J33 (140 kDa) and 72 (155 kDa). To further examine the observed proteolytic processing, the kinetics of the process were examined in cultured keratinocytes. Cells were pulse labeled and chased for various times. The results indicated that (1) synthesis of (~3 might be a ratelimiting step in iaminin 5 assembly; (2) processing of (~3 to 165 kDa began 10 min. after labeling, approximately corresponding to the time of secretion; (3) processing of 72 to 105 kDa occurred later, 3 hours after labeling; (4) processing of ~3 and 72 did not continue after release into the culture medium. Extracellular laminin 5 was shown to be concentrated in contacts with the plastic substrate by immunofluorescence, and was fully processed. EDTA and o-phenanthroline inhibited the processing. Unprocessed laminin 5, showed high affinity for heparin through the (z3 and 72 chains. These results suggest that both integrins and cell surface heparan sulfate proteoglycans can bind laminin 5, may participate in the extracellular processing or transport of the fully processed laminin 5 molecules to anchoring contacts. The processing of (z3 and 72 are temporally separate and therefore are likely to be due to the actions of different enzymes.
163
superficial split lines of articular cartilage. The split line pattern was demonstrated on the femoral and tibial condyles of canine knee. The optical path difference (= retardation, 7) and the cartilage zone thickness analysis of the sections was performed with a computerized analysis of the images of a polarized light microscope (Leitz Ortholux ® 2 POL) connected to a thermoelectrically cooled camera (Photometrics CH250/A). Fresnel's general equation was utilized to relate the intensity of incident light to the intensity of emergent light as altered by the retardation of the object and compensator. The mean birefrigence [= areaadjusted integrated retardation value (AIR) (nm/l~m2)] of collagen fibrils and thickness (mm or %) of articular cartilage zones were determined. The mean values of AIR were higher in the femoral than in the tibial cartilage. The mean thickness of the superficial zone, and the proportion of the superficial zone thickness to the whole thickness of the uncalcified cartilage, were higher at femoral than tibial test points. The specimen orientation related to the split lines affected both the AIR as well as the thickness of articular cartilage zones. Polarized light microscopy is a sensitive method and it gives a good overall insight into the general pattern of collagen fibril network of tissue. The orientation of the specimen relative to the superficial split lines is, however, critical since sections cut parallel to the artificial split lines show higher birefringence and thickness of the superficial zone than sections cut perpendicularly. Threfore, determination of split line direction is recommended prior to microscopy.
Running Activity of Mice Monitored with a Computer-Assisted System. Birefringence and Collagen in Cartilage.
Arrangement
of
J.P.A. Arokoski *t, M.M. Hyttinen*, M. EIo*, K. Kiraly, T. Lapvetel&inen, H.J. Helminen* * Department of Anatomy, University of Kuopio, P.O. Box 1627, FIN-70211, Kuopio, Finland; tDept, of Physical and Rehabilitation Medicine, Kuopio Univ. Hospital, Finland. We examined the collagen fibril network of articular cartilage from unstained histological sections cut parallel, perpendicular, or at 45° angles to the
T. Lapvetel,~inen, A. Tilhonen*, T. Nevalainen t, J. Linkblom, K. Kiraly, P. Halonen~, H.J. Helminen Department of Anatomy, University of Kuopio, Kuopio, Finland; * Technical Centre, t National Laboratory Animal Center, ~Computer Center, Univesity of Kuopio, Finland. We have taken into use a computer-based follow-up system of mice to record the running activity of the animals. It was anticipated that the running activity would be altered when a mutated gene of the musculoskeletal system affects the development and function of the Iocomotory system. Thus, running activity would be one