A Direct Spectrophotometric Microassay for Sulfated Glycosaminoglycans in Cartilage Cultures

Farndale, Richard W.; Sayers, C A; Barrett, Alan J.

doi:10.3109/03008208209160269

Cited by 1,268 publications

(794 citation statements)

References 6 publications

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“…Cartilage breakdown was measured by the release of proteoglycan fragments into cartilage-conditioned media using the dimethylmethylene blue dye-binding assay (25), which was modified to allow direct measurement with a 96-well microtiter plate and an automatic spectrophotometer, as described previously (6). Briefly, a 150-pl sample was combined with 100 p1 [v/v] Measurement of PA activity.…”

Section: Methodsmentioning

confidence: 99%

Effects of tumor necrosis factor α and β on resorption of human articular cartilage and production of plasminogen activator by human articular chondrocytes

Campbell

Piccoli

Roberts

et al. 1990

Arthritis & Rheumatism

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We examined the effects of recombinant human tumor necrosis factor (TNF) on human articular cartilage and chondrocytes in culture. Both TNFa and TNFP stimulated cartilage matrix breakdown during prolonged culture and elevated the levels of plasminogen activator (PA) activity in both the supernatants and cell layers of cultured chondrocytes. Characterization of the PA activities by immunochemistry and by zymography following gel electrophoresis indicated that human chondrocytes produce both urokinase-type PA and tissuetype PA in response to TNF. The addition of both interleukin-1 and TNFa or TNFP to chondrocyte cultures demonstrated a synergism between these cytokines in the generation of PA activity in the culture supernatants and cell layers. Our results suggest that both activated lymphocytes and monocytes may contribute to the cartilage destruction of inflammatory arthritis through their stimulation of chondrocytes with TNFP and TNFa, respectively. Since PA is the only neutral proteinase reported to be elevated in TNF-stimulated chondrocyte cultures, it could have an important role in TNF-mediated cartilage destruction.

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Section: Methodsmentioning

confidence: 99%

Effects of tumor necrosis factor α and β on resorption of human articular cartilage and production of plasminogen activator by human articular chondrocytes

Campbell

Piccoli

Roberts

et al. 1990

Arthritis & Rheumatism

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“…The cartilage was dissolved in papain buffer (125 pglml papain (Sigma, St. Louis, MO), 5 mM L-cysteine (Sigma), 5 mM phosphate-buffered EDTA (Sigma)) at 60 "C for 18 h and subsequently stored at -70 "C. GAG content was determined with dimethylmethylene blue (Fluka, Milwaukee. WI) in a colorimetric assay, using chondroitin sulfate as the standard [9,10,14]. DNA content in the harvested tissue was determined using Hoechst 33258 dye (bisbenzimide) (Amersham Pharmacia Biotech, Piscataway.…”

Section: Mic~rosc~oijic Tr/itr/j~si\mentioning

confidence: 99%

Reducing joint destruction due to septic arthrosis using an adenosine_2A receptor agonist

Cohen

Gill

Baer

et al. 2004

Journal Orthopaedic Research

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We assessed the efficacy of a new adenosine AZA agonist ATL146e, a potent inhibitor of white blood cell chemotaxis, to reduce cartilage damage in the treatment of septic arthrosis. A live septic arthrosis model was created using Staphylococcus aureus in rabbit knees. Animals were divided into five treatment groups: (1) untreated infected control, (2) antibiotics control, and antibiotics plus ATL146e for (3) 24, (4) 48, or (5) 72 h and assessed at I , 4, and 7 days.Knees in all ATL 146e treated animals exhibited no detectable effusion, and histologic examination revealed near normal cartilage and diminished synovial inflammatory response. Synovial WBC counts decreased with the addition of ATL146e when compared to infected and antibiotic controls. Histologic grading of osteochondral specimens demonstrated improved scores for animals treated with ATLl46e compared to infected (p < 0.00004) and antibiotics controls (p < 0.05). Analysis of glycosaminoglycan content revealed significantly decreased loss of articular cartilage following infection in the ATL 146e groups when compared to infected (p < 0.03) and antibiotics controls (p < 0.05).Addition of an adenosine AZA agonist to antibiotic therapy decreases joint inflammation and articular cartilage destruction without compromising bacterial clearance in rabbit knees following intraarticular bacterial infection. The use of adenosine agonists selective to the AZA receptor to augment conventional treatment of joint sepsis may be chondroprotective and ultimately help prevent arthrosis.

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“…We further modified the method developed by Farndale et al 39 and modified by Poorthuis et al 10 About 50 mg of tissue was homogenized in 0.5 ml of buffer (50 mm TrisHCl, pH 7.5, 10 mm MgCl 2 , 2 mm Ca 2 Cl), digested with proteinase K for 4 h at 55°C, boiled for 15 min and treated with DNase I (20 U/ml) for 1 h at 37°C. After chloroform extraction, 100 l of the aqueous phase was passed through a 250 l Sephadex G-25 spin column.…”

Section: Gag Determinationsmentioning

confidence: 99%

“…This removed contaminating small molecules that interfere with the dye-binding assay. The flow-through from the spin column was added to 900 l of dimethylmethylene blue reagent 39 and the absorbancy at 535 nm was measured. Experimental values were compared with chondroitin sulfate C (shark) standards.…”

Section: Gag Determinationsmentioning

confidence: 99%

Treatment of lysosomal storage disease in MPS VII mice using a recombinant adeno-associated virus

Watson¹,

Sayles²,

Chen³

et al. 1998

Gene Ther

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Mucopolysaccharidosis type VII (MPS VII) is a lysosomalproduced low GUS activity in several tissues. This latter storage disease caused by a genetic deficiency of ␤-glucutreatment of MPS VII mice reduced glycosaminoglycan ronidase (GUS). We used a recombinant adeno-associalevels in the liver to normal and reduced storage granules ted virus vector (AAV-GUS) to deliver GUS cDNA to MPS dramatically. We show that a single administration of VII mice. The route of vector administration had a dramatic AAV-GUS can provide sustained expression of GUS in a effect on the extent and distribution of GUS activity. Intravariety of cell types and is sufficient to reverse the disease muscular injection of AAV-GUS resulted in high, localized phenotype at least in the liver. production of GUS, while intravenous administration

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A Direct Spectrophotometric Microassay for Sulfated Glycosaminoglycans in Cartilage Cultures

Cited by 1,268 publications

References 6 publications

Effects of tumor necrosis factor α and β on resorption of human articular cartilage and production of plasminogen activator by human articular chondrocytes

Effects of tumor necrosis factor α and β on resorption of human articular cartilage and production of plasminogen activator by human articular chondrocytes

Reducing joint destruction due to septic arthrosis using an adenosine_2A receptor agonist

Treatment of lysosomal storage disease in MPS VII mice using a recombinant adeno-associated virus

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A Direct Spectrophotometric Microassay for Sulfated Glycosaminoglycans in Cartilage Cultures

Cited by 1,268 publications

References 6 publications

Effects of tumor necrosis factor α and β on resorption of human articular cartilage and production of plasminogen activator by human articular chondrocytes

Effects of tumor necrosis factor α and β on resorption of human articular cartilage and production of plasminogen activator by human articular chondrocytes

Reducing joint destruction due to septic arthrosis using an adenosine2A receptor agonist

Treatment of lysosomal storage disease in MPS VII mice using a recombinant adeno-associated virus

Contact Info

Product

Resources

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Reducing joint destruction due to septic arthrosis using an adenosine_2A receptor agonist