Collagen and related research最新文献

Pseudoxanthoma elasticum is a genetic disease characterized by progressive mineralization of elastic fibers. Previous studies suggested that other components, apart from elastin, might be involved in the alterations of this connective tissue disorder (Martinez-Hernandez and Huf f er, 1974; Pasquali Ronchetti et al., 1981; 1986). Evidence is presented that proteoglycan metabolism is altered in PXE-affected patient. Urinary GAGs suggests an increased degradation of glucosamine-containing GAGs in the patient. Pulse and chase experiments on in vitro skin fibroblasts indicated a decreased rate of synthesis of ³⁵SO₄ containing GAGs or an increase of their turnover rate in PXE. Moreover, when PGs produced from skin fibroblasts were identified by ultracentrifugation and gel filtration in associative conditions, PXE fibroblasts produced a significantly higher amount of the high molecular weight fraction of sulfated PGs. This high molecular weight material was present both in the medium and in the matrix and disappeared under dissociative conditions or after treatment with hyaluronidase or with pancreas elastase. By electron microscopy, PXE fibroblasts appeared to produce and secrete an enormous amount of toluidine blue 0 positive material organized as filaments and amorphous masses. These data are in agreement with previous observations of the presence of abnormal masses of microfilaments, in the dermis of PXE patients, which were sensitive to hyaluronidase and partially to trypsin and elastase (Pasquali Ronchetti et al., 1986). The results seem to confirm that at least some of the alterations of connective tissues in PXE are due to abnormal PGs metabolism and to their tendency to form abnormal aggregates in the extracellular space.

Monoclonal antibodies have been produced against porcine synovial collagenasewhich recognize both the active enzyme and its inactive precursor. These antibodies inhibited the collagenolytic activity of collagenase, but not its activity with a synthetic peptide substrate. The antibodies were also able to recognize human synovial, human skin fibroblast and human chondrocyte collagenase but not the enzyme from human granulocytes.

One of the monoclonal antibodies was successfully used for the immunopurificationof the porcine enzyme and these experiments led to the demonstration of an endogenous activator of procollagenase in the synovial cell culture medium.

Normal articular cartilage from adult dogs was analyzed for hyaluronate,hexuronate and hydroxyproline. The low weight-bearing areas of both tibial plateaus and femoral condyles displayed a higher collagen content and a lower proteoglycan content than the regions of maximum contact. Both superficial and deeper layers contained more hyaluronate in areas of maximum than of minimum contact. On the other hand, in each weight-bearing area, the proportion of hyaluronate relative to total proteoglycan content appeared twice as much in the superficial layers than in their corresponding underlying zones. The molecular weight and in vitro aggregating capacity of the hyaluronate molecules were however quite similar in the different topographical areas of the articular tissue.

This is the first study concerning the extent to which relative collagen production (RCP) in rat periodontal tissues is affected by diabetes. Determination of RCP, rather than individual production rates for collagen or for non-collagen protein, was deemed necessary because saturation of all proline pools in tissues of diabetics (and non-diabetic controls) was not achieved. Such non-saturation occurred despite the injection of a poolexpanding dose of proline (400–1150 mg/rat), non-saturation indicated by the lesser specific radioactivity (S.R.) of free- [³H]proline in tissues than that of the injected solution. RCP was decreased in five periodontal tissues (incisor and molar gingiva, incisor and molar periodontal ligament, antemolar palatal mucosa) and in skin. Diabetes-decreased RCP seems to result from decreased collagen synthesis and increased intracellular degradation, although some evidence is presented for increased extracellular degradation of recently secreted collagen.

The intracellular degradation of interstitial collagen is accomplished by two neutral metalloproteases, collagenase and gelatinase. Both enzymes are inhibited by metal chelating agents, by certain sulfhydryl reagents, and by similar protein inhibitors. Here, we demonstrate that the dye eriochrome black T (EBT) appears to be unique in its capacity to inhibit collagenase but not gelatinase. Using native reconstituted helical collagen in gel form at 37°C, half-maximal inhibiton of collagenase activity by EBT occurs at ~ 45 [tM. EBT more effectively inhibits the breakdown of native collagen in solution, with a K_I of ~ 8 ¼M. Using a newly-developed spectrophotometric substrate, AcProLeuGly-S-LeuLeuGly-OC₂H₅, a K_I of 1.4 ¼M was calculated for EBT on collagenase. Although this same thiopeptolide serves as a substrate for gelatinase with kinetics similar to those of collagenase, no inhibition by EBT was observed. EBT also did not inhibit the gelatinase-mediated breakdown of the natural substrate, gelatin. The data suggest that EBT may have significant potential for allowing the differentiation in biological fluids of two metalloproteases with similar cleavage site specificities.

[¹²⁵1]-labelled rabbit antibodies against rat type I collagen and non-immune IgG were injected into rat circulation. The kinetics of their clearance and the biodistribution in different organs were studied. Both preparations showed very similar clearance rate, the kinetics fitting bi-exponential approximation with characteristic parameters t_1,1/2 = 201 ± 20 min before 320 min and t_1,1/2 = 1350 ± 450 min at times over 320 min for antibodies and 258 ± 45 min and 890 ± 140 min for IgG. The specific affinity of the circulating antibodies did not decrease within 24 hours. The antibodies were specifically accumulated in spleen, where their accumulation was 5-fold higher than that of nonimmune IgG. Accumulation of antibodies was maximal 3 hours after the injection. The localization ratio (i.e. the ratio of the amount of the antibodies per g of tissue to that per g of blood) reached a maximum 24 hours after the injection and remained stable for 120 hours. Immunofluorescent staining of spleen sections resulted in a bright fluorescence of dense collagenous structures in the trabeculae and in the wall of the central follicular arterium, bright spot fluorescence in the marginal zone of the follicle, and diffuse fluorescence in the red pulp. These findings suggest an unusually high accessibility of collagen type I in spleen to circulating blood plasma components.

The composition of the weight bearing portion of the femoral head articular cartilage from both sides of greyhounds 11–32 months of age was studied with regard to variability of proteoglycans. Extractability of proteoglycans was very similar for all samples. Other parameters, i.e. contents of proteoglycans in the cartilage, hyaluronic acid content of the proteoglycan fraction, as well as contents of keratan sulfate and chondroitin sulfate, size of proteoglycans and ability of proteoglycans to aggregate with hyaluronic acid, were similar in samples from the right and left side of each dog. The variability from one dog to another was large and not related to age. The data show that in experimental studies of joint disease the contralateral joint can be used as a control to a treated joint, while the large interindividual variability makes comparison difficult between individuals.

Quantitative and qualitative variations in bone composition were studied in postmortem iliac crest bone specimens from 32 women aged 60 to 75, grouped according to the presence of osteoarthritis at the hand joints. The EDTA extraction yield of macromolecules was significantly greater in the osteoarthritis group. Collagen and osteocalcin content were significantly increased and proteoglycan content decreased in the osteoarthritis group. No difference in sialoprotein content was found.

In a clinical study of 20 patients with generalised osteoarthritis, we found compared to 15 controls also significantly increased serum levels of osteocalcin.

These results indicate in an unselected group of cases that differences in bone matrix constituents exist between elderly women with and without an osteoarthritic constitution. The finding that these alterations are present even far away from the articular lesion, and the fact that the serum osteocalcin levels are elevated, indicate that osteoarthritis is part of a more generalised bone disease and not a purely cartilage disease.