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  • mineralization  (3)
  • Alkaline phosphatase  (1)
  • 1
    ISSN: 1432-0827
    Schlagwort(e): Matrix vesicles ; 32P-phosphate and45Ca-metabolism ; Epiphyseal cartilage ; Calcification ; Alkaline phosphatase
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie , Medizin , Physik
    Notizen: Summary Matrix vesicle-enriched fractions were isolated from different zones of epiphyseal cartilage by nonenzymatic methods involving tissue homogenization, differential centrifugation, and isosmotic Percoll gradient fractionation. Uptakes of both32Pi and45Ca were studied concomitantly over periods from 20 min to 24 h. Percoll density gradients separated epiphyseal microsomes into two alkaline phosphatase-rich fractions: a low-density noncalcifiable fraction (P-I), and a higher-density fraction (P-II) which readily mineralized. The P-II fraction was found only in calcifying regions of the growth plate. Based on chemical and physical properties and enzyme activities, both fractions were similar except that P-II contained significantly higher levels of mineral ions than did P-I, and had lower levels of alkaline phosphatase. The mineral appeared to be primarily in a noncrystalline form. Metabolism of32Pi and45Ca by P-II followed a complex kinetic pattern in which accumulation of large amounts of both ions was preceded by an initial limited burst of uptake and a lag-phase of variable duration. During mineral ion loading, the density of the P-II fraction progressively increased as evidenced by co-migration of45Ca,32Pi, and alkaline phosphatase to increasingly higher densities. During the period of early mineral deposition (1–5 h), Ca/P uptake ratios were very low (1.0–1.2) and X-ray diffraction patterns showed a predominantly amorphous pattern. This suggests that the mineral accumulated in matrix vesicles is initially some form of noncrystalline calcium monohydrogenphosphate. L-tetramisole, a potent inhibitor of alkaline phosphatase, inhibited accumulation of both45Ca and32Piin the absence of organic P substrates,32Pi being preferentially inhibited over45Ca. This finding, coupled with recent studies on the behavior of alkaline phosphatase at physiological pH, suggests that the protein is not acting as a phosphohydrolase, but rather as a Pi-binding or transport agent in vesicle-mediated calcification.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 2
    ISSN: 0730-2312
    Schlagwort(e): chondrocytes ; cell culture ; mineralization ; calcospherites ; Ca and P mapping ; matrix vesicles ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Chemie und Pharmazie , Medizin
    Notizen: Advances in the culture of mineralizing growth plate chondrocytes provided an opportunity to study endochondral calcification under controlled conditions. Here we report that these cultures synthesize large amounts of proteins characteristically associated with mineralization: type II and X collagens, sulfated proteoglycans, alkaline phosphatase, and the bone-related proteins, osteonectin and osteopontin. Certain chondrocytes appeared to accumulate large amounts of Ca2+ and Pi during the mineralization process: laser confocal imaging revealed high levels of intracellular Ca2+ in their periphery and X-ray microanalytical mapping revealed the presence of many Ca2+- and Pi-rich cell surface structures ranging from filamentous processes 0.14 ± 0.02 μm by 0.5-2.0 μm, to spherical globules 0.70 ± 0.27 μm in diameter. Removal of organic matter with alkaline sodium hypochlorite revealed numerous deposits of globular (0.77 ± 0.19 μm) mineral (calcospherites) in the lacunae around these cells. The size and spatial distribution of these mineral deposits closely corresponded to the Ca2+-rich cell surface blebs. The globular mineral progressively transformed into clusters of crystallites. Taken with earlier studies, these findings indicate that cellular uptake of Ca2+ and Pi leads to formation of complexes of amorphous calcium phosphate, membrane lipids, and proteins that are released as cell surface blebs analogous to matrix vesicles. These structures initiate development of crystalline mineral. Thus, the current findings support the concept that the peripheral intracellular accumulation of Ca2+ and Pi is directly involved in endochondral calcification.
    Zusätzliches Material: 13 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 3
    ISSN: 0730-2312
    Schlagwort(e): chondrocytes ; osteogenic protein-1 ; retinoic acid ; mineralization ; ALP ; proteoglycans ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Chemie und Pharmazie , Medizin
    Notizen: Osteogenic protein-1 (OP-1), a member of the TGF-β family of proteins, induces endochondral bone formation. Here we studied the effect of OP-1 on the development of primary cultures of avian growth plate (GP) chondrocytes in either serum-free or serum-containing medium, in the absence or presence of retinoic acid (RA). OP-1 was added on day 7 of culture and continued for 7 days, or until the cultures were harvested, typically on day 21. Alone, OP-1 caused ∼2-fold increase in proteoglycan synthesis into both the medium and the cell:matrix layer. Additionally, OP-1 caused a dosage-dependent increase in alkaline phosphatase (ALP) activity, and an increase in protein, when given from days 7-14 and examined on day 14. This stimulation was greater in cells grown in serum-free than in serum-containing media (3-5-fold vs. 2-3-fold increase in ALP; ∼40% vs. ∼20% increase in protein). Such stimulation of ALP activity and proteoglycan (PG) synthesis in cultured GP cells indicates that OP-1 elicits differentiation of chondrocytes. OP-1 minimally affected cell division (DNA content); however, a slight increase was seen when examined early in the culture. Alone, OP-1 increased mineral (Ca and Pi) content of the cultures by ∼2-fold in both types of media. As early as day 14, clusters of mineral encircled many of the OP-1 treated cells. Thus, as in vivo, OP-1 strongly promoted mineral formation by the cultured GP chondrocytes. When present together, OP-1 and RA generally blocked the action of the other. Separately OP-1 and RA each stimulated protein synthesis, ALP activity, and Ca2+ deposition; together they were inhibitory to each. Also, RA blocked the stimulation of PG synthesis induced by OP-1; whereas OP-1 decreased cell division engendered by RA. Thus, this GP chondrocyte culture system is a good model for studying factors that influence differentiation and mineral deposition during bone growth in vivo. J. Cell. Biochem. 67:498-513, 1997. © 1997 Wiley-Liss, Inc.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 4
    Digitale Medien
    Digitale Medien
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 68 (1998), S. 110-120 
    ISSN: 0730-2312
    Schlagwort(e): cadmium ; zinc ; cell culture ; mineralization ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Chemie und Pharmazie , Medizin
    Notizen: Following exposure to cadmium or zinc, chickens were sacrificed and the liver, kidney, and bone epiphyseal growth plates harvested. When cytosolic extracts of the growth plate cartilage were fractionated by gel filtration chromatography, a protein with high metal-binding capacity and low ultraviolet (UV) absorbance eluted in the same position as liver metallothionein (MT) and a MT standard. Cd or Zn treatment resulted in a 25-fold or 5-fold induction in growth plate MT, respectively. In liver the greatest level of MT induction was seen with short-term Cd exposures. In contrast, MT levels in the growth plate increased as the duration of Cd exposure increased. Induction of MT in growth plate chondrocyte cell cultures was observed for media Cd concentrations of ≥0.1 μM and Zn concentrations of ≥100 μM. Basal and inducible levels of MT declined through the culture period and were lowest in the terminally differentiated mineralized late stages of the culture. Alkaline phosphatase activity was also lowest in the late-stage cultures, while total cellular protein increased throughout the culture period. Treatment of chondrocytes with Zn prior to Cd exposure resulted in a protective induction of MT. Pre-treatment of chondrocytes with dexamethasone resulted in suppressed synthesis of MT upon Cd exposure and greater Cd toxicity. Both Cd and Zn resulted in significantly increased levels of MT mRNA in chondrocyte cell cultures. Dexamethasone treatment resulted in an approximate 2- to 3-fold increase in MT mRNA. This is contrary to the finding that MT protein levels were decreased by dexamethasone. The findings suggest that an increased rate of MT degradation in dexamethasone-treated and late-stage chondrocyte cultures may be associated with the terminally differentiated phenotype. J. Cell. Biochem. 68:110-120, 1998. © 1998 Wiley-Liss, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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