Matrix (Stuttgart, Germany)最新文献

Type I procollagen biodynthesis and matrix deposition were studied in cultured fibroplasts of four probands affected by Osteogenesis Imperfecta and in whom the mutations have been characterized. The mutations along the trip1e helix altered all biochemical parameters considered, i.e.thermal stability, kinetics ofprocollagen secretion and rate of maturation from procollagen to collagen. The biochemical findings were peculiar for each case considered, but there was no correlation between biochemical parameters an clinical phenotype. In a our probands, regardless of the clinical severity, mutant chains appeared in the insoluble matrix formed by fibroblasts cultured in the presence of dextran sulfate. The densitometric scanning revealed a relative increase amount of fibronectin, suggesting that the matrix contained a lower quantity of type I collagen. Furthermore, the amount of mutant chains found in the insoluble fraction was clearly less than expected, considering that 75% of new synthesized trimers are abnormal. Therefore, in the presence of a mutation, the protein available for extracellular matrix formation is reduced and the mutant trimers incorporated in the matrix probably interfere with normal fibril performance. The abnormal fibril morphology has a dramatic effect in bone, interfering presumably with a correct mineral deposition and interactions with non/collagenous bone proteins.

As part of a muticenter study on atherosclerosis, we examine defined segments of thoracic and abdominal aortas from 118 white males, age 15–34 years, who died from external causes. One half o each aorta specimen was graded or lesions. Intima-media preparations were assayed for collagen an cholesterol in two standardized regions (dorsal and ventral) derived from the alternate half of each segment. Even though the mean extent of intimal surface involvement with raise lesions remained minimal (0–6%), the data revealed a remarkable transition in vessel wall chemistry over this time span. For example, the amount of collagen per unit surface area increases with age in all vessel segments except the ventral domain of the thoracic aorta. The amount of collagen as a percent of total vessel protein rises with age only in the ventral and dorsal regions of the abdominal aorta. Free and esterified cholesterol levels per unit surface area increase with age in all vessel segments. There is a significant correlation between collagen and esterified cholesterol per unit surface area in all vessel regions with the exception of the abdominal ventral segment. In the latter segment increases in collagen per unit surface area occur without a corresponding increase in cholesterol level suggesting that connective tissue proliferation may actually precede lipid deposition in the genesis of atherosclerosis. Esterified cholesterol is present at higher levels in the dorsal domains of the thoracic and abdominal aortas than in the ventral domains. These findings provide chemical data confirming that the dorsal domain is the most lesion-prone region of these vessel segments. The most pronounced changes in aorta chemistry become apparent in the 25–29 year age range. Thus, the results suggest that for white males, preventive measures for atherosclerosis such as dietary restrictions should be instituted at the beginning of the third decade.

As part of an investigation of the effect of sepsis on the sinusoidal cells of liver we studied the influence of conditioned media from Kupffer cells (KC) and liver endothelial cells (LEC) from normal rats and animals pretreated with endotoxin (ET) on the hyaluronan (HA) production by stellate cells (SC) in culture. SC proliferation, as measure by direct cell counting and [³H]thimidine incorporation, was also recorded. An SC from an ET-treated rat produces similar amounts of HA as a normal SC when grown in culture medium containing 10% fetal calf serum (FCS). When animals were treated with ET, there was, however, an increase in the yield of isolated SC and thus the whole cell population in ET-treated rats has a potential to produce a larger amount of the polysaccharide. Factors present particularly in the KC conditioned media supplemented with 10% FCS from normal and endotoxemic animals greatly stimulated the proliferation and synthesis of HA in SC cultures, after a lag-phase which was four days for normal SC and three days from ET-treated animals. The rate of HA synthesis was closely related to the increase in the amount of thymidine incorporation into DNA. Conditioned media containing 10% FCS obtained from LEC cultures were also stimulatory but were less effective in inducing cell proliferation and prodution of HA. We suggest that the elevation of plasma HA found in severe endotoxemia and sepsis may be cause in part by the: (i) increased numer of SC; (ii) an in vivo activation of SC population which predisposes for HA-production; and, (iii) action of factors or mediators from cells of the retilculoendothelial system of the liver on the cell proliferation and on the HA synthesis in the SC population.

Thermolysin digests of human elastins were examined for elastin peptide markers as determined by HPLC followed by amino acid sequencing of promising peaks. The tetrapeptide VAPG was found to occur in the early portion of the chromatogram highly fashion. The peptide appears to be significantly amplified, when compared with the other peptides, in that it is derived from the hexapeptide repeat in e1astin, VGVAPG, which repeats itself in two three-pieceegments in the c-terminal portion of the tropoelastin molecule. VAPG serves as a highly reliable quantitative measure for human elastins, allowing sensitivities to less than a microgram. Thus, it is a significantly more accurate measure than other existing methods. Precision also appears to be enhanced because of the directness of the measurements. The use of VAPG as a quantitative marker for human elastin has clinical application in the study of elastin-based connective tissue diseases.

A reconstituted basement membrane (matrigel) and/or fibroblasts promote the growth of human breast tumors in athymic nude mice. We have investigated in vitro the effect of matrigel or purified glycoproteins (laminin and fibronectin) on tumoral MCF7 cells-fibroblasts interactions. In coculture on matrigel, MCF7 cells organized into clusters attached on top of fibroblasts aggregates. During the process resulting in tumor cells-fibroblasts aggregation, fibroblasts actively migrated while MCF7 cells were passively transported. Using purified proteins, specific antibodies and synthetic peptides, we show that cell aggregation induced by immobilized and soluble laminin is antagonized by exogenous fibronectin or fibronectin synthesized by f fibroblasts.

A protein present as a M_r 62 k monomer and as several differently sized disulfide-bonded oligomers has been isolated from rat one mineralized matrix. Its overall tissue distribution determined by ELISA immunoassays showed the protein present only in bone, tooth and in serum while aorta, cartilage, intestine, kidney, liver, muscle, skin, spleen an tendon were all negative. Despite that the 62 kDa protein was abundant and selectively found in bone, no positive cDNA clone could be identified in several rat bone libraries. Positive clones were, however, identified in a rat liver expression library. A cDNA clone of 1.3 kb hybridized in a Northern blotting assays to a 1.8 mRNA in rat liver. No hybridization signa1 was detected with RNA from bone, brain, lung, muscle, spleen and kidney. Sequence analysis of the isolated cDNA clone revealed a 50-bp untranslated region followed by an open reading frame of 357 amino acids. The open reading framce can be divided into a 17-amino acid signal peptide followed by the mature protein of 340 amino acids with alanine as its N-terminal amino acid. A short N-terminal amino acid sequence from the isolated 62-kDa bone protein verified the molecular identity of the cDNA clone. The primary structure of the 62-kDa liver protein was identical to a that of a 63-kDa phosphorylated glycoprotein (pp⁶³) from liver.

A 200-amino acid long motif first recognized in von Willebrand Factor (type A module) has been found in components of the extracellular matrix, hemostasis, cellular adhesion, and immune defense mechanisms. At present the extracellular matrix is the predominant site of expression of type A modules since at least four non-fibrillar collagens and two non-collagenous proteins contain a variable number of modules ranging from one to twelve. The medules conform to a consensus motif made of short conserved subregions separated by stretches of variable length. The proteins that incorporate type A modules participate in numerous biological events such as cell adhesion, migration, homing, pattern formation, and signal transduction after interaction with a large array of ligands.

Mouse 3T6 i ro asts deposited an organize co agenous extrace u ar matrix during longterm culture in the presence of ascorbic acid. The matrix produced by the cells had a similar comprising distribution of collagen types as the mouse dermal matrix, comprising predominantly type I with smaller amounts of types III aand V collagens. By day more than 70% of the collagen in the 3T6 matrix was involved in covalent crosslinkages and required pepsin digestion for extraction. Incorporation of NaB³H₄ into reducible crosslinks and aldehydes directly demonstrated the involvement of the α1(I)CB6 an α2(I)CB3.5 in crosslinks. The pattern of reducible crosslinks in the in vitro 3T6 matrix was similar to that in mouse skin suggesting a comparable fibril organization. Processing of procollagen to collagen occurred efficiently throughout the culture period and the rate of collagen production was unaltered during 15 days of culture, indicating that the development of a collagenous matrix does not directly play a role in procollagen processing or biosynthetic regulation. The existence of a preformed matrix did however, increase the efficiency with which newly synthesized collagen was incorporated into the pericellular matrix. At day 0, when there was no measurable a matrix present, 29% of the collagen synthesised wad deposited, while by 15,88% of the collagen was laid down in the matrix. The development of this 3T6 culture system, where collagen is efficiently incorporated into an organized extracellular matrix, will facilitate detailed studies on matrix organization and regulation and provide a system in which protein-engineered mutant collagens can be expressed to determine their effects on the production of a functional extracellular matrix.

Interstitial fibrosis is a common feature o renal aging.

The steady-state levels of type I an type III collagen mRNAs as we as DNA, protein and collagen deposition were investigated in the cortex, inner and outer medulla of aged (22 months old) rats in comparison to young (5 months old) controls.

Our data show that the cortex an outer medulla of old rats expressed significantly higher percentage of type I collagen mRNA compared to the respective regions in the rat kidneys. Moreover, within the group of the old rats, the cortex expressed significantly higher percentage of type I collagen mRNA compared to the inner medulla whereas in the group of the young rats the expression was similar in all kidney regions.

The ratio of extracellular collagen to DNA was significantly higher in the cortex, inner and outer medulla of old compared to young rats. The ratio of collagen to total protein, although showing difference, attained statatistical significance in the cortex only.

Thus, the present study indicates a close relationship between the expression of the mRNA for type I collagen, the major structuralconstituent of fibrofic tissues, the deposition of collagen in both the cortex and outer medulla of the kidney. Moreover, the clear differences found between old and young rat kidneys can serve as markers for renal aging and might explain at least some of the kidney impairments caused by fibrosis during senescence.