Quantitative study of the lactate dehydrogenase-elevating virus in mouse embryo cultures.
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Biomedical subjects
Publications and source records attributed to H C Anderson.
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Chondrocytes obtained from collagenase-digested epiphyseal growth plate cartilage of rachitic rats were grown in multilayer cultures. The cultured chondrocytes produced a metachromatic matrix and further electron microscopic examination revealed typical features of cartilage matrix collagen fibrils and matrix vesicles. The alkaline phosphatase activity in cultures was high during the entire 3-week culture period. Acid phosphatase showed a marked increase in activity during the first week of culture. The appearance of apatite crystals in the synthesized matrix was monitored by electron microscopy over a 3-week period. First crystals were consistently found to be associated with matrix vesicles, and in the older cultures calcification spread into the surrounding matrix. No collagen fibrils associated with mineralization were observed during the early culture period. This study clearly demonstrates that in chondrocyte cultures the first mineral crystals were found within or in close association with matrix vesicles. This gives further support to the hypothesis that matrix vesicles are the primary site of mineralization in cartilage. In addition to calcification studies it is suggested that this model is suitable for studying the effects of hormones or other agents on rachitic chondrocytes in vitro.
The preparation of mineralizing tissues for electron microscopy by methods that involve the use of aqueous solvents may result in translocation and/or removal of mineral, leading to the possible erroneous interpretation of initial calcifying loci. The failure of a recent cryoultramicrotomy study to detect a mineral phase associated with matrix vesicles raised doubts about the role of the matrix vesicle in initial mineralization and prompted us to reexamine the ultrastructure of calcifying cartilage using three different anhydrous preparatory techniques. Matrix vesicles or structures similar in profile and location were identified in rat epiphyseal growth plate cartilages that had been subjected to freeze-substitution, cryoultramicrotomy, or ethylene glycol fixation. Furthermore, mineral deposits, as confirmed by energy dispersive x-ray analysis, were observed within or in close association with the matrix vesicles in the extracellular matrix of the growth plate cartilage by all three methods. No evidence of mineral deposition at other sites preceding matrix vesicle calcification was found. Our studies support the conclusion of many investigations that matrix vesicles are the initial site of growth plate calcification.
The plasma membranes of epithelial cells are divided into apical and basolateral domains. These two surfaces are characterized by markedly different protein compositions, reflecting the ability of the cell to target newly synthesized membrane proteins to specific regions of the cell surface. This targeting capability is also apparent in the polarized release of secretory products. Recent studies using canine renal tubule (MDCK) cells have suggested that distinct sets of secretory proteins are released from their apical and basolateral poles. We report experiments designed to examine secretory protein sorting by MDCK cells. We have shown that secretion of basement membrane components (laminin and heparan sulphate proteoglycan (HSPG] takes place from the basolateral cell surface and that this polarized release results from active sorting. The sorting process which mediates this polarized secretion requires an acidic intracellular compartment. MDCK cells treated with NH4Cl to raise the pH of their intracellular compartments, secrete laminin and HSPG by a default pathway which leads to their release in roughly equal quantities into the medium of both the apical and basolateral compartments.