Isolation and Partial Characterization of Precursors to Minor Cartilage Collagens

Clark, Charles C.; Richards, Charlotte F.

doi:10.1016/s0174-173x(85)80011-x

Cited by 30 publications

(18 citation statements)

References 51 publications

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“…After 18 h at 4 ~ C, the combined material was dialysed against deionized water and then against 0.4 M NaC1-0.1 M Tris. Chymotrypsin (0.3 mg/ml) was added for 6 h at 10 ~ C, after which N-tosyl-L-phenylalanine chloromethyl ketone (0.1 mg/ml) was added overnight to stop the reaction (Clark and Richards 1985). The soluble material was dialysed against 0.5 M acetic acid (4 ~ C).…”

Section: Determination Of Collagen Types Intestinal Smooth Muscle Cementioning

confidence: 99%

Collagen production by human smooth muscle cells isolated during intestinal organogenesis

et al. 1992

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The extracellular matrix influences organogenesis by modulating cell behavior. In humans, collagen is the major matrix constituent of the adult intestinal wall and is synthesized by smooth muscle cells. The objective of the current study was to examine collagen production by fetal human intestinal smooth muscle cells isolated during intestinal morphogenesis. Techniques were developed for the isolation and culture of human fetal intestinal smooth muscle cells. The cultured cells were confirmed as muscle by immunohistochemical stains for cytoskeletal filaments and documentation of contractile behavior. In culture, these cells stained for mesenchymal and muscle cytoskeletal proteins: vimentin, actin, and desmin, and did not stain for neural or epithelial markers. The muscle cells contracted in response to acetylcholine, in contrast to human fetal dermal fibroblasts which did not contract appreciably. Collagen production was assayed by the uptake of [3H]-proline into collagenase-digestible protein. Collagen production was greatest at 11 weeks gestation, the youngest age studied. By 20 weeks gestation, collagen production had decreased to adult levels. However, when compared to another matrix-producing fetal mesenchymal cell, the dermal fibroblast, intestinal smooth muscle cells produced twice as much collagen. Collagen types were determined by polyacrylamide slab gel electrophoresis. Smooth muscle cells predominantly produced types I and III collagen alpha chains. Therefore, collagen production is a significant function of human fetal intestinal smooth muscle cells, and probably plays a major role in the development of intestinal structure. The in vitro model presented here provides a means of studying the regulation of this collagen production throughout intestinal organogenesis.

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Section: Determination Of Collagen Types Intestinal Smooth Muscle Cementioning

confidence: 99%

Collagen production by human smooth muscle cells isolated during intestinal organogenesis

et al. 1992

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“…The data were statistically analyzed for differences among groups. Aliquots of the [ ''C]proline-labeled samples were digested with chymotrypsin and prepared for SDS-polyacrylamide gel electrophoresis (PAGE) as described previously (7). The resulting fluorograms were analyzed densitometrically .…”

Section: Partmentioning

confidence: 99%

Proliferation and macromolecular synthesis by rat calvarial bone cells grown in various oxygen tensions

Brighton

Schaffer

Shapiro

et al. 1991

Journal Orthopaedic Research

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Perinatal rat calvarial bone cells were isolated by sequential collagenase digestion and grown in oxygen tensions ranging from 1 to 60% O2. Cell proliferation as determined by automated cell counting and DNA content was greatest in the lower oxygen tensions (less than or equal to 9% O2), whereas alkaline phosphatase activity and [35S]sulfate and [14C]proline incorporation were greatest in the higher oxygen tensions (greater than or equal to 13% O2). It is concluded that lower oxygen concentrations favor bone cell proliferation, whereas higher oxygen concentrations favor macromolecular synthesis. These findings, when related to the known pO2 of the fracture callus, suggest the following sequence of events: first, at the time of fracture an ingrowth of osteoprogenitor cells, capillary buds, and primitive mesenchymal cells occurs in the fracture site, a region of low pO2; second, a great increase in cellular proliferation accompanied by an initiation of macromolecular synthesis follows; finally, as the pO2 levels begin to increase, cellular proliferation decelerates, accompanied by an increase in macromolecular synthesis.

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“…Chondrocytes were isolated from 17-day chick-embryo sterna and cultured in suspension as described previously (Clark & Richards, 1985;Clark et al, 1987). In most of the experiments, the culture medium, which contained ascorbate and ,aminopropionitrile (50 jug/ml each) and 300 #uM-Na2SO4 was further supplemented with 1 mM-L-glutamine (Speight et al, 1978).…”

Section: Chondrocyte Isolation and Culturementioning

confidence: 99%

Post-translational alterations in newly synthesized cartilage proteoglycans induced by the glutamine analogue 6-diazo-5-oxo-l-norleucine. Time course of inhibition and recovery

Clark

Richards

Iozzo

1991

Biochemical Journal

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Incorporation of [35S]sulphate by cultures of matrix-free cells from chick embryo sterna in the presence of the glutamine analogue 6-diazo-5-oxo-L-norleucine (0.58 mM) was inhibited in a time-dependent manner to less than 15% of that in control cultures after 2 h. Characterization of the major cartilage proteoglycan synthesized under these conditions showed that it contained few, if any, normal-sized chondroitin sulphate chains and only about half of the normal complement of substituted serine residues. Subsequent addition of D-glucosamine hydrochloride (final concn. 2 mM) resulted in a time-dependent recovery of [35S]sulphate incorporation to 90% of control cultures after 2 h, but restored the chondroitin sulphate chains to normal size within 15 min. On the basis of these results, it is concluded that a 2 h preincubation is necessary to deplete the chondrocytes of the endogenous supply of UDP-N-acetyl-D-glucosamine required for optimal glycoconjugate synthesis, and that this situation results in the synthesis of a chondroitin sulphate proteoglycan with significantly altered properties, owing to the paucity of glycosaminoglycan chains; however, this condition is completely reversible if the D-glucosamine pool is repleted.

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Isolation and Partial Characterization of Precursors to Minor Cartilage Collagens

Cited by 30 publications

References 51 publications

Collagen production by human smooth muscle cells isolated during intestinal organogenesis

Collagen production by human smooth muscle cells isolated during intestinal organogenesis

Proliferation and macromolecular synthesis by rat calvarial bone cells grown in various oxygen tensions

Post-translational alterations in newly synthesized cartilage proteoglycans induced by the glutamine analogue 6-diazo-5-oxo-l-norleucine. Time course of inhibition and recovery

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