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A minicollagen containing the COL1 and NC1 domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triple-helical domain in which the cysteines are not involved in inter- but in intrachain bonds. In reducing conditions, providing that the triple-helix is maintained, disulfide exchange between intra- and interchain bonding is observed, suggesting that the triple-helix forms first and that in favorable redox conditions interchain bonding occurs to stabilize the molecule. This hypothesis is verified by in vitro reassociation studies performed in the presence of reducing agents, demonstrating that the formation of interchain disulfide bonds is not a prerequisite to the trimeric association and triple-helical folding of the collagen XII molecule. Shortening the COL1 domain of minicollagen XII to its five C-terminal GXY triplets results in an absence of trimers. This can be explained by the presence of a collagenous domain that is too short to form a stable triple-helix. In contrast, the presence of five additional C-terminal triplets in COL1 allows the formation of triple-helical disulfide-bonded trimers, suggesting that the presence of a triple-helix is essential for the assembly of collagen XII.A minicollagen containing the COL1 and NC1 domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triple-helical domain in which the cysteines are not involved in inter- but in intrachain bonds. In reducing conditions, providing that the triple-helix is maintained, disulfide exchange between intra- and interchain bonding is observed, suggesting that the triple-helix forms first and that in favorable redox conditions interchain bonding occurs to stabilize the molecule. This hypothesis is verified by in vitro reassociation studies performed in the presence of reducing agents, demonstrating that the formation of interchain disulfide bonds is not a prerequisite to the trimeric association and triple-helical folding of the collagen XII molecule. Shortening the COL1 domain of minicollagen XII to its five C-terminal GXY triplets results in an absence of trimers. This can be explained by the presence of a collagenous domain that is too short to form a stable triple-helix. In contrast, the presence of five additional C-terminal triplets in COL1 allows the formation of triple-helical disulfide-bonded trimers, suggesting that the presence of a triple-helix is essential for the assembly of collagen XII.

Fibromodulin belongs to the family of small, leucine-rich proteoglycans which have been reported to interact with collagens and to inhibit type I collagen fibrillogenesis. Decorin and fibromodulin exhibit a noticeable degree of sequence similarity. However, as previously reported [Font, B., Eichenberger, D., Rosenberg, L. M. & van der Rest, M. (1996) Matrix Biol. 15, 341-348] the domains of these molecules implicated in the interactions with type XII and type XIV collagens are different, these being the dermatan sulphate/chondroitin sulphate chain for decorin and the core protein for fibromodulin. At the present time the fibromodulin domains implicated in the interactions with fibrillar collagens remain unknown. In experiments reported here, we have sought to identify the structural requirements for fibromodulin interaction with collagen and for the control of type I collagen fibrillogenesis. Circular dichroism spectra and fibrillogenesis inhibition studies show that fibromodulin structure and its collagen fibrillogenesis control function are strictly dependent on the presence of intact disulphide bridge(s). In addition, we show that the binding of fibromodulin (or fibromodulin-derived fragments) to type I collagen is not necessarily correlated with fibrillogenesis inhibition. To isolate fibromodulin domains, the native proteoglycan was submitted to mild proteolysis. We have isolated an alpha-chymotrypsin-resistant fragment which contains the bulk of the N-terminal and central region of the molecule including the leucine-rich repeats 4 and 6 reported for decorin to be involved in type I collagen binding. This fragment does not bind to type I collagen. Using enzymes with different specificities, a number of large fragments of fibromodulin were obtained, suggesting a compact structure for this molecule which is relatively resistant to proteolysis. None of these N-glycosylated fragments were able to bind to type I collagen in co-sedimentation experiments. Taken together these results suggest that fibromodulin-type I collagen interactions leading to fibrillogenesis inhibition require more than one binding domain. One of these domains could be the C-terminal end of the molecule containing the disulphide loop which is absent in the chymotrypsin-resistant fragment.Fibromodulin belongs to the family of small, leucine-rich proteoglycans which have been reported to interact with collagens and to inhibit type I collagen fibrillogenesis. Decorin and fibromodulin exhibit a noticeable degree of sequence similarity. However, as previously reported [Font, B., Eichenberger, D., Rosenberg, L. M. & van der Rest, M. (1996) Matrix Biol. 15, 341-348] the domains of these molecules implicated in the interactions with type XII and type XIV collagens are different, these being the dermatan sulphate/chondroitin sulphate chain for decorin and the core protein for fibromodulin. At the present time the fibromodulin domains implicated in the interactions with fibrillar collagens remain unknown. In experiments reported here, we have sought to identify the structural requirements for fibromodulin interaction with collagen and for the control of type I collagen fibrillogenesis. Circular dichroism spectra and fibrillogenesis inhibition studies show that fibromodulin structure and its collagen fibrillogenesis control function are strictly dependent on the presence of intact disulphide bridge(s). In addition, we show that the binding of fibromodulin (or fibromodulin-derived fragments) to type I collagen is not necessarily correlated with fibrillogenesis inhibition. To isolate fibromodulin domains, the native proteoglycan was submitted to mild proteolysis. We have isolated an alpha-chymotrypsin-resistant fragment which contains the bulk of the N-terminal and central region of the molecule including the leucine-rich repeats 4 and 6 reported for decorin to be involved in type I collagen binding. This fragment does not bind to type I collagen. Using enzymes with different specificities, a number of large fragments of fibromodulin were obtained, suggesting a compact structure for this molecule which is relatively resistant to proteolysis. None of these N-glycosylated fragments were able to bind to type I collagen in co-sedimentation experiments. Taken together these results suggest that fibromodulin-type I collagen interactions leading to fibrillogenesis inhibition require more than one binding domain. One of these domains could be the C-terminal end of the molecule containing the disulphide loop which is absent in the chymotrypsin-resistant fragment.

The distinction between benign and malignant smooth muscle tumours relying on histological features such as the mitotic index and pleomorphism remains a generally acknowledged difficulty in modern pathology. A cell image processor was therefore used to quantitatively assess the desmin and vimentin immunostain in 39 smooth muscle tumours which included 26 benign (leiomyomas) and 13 malignant (leiomyosarcomas) cases. The 13 leiomyosarcomas were primary (non-recurrent and non-metastatic). Ploidy level and cell density were also assessed on each of these 39 tumours by means of the computer-assisted microscopic analysis of 5-microns thick Feulgen-stained histological sections. The results show that while neither the ploidy level determination nor the quantitative assessment of the vimentin immunostain made it possible to distinguish between leiomyomas and leiomyosarcomas, cell density determination and the quantitative assessment of the desmin immunostain enabled such a distinction to be made. Indeed, the leiomyomas exhibited a much higher level of desmin positivity than the leiomyosarcomas, as did diploid tumours as compared to the aneuploid (benign or malignant) ones. Furthermore, the leiomyoma group exhibited a significantly lower mean cell density value than the leiomyosarcoma group. The present study further confirms the lack of relationship between ploidy level and cytological malignancy in smooth muscle tumours.

Thirty-five lipomatous tumors were quantitatively described using 47 variables generated by means of computer-assisted microscope analysis. Of these 47 quantitative variables, 27 were computed on Feulgen-stained specimens (25 on cytologic and 2 on histologic samples) and, of the remaining 20, 8 related to vimentin and S-100 protein immunostaining patterns and the other 12 to the glycohistochemical staining patterns of peanut agglutinin, succinylated wheat germ agglutinin, and concavalin A agglutinin. The 35 lipomatous tumors included 6 atypical lipomas and 8 well differentiated, 5 dedifferentiated, 6 myxoid, and 10 pleomorphic liposarcomas. The actual diagnostic value contributed by each of the 47 variables with respect to the 5 lipomatous tumor groups was determined by means of the decision tree technique, an artificial intelligence-related algorithm that forms part of the supervised learning algorithms. Of the 47 quantitative variables, the decision tree technique retained 8: i.e., 2 tissue architecture-, 2 DNA ploidy level-, 2 morphonuclear-, 1 lectin histochemical-, and 1 vimentin immunostain-related variables. The decision tree technique made use of these 8 variables to set up logical rules that make it possible to identify atypical lipomas from well differentiated liposarcomas, on the one hand, and dedifferentiated liposarcomas from those that are well differentiated and pleomorphic, on the other. Thus, the combination of an artificial intelligence algorithm analyzing quantitative variables generated by means of the computer-assisted microscope analysis of cytologic and histologic samples from lipomatous tumors can be considered an expert system contributing significant diagnostic information to conventional diagnosis.

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