Matrix (Stuttgart, Germany)最新文献

The effects of sulfonate detergents on fibril formation by type I (rat tail tendon) collagen, untreated and pepsin-treated, were studied at two temperatures. Fluorescent probes and CD spectra were used to study possible conformational changes in the collagen. With untreated collagen, the rate of fibril formation was reduced by the presence of the detergents, particularly at the low temperature and when the detergent molecules had longer aliphatic chains. With pepsine-treated collagen, however, the rate was increased by the longer-chain detergent at the higher temperature. These observations are discussed in the light of present knowledge of the role of telopeptides and hydrophobic interactions in fibril formation.

Lyophilized mid-wall left ventricular myocardial tissues of the long-tailed non-human primate, Macaca fascicularis, were examined for the presence of glycosaminoglycans and chondroitin/dermatan sulfate proteoglycans. Mean uronic acid concentration in all samples was 0.97 ± 0.27 μg per mg dry weight myocardium. The distribution of the glycosaminoglycans in the myocardium, determined by cellulose acetate strip electrophoresis was 62 ± 4% heparan sulfate, 20 ± 6% hyaluronan, and 16 ± 5% chondroitin/dermatan sulfate. The analysis of chondroitin/ dermatan sulfate proteoglycans, done directly on the extracts of lyophilized myocardium using agarose-acrylamide gel electrophoresis and Western blotting with monoclonal antibodies to various carbohydrate epitopes and with polyclonal antibodies to the protein core, showed the presence of biglycan and decorin. That these two and no other chondroitin/dermatan sulfates were present was established by core protein analysis using SDS PAGE and Western blotting. Quantification of chondroitin/dermatan sulfate proteoglycans uncovered high individual specific variability of the chondroitin/dermatan sulfate epitopes, but only moderate variability of biglycan and decorin core proteins. The variability of the chondroitin/dermatan sulfate epitopes is most likely related to individual specific differences in chain number, iduronate content and sulfation patterns of biglycan and decorin.

Age-related changes in connective tissues can alter their functions of elasticity, compressibility and support. Matrix Gla protein (MGP) is a vitamin K-dependent connective tissue component of unknown function. We have purified bovine MGP, and developed a specific radioimmunoassay for it. Since it is found in highest concentration in cartilage, we have developed quantitative extraction methods for MGP, and examined the age-related changes of MGP relative to other components found in the cartilage matrix. The ratio of hydroxyproline to MGP increases with age, while the ratio of glycosaminoglycan to MGP is constant. No effect is seen for MGP in the dietary restricted rat with prolonged lifespan, while both hydroxyproline and glycosaminoglycan contents of tracheal cartilage are significantly increased by dietary restriction (p ≤ .05). These data show that MGP and glycosaminoglycan concentration are relatively constant in rats from 6 to 30 months of age, while hydroxyproline concentration increases with age.

Polycystic kidney disease (PKD) is a life-threatening disease characterized by focal dilatations or cysts in certain kidney tubules. Changes (i. e. thickening) in the support structure for these tubules, the basement membrane, have been related to the development of the cysts. Analysis of changes in basement membranes of humans with PKD is difficult, however, due to the restricted amount of material available for study. Several genetic and induced animal models, including diphenylamine-treated rats, have been employed to study the effects of PKD on basement membrane synthesis. While all these studies agree that PKD has a significant influence on basement membranes, no clear understanding as to how PKD effects basement membrane composition has emerged. Here, we report our findings of the effect of diphenylamine treatment on the composition of the basement membrane. Our immunohistological studies indicate that bamin, a recently described glycoprotein associated with glomerular basement membranes (Robinson et al., 1989), is not present in the glomerular basement membranes of diphenylaminetreated mice. This finding was confirmed by analysis of the composition of the basement membrane matrix synthesized by EHS tumors grown in control and diphenylamine-treated mice. The possible role of bamin in the pathogenesis of renal cysts is discussed.

The effects of recombinant human interleukin-1α(IL-1α) and murine epidermal growth factor (EGF) on the release of collagenase were studied in an in vitro model system using periosteal explants from rabbit calvariae. Following an incubation period of 72 h it was shown that IL-1α in combination with EGF (IL-1α + EGF) induced a synergistic increase in the amount of collagenase released by periosteal explants. This increase appeared to be at least 10-fold. Most of the enzyme was present in a latent form since the increase in enzyme activity was only detectable after activation by APMA and the molecular weight as determined in immunoblots corresponded to the latent form of this enzyme. Incubations carried out with IL-1α alone resulted in a 2- to 4-fold increase of total enzyme activity, whereas the amount of collagenase in media of EGF-treated periostea did not surpass control values. A neutralizing anti-IL-1α antibody completely blocked the enhanced release of collagenase as induced both by IL-1α and by IL-1α + EGF. Indomethacin partially prevented the IL-1α + EGF-induced increase in enzyme release, suggesting the involvement of prostaglandins.

The amount of tissue inhibitor of metalloproteinases (TIMP) as determined by ELISA was slightly elevated in culture media obtained from all cytokine-treated explants. Comparable results were obtained by Western blot analysis as well as by a functional bioassay.

It is suggested that the concomitant presence of the cytokines IL-1α and EGF may play an important role in collagenase-mediated degradation of collagen.

A recombinant system was developed for the production of homotrimeric type I collagen in stably transfected HT1080 cells. A DNA construct (COL1A1-CMV) was prepared that contained the cDNA for the human COL1A1 gene under the transcriptional control of the promoter and enhancer of the immediate early gene of CMV. The construct, which also contained a neomycin-resistance gene, was transfected into HT1080 cells, a human fibrosarcoma cell line that synthesizes type IV collagen but does not normally synthesize any of the fibrillar collagens. Cells derived from the neomycin-resistant tranfectants were then screened using a polyclonal antibody specific for human proal (I) chains in order to identify clones that secreted high levels of the proa(I) chain of type I procollagen. About 2% of neomycin-resistant clones secreted procollagen that consisted of a homotrimer of proα1(1) chains. The procollagen was post-translationally over-modified as judged by slower migration on SDS-polyacrylamide gel electrophoresis of the proα1(1) chains compared to proα1(1) chains of normal type I procollagen. The procollagen was triple helical as assayed by protease digestion with a variable cleavage at 38 °C and a thermal transition of both the intact and partially cleaved protein of about 41 °C. The system provides a method of expressing genes for fibrillar procollagens so that fully recombinant proteins are generated and easily isolated.

Osteopontin (OPN) is a phosphorylated, sialic acid containing glycoprotein that can be extracted from the mineralized extracellular matrix of bone. In the present study we determined the expression patterns of OPN in dental tissues and alveolar bone of 1–3 day old (neonatal) rats by means of 1) immunohistochemistry, 2) Northern blotting and 3) in situ hybridization. We compared these patterns with those of type I collagen.

We localized collagen typeI expression in osteoblasts adjacent to alveolar bone and in odontoblasts lining predentin/dentin, but not in the epithelial ameloblasts. For OPN, we observed a weak antigenicity in predentin. Although generally no cellular immunostaining was found, very occasionally a minor immunoreactivity was detected in a small number of premineralizing incisor odontoblasts. On the mRNA level, however, no OPN transcripts could be detected in odontoblasts, either by in situ or by Northern hybridization analyses. Also the odontoblasts of the bone-like dentin (osteodentin) region in the tip of incisors were negative for OPN. In contrast, however, osteoblasts of alveolar bone showed strong positive signals with all three techniques, confirming the sensitivity and specificity of the detection methods.

From the data obtained in this study, it can be concluded that during early stages of dentinogenesis OPN presumably is not expressed in developing rat tooth germs. The weak immunostaining observed sporadically in some young odontoblasts is probably due to resorption of OPN of non-dental origin entrapped in the predentin.

Monoclonal antibodies have been developed that recognize epitopes in native chondroitin sulfate chains. One of these antibodies, CS-56, reportedly recognizes chondroitin 4- and 6sulfates. However, this antibody, and four other anti-chondroitin sulfate antibodies, 40, 4D3, 60 and 7D4, do not recognize epitopes in chondroitin sulfate chains from Swarm rat chondrosarcoma proteoglycan, an indication that native chondroitin sulfate epitopes are more structurally complex than the standard 0-, 4-, and 6-sulfated disaccharide repeats that constitute the backbone of chondroitin sulfate chains. A series of limited chondroitinase digestions was performed on the large aggregating proteoglycan monomer extracted from embryonic chick chondrocyte cultures to identify the digestion parameters required to release the different native chondroitin sulfate epitopes. Some epitopes were more accessible to enzymatic digestion than other epitopes. The approximate location of epitopes was determined by measuring the size of undigested oligosaccharides retained on the core protein following a limited digestion, and correlating this with the level of immunoreactivity for the different antibodies. These analyses identified the locations of three different antigenic domains. Domain 1 resides at the linkage region and contains epitopes for two of the five antibodies, and a portion of the epitopes for a third antibody. Domain 2 lies in the interior of the chain and contains epitopes for three of the five antibodies. Domain 3 resides at the non-reducing terminus and does not contain epitopes for any of the anti-chondroitin sulfate antibodies used in this study. These results indicate that specific native chondroitin sulfate epitopes are non-randomly distributed within the linear framework of chondroitin sulfate chains.

Antibodies against a synthetic peptide representing the 14 C-terminal amino acids of the N-terminal propeptide of rat and bovine procollagen type III were raised in rabbits and used to develop a radioimmunoassay. N-Terminal propeptide of procollagen type III, purified from calf skin, served as standard and tracer material. The IC₅₀ of the standard inhibition curve was 2.1 μ/I, the lower limit of detection about 0.4 μg/1, interassay variation was 8.5% and the intraassay variation 6.6% in typical experiments. Three peaks of antigenicity were detected in rat serum after gel chromatography. One peak coeluted with purified N-terminal propeptide of procollagen type III, one peak contained material approximately twice this size, and one peak eluted close to the void volume of the column. The antigen concentration in rat serum decreased in an age dependent manner. Rat, bovine, sheep and minipig serum antigen was sufficiently crossreactive to allow the application of the assay to these species, whereas human, goat and guinea pig samples were not. The degradation product Col 1, causing non-parallel inhibition in commercially available assays for human samples was not recognized since it does not contain the epitope represented by the synthetic peptide. The assay was used to study the half-life of bovine and endogenous N-terminal propeptide of procollagen type III in isolated perfused rat liver. [¹²⁵I]-labeled antigen was cleared rapidly from the perfusate (t_1/2 less than 5 min). The bovine antigen was removed from the perfusate with a half-life of 15 ± 4 min. Endogenous propeptide was perfused for 120 min with little change in concentration. The data indicate that the studies of elimination of peptides by the liver may be influenced by species differences, and that iodinated proteins are not a suitable tool for such studies.