Matrix Biosynthesis Research Articles

37] Assays of growth factor stimulation of extracellular matrix biosynthesis

The chapter presents some of the more useful methods for the quantitative and qualitative measurements of extracellular matrix components affected by growth factors. In primary culture a strong, positive correlation between the growth stimulating potencies of growth factors and their abilities to differentially amplify basement membrane protein–laminin, collagen IV, and proteoglycan, synthesis has been established. Additionally, when mammary cells are cultured on different types of extracellular matrix proteins, they display differing requirements for growth factors. These observations suggest that at least part of the mechanism by which growth factors act is by enhancing the biosynthesis of extracellular matrix proteins. This is especially true in the case of primary cell cultures in which cell selection for culture conditions has not taken place. Thus quantitation of growth factor requires analysis of cells and growth medium. Growth factors have been shown to alter both the biosynthetic and degradative rates of matrix proteins, especially of collagen, the component that has been studied in greatest detail.

An immunofluorescence study of extracellular matrix associated with cytotrophoblast of the chorion laeve

Using immunofluorescence we have studied the distribution within and beneath the cytotrophoblast of chorion laeve of five extracellular matrix components: types I, III and IV collagen, fibronectin and laminin. Fibronectin, laminin and types I and IV collagen are located in a network of extracellular matrix which encapsulates cells of the cytotrophoblast multilayer. The results suggest that cytotrophoblast in all layers is active in matrix biosynthesis. The pseudo-basement membrane, which links this intercellular matrix to the underlying reticular layer, contains rich deposits of fibronectin and type III collagen along with more restricted foci of laminin and type IV collagen. The reticular layer contains fibronectin, type III collagen and small amounts of type I collagen, but no laminin or type IV collagen. Type III collagen is also found in the maternal decidua. Extracellular matrix markers may be useful in defining trophoblast differentiation pathways. Possible reasons are discussed for the unusual architecture of extracellular matrix of cytotrophoblast of the chorion laeve. Cytoplasmic actin occurs in cells throughout the chorion laeve, while cytokeratin appears only in cytotrophoblast. Plasminogen also occurs throughout cytotrophoblast in pericellular locations.

Synovial factors and chondrocyte-mediated breakdown of cartilage: inhibition by hydrocortisone.

Cartilage-synovium interactions were explored in a model culture system. Bovine nasal-cartilage discs were cocultured with minced rheumatoid synovium or synovium-conditioned media (SCM) in the presence or absence of hydrocortisone. Cartilage breakdown was assessed by the release of proteoglycan (PG) and hydroxyproline, and matrix biosynthesis by [35S]sulfate incorporation during pulse labeling. Chondrocyte-dependent breakdown in response to synovial factors (i.e., "catabolin" activity) was assessed by the difference in PG release between living and dead cartilages. Short-term contact with minced synovial membrane or exposure to its products released at a distance was sufficient to induce cartilage degradation in coculture; continued exposure was not required for breakdown to persist. Conditioned media from short-term synovial culture were similarly potent, and the induced breakdown was chondrocyte dependent. Matrix biosynthesis was inhibited in exposed cartilage but could be rapidly restored to normal on synovium removal despite the persistence of cartilage breakdown. Early hydrocortisone treatment suppressed the initiation of cartilage breakdown in cocultures and largely abolished the appearance of inductive factors in SCM. Later applications had little effect either in cocultures or in catabolin assays. We conclude that synovium-induced breakdown is an early event and that chondrocyte catabolic mechanisms once they have been activated are sufficient to maintain breakdown at a high level. Hydrocortisone, as well as limiting proteolysis, inhibits early tissue interactions at the level of synovial catabolin production or release.

Influence of colchicine on the synthesis and secretion of proteoglycans and collagen by fetal guinea pig chondrocytes

Fetal guinea-pig epiphyseal chondrocytes were cultured in monolayers and as aggregates in the presence of antimicrotubular agents. Colchicine and vinblastine caused a dissociation of the Golgi complex, in addition to the disappearance of microtubules. Synthesis and secretion of proteoglycans and collagen were studied using radioactive precursors. Colchicine inhibited the synthesis of proteoglycans. The drug also inhibited secretion with an intracellular accumulation of these molecules. The proteoglycans secreted by the colchicine-treated cells had a smaller molecular size and contained a smaller proportion of aggregated molecules than proteoglycans in control cultures. However, there was no difference in the average size of the chondroitin sulfate side chains of the proteoglycan molecules. Nor was there any increase in the breakdown of proteoglycans in colchicine-treated cultures. Vinblastine was also found to inhibit synthesis and secretion of proteoglycans. Deuterium oxide also inhibited the synthesis of these molecules but stimulated their secretion into the medium. Colchicine caused an inhibition of both synthesis and secretion of collagen. It is suggested that the quantitative and qualitative effects of colchicine could be the result of disturbances in the Golgi complex, possibly in combination with a retarded translocation of secretory vacuoles. However, as the colchicine-treated chondrocytes were still able to continue a large part of their matrix biosynthesis with only moderate changes in the structure of the secreted molecules, it is probable that alternative pathways for the secretion of matrix molecules exist and/or the Golgi complex is able to retain a major part of its function despite the structural alterations.

Reaction of the cytosine nucleus with hydrazine in the presence of bisulphite

1. Introduction The presence of a reactive hydrazine group in ti- amino cytosine provides ample opportunities for the specific introduction of labels (fluorescent or radio- active), combinations with carbonyl-containing carriers (cellulose or Sephadex) and for other specific secondary reactions. Also, p-amino cytidine is an effective mutagen [ 11, this effect being evidently caused by the double functional specificity of this compound in the systems of matrix biosynthesis similar to p-hydroxy- and fl-methoxy-derivatives of cytidine and adenosine [2]. By hydrazine action in aqueous neutral solutions the conversion of cytosine into the p-amino cytosine is slow even in rather vigorous conditions [ 3] . As was shown earlier [4-81 , the nucleophilic substitution of the exocyclic amino group of cytosine is greatly facilitated by the presence of bisulphite. In this paper it is shown that p-amino cytosine derivatives can be obtained in mild conditions when cytidine is treated with a bisulphite-hydrazine mix- ture and some kinetic characteristics of this reaction are adduced. 2. Materials and methods Sodium metabisulphite NazSzOs was prepared as described earlier [8] . Hydrazine hydrate was an analytically pure preparation. The reaction was followed by the changes of u.v.- spectra in lOO-fold water dilutions of portions of the reaction mixture. 212 7 mg of 2’-desoxycytidine-5’-phosphate were dissolved in 2 ml of 1 M sodium metabisulphite solution containing hydrazine (hydrazine concentration, pH values and temperature are given in table 1). Immedi- ately after nucleotide dissolution and then after fixed intervals of 0.1 ml portions were taken and diluted in 10 ml of wafer. The spectra were measured lo- 15 minutes after dilution. To study the velocity of the reverse reaction, the mixtures were incubated until no further spectral changes occurred and then portions were diluted SO-fold with 0.5 M buffer solutions (pH 5.0 - sodium acetate, pH 6.0 and 7.0 - sodium phosphate, pH 7.9 - TrisHCl, pH 9.0 - glycine-NaOH). The diluted mixtures were incubated at the required temperature and their spectra measured periodically. The constants of reaction velocity were calculated according to the formula: +? lgDDOI;m t 00

Distortions of normal bone cell metabolism induced in multiple myeloma

Samples of bone marrow infiltrated with myeloma cells derived from iliac crest biopsies taken from 12 patients with multiple myeloma were studied in vitro in an attempt to discover the cause of the bone lesions characteristic of the disease. The relative affinities of the two cell types for glucose and proline were examined and found to be similar making inhibition of bone matrix biosynthesis due to competion for substrate unlikely as a cause for bone destruction. Measurements of incorporation of proline into bone cells and matrix revealed increased retention of label in cells (seemingly in a macromolecule) but generally normal incorporation into collagen. Parallel studies of myelomatous marrow suggested that these changes involved a change in bone cell metabolism per se and were not the result of marrow contamination. Collagenase activity was normal or low in the bone cells of four patients and virtually absent from marrow and plasmacytomas, thus ruling out abnormal biosynthesis of collagenase as a cause of bone destruction. These data are interpreted as indicating a redirection and stimulation of bone cell biosynthetic mechanisms as a result of the invasion of the marrow by Myeloma cells. The nature of the material (presumed to be protein) made is unknown.

Collagen synthesis and carbohydrate metabolism of rachitic bone.

1. This paper reports studies on the metabolism of bone from normal chicks and from chicks with vitamin D-deficiency rickets. Both in vitro and in vivo there was an increased incorporation of [(14)C]proline into collagen hydroxyproline by rachitic bone. The proportion of the collagen that was soluble in cold salt solutions was greater with the rachitic bone. These results show that in rickets there is an increased synthesis of bone collagen, but they do not provide any evidence of a defect in the maturation of collagen. 2. Rachitic bone incubated aerobically in vitro consumed more glucose and released more lactate than normal bone. Bone from rachitic chicks treated with vitamin D 48hr. previously had rates of glycolysis that were nearly normal. Though we were unable to show any direct action of vitamin D in vitro, we consider that vitamin D probably has a direct action on bone, possibly related to matrix biosynthesis.

Bone Matrix Biosynthesis in Vitro: I. LABELING OF HEXOSAMINE AND COLLAGEN OF NORMAL BONE

Bone Matrix Biosynthesis in Vitro: I. LABELING OF HEXOSAMINE AND COLLAGEN OF NORMAL BONE

Bone Matrix Biosynthesis in Vitro: II. EFFECTS OF PARATHYROID HORMONE

Bone Matrix Biosynthesis in Vitro: II. EFFECTS OF PARATHYROID HORMONE