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Query: UNIPROT:P00790 (PGA)
 2,475 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Type X collagen is a short-chain, network-forming collagen found in hypertrophic cartilage in the growth zones of long bones, vertebrae, and ribs. To obtain information about the structure and assembly of mammalian type X collagen, we generated recombinant human type collagen X by stable expression of full-length human alpha1(X) cDNA in the human embryonal kidney cell line HEK293 and the fibrosarcoma cell line HT1080. Stable clones were obtained secreting recombinant human type X collagen (hrColX) in amounts of 50 microg/ml with alpha1(X)-chains of apparent molecular mass of 75 kDa. Pepsin digestion converted the native protein to a molecule migrating as one band at 65 kDa, while bands of 55 and 43 kDa were generated by trypsin digestion. Polyclonal antibodies prepared against purified hrColX reacted specifically with type X collagen in sections of human fetal growth cartilage. Circular dichroism spectra and trypsin/chymotrypsin digestion experiments of hrColX at increasing temperatures indicated triple helical molecules with a reduced melting temperature of 31 degrees C as a result of partial underhydroxylation. Ultrastructural analysis of hrColX by rotary shadowing demonstrated rodlike molecules with a length of 130 nm, assembling into aggregates via the globular noncollagenous (NC)-1 domains as reported for chick type X collagen. NC-1 domains generated by collagenase digestion of hrColX migrated as multimers of apparent mass of 40 kDa on SDS-polyacrylamide gel electrophoresis, even after reduction and heat denaturation, and gave rise to monomers of 18-20 kDa after treatment with trichloroacetic acid. The NC-1 domains prepared by collagenase digestion comigrated with NC-1 domains prepared as recombinant protein in HEK293 cells, both in the multimeric and monomeric form. These studies demonstrate the potential of the pCMVsis expression system to produce recombinant triple helical type X collagens in amounts sufficient for further studies on its structural and functional domains.
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PMID:Characterization of human type X procollagen and its NC-1 domain expressed as recombinant proteins in HEK293 cells. 946 10

New cartilage formation has been successfully achieved by technology referred to as tissue engineering. Polymers and hydrogels such as poly(glycolic acid), calcium alginate, and poly(ethylene) and poly(propylene) hydrogels have been used as cell carriers to regenerate cartilage in the nude mouse model. The next step toward human applications of engineered cartilage is to demonstrate their potential in immunocompetent animal models. This study compared the suitability of three polymers for generating tissue engineered elastic cartilage using autologous cells in an immuno-competent porcine animal model. Auricular cartilage was obtained from pigs. Chondrocytes were isolated onto fiber based poly(glycolic acid) (PGA) scaffolds or suspended in calcium alginate or pluronic F127 gel at constant concentrations. Chondrocyte-polymer constructs were either implanted (PGA) or injected (calcium alginate and pluronic) as autologous implants subcutaneously into the pigs from which the cells had been isolated. Specimens were harvested and analyzed grossly and historically after 6 weeks in vivo. All explants demonstrated cartilage formation to a variable degree. When using PGA or calcium alginate, the overall histological appearance of the tissue formed is that of fibrocartilage with thick bundles of collagen dispersed in the tissue. When using pluronics as scaffold, histologic features resemble those of native elastic cartilage, showing a more organized arrangement of the cells, which seems to correlate to functional properties as elastin presence in the tissue engineered cartilage. Elastic cartilage engineered in an immunocompetent animal model varies with the type of polymer used. The behavior of the cell-polymer constructs is not fully understood and outcome seems to be related to several factors, including inflammatory reaction. Further studies with similar models are needed to determine the feasibility of engineering tissue generated from different cell-polymer constructs prior to human application.
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PMID:Comparative study of the use of poly(glycolic acid), calcium alginate and pluronics in the engineering of autologous porcine cartilage. 964 28

New cell culture techniques raise the possibility of creating cartilage in vitro with the help of tissue engineering. In this study, we compared two resorbable nonwoven cell scaffolds, a polyglycolic acid/poly-L-lactic acid (PGA/PLLA) (90/10) copolymer (Ethisorb) and pure PLLA (V 7-2), with different degradation characteristics in their aptitude for cartilage reconstruction. Chondrocytes were isolated enzymatically from human septal cartilage. The single cells were resuspended in agarose and transferred into the polymer scaffolds to create mechanical stability and retain the chondrocyte-specific phenotype. The cell-polymer constructs were then kept in perfusion culture for 1 week prior to subcutaneous transplantation into thymusaplastic nude mice. After 6, 12, and 24 weeks, the specimens were explanted and analyzed histochemically on the presence of collagen (azan staining), proteoglycans (Alcian blue staining), and calcification areas (von Kossa staining). Furthermore, different collagen types (collagen type I, which is found in most tissues, but not in hyaline cartilage matrix; and collagen type II, which is cartilage specific) were differentiated immunohistochemically by the indirect immunoperoxidase technique. Vascular ingrowth was investigated by a factor VIII antibody, which is a endothelial marker. Quantification of several matrix components was performed using the software Photoshop. Significant differences were found between both nonwoven structures concerning matrix synthesis and matrix quality as well as vascular ingrowth. Ethisorb, with a degradation time of approximately 3 weeks in vitro, showed no significant differences from normal human septal cartilage in the amount of collagen types I and II 24 weeks after transplantation. Thin fibrous tissue layers containing blood vessels encapsulated the transplants. V 7-2 constructs, which did not show strong signs of degradation even 24 weeks after transplantation, contained remarkably smaller amounts of cartilage-specific matrix components. At the same time, there was vascular ingrowth even in central parts of the transplants. In conclusion, polymer scaffolds with a short degradation time are suitable materials for the development of cartilage matrix products, while longer stability seems to inhibit matrix synthesis. Thus, in vitro engineering of human cartilage can result in a cartilage-like tissue when appropriate nonwovens are used. Therefore, this method could be the ideal cartilage replacement method without the risk of infection and with the possibility of reconstructing large defects with different configurations.
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PMID:Cartilage reconstruction in head and neck surgery: comparison of resorbable polymer scaffolds for tissue engineering of human septal cartilage. 978 96

Previously we reported that cultured rat Schwann cells secrete p200, a collagen-like heparin-binding adhesive glycoprotein with a restricted pattern of expression. Here we report that p200 is secreted as a stable trimer, but only after treatment of Schwann cells with ascorbic acid, and was deposited in the fibrillar extracellular matrix. Heparin and heparitinase treatment inhibited incorporation of p200 into extracellular matrix, suggesting the involvement of Schwann cell heparan sulfate proteoglycans in this process. Pepsin digestion revealed that p200 secreted by ascorbate-treated cells contains a collagenous domain of approximately 140 kDa. Immunofluorescent staining of rat embryos at different ages showed that p200 first appeared between embryonic days 15 and 18, and was confined to peripheral nerves. Staining of adult peripheral nerve was negative, but p200 expression was induced in adult sciatic nerve following nerve transection. These data suggest that p200 carries out unique functions during peripheral nerve development and regeneration and that its expression by Schwann cells is regulated by axon-Schwann cell interaction.
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PMID:p200, a collagen secreted by Schwann cells, is expressed in developing nerves and in adult nerves following axotomy. 1033 58

Pepsin-solubilized elastin (PSE)-conjugated collagen film was prepared from a collagen matrix with PSE by drying it and crosslinking the constituents with water-soluble carbodiimide or microbial transglutaminase to improve the physical properties of the collagen film. The crosslinking reduced the solubility and improved the thermal stability, the thermal transition properties, and the elasticity of the control film in water. In particular, water-soluble carbodiimide strongly influenced these properties. The PSE-conjugated collagen film showed good permeation by water-soluble tasting substances such as oligosaccharides and amino acids, but poor permeation by polysaccharide, protein, and hydrophobic substances such as retinol and cholesterol.
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PMID:Improvement of the physical properties of pepsin-solubilized elastin-collagen film by crosslinking. 1066 47

Anticoagulative effect of pepsin is observed in vitro when its concentration is 36 microM and higher. This effect is due to inhibition of fibrin monomer polymerization. Protamine abolishes anticoagulative effect of pepsin. Pepsin does not influence platelet aggregation induced by ADP and collagen.
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PMID:Anticoagulative effect of pepsin. 1069 79

The functions of aminotelopeptide and N-terminal cross-linking of collagen I were examined. Acetic acid-soluble collagen I (ASC) was purified from neonatal bovine skin and treated with three kinds of proteases. The amino acid sequencing analysis of the N terminus showed that ASC contained a full-length aminotelopeptide. Pepsin and papain cleaved the aminotelopeptide of the alpha1 chain at the same site and the aminotelopeptide of the alpha2 chain at different sites. Proctase-treated ASC lost the whole aminotelopeptide, and the N-terminal sequence began from the tenth residue inside the triple helical region. The rates of fibril formation of pepsin-treated ASC and proctase-treated ASC were the same and were slower than that of ASC. The denaturation temperatures, monitored by CD ellipticity at 221 nm, of ASC, pepsin-treated, or papain-treated collagens were the same at 41.8 degrees C. Proctase-treated ASC showed a lower denaturation temperature of 39.9 degrees C. We also observed the morphology of the collagen fibrils under an electron microscope. The ASC fibrils were straight and thin, whereas the fibrils of pepsin-treated ASC were slightly twisted, and the fibrils from papain- and proctase-treated ASC were highly twisted and thick. When the collagen gel strength was examined by a modified method of viscosity-measurement, ASC was the strongest, followed by pepsin-treated ASC, and papain- and proctase-treated ASCs were the weakest. These results suggest that the aminotelopeptide plays important roles in fibril formation and thermal stability. In addition, the functions of intermolecular cross-linking in aminotelopeptides may contribute to the formation of fibrils in the correct staggered pattern and to strengthening the collagen gel.
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PMID:Possible involvement of aminotelopeptide in self-assembly and thermal stability of collagen I as revealed by its removal with proteases. 1085 Dec 40

The brittleness of bone in patients with osteogenesis imperfecta (OI) has been attributed to an aberrant collagen network. However, the role of collagen in the loss of tissue integrity has not been well established. To gain an insight into the biochemistry and structure of the collagen network, the cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) and the level of triple helical hydroxylysine (Hyl) were determined in bone of OI patients (types I, III, and IV) as well as controls. The amount of triple helical Hyl was increased in all patients. LP levels in OI were not significantly different; in contrast, the amount of HP (and as a consequence the HP/LP ratio and the total pyridinoline level) was significantly increased. There was no relationship between the sum of pyridinolines and the amount of triple helical Hyl, indicating that lysyl hydroxylation of the triple helix and the telopeptides are under separate control. Cross-linking is the result of a specific three-dimensional arrangement of collagens within the fibril; only molecules that are correctly aligned are able to form cross-links. Inasmuch as the total amount of pyridinoline cross-links in OI bone is similar to control bone, the packing geometry of intrafibrillar collagen molecules is not disturbed in OI. Consequently, the brittleness of bone is not caused by a disorganized intrafibrillar collagen packing and/or loss of cross-links. This is an unexpected finding, because mutant collagen molecules with a random distribution within the fibril are expected to result in disruptions of the alignment of neighboring collagen molecules. Pepsin digestion of OI bone revealed that collagen located at the surface of the fibril had lower cross-link levels compared with collagen located at the inside of the fibril, indicating that mutant molecules are not distributed randomly within the fibril but are located preferentially at the surface of the fibril.
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PMID:Pyridinium cross-links in bone of patients with osteogenesis imperfecta: evidence of a normal intrafibrillar collagen packing. 1089 81

Although the triple-helical structure of fibrillar collagen is regarded in general as being quite similar, each type of collagen molecule has inherent characteristics in the triple-helical domain. Few studies have ever been performed in terms of the aggregate structure of the triple-helical domain of fibrillar collagen. Reconstituted aggregates from the purified triple-helical domain of each type of fibrillar collagen might amplify the subtle differences in the structural characteristics of each type of collagen molecule. In this study, the reconstituted aggregate structure of pepsin-treated type V collagen (type Vp collagen), that is, virtually its triple-helical domain was characterized by transmission electron microscopy. Pepsin-treated type I (type Ip) and type II (type IIp) collagen were compared with type Vp collagen. Unique features of the aggregate structure of the triple-helical domain of the type V collagen can be summarized as follows:These results suggested that the lateral packing of the triple-helical domain of type V collagen is determined by its molecular structure. The characteristics of type Vp collagen fibrils might be explained by their characteristic amino acid composition. A significant feature of the triple-helical domain of type V collagen is the high content of glycosylated hydroxylysine residues. Molecular model building of the collagenous structure suggests that a change in surface roughness is conspicuous by incorporating the glycosylated hydroxylysine residues. More than a ten-fold content of bulky glycosylated hydroxylysine residues in type V collagen compared to that of type I might have a significant influence on both the intermolecular and interfibrillar interactions of the triple-helical domain of type V collagen molecule.
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PMID:The fibril structure of type V collagen triple-helical domain. 1100 11

To understand the reparative process of medial collateral ligament (MCL), fibrillar collagen and their relative ratios in healing MCL with anterior cruciate ligament (ACL) reconstruction were analyzed. Skeletally mature New Zealand white rabbits were subjected to a mop-end tear of MCL without repair with ACL reconstruction. Rabbits were killed 6 and 52 weeks after injury. Ligamentous tissues from the injury site and sham controls were soaked in 0.5 M acetic acid for 24 h, minced, and treated with pepsin to solubilize collagen. Pepsin solubilized about 80% of the total collagen as determined by hydroxyproline analysis of the pepsin residues. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the solubilized collagen revealed presence of fibrillar collagen types I, III, and V. Densitometric scanning of the protein bands corresponding to types I, III, and V collagen indicated that in sham controls types III and V collagen represented about 8% and 12%, respectively, of the type I collagen whereas the healed MCL ligaments at 6 weeks showed significant increase in type III and V collagen to about 19% and 24%, respectively. By 52 weeks type III collagen in the healed MCL had returned to that of sham controls while type V collagen remained elevated at approximately 18%. These data suggest that presence of type V collagen in high concentration in healing ligaments may have an influence on collagen fibril diameters seen in healed ligament and should be included in the analysis when evaluating ligament healing.
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PMID:Type V collagen is increased during rabbit medial collateral ligament healing. 1106 Dec 96


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