scholarly journals Cartilage matrix protein forms a type II collagen-independent filamentous network: analysis in primary cell cultures with a retrovirus expression system.

1995 ◽  
Vol 6 (12) ◽  
pp. 1743-1753 ◽  
Author(s):  
Q Chen ◽  
D M Johnson ◽  
D R Haudenschild ◽  
M M Tondravi ◽  
P F Goetinck
Keyword(s):  
Growth Factor ◽  
Matrix Protein ◽  
Expression System ◽  
Type Ii Collagen ◽  
Cartilage Matrix ◽  
Type Ii ◽  
Tail Domain ◽  
Filament Formation ◽  
Wild Type ◽  
Epidermal Growth

Cartilage matrix protein (CMP) is expressed specifically in mature cartilage and consists of two von Willebrand factor A domains (CMP-A1 and CMP-A2) that are separated by an epidermal growth factor-like domain, and a coiled-coil tail domain at the carboxyl terminal end. We have shown previously that CMP interacts with type II collagen-containing fibrils in cartilage. In this study, we describe a type II collagen-independent CMP filament and we analyze the structural requirement for the formation of this type of filament. Recombinant wild-type CMP and two mutant forms were expressed in chick primary cell cultures using a retrovirus expression system. In chondrocytes, the wild-type virally encoded CMP is able to form disulfide bonded trimers and to assemble into filaments. Filaments also form with CMP whose Cys455 and Cys457 in the tail domain were mutagenized to prevent interchain disulfide bond formation. Therefore, intermolecular disulfide bonds are not necessary for the assembly of CMP into filaments. Both the wild-type and the double cysteine mutant also form filaments in fibroblasts, indicating that chondrocyte-specific factors are not required for filament formation. A truncated form of CMP that consists only of the CMP-A2 domain and the tail domain can form trimers but fails to form filaments, indicating that the deleted CMP-A1 domain and/or the epidermal growth factor domain are necessary for filament assembly but not for trimer formation. Furthermore, the expression of the virally encoded truncated CMP in chondrocyte culture disrupts endogenous CMP filament formation. Together these data suggest a role for CMP in cartilage matrix assembly by forming filamentous networks that require participation and coordination of individual domains of CMP.

scholarly journals Tailless keratins assemble into regular intermediate filaments in vitro

1990 ◽  
Vol 97 (2) ◽  
pp. 317-324
Author(s):  
M. Hatzfeld ◽  
K. Weber
Keyword(s):  
Intermediate Filaments ◽  
Consensus Sequence ◽  
In Vitro Mutagenesis ◽  
Type I ◽  
Type Ii ◽  
Tail Domain ◽  
Filament Formation ◽  
Wild Type ◽  
Keratin 8

To study the influence of the non alpha-helical tail domain of keratins in filament formation, we prepared a truncated keratin 8 mutant, K8/tailless. Using site-directed in vitro mutagenesis we introduced a stop codon in the position coding for amino acid number 417 of the K8/wild-type sequence, thereby deleting 86 amino acids of the non alpha-helical tail domain but leaving the consensus sequence at the end of the rod domain intact. Expression of the truncated keratin 8 in Escherichia coli allowed us to purify the protein by a two-step procedure. The filament-forming capacity of the truncated K8 with wild-type K18 and K19 was analyzed using in vitro reconstitution. The in vitro assembly studies with K8/tailless and K18 wild-type indicate that the C-terminal tail domain of a type II keratin, including the homologous subdomain H2, is not required for filament formation. Moreover, reconstitution experiments with K8/tailless and K19, a naturally occurring tailless keratin I, show that the tail domains of type I as well as type II keratins are not an essential requirement for in vitro filament formation. Our results suggest that in vitro filament elongation does not depend on interactions between head and tail domains, although the tail domain might have a role in stabilization of intermediate filaments arising from certain keratin pairs.

Gene regulation during cartilage differentiation: temporal and spatial expression of link protein and cartilage matrix protein in the developing limb

Development ◽  
1989 ◽  
Vol 107 (1) ◽  
pp. 23-33
Author(s):  
N.S. Stirpe ◽  
P.F. Goetinck
Keyword(s):  
Matrix Protein ◽  
Core Protein ◽  
Type Ii Collagen ◽  
Cartilage Matrix ◽  
Type Ii ◽  
Spatial Expression ◽  
Link Protein ◽  
Genes Encoding ◽  
Temporal And Spatial ◽  
And Cartilage

The temporal and spatial expression of link protein and cartilage matrix protein genes was defined during chondrogenesis in the developing chick embryonic wing bud, using RNA in situ hybridization. For comparison, the expression of genes encoding type II collagen and cartilage proteoglycan core protein was also examined. Link protein transcripts are first detected at stage 25 of Hamburger and Hamilton, together with proteoglycan core protein transcripts. Type II collagen transcripts were first detected as early as stage 23 whereas cartilage matrix protein transcripts could not be detected before stage 26. The results of the study indicate that the temporal expression of the genes for cartilage matrix protein and type II collagen are independent of each other and also independent of that for link protein and proteoglycan core protein.

scholarly journals Oligomeric forms of the 148 kDa cartilage matrix protein

1997 ◽  
Vol 328 (2) ◽  
pp. 665-668 ◽  
Author(s):  
Reema ZEINELDIN ◽  
Suzanne EKBORG ◽  
John BAKER
Keyword(s):  
Molecular Mass ◽  
Matrix Protein ◽  
Type Ii Collagen ◽  
Cartilage Matrix ◽  
Type Ii ◽  
Age Related ◽  
Sds Page ◽  
Self Association ◽  
Chromatography Step ◽  
Oligomeric Forms

The 148 kDa cartilage matrix protein (CMP), composed of three disulphide-bonded subunits, is a cartilage-specific glycoprotein found in association with fibrils of type II collagen and possibly with aggrecan. It is probable that CMP serves a structural role. As cartilage ages, an increasing proportion of the CMP becomes insoluble and resistant to extraction. In the present study, the isolation of CMP has been improved by inclusion of a hydrophobic chromatography step, thereby removing the remaining traces of collagen and proteoglycan. Evidence of self-association of CMP is presented. Higher-molecular-mass forms of CMP, ranging in apparent molecular mass from 270 to 510 kDa and separated by SDS/PAGE, were located using a specific anti-CMP monoclonal antibody. Both CMP and its oligomeric forms are reducible to 52 kDa subunits, and only trace amounts of other proteins. The formation of oligomers, which may constitute 23% of the total cartilage matrix protein, could occur as a byproduct of the normal biosynthetic trimerization of subunits. Alternatively, the oligomers may represent a step toward the age-related cross-linking and insolubilization of CMP.

scholarly journals Chemotherapy versus erlotinib as second-line treatment in patients with advanced non-small cell lung cancer and wild-type epidermal growth factor receptor: an individual patient data (IPD) analysis

ESMO Open ◽  
2018 ◽  
Vol 3 (6) ◽  
pp. e000327
Author(s):  
Marina Chiara Garassino ◽  
Tomoya Kawaguchi ◽  
Vanesa Gregorc ◽  
Eliana Rulli ◽  
Masahiko Ando ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Individual Patient Data ◽  
Growth Factor Receptor ◽  
Line Treatment ◽  
Second Line ◽  
Wild Type ◽  
Mean Survival Time ◽  
Epidermal Growth

The efficacy of second-line treatment in patients with epidermal growth factor receptor (EGFR) wild-type tumours is still debatable. We assessed the efficacy of a standard second-line chemotherapy compared with erlotinib in an individual patient data approach for meta-analysis. The primary endpoint was overall survival (OS), and secondary endpoint was progression-free survival (PFS). Both were compared by log-rank test. The ‘restricted mean survival time’ (RMST) was estimated in each study and the difference in mean survival time up to the last available time point was calculated. The Cox proportional hazards model was used on survival analyses to provide HRs, to adjust for confounding variables and to test possible interaction with selected factors. Three randomised trials comparing chemotherapy versus erlotinib were analysed, including 587 randomised patients. Overall, 74% of patients included in the original trials were considered. 464 deaths and 570 progressions or deaths were observed. Compared with erlotinib, chemotherapy was associated to a decreased risk of progression (29%; HR: 0.71, 95% CI: 0.60 to 0.84, p< 0.0001;) but with no statistical significant reduction in OS (HR: 0.89, 95% CI: 0.74 to 1.06; p<0.20). No heterogeneity was found in both analyses. Patients treated with chemotherapy gained an absolute 1.5 and 1.6 months, respectively, in PFS and lifetime (RMST 95% CI: PFS 0.49 to 2.44; OS 95% CI: −1.04 to 4.25). These results showed that patients without a constitutively activated EGFR had better PFS with chemotherapy rather than with erlotinib while no statistical difference was observed in OS.

scholarly journals A Novel High Sensitivity Type II Collagen Blood-Based Biomarker, PRO-C2, for Assessment of Cartilage Formation

2018 ◽  
Vol 19 (11) ◽  
pp. 3485 ◽  
Author(s):  
Yunyun Luo ◽  
Yi He ◽  
Ditte Reker ◽  
Natasja Gudmann ◽  
Kim Henriksen ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Growth Factor ◽  
Knee Osteoarthritis ◽  
High Sensitivity ◽  
Type Ii Collagen ◽  
Cartilage Explants ◽  
Type Ii ◽  
Serum Samples ◽  
Human Cartilage ◽  
Cartilage Formation

N-terminal propeptide of type II collagen (PIINP) is a biomarker reflecting cartilage formation. PIINP exists in two main splice variants termed as type IIA and type IIB collagen NH2-propeptide (PIIANP, PIIBNP). PIIANP has been widely recognized as a cartilage formation biomarker. However, the utility of PIIBNP as a marker in preclinical and clinical settings has not been fully investigated yet. In this study, we aimed to characterize an antibody targeting human PIIBNP and to develop an immunoassay assessing type II collagen synthesis in human blood samples. A high sensitivity electrochemiluminescence immunoassay, hsPRO-C2, was developed using a well-characterized antibody against human PIIBNP. Human cartilage explants from replaced osteoarthritis knees were cultured for ten weeks in the presence of growth factors, insulin-like growth factor 1 (IGF-1) or recombinant human fibroblast growth factor 18 (rhFGF-18). The culture medium was changed every seven days, and levels of PIIBNP, PIIANP, and matrix metalloproteinase 9-mediated degradation of type II collagen (C2M) were analyzed herein. Serum samples from a cross-sectional knee osteoarthritis cohort, as well as pediatric and rheumatoid arthritis samples, were assayed for PIIBNP and PIIANP. Western blot showed that the antibody recognized PIIBNP either as a free fragment or attached to the main molecule. Immunohistochemistry demonstrated that PIIBNP was predominately located in the extracellular matrix of the superficial and deep zones and chondrocytes in both normal and osteoarthritic articular cartilage. In addition, the hsPRO-C2 immunoassay exhibits acceptable technical performances. In the human cartilage explants model, levels of PIIBNP, but not PIIANP and C2M, were increased (2 to 7-fold) time-dependently in response to IGF-1. Moreover, there was no significant correlation between PIIBNP and PIIANP levels when measured in knee osteoarthritis, rheumatoid arthritis, and pediatric serum samples. Serum PIIBNP was significantly higher in controls (KL0/1) compared to OA groups (KL2/3/4, p = 0.012). The hsPRO-C2 assay shows completely different biological and clinical patterns than PIIANP ELISA, suggesting that it may be a promising biomarker of cartilage formation.

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