Collagen and related research最新文献

Dentinogenesis offers a unique system for the study of changes in collagen structureoccurring simultaneously with mineralization. Bovine dentin was found to contain about one reducible crosslink per collagen molecule; rat dentin contained twice this amount. In contrast, bovine dentin contained twice as much pyridinium crosslink as did rat dentin collagen. These results indicate that the collagen in rat teeth is less mature and again emphasize the difference in composition between the organic matrices of rat and bovine dentin. In dissected bovine predentin, the unmineralized precursor of dentin, the content of reducible crosslinks was almost double that of dentin. Only minute amounts of non-reducible crosslinks were found in predentin, whereas both pyridinoline and deoxy-pyridinoline were present in collagen from mineralized dentin. The observed differences in crosslinking between predentin and dentin of the same teeth may indicate some alterations within the area of mineralization.

The abnormal cauliflower collagen fibrils present in skin of patients with Ehlers-Danlostype I were studied by electron microscopy and computerized image analysis. The size control of the few fibrils displaying a cauliflower shape is apparently lost, eventuating in an increased amount of collagen polymers constitutive of these fibrils. This situation is fundamentally different from that occuring in dermatosparaxis, an animal disease considered as a model for Ehlers-Danlos syndrome.

In situ hybridization with ³²P- or ³¹S-labeled double-stranded DNA or single-strandedRNA probes was used to investigate the temporal and spatial distribution of cartilage-characteristic type II collagen mRNA during embryonic chick limb development and cartilage differentiation in vivo. When the type II collagen probes were hybridized to sections through embryonic limb buds at the earliest stages of their development (stages 18–25), an accumulation of silver grains representing type II collagen mRNA first became detectable in the proximal central core of the limb coincident with the prechondrogenic condensation of mesenchymal cells that characterizes the onset of cartilage differentiation. At later stages of development (stage 32; 7 days) intense hybridization signals with the type II collagen probes were localized over the well differentiated cartilage rudiments, whereas few or no silver grains above background were observed over the non-chondrogenic tissues. In contrast, sections hybridized with a probe complementary to mRNA for the α1 chain of type I collagen exhibited an intense hybridization signal over the perichondrium and little or no signal over the cartilage primordia. At all stages of development examined, ³²P-labeled double-stranded DNA probes or single-stranded RNA probes labeled with either ³²P or ³⁵S provided adequate hybridization signals. Several experimental protocols were employed to control for the potential cross-hybridization and non-specific hybridization of the type II collagen probes. These included the utilization of labeled noncomplementary “sensestrand” type II collagen RNA as a control probe for nonspecific background, and prehybridization with a large excess of appropriate unlabeled RNA to block sequences in heterologous collagen RNAs that might cross-hybridize to the specific labeled probe.

Dermatan sulfate (DS) proteoglycans (PGs) were extracted from human post-burnscar (Sc) tissues with 4M guanidinium chloride and isolated from the extracts by DEAE-cellulose chromatography and by differential ethanol precipitation. The DS.PGs were further purified by Sepharose CL-6B column chromatography. The average molecular weight (M_r) of hypertrophic scar (HSc) tissue DS.PGs was 39,000 based on sedimentation equilibrium measurements. Alkaline borohydride treatment of DS.PGs liberated glycosaminoglycan (GAG) chains and the presence of xylitol indicated that these chains were attached to protein core by xylosyl residues. The average M_r of the DS.GAG chain from HSc and normal scar (NSc) samples were 23,500 and 20,000 respectively. After digestion of the HSc and NSc, DS.PGs with chondroitinase ABC in the presence of proteinase inhibitors, two peptide components with M_r values of 21,500 and 17,000 were detected by SDS-polyacrylamide gel electrophoresis using reducing conditions. Analysis of the protein core fractions derived from NSc and HSc DS.PGs by Sepharose CL-6B column chromatography showed the presence of a single NH₂-terminal amino acid (aspartic acid) and also that the fractions with different K_AV values had an identical NH₂-terminal sequence (A₁-A_S). The A₁-A₂₃ sequence of NSc DS.PG (major fraction, C) : NH₂Asp-Glu-Ala-O-Gly-Ile-Gly-Pro-Glu-Val-Pro-Asp-Asp-Arg-Asp-Phe-Glu-Pro-Ser-Leu-Gly-Pro-Val was the same as reported for a DS.PG isolated from human fetal membrane (HFM) tissue (Brennan et al., 1984).

ELISA inhibition assay using monoclonal antibodies raised in rabbit against the NH₂-terminal peptide (containing 15 amino acids) of human fetal membrane tissue were found to cross-react with HSc and NSc DS.PGs. Monoclonal antibodies to bovine skin DS.PGs protein core (Pearson et al., 1983) did not show any cross-reactivity with scar DS.PGs. These results show that the scar DS.PGs described here are different from normal bovine skin DS.PGs in the size and type of the protein core, and that in all the samples, the peptide components have the same NH₂-terminal amino acid sequence.

Cyanogen bromide (CNBr) peptides of ³H-labelled type I collagen isolated fromyoung rat skin were injected into adult rats via the tail vein. After 1, 5 and 10 minutes, the fate of the circulating radioactive peptides was investigated by measurement of the tissue distribution of radioactivity. Rapid removal of peptides from the circulation and their concomitant accumulation in the kidney proximal tubule epithelia was confirmed by autoradiography of kidney sections at the light microscope level. Immunopositive material was also identified and localized in proximal tubules using antibodies directed against denatured type I collagen chains and their CNBr degradation products. Ultrastructural autoradiography indicated that rapid removal of collagen peptides from the glomerular filtrate occurs via the brush border, apical vacuoles, and lysosomes.

Comparison of the proteins secreted by early and late passage cultures of humanfibroblasts reveals enhanced production of three proteins (M_r = 55,000, 58,000, and 61,000) in the late passage culture conditioned medium. The 55,000 and 58,000 Da proteins react with anticollagenase antibodies and are identified as procollagenase (Sottile, J. M. and Millis, A. J. T., J. Cell Biol. 106: 1518a, 1987). The production of immunoreactive collagenase was assayed using immunoprecipitation and immunofluorescence. Immunofluores cence microscopy revealed that each culture was heterogenous. The percentage of collagenase positive cells ranged from 1% (early passage) to 35% (late passage). Late passage cultures also secreted higher levels of immunoprecipitable collagenase into the culture medium than early passage cultures. High levels of collagenase production were also observed in fibroblasts cultured from donors with Werner's syndrome and from an aged donor. Media conditioned by the growth of human fibroblasts contained collagenase stimulating activity. In the presence of conditioned medium, the percentage of collagenase positive cells was increased to 10–20% (early passage) and 85% (late passage), indicating that each culture contains both responding and non-responding cells.

Intravenously administered ¹²⁵I -labelled bovine aminoterminal propeptide of typeIII procollagen (¹²⁵I -BPIIINP) had a half-life in blood of about 2 minutes. Low molecular weight degradation products appeared in the circulation about 5 minutes after injection. BPIIINP coupled to ¹²⁵I -labelled tyramine cellobiose (¹²⁵I -TC-BPIIINP) was administered intravenously to determine the cellular site of uptake. TC is non-degradable and is therefore accumulated intralysosomally. With this ligand I could show that PIIINP is taken up mainly by the liver endothelial cells (LEC), with very low uptake in other types of liver cells and at extrahepatic sites. Studies on binding and endocytosis of labelled PIIINP in cultures of purified populations of liver cells can be summarized as follows: 1) Uptake and degradation were observed mainly in LEC; 2) PIIINP associated with Kupffer and parenchymal cells, but degradation was very low; 3) Serum was not required for binding of PIIINP to LEC; 4) Binding was specific, that is, other ligands, such as collagen type III, hyaluronan, chondroitin sulfate, formaldehyde-treated albumin, and mannose, that are recognized by distinct receptors on LEC, did not compete with PIIINP for binding; 5) BPIIINP, TC-BPIIINP, and rat PIIINP (RPIIINP) were recognized with the same specificity by LEC; 6) BPIIINP bound to LEC with high affinity (dissociation constant = 1 nM), and about 4.2 × 10⁵, 3.2 × 10⁵, and 1.6 × 10⁵ molecules of BPIIINP, TC-PIIINP, and R-PIIINP, respectively were bound per cell; 7) PIIINP could not be degraded by conditioned medium from cultured Kupffer cells; 8) Leupeptin, which is a strong inhibitor of lysosomal collagenolysis, only weakly inhibited degradation of PIIINP; 9) Binding and endocytosis of PIIINP was not Ca⁺⁺-dependent; 10) Agents that inhibit the endocytic machinery inhibited uptake and degradation of PIIINP. In conclusion, the present results suggest that PIIINP is rapidly eliminated from the circulation by receptor-mediated endocytosis in LEC.

The near UV absorption of type I collagen can be used to measure its tyrosine contentand, therefore, the integrity of the nonhelical telopeptides. The far UV absorption of the protein can be used to determine its concentration and to monitor its thermal denaturation. The concentration, when determined along with low speed centrifugation to precipitate collagen fibrils, allows monitoring of the in vitro assembly of collagen fibrils. These methods are examined in detail and compared with other available techniques in terms of the advantages, drawbacks, and pitfalls in their usages.

The molecular mechanism involved in the expression of collagens by hepatocytes wereinvestigated in both pure and co-culture with another rat liver epithelial cell type (RLEC). We measured the steady-state levels of mRNAs coding for proαl(I) and proαl (IV) chains by Northern analysis and by dot blotting, using specific recombinant cDNA probes. In freshly isolated hepatocytes, only small amounts of proαl (I) and proαl (IV) mRNAs were detected by dot-blot analysis. After 3 days in culture, the proαl (I) and proαl (IV) mRNA levels increased 2 to 5 times. The amount of proαl (IV) mRNAs was identical in hepatocyte cultured with RLECs while the proαl (I) mRNA level was 5 times that in pure hepatocyte culture. Hydrocortisone reduced proαl (I) mRNA in hepatocyte cultures, but had no effect on co-cultured cells. In both culture systems, this glucocorticoid did not act on the steady-state proαl (IV) mRNA level. Whatever the age and the type of culture (pure or mixed) RLECs exhibited the highest levels of proα1(I) and proα1(IV) mRNAs, which were reduced by hydrocortisone. These results show that procollagen gene expression by hepatocytes is not directly correlated with their functional state and that corticosteroids differently affect the expression of different collagen genes and collagen deposition.