RESUMEN

3-nitrotyrosine (3NT) is regarded as a "footprint" of nitric oxide generation. The study aimed at documenting the presence and distribution of 3-nitrotyrosine (3NT) in muscle tissue samples from patients with idiopathic inflammatory myopathies (IIM) as well as from those with non-inflammatory myopathies to consider whether polymyositis (PM) and dermatomyositis (DM) could be distinguished based on 3NT immunohistochemistry in muscle biopsy. Cryosections prepared from muscle biopsies of 54 patients with either IIM, i.e., PM and DM, or various non-inflammatory myopathies were immunostained using monoclonal antibody against 3NT. The 3NT immunostaining was localized to endothelial cells and their close surroundings in muscle biopsies of DM and PM patients but only in those areas of tissue sections where inflammatory cell infiltrates were present. No 3NT positivity was found in tissue sections of IIM patients without inflammatory infiltrates in the studied sample as well as in muscle tissue sections of patients with non-inflammatory myopathies. However, the endothelial cells were also positive in cases of confirmed non-inflammatory myopathies with secondary lymphocytic infiltration (myodystrophies, myasthenia gravis). Despite the pathogenetic significance, the 3NT immunohistochemistry is of low diagnostic value for the differential diagnosis of IIM in muscle biopsy.

Asunto(s)

Músculo Esquelético/metabolismo , Miositis/metabolismo , Tirosina/análogos & derivados , Adulto , Anciano , Dermatomiositis/metabolismo , Dermatomiositis/patología , Endotelio/metabolismo , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Músculo Esquelético/patología , Polimiositis/metabolismo , Polimiositis/patología , Unión Proteica , Tirosina/metabolismo

RESUMEN

OBJECTIVE: To investigate the utility of serum COMP level measurements as a predictor of future damage of the weight-bearing (large) joints in RA patients participating in intensive exercise. METHODS: Data of the 281 completers of a 2-yr randomized controlled trial (Rheumatoid Arthritis Patients In Training; RAPIT) comparing the effects of usual care physical therapy with high-intensity weight-bearing exercises were analysed. The primary outcome variable was defined as the change in radiological joint damage (Larsen score) of the large joints. Potential predictors of outcome were defined: baseline and change in serum level of COMP after 3 months, baseline radiological damage of the large and small joints, number of months on glucocorticoids, change in disease activity and in physical capacity (aerobic fitness and muscle strength) after 2 yrs, and participation in the exercise group. RESULTS: In cross-sectional evaluation of baseline data, we found strong association between the high serum COMP level and current damage of the large joints. Serum COMP level at baseline, however, was not associated with an increased rate of radiological joint damage after 2 yrs of follow-up. Furthermore, neither interaction between baseline COMP level and participation in exercises, nor change in COMP level after 3 months of exercising were associated with future damage of the large joints. CONCLUSION: Neither baseline serum COMP level nor its individual change after 3 months from start of intensive exercise predict longitudinal progression of damage of the large joints in this population.

Asunto(s)

Artritis Reumatoide/diagnóstico , Terapia por Ejercicio/efectos adversos , Proteínas de la Matriz Extracelular/sangre , Glicoproteínas/sangre , Adulto , Artritis Reumatoide/diagnóstico por imagen , Artritis Reumatoide/rehabilitación , Biomarcadores/sangre , Proteína de la Matriz Oligomérica del Cartílago , Estudios Transversales , Progresión de la Enfermedad , Terapia por Ejercicio/métodos , Femenino , Humanos , Masculino , Proteínas Matrilinas , Persona de Mediana Edad , Selección de Paciente , Pronóstico , Radiografía , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Soporte de Peso

RESUMEN

This study was designed to assay and compare cartilage oligomeric matrix protein (COMP) in horse sera, in samples from normal and joint diseased horses, and to investigate the relationships between COMP in sera and synovial fluids (SF) with keratan sulphate (KS) data. Sera from 38 horses free of any joint pathology (controls) and from horses with aseptic joint disease (AJD horses, n = 40) were assayed for COMP and KS concentrations. Of the 78 horses in the study, 53 were also assayed for COMP and KS concentrations in SF. COMP and KS were measured by inhibition ELISA, using monoclonal antibodies 12C4 and 5D4, respectively. The COMP concentration in sera from AJD horses (mean +/- s.d. 10.7 +/- 7.4 microg/ml) was significantly (P<0.02) lower than in control sera (14.8 +/- 7.8 microg/ml). The joint disease sera also had significantly lower (P<0.01) KS levels (180.5 +/- 61.8 ng/ml) than controls (237.1 +/- 116.1 ng/ml). A significant correlation (r = 0.52, n = 53, P<0.001) was seen between serum and SF in COMP levels; no such relationship was seen in KS levels. It is possible that serum COMP concentration could be a more specific marker of equine joint disease than any other described to date.

Asunto(s)

Proteínas de la Matriz Extracelular/sangre , Glicoproteínas/sangre , Enfermedades de los Caballos/sangre , Sulfato de Queratano/sangre , Osteoartritis/veterinaria , Animales , Anticuerpos Monoclonales/análisis , Biomarcadores/sangre , Western Blotting/veterinaria , Estudios de Casos y Controles , Electroforesis en Gel de Poliacrilamida/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Enfermedades de los Caballos/diagnóstico , Caballos , Artropatías/sangre , Artropatías/diagnóstico , Artropatías/veterinaria , Proteínas Matrilinas , Osteoartritis/sangre , Osteoartritis/diagnóstico , Líquido Sinovial/química

RESUMEN

OBJECTIVE: To examine the cross-sectional relationship between serum cartilage oligomeric matrix protein (COMP) and hip and knee clinical signs and symptoms in a sample of adults without radiographic hip or knee osteoarthritis (OA). DESIGN: A total of 145 persons with available sera and no evidence of radiographic hip or knee OA (Kellgren-Lawrence grade 0) were randomly selected from the Caucasian participants of the Johnston County Osteoarthritis Project. COMP was quantified by a competitive ELISA assay with a monoclonal antibody 17-C10. Hip and knee clinical signs and symptoms were assessed by physical examination and interview, and their associations with Ln COMP analysed with general linear models. RESULTS: After adjustment for age, gender, body mass index (BMI), and other symptomatic joints, mean Ln COMP was statistically significantly higher among persons with hip-related clinical signs (P=0.018), among those with hip-related symptoms (P=0.046), and among individuals meeting American College of Rheumatology clinical criteria for hip OA (P=0.021). There were no statistically significant associations between any of the knee-related clinical signs and symptoms and Ln COMP. CONCLUSION: Serum COMP may be useful as a biomarker of pre-radiographic hip joint pathology; its utility as a biomarker of pre-radiographic knee joint pathology is unclear.

Asunto(s)

Proteínas de la Matriz Extracelular/sangre , Glicoproteínas/sangre , Articulación de la Cadera/patología , Articulación de la Rodilla/patología , Anciano , Anciano de 80 o más Años , Proteína de la Matriz Oligomérica del Cartílago , Cartílago Articular/diagnóstico por imagen , Cartílago Articular/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Articulación de la Cadera/diagnóstico por imagen , Humanos , Articulación de la Rodilla/diagnóstico por imagen , Masculino , Proteínas Matrilinas , Persona de Mediana Edad , Radiografía

RESUMEN

OBJECTIVE: To evaluate the prognostic utility of serum COMP level measured with a new sandwich ELISA, by correlating COMP level with outcome measures of osteoarthritis (OA) progression. DESIGN: Patients (N=48) had symptomatic primary knee OA of Kellgren-Lawrence (K-L) grade I-III and met ACR criteria. These patients were evaluated prospectively as part of a double-blind drug trial of 3 years' duration and represented the placebo arm of the study. Serum COMP levels were measured by sandwich ELISA with monoclonal antibodies 16-F12 and 17-C10 at baseline and at study end and levels were correlated with changes in (1) joint space width (JSW), (2) K-L grade, (3) Lequesne, and (4) WOMAC indices, over 3 years. RESULTS: The change in JSW over 3 years, summed for both knees, correlated positively with serum COMP level at baseline as well as at study end. Patients were sorted by level of progression based upon a change in K-L grade summed for both knees over 3 years; patients who progressed by two K-L grades were shown to have had significantly higher COMP levels at baseline as well as at study end. Baseline and study end COMP levels did not correlate with the change of Lequesne or WOMAC indices. Baseline COMP levels correlated strongly with end serum COMP levels. CONCLUSION: Serum COMP has the potential to be a prognostic marker of disease progression. High COMP levels, persisting over the 3-year study period in the patients with radiographic progression, indicated differences in disease activity detectable throughout the entire follow-up interval.

Asunto(s)

Proteínas de la Matriz Extracelular/sangre , Glicoproteínas/sangre , Osteoartritis de la Rodilla/diagnóstico por imagen , Anciano , Proteína de la Matriz Oligomérica del Cartílago , Cartílago Articular/diagnóstico por imagen , Cartílago Articular/patología , Método Doble Ciego , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Articulación de la Rodilla/diagnóstico por imagen , Articulación de la Rodilla/patología , Masculino , Proteínas Matrilinas , Persona de Mediana Edad , Osteoartritis de la Rodilla/sangre , Osteoartritis de la Rodilla/patología , Estudios Prospectivos , Radiografía

RESUMEN

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a component of articular cartilage whose serum levels show a strong correlation with radiographic osteoarthritis (OA) status. It has recently been found, however, that COMP is also produced in synovium. To assess the hypothesis that synovitis affects serum COMP levels in patients with knee OA, we collected sera for COMP simultaneous with a clinical examination for synovitis. DESIGN: Sera were collected from OA patients who fulfilled the American College of Rheumatology criteria for knee OA. Radiographs were classified according to the grading system of Kellgren and Lawrence. Synovitis was diagnosed clinically by joint tenderness plus swelling and/or increased warmth over the joint. COMP levels in sera were measured by inhibition ELISA with monoclonal antibody (mAb) 17-C10. RESULTS: Serum COMP levels were significantly correlated with age, synovitis and an interaction of synovitis and OA severity. Synovitis showed the strongest effect on COMP levels (R=0.1587, P< 0.01), in contrast to C-reactive protein, duration of OA and OA severity score which showed no significant effect on COMP levels. Individual signs of synovitis, namely, joint tenderness and warmth had a significant effect on serum COMP levels while swelling alone did not. CONCLUSION: Synovitis exerts a significant effect on serum COMP levels measured with mAb 17-C10 in OA patients. These findings underscore the importance of the clinical joint examination to assess for synovitis, when attempting to apply objective measures, such as COMP, to the clinical setting.

Asunto(s)

Proteínas de la Matriz Extracelular/sangre , Glicoproteínas/sangre , Osteoartritis de la Rodilla/sangre , Sinovitis/sangre , Adulto , Factores de Edad , Anciano , Análisis de Varianza , Anticuerpos Monoclonales , Biomarcadores/sangre , Proteína C-Reactiva/análisis , Proteína de la Matriz Oligomérica del Cartílago , Intervalos de Confianza , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Modelos Lineales , Masculino , Proteínas Matrilinas , Persona de Mediana Edad , Distribución Normal , Osteoartritis de la Rodilla/complicaciones , Sensibilidad y Especificidad , Índice de Severidad de la Enfermedad , Estadísticas no Paramétricas , Sinovitis/etiología

RESUMEN

OBJECTIVE: This study was designed to assay cartilage oligomeric matrix protein (COMP) in equine synovial fluids and to compare the concentration in synovial fluids from normal horses with joint diseased horses. The relationship between the COMP degradation and the matrix metalloproteinase activity in synovial fluids was also investigated. DESIGN: Using COMP antigen prepared from equine articular cartilage and murine monoclonal antibody (12C4) raised against human COMP, an inhibition ELISA was developed. COMP in equine synovial fluids from normal and diseased joints was quantified. Metalloproteinase activities were evaluated in the same synovial fluids by a gelatin degradation ELISA. COMP fragments were evaluated qualitatively by Western blotting. RESULTS: The COMP inhibition ELISA was reliable at concentrations of equine COMP between 62.5 and 2000 ng/ml. COMP values in joint fluids in both aseptic and septic joint disease (19.7+/-15.3 and 16.1+/-11.2 microg/ml, respectively) were significantly (P < 0.001) lower than normal (53.2+/-29.0 microg/ml). The molecular sizes of COMP on immunoblots were different between normal and diseased synovial fluids; more fragments were seen in diseased fluids. The aseptic (26.6 +/- 20.6%) and septic joint disease synovial fluids (36.1 +/- 37.5%) had significantly higher (P < 0.02 and 0.002, respectively) gelatinolytic activities than normal (13.6 +/- 13.7%). There was a negative correlation (R = -0.31, P < 0.002) between COMP level and gelatinase activity. Conclusions We conclude that the fragment pattern and the absolute COMP concentration maybe useful for monitoring joint disease, and that COMP degradation in synovial fluids from progressed joint disease may be due to MMP gelatinolytic activity.

Asunto(s)

Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Enfermedades de los Caballos/diagnóstico , Artropatías/diagnóstico , Articulaciones/metabolismo , Líquido Sinovial/química , Animales , Western Blotting/métodos , Proteína de la Matriz Oligomérica del Cartílago , Ensayo de Inmunoadsorción Enzimática/métodos , Gelatina/metabolismo , Miembro Posterior , Caballos , Artropatías/veterinaria , Proteínas Matrilinas

RESUMEN

Damage to the meniscus can lead to posttraumatic osteoarthritis. Early markers of joint injury and tissue disease may be useful in developing and administering clinical treatment. We investigated the effects of total medial meniscectomy on biomarkers measured serially in synovial lavage fluid each month for 3 months. Following meniscectomy in dogs, four biomarkers were evaluated: cartilage oligomeric matrix protein, keratan sulfate epitope (5D4), the 3B3(-) neoepitope of chondroitin-6-sulfate, and the 3B3(+) chondroitinase-generated epitope of chondroitin-6-sulfate. Meniscectomy led to statistically significant elevations of all four biomarkers, with levels peaking at 4 weeks. By 12 weeks, the level of the 5D4 epitope returned to the preoperative baseline level whereas that of cartilage oligomeric matrix protein, 3B3(-), and 3B3(+) remained above the baseline. Concentrations of these biomarkers in the knees not operated on did not change significantly from the baseline. The levels of cartilage oligomeric matrix protein and 3B3(-) relative to 3B3(+) remained constant in all knees. In contrast, the level of 5D4 relative to 3B3(+) declined over time in the knee operated on but remained constant in the knee not operated on. These results demonstrate a quantitative change in the molecular components of synovial fluid after meniscectomy, as well as a qualitative change evinced by an alteration in the relative proportions of these epitopes. Extensive analyses showed a strong correlation between serum levels of 3B3(-) from the femoral and cephalic veins; however, serum 3B3(-) was not correlated with synovial fluid 3B3(-). These findings support the hypothesis that the concentrations of select cartilage biomarkers in synovial fluid are altered following meniscectomy and are promising tools for objectively monitoring the induction of osteoarthritis in this model system.

Asunto(s)

Proteínas Bacterianas/análisis , Sulfatos de Condroitina/análisis , Modelos Animales de Enfermedad , Sulfato de Queratano/análisis , Proteínas de la Membrana , Meniscos Tibiales/cirugía , Osteoartritis/metabolismo , Líquido Sinovial/química , Transferasas , Animales , Biomarcadores , Cartílago Articular/patología , Perros , Epítopos , Masculino , Osteoartritis/patología

RESUMEN

OBJECTIVE: To characterize serum cartilage oligomeric matrix protein (COMP) levels by age and gender for a radiographically defined population free of hip and knee osteoarthritis (OA), and to examine the potential utility of COMP as a diagnostic biomarker for knee OA. METHODS: Serum samples and knee and hip radiographs were obtained at a baseline evaluation as part of the Johnston County Osteoarthritis Project, a population-based study of OA in rural North Carolina. A total of 291 Caucasian participants were randomly selected for COMP analysis, 143 patients with radiographic knee OA (Kellgren/Lawrence [K/L] grade > or = 2) and 148 controls with neither hip nor knee OA (K/L grade 0), evenly distributed by age and gender. COMP was quantified by competitive enzyme-linked immunosorbent assay with monoclonal antibody 17-C10. The natural log-transformed COMP data were analyzed using general linear models. RESULTS: Serum COMP levels were significantly elevated (P = 0.0001) in the age > or = 65 group (mean +/- SD 1,302.1 +/- 496.7 ng/ml) versus the age 45-54 and age 55-64 groups (1,058.1 +/- 432.4 and 1,038.6 +/- 313.3, respectively). Serum COMP levels of the OA group were significantly higher than those of the control group (1,208.57 +/- 487.47 ng/ml versus 1,061.83 +/- 370.58 ng/ml; P = 0.0093). Serum COMP levels also increased significantly with knee OA K/L grade (P = 0.0047), knee OA laterality (P = 0.0043), and number of knee and hip joints involved (P = 0.0001). There was no significant difference in serum COMP levels by gender or obesity. CONCLUSION: We demonstrate that in a population-based sample, serum COMP levels can distinguish an OA-affected subgroup from an unaffected subgroup and can reflect disease severity and multiple joint involvement in OA.

Asunto(s)

Proteínas de la Matriz Extracelular/sangre , Glicoproteínas/sangre , Osteoartritis de la Rodilla/sangre , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Proteína de la Matriz Oligomérica del Cartílago , Progresión de la Enfermedad , Femenino , Articulación de la Cadera/diagnóstico por imagen , Humanos , Articulación de la Rodilla/diagnóstico por imagen , Masculino , Proteínas Matrilinas , Persona de Mediana Edad , Osteoartritis de la Rodilla/diagnóstico por imagen , Radiografía

RESUMEN

We investigated the expression of cartilage oligomeric matrix protein (COMP) in normal and rheumatoid arthritis (RA) synovial fibroblasts. In situ hybridization (ISH) was conducted on synovial specimens from five RA patients applying specific probes for COMP or fibroblast collagen type I. ISH was combined with immunohistochemistry, applying antibodies to the macrophage marker CD68. Ribonuclease protection assay (RPA) and rapid amplification of 3'-cDNA ends (3'-RACE) were performed on total RNA from normal and RA synovial fibroblast cultures. Protein extracts from fibroblasts and culture supernatants were compared with synovial fluids and protein extracts from isolated chondrocytes by Western blot utilizing polyclonal and monoclonal antibodies (18-G3 mAb) to COMP. COMP mRNA was detected in fibroblasts of RA synovium by ISH, and in normal and RA synovial fibroblast cultures by RPA. 3'-RACE demonstrated sequence homology of chondrocyte and synovial fibroblast COMP along the coding sequence. COMP protein was detected in synovial fibroblasts and culture supernatants by immunoblot. Using polyclonal antibodies, the major portion of COMP from fibroblasts and culture supernatants was present as low-molecular-weight (LMW) bands, corresponding to those found in synovial fluids. These LMW COMP bands, however, were not detected in any of the cells or tissues tested using 18-G3 mAb. In protein extracts from chondrocytes and in COMP purified from cartilage, these LMW bands could not be detected. In conclusion, the data suggest that certain forms of COMP detected in synovial fluid are secreted from synovial fibroblasts and could be distinguished by specific mAbs from COMP secreted by chondrocytes.

Asunto(s)

Cartílago Articular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Western Blotting , Proteína de la Matriz Oligomérica del Cartílago , Cartílago Articular/patología , Células Cultivadas , Condrocitos/metabolismo , Condrocitos/patología , Cartilla de ADN/química , Proteínas de la Matriz Extracelular/genética , Fibroblastos/metabolismo , Glicoproteínas/genética , Humanos , Hibridación in Situ , Proteínas Matrilinas , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Sinovectomía , Membrana Sinovial/patología

RESUMEN

Cartilage oligomeric matrix protein (COMP) is a high-molecular-weight glycoprotein found at a high concentration in articular cartilage. Recent studies have shown that the joint fluid and serum levels of antigenic COMP, measured by an enzyme-linked immunosorbent assay (ELISA) which uses a polyclonal antiserum raised against bovine COMP, provide important information about metabolic changes occurring in the cartilage matrix in joint disease. In this report, we describe the specificity of three monoclonal antibodies (mAbs) to human COMP and their usefulness in quantifying antigenic COMP fragments in body fluids. Two of the mAbs (16-F12 and 18-G3) recognized both oligomeric and monomeric forms of COMP, but the third (17-C10) reacted positively only with the former. Immunoblots of human COMP, predigested with trypsin for up to 6 h, showed that the three mAbs are directed against different epitopes identified on small tryptic fragments of 30 kDa (16-F12), 25 kDa (17-C10), and 40 kDa as well as 30 kDa (18-G3), respectively. The antibodies also recognized a different pattern of fragments in human pathological synovial fluids. This was particularly striking in the case of the medium size fragments (16-F12: 90 and 110 kDa; 17-C10: 70 and 90 kDa; 18-G3: up to five bands from 70 to 130 kDa). Competitive indirect inhibition ELISAs developed with mAbs 16-F12 and 17-C10 revealed further differences in the specificities of these antibodies. Thus, while mAb 16-F12 can be used only to quantify antigenic COMP in human synovial fluid and serum, mAb 17-C10 is useful in addition when analyzing canine and horse synovial fluid as well as canine serum. The results of analyses of synovial fluid samples from patients with osteoarthritis and rheumatoid arthritis provided preliminary evidence in support of the contention that measurement of the different COMP epitopes recognized by these mAbs in body fluids could prove useful in the clinical assessment of patients with joint disease.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Proteínas de la Matriz Extracelular/inmunología , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Líquido Sinovial/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos/inmunología , Antígenos/metabolismo , Artritis Reumatoide/metabolismo , Unión Competitiva , Western Blotting , Cartílago/inmunología , Proteína de la Matriz Oligomérica del Cartílago , Densitometría , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Proteínas de la Matriz Extracelular/química , Femenino , Glicoproteínas/química , Humanos , Proteínas Matrilinas , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Líquido Sinovial/química , Líquido Sinovial/inmunología , Tripsina/metabolismo

RESUMEN

Proteoglycans extracted with 4 M guanidinium chloride from young (mean 20 years) or old (mean 79 years) macroscopically normal human articular cartilage were separated by density gradient centrifugation and Q-Sepharose chromatography and characterized by gradient gel SDS/PAGE and immunodetection before and after removal of glycosaminoglycan chains. The extracts contained two large populations of aggrecan, a population of small N-terminal aggrecan fragments, as well as decorin, biglycan and fibromodulin. The distribution of all these species in density gradient fractions has been determined. The large aggrecan populations comprised four different chondroitin sulphate-bearing core proteins while the population of smaller fragments comprised eight different components. The two smallest fragments (35 and 42 kDa), identified as the first globular domain of aggrecan (N-terminal) (G1) and containing no glycosaminoglycan, were detected only in extracts of old cartilage. A 55 and a 70 kDa fragment of G1 were present in both keratan sulphate-containing and non-keratan sulphate-containing forms. Four other fragments, each containing keratan sulphate epitopes, were identified and these contained either G1 epitopes (one 95 kDa species), or G1 and G2 epitopes (three species). These results have suggested that proteolytic processing at the N-terminus is more extensive than has previously been recognized and raises the possibility that more than one proteinase may be involved in aggrecan degradation in vivo. With the exception of the two smallest G1 fragments, the repertoire of proteoglycan fragments found in young and old human articular cartilage is essentially the same, although the relative abudnance of various species differed. The older tissue contains a larger proportion of C-terminally truncated aggrecan fragments and a significantly decreased content of decorin and biglycan.

Asunto(s)

Envejecimiento/metabolismo , Cartílago Articular/metabolismo , Proteínas de la Matriz Extracelular , Ácido Hialurónico/metabolismo , Proteoglicanos/aislamiento & purificación , Proteoglicanos/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Agrecanos , Cartílago Articular/química , Catálisis , Centrifugación por Gradiente de Densidad , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/metabolismo , Humanos , Ácido Hialurónico/análisis , Lectinas Tipo C , Proteoglicanos/análisis

RESUMEN

Approx. 10% of the total proteoglycan content of normal young human articular cartilage was extracted under associative conditions with Dulbecco's PBS. Proteoglycans isolated from the extract by Q-Sepharose chromatography were separated by gel chromatography and characterized by gradient gel SDS/PAGE and immunoblotting. Three species of small proteoglycans, two main populations of aggrecan and a population of its smaller fragments were identified. The major populations of aggrecan contained chondroitin sulphate chains, all or part of the N-terminal G1 and G2 domains and, therefore, intact keratan sulphate domains. The larger population was estimated by gradient SDS/PAGE to have a molecular mass of approx. 600 kDa or greater. The second population had an apparent molecular mass of approx. 300-600 kDa. Core proteins derived from these populations of proteoglycans separated on SDS/PAGE into several clusters of bands in the range from 120 to approx. 360 kDa. The extract further contained smaller fragments which lacked chondroitin sulphate but reacted with antibodies against keratan sulphate, and against epitopes present in the G2 domain of aggrecan. The presence of the G2 domain in a broad range of populations of decreasing size indicated extensive cleavage of the aggrecan core protein within its chondroitin sulphate domain. These findings suggest that fragmentation of aggrecan probably occurs in vivo in normal articular cartilage of young individuals. Associative extracts also contained decorin, biglycan and fibromodulin. These were resolved from aggrecan by gel chromatography and identified by immunodetection.

Asunto(s)

Cartílago Articular/metabolismo , Proteínas de la Matriz Extracelular , Proteoglicanos/aislamiento & purificación , Agrecanos , Anticuerpos Monoclonales , Fraccionamiento Químico , Condroitín/química , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Lectinas Tipo C , Peso Molecular , Proteoglicanos/química , Proteoglicanos/metabolismo

RESUMEN

This study demonstrates a relationship between the biochemical properties of the extracellular matrix of a cartilaginous model of long bone and the fusion of mononuclear clastic elements into multinucleate chondroclasts. The results suggest that the chondroitin sulphate level in the extracellular matrix has an inhibitory effect on this process.

Asunto(s)

Cartílago/química , Células Madre Hematopoyéticas/citología , Animales , Fusión Celular , Embrión de Pollo , Pollos

RESUMEN

Human cumuli-oophori were cultured in vitro in the presence of radioactive protein and polysaccharide precursors. The time course of the cumulus cell secretion was traced by histoautoradiography. Matrix solubilization, and sodium dodecyl sulphate polyacrylamide gel electrophoresis and high-performance liquid chromatography showed that proteoglycan (Mr greater than 1,700,000) was the main cumulus cell product that was prevailingly deposited in the cumulus intercellular matrix and partly released into the culture medium. It was capable of accelerating the conversion of proacrosin to acrosin and this activity was abolished by enzymatic removal of chondroitin sulphate, the predominant glycosaminoglycan of this proteoglycan fraction. None of the other fractions, including a proteoglycan of Mr 80,000-90,000, containing heparan sulphate, accelerated the conversion of proacrosin to acrosin under the conditions used. The results suggest that chondroitin sulphate is the active component of the high-Mr proacrosin activator of the human cumulus-oophorus.

Asunto(s)

Acrosina/biosíntesis , Acrosina/metabolismo , Sulfatos de Condroitina/metabolismo , Precursores Enzimáticos/metabolismo , Matriz Extracelular/metabolismo , Folículo Ovárico/metabolismo , Proteoglicanos/biosíntesis , Autorradiografía , Células Cultivadas , Cromatografía Líquida de Alta Presión , Activación Enzimática/fisiología , Matriz Extracelular/química , Femenino , Humanos , Microscopía Electrónica de Rastreo , Folículo Ovárico/química , Folículo Ovárico/citología , Proteoglicanos/análisis

RESUMEN

A procedure is described for the preparation of 1.32-10% polyacrylamide gradient gels. Loose polyacrylamide gel on the top side of the gradient was stabilized with a layer of 0.4% agarose gel which also formed sample wells. The upper limit of separation achieved in these gels was estimated to be approximately 2 X 10(6) using globular protein standards. However, large aggregating proteoglycans from cartilage which have a molecular weight range of 1-4 X 10(6) penetrate and separate in these gels. A simple one-step procedure is also described for simultaneous staining of proteins and large proteoglycans in polyacrylamide gels.

Asunto(s)

Electroforesis en Gel de Poliacrilamida/métodos , Proteoglicanos/aislamiento & purificación , Coloración y Etiquetado/métodos , Animales , Bovinos , Estudios de Evaluación como Asunto , Humanos , Peso Molecular , Proteínas/aislamiento & purificación

RESUMEN

Two specimens of human articulage were successively extracted with solutions of phosphate-buffered saline (PBS), 7 M-urea and 4 M-guanidine hydrochloride (Gdn-HCl). Proteoglycans from individual extracts were fractionated by DEAE-Sephacel chromatography and gel chromatography on Sephacryl S-400. The presence of three populations of large proteoglycans was demonstrated in all three extracts by composite agarose/polyacrylamide-gel electrophoresis (CAPAGE). The population corresponding to the fastest CAPAGE band of aggregating proteoglycans was shown to be extremely polydisperse, having Mr (as estimated by SDS/PAGE) decreasing continuously from more than 300,000 to the size corresponding to 'free' hyaluronic acid-binding region (HABR) (about 70,000). A rather polydisperse set of HABR-containing fragments which spanned a broad range of sizes, and also differed in their keratan sulphate contents, was isolated from both 7 M-urea and 4 M-Gdn-HCl extracts. PBS and 7 M-urea extracts, but not the Gdn-HCl extract, further contained small proteoglycans, identified as fast-migrating bands on CAPAGE electrophoretograms. One of those small species was recognized with an antibody against the small proteoglycan PG II; the other two remain to be positively identified. However, the glycosaminoglycan of the small species which was present exclusively in the PBS extract was identified as keratan sulphate; this species may thus belong to the family of small keratan sulphate-containing proteolygans.

Asunto(s)

Proteínas Portadoras/aislamiento & purificación , Cartílago Articular/química , Proteoglicanos/aislamiento & purificación , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Portadoras/metabolismo , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Glicosaminoglicanos/aislamiento & purificación , Humanos , Receptores de Hialuranos , Ácido Hialurónico/metabolismo , Immunoblotting , Peso Molecular

RESUMEN

The authors investigated in articular cartilage the presence and amount of the free fragment of the protein nucleus of the proteoglycan monomer containing the area of the bond with hyaluronic acid (HABR) and the presence of the small dermatan sulfate proteoglycan in relation to age. The articular cartilage of older subjects contains a much higher ratio of the protein fragment with functional HABR, as compared with young adult tissue. The more ready extractability of this fragment suggests an impaired bond between the proteoglycan monomer and hyaluronic acid. The ratio of the small dermatan sulfate proteoglycan is significantly reduced in the articular cartilage of older subjects. In the articular cartilage of a very old person (87 years) this proteoglycan was not detected or its content is at the borderline of detection of the methods used.

Asunto(s)

Envejecimiento/metabolismo , Cartílago Articular/metabolismo , Glicosaminoglicanos/metabolismo , Proteoglicanos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Portadoras/metabolismo , Dermatán Sulfato/metabolismo , Humanos , Receptores de Hialuranos , Persona de Mediana Edad

Asunto(s)

Cartílago Articular/metabolismo , Interleucina-1/farmacología , Proteoglicanos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Adulto , Envejecimiento , Cartílago Articular/efectos de los fármacos , Cartílago Articular/crecimiento & desarrollo , Células Cultivadas , Niño , Humanos , Cinética , Persona de Mediana Edad , Proteínas Recombinantes/farmacología

RESUMEN

The review presents a survey of published findings concerning the mechanism of luminol-dependent chemiluminescence in biological systems. The potential of various oxygen species (superoxide anion, hydrogen peroxide, hydroxyl radical) to react with luminol is discussed. The ability of commonly used enzymes (superoxide dismutase, catalase), inhibitors, and oxygen radical scavengers to discriminate between individual oxygen species is assessed together with the potential of a variety of substances encountered in biological systems to interfere in luminol-dependent chemiluminescence reactions. It is concluded that luminol-dependent chemiluminescence gives at present very little ability to discriminate between individual oxygen or radical species. Furthermore, luminol-dependent chemiluminescence used in biological systems is extremely prone to many interferences, which are very difficult to control.

Asunto(s)

Fagocitos/fisiología , Animales , Humanos , Peróxido de Hidrógeno/metabolismo , Mediciones Luminiscentes , Luminol , Peroxidasa/metabolismo