Collagen and related research最新文献

It is generally accepted that collagenase from human fibroblast cultures consists of two proenzymes (M_r 60,000 and 55,000) and two active forms (M_r 50,000 and 43,000). We demonstrated previously that epidermal growth factor (EGF) as well as a number of other growth factors induced the secretion of procollagenase (M_r 60,000, M_r 55,000) into the medium of human fibroblast cultures (Chua et al., 1985). In the presence of tunicamycin and EGF, the secretion of the larger form of procollagenase was suppressed preferentially with concomitant appearance of a new band, M_r 40,000. This M_r 40,000 band could be specifically immunoprecipitated by antibody raised against human collagenase. By two-dimensional peptide mapping, the M_r 40,000 material appeared to have similar composition as the M_r 60,000 band. In a time course study, the M_r 55,000 procollagenase band was the earliest protein to appear in the medium after 1 hour labeling with [³⁵S] -methionine. The M_r 60,000, 50,000 and 43,000 bands appeared after a 2 hour labeling period. Our results indicate that human collagenases are glycosylated proteins and are synthesized via the dolichol phosphate pathway.

Frozen sections of chick tissues were exposed to affinity-purified monoclonal antibodies raised against chick gp 115 and to affinity-purified antibodies raised against chick tropoelastin to study the distribution pattern of the corresponding antigens by the avidin-biotin immunoperoxidase technique. Laminin and fibronectin antibodies were used for comparison. Gp 115 and tropoelastin antibodies localized to the same structure in several of the tissues examined. The endothelial membrane of the cornea and Bruch's membrane in the choroid were positive, while the corneal epithelial membrane was negative. Both antibodies displayed a peculiar punctate reactivity in the corneal stroma and a very fine fibrillar pattern in the conjunctiva and at the corneal-scleral junction. Liver, heart and large vessels, striated muscle and skin showed a similar pattern both for tropoelastin and gp 115 antibodies. Few differences were seen in the distribution of the reactivity: the pericellular matrix of intestinal smooth muscle cells was stained by gp 115 but not by tropoelastin antibodies. However, the reactivity of gp 115 and tropoelastin antibodies was similarly distributed in the lung smooth muscle cell clusters. The peritubular matrix in the kidney did also not react with tropoelastin antibodies as did the brain intraparenchymal vessels; whereas gp 115 antibody reactivity was present in both sites. We interpret these lack of apparent codistribution in some tissues as a variation in the relative availability of the target antigen for the reaction with the antibody and not as a consequence of a qualitative difference in the distribution of gp 115 and tropoelastin. By the use of anti gp 115 monoclonal antibodies that do not cross-react, and presumably recognize different epitopes, it was shown that some but not all antibodies, react with brain intraparenchymal blood vessels; whereas the pattern of distribution in other tissues was the same. This suggests that in vessels with an undetectable level of elastin, certain epitopes of gp 115 molecule might not be recognized as a result of being masked by other components or by a different conformation of the molecule.

Poly A⁺ RNA, isolated from a single 210 day fetal bovine nuchal ligament, was used to synthesize cDNA by the RNase H method, using AMV reverse transcriptase for first strand synthesis and DNA polymerase I for the second strand. The cDNA was inserted into λ10 using EcoRI linkers, and recombinant phage containing elastin sequences were identified by hybridization with a 1.3 kb sheep elastin cDNA clone, pcSELI (Yoon, K. et al., Biochem. Biophys. Res. Comm. 118: 261-265, 1984). Three clones containing the largest inserts of 2.9, 2.8, and 2.6 kb were selected for further study. The complete sequence analysis of the 3 clones was correlated with the sequence of 10.2 kb of the bovine elastin gene. The analyses: (1) showed that the cDNA encompassed the great majority of the translated sequence, (ii) ordered the tryptic peptides of porcine tropoelastin, (iii) determined new amino acid sequences not previously found in the porcine peptides and (iv) demonstrated that alternative splicing of the primary transcript leads to significant variation in the sequence of the translated portion of the mRNA.

We investigated the effects of 1,25-dihydroxyvitamin D3 on the synthesis of osteopontin, a phosphorylated cell attachment glycoprotein, in ROS 17/2.8 cells, a clonal osteoblast-like rat osteosarcoma cell line. We observed a dose dependent increase in uptake of [³²P0₄] into osteopontin secreted into the medium. An increased incorporation of [³⁵S] -methionine into secreted osteopontin suggested the effect was that of increased protein biosynthesis. Using a radioimmunoassay we demonstrated a dose dependent increase in the amount of secreted osteopontin, an increase which could be blocked by Actinomycin D, in response to 1,25-dihydroxyvitamin D3. These results suggest that the hormonal form of vitamin D regulates the biosynthesis of osteopontin, possibly at the level of transcription.

The evidence of a adsorption phenomenon of the acid-soluble collagen molecules on the glass material is shown. A way of calculation is proposed from viscosimetric measurements. It could be pointed out that a small amount corresponding to 4 to 5% of the collagenous concentration is involved in such a adsorption process, at 25 °C. This adsorption phenomenon is temperature-dependent and weakened by a previous NaOH-treatment of the collagen solutions.

It is confirmed that intermolecular associations may take place in acidic conditions and they are minimized for a pH range corresponding to the pK area of aspartic and glutamic acid residues. The influence of temperature showed that ionic interactions are reinforced by hydrophobic effects. The modification of the ionization and the removal of the telopeptidic regions from the native molecules are responsible for the decrease in these associations. On the other hand, it was shown that the relative flexibility of the molecule may explain the modifications in the {η} values depending on the pH and ionic-strength conditions for the molecular solutions.

Extracts of demineralized rat bones which contained factors stimulating bone induction were reconstituted with highly purified human type I collagen to provide a suitable and easily manipulated delivery system for surgical implantation. When implanted subcutaneously in rats, the implants governed and delineated the dimensions of the resulting bony tissue. It is proposed that this implant system has clinical application in the filling of osseous defects within the scope of orthopaedic and oral and maxillofacial surgery. It is presented here as a potential improvement over conventional implant materials without osteoinductive properties.

Sulfated proteoglycans of fetal, newborn and adult bovine tendon are extracted with neutral salt and then with 4 M guanidine HCI in the presence of proteinase inhibitors. Dermatan sulfate containing small proteoglycans are separated from chondroitin sulfate rich proteoglycans by CsCl-gradient centrifugation, by affinity chromatography using concanavalin A and by molecular sieve chromatography. These proteoglycans are comprised of a core protein with an average Mr of about 53,000 on polyacrylamide gel electrophoresis with SDS and of dermatan sulfate side chains with M_r 25,000–38,000 by gel chromatography. The tryptic peptides patterns observed in the small proteoglycans from newborn calf and adult bovine tendon are identical but are distinct from those of embryonic calf tendon. The patterns of the tryptic peptides of the core protein from embryonic calf tendon are similar to those from the dermatan sulfate proteoglycans of calf skin. These results indicated that genetically independent genes of the core proteins from dermatan sulfate proteoglycans in tendon tissues were expressed by aging.

The action of IGF-I (insulin-like growth factor I) on the synthesis of tropoelastin in chick embryonic aortae was examined. Maximal and selective stimulation of the relative (40%) and absolute (145%) rates of tropoelastin synthesis over control occurred at an IGF-I concentration of 100 ng/ml of medium. Parallel to the increase in synthesis was a 92% increase in the amount of tropoelastin activity per 100 ng of poly (A)⁺ RNA translated in a cell-free system. The relative rate of tropoelastin synthesis achieved at maximal stimulation is greater than that observed during normal aortic embryogenesis. The stimulatory action of the hormone on elastin synthesis appears to be at a pretransitional level perhaps involving increased transcription or stabilization of the tropoelastin mRNAs. These results suggest that IGF-I may play a key role in the regulation of elastogenesis in arterial tissue.

Bone, calvaria and dentine collagens were incubated with crude preparations of lysosomal cathepsins obtained from liver, spleen and bone cells. Degradation was most rapid near or below pH 4 and the rate of degradation was increased two-to four-fold in the presence of 50–75 mM CaCl₂. This concentration of Ca²⁺ ions was close to the saturating level of ions released from calcium hydroxyapatite in the pH range 3.5–4.0. Purified cathepsins B and L were very much less effective than the crude extracts in degrading the hard-tissue collagens. Cathepsin B was equally sensitive to the inclusion of 50 mM CaCl₂ but cathepsin L activity was only slightly increased. The activating effect of Ca²⁺ ions was not specific as Mg²⁺ ions were equally effective. A partially-purified preparation of cathepsin N gave results similar to those obtained for the crude, mixed enzyme preparations. Spleen and bone cell extracts were much more effective than those of liver despite a lower content of cathepsins B and L. These findings suggest that a third enzyme, cathepsin N, which is known to be more abundant in spleen than liver, had contributed more of the collagenolytic activity in the spleen and bone cell extracts. Therefore in osteoclastic bone resorption the major collagen-degrading enzyme could be cathepsin N.

Tendon collagen was degraded very rapidly by the crude and pure preparations of lysosomal cathepsins in the CaCl₂-free buffers. However, when 50 mM CaCl₂ was included in the incubation mixtures the reaction was strongly inhibited. The effect of the added CaCl₂ appeared to be on the substrate since the activity of cathepsins B and L, was depressed by CaCl₂ in the fluorimetric peptidase assays for these enzymes.