Publications (9)29.62 Total impact
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Article: Expression of recombinant ADAMTS in insect cells.
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ABSTRACT: The "a disintegrin and metalloproteinase with thrombospondin motifs" (ADAMTS) enzymes are secreted proteinases involved in development, blood clotting and the turnover of extracellular matrix. Manufacturing recombinant enzyme presents quite a challenge due to the presence of disulphide bridges, the large size and modular structure. A sub-group of these enzymes are known as "aggrecanases" and it is likely that they are involved in a number of pathologies related to increased turnover of the extracellular matrix, particularly in tissues where the concentration of proteoglycans is high, such as cartilage and the central nervous system. We have expressed three of these enzymes, ADAMTS-1, -4 and -5, in insect cells using plasmid-based systems.Methods in molecular biology (Clifton, N.J.) 01/2010; 622:83-98. -
Article: The regulation of aggrecanase ADAMTS-4 expression in human Achilles tendon and tendon-derived cells.
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ABSTRACT: Several members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family have been identified as aggrecanases, whose substrates include versican, the principal large proteoglycan in the tendon extracellular matrix. We have characterized the expression of ADAMTS-4 in human Achilles tendon and tendon-derived cells. ADAMTS-4 mRNA levels were higher in ruptured tendon compared with normal tendon or chronic painful tendinopathy. In tissue extracts probed by Western blotting, mature ADAMTS-4 (68 kDa) was detected only in ruptured tendons, while processed ADAMTS-4 (53 kDa) was detected also in chronic painful tendinopathy and in normal tendon. In cultured Achilles tendon cells, transforming growth factor-beta (TGF-beta) stimulated ADAMTS-4 mRNA expression (typically 20-fold after 24 h), while interleukin-1 induced a smaller, shorter-term stimulation which synergised markedly with that induced by TGF-beta. Increased levels of immunoreactive proteins consistent with mature and processed forms of ADAMTS-4 were detected in TGF-beta-stimulated cells. ADAMTS-4 mRNA was expressed at higher levels by tendon cells in collagen gels than in monolayer cultures. In contrast, the expression of ADAMTS-1 and -5 mRNA was lower in collagen gels compared with monolayers, and these mRNA showed smaller or opposite responses to growth factors and cytokines compared with that of ADAMTS-4 mRNA. We conclude that both ADAMTS-4 mRNA and ADAMTS-4 protein processing may be differentially regulated in normal and damaged tendons and that both the matrix environment and growth factors such as TGF-beta are potentially important factors controlling ADAMTS aggrecanase activities in tendon pathology.Matrix Biology 07/2008; 27(5):393-401. · 3.30 Impact Factor -
Article: The role of proteases in pathologies of the synovial joint.
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ABSTRACT: Synovial (diarthrodial) joints are employed within the body to provide skeletal mobility and have a characteristic structure adapted to provide a smooth almost frictionless surface for articulation. Pathologies of the synovial joint are an important cause of patient morbidity and can affect each of the constituent tissues. A common feature of these pathologies is degenerative changes in the structure of the tissue which is mediated, at least in part, by proteolytic activity. Most tissues of the synovial joint are composed primarily of extracellular matrix and key pathological roles in the degeneration of this matrix are performed by metalloproteinases such as matrix metallproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS). However, other proteases such as cathepsin K are likely to play an important role, especially in bone turnover. In addition to the cleavage of structural proteins, proteolytic activities are employed to regulate the activity of other proteases, growth factors, cytokines and other inflammatory mediators. Proteases combine to form complex regulatory networks, the correct functioning of which is required for tissue homeostasis and the imbalance of which may be a feature of pathology. A precise understanding of the proteases involved in these networks is required for a true understanding of the associated pathology.The International Journal of Biochemistry & Cell Biology 02/2008; 40(6-7):1199-218. · 4.63 Impact Factor -
Article: Expression profiling of metalloproteinases and tissue inhibitors of metalloproteinases in normal and degenerate human achilles tendon.
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ABSTRACT: To profile the messenger RNA (mRNA) expression for the 23 known genes of matrix metalloproteinases (MMPs), 19 genes of ADAMTS, 4 genes of tissue inhibitors of metalloproteinases (TIMPs), and ADAM genes 8, 10, 12, and 17 in normal, painful, and ruptured Achilles tendons. Tendon samples were obtained from cadavers or from patients undergoing surgical procedures to treat chronic painful tendinopathy or ruptured tendon. Total RNA was extracted and mRNA expression was analyzed by quantitative real-time reverse transcription-polymerase chain reaction, normalized to 18S ribosomal RNA. In comparing expression of all genes, the normal, painful, and ruptured Achilles tendon groups each had a distinct mRNA expression signature. Three mRNA were not detected and 14 showed no significant difference in expression levels between the groups. Statistically significant (P < 0.05) differences in mRNA expression, when adjusted for age, included lower levels of MMPs 3 and 10 and TIMP-3 and higher levels of ADAM-12 and MMP-23 in painful compared with normal tendons, and lower levels of MMPs 3 and 7 and TIMPs 2, 3, and 4 and higher levels of ADAMs 8 and 12, MMPs 1, 9, 19, and 25, and TIMP-1 in ruptured compared with normal tendons. The distinct mRNA profile of each tendon group suggests differences in extracellular proteolytic activity, which would affect the production and remodeling of the tendon extracellular matrix. Some proteolytic activities are implicated in the maintenance of normal tendon, while chronically painful tendons and ruptured tendons are shown to be distinct groups. These data will provide a foundation for further study of the role and activity of many of these enzymes that underlie the pathologic processes in the tendon.Arthritis & Rheumatism 04/2006; 54(3):832-42. · 7.87 Impact Factor -
Article: ADAMTS proteinases: potential therapeutic targets?
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ABSTRACT: ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteinases have been implicated in a number of connective tissue pathologies including Ehlers-Danlos syndrome type VII C, Weill-Marchesani syndrome, encephalomyelitis, and arthritis. These proteinases therefore represent potential therapeutic targets for the treatment of such conditions. The synthesis and activity of ADAMTS proteinases is regulated at multiple levels: transcription, RNA splicing, translation, proteolytic processing, cofactor stimulation and inhibition, each of which represents a possible point of therapeutic intervention. Recent research suggests that, in addition to the direct inhibition of ADAMTS proteinases with low molecular weight non-peptidic inhibitors, targeting the transcription and protein processing of these enzymes could be effective therapeutic approaches.Current Pharmaceutical Biotechnology 03/2006; 7(1):25-31. · 2.81 Impact Factor -
Article: ADAMTS proteinases: a multi-domain, multi-functional family with roles in extracellular matrix turnover and arthritis.
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ABSTRACT: Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family are known to influence development, angiogenesis, coagulation and progression of arthritis. As proteinases their substrates include the von Willebrand factor precursor and extracellular matrix components such as procollagen, hyalectans (hyaluronan-binding proteoglycans including aggrecan), decorin, fibromodulin and cartilage oligomeric matrix protein. ADAMTS levels and activities are regulated at multiple levels through the control of gene expression, mRNA splicing, protein processing and inhibition by TIMP (tissue inhibitor of metalloproteinases). A recent screen of human cartilage has shown that multiple members of the ADAMTS family may be important in connective tissue homeostasis and pathology.Arthritis Research & Therapy 02/2005; 7(4):160-9. · 4.45 Impact Factor -
Article: A method for the non-covalent immobilization of heparin to surfaces.
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ABSTRACT: The interaction of heparan sulfate (HS) with specific proteins facilitates a wide range of fundamental biological processes including cellular proliferation and differentiation, tissue homeostasis, and viral pathogenesis. This multiplicity of function arises through sequence diversity within the HS chain. Heparin, which is very similar in structure to the sulfated regions of HS, is an excellent model for studying HS-protein interactions. The development of high-throughput enzyme-linked immunosorbent-like assays using surface-immobilized heparin has been hindered by the inability of this glycosaminoglycan to adhere to microtiter surfaces. Here we report the passive noncovalent adsorption of heparin onto microtiter wells following their treatment by plasma polymerization; there was no detectable binding of functional heparin onto untreated plates. Heparin immobilized in this way was able to interact with four different heparin-binding proteins tested, i.e., TSG-6, chemokines IL-8 and KC, and complement factor H. Heparin preparations ranging in size from high molecular weight to a defined decasaccharide could be adsorbed onto these plates in a functionally active form. Since plasma polymerization is possible for virtually any surface, this technique is likely to be of general use in the identification and characterization of heparin/HS-binding proteins in a wide range of applications.Analytical Biochemistry 08/2004; 330(1):123-9. · 3.00 Impact Factor -
Article: Selective inhibition of ADAMTS-1, -4 and -5 by catechin gallate esters.
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ABSTRACT: Three mammalian ADAMTS enzymes, ADAMTS-1, -4 and -5, are known to cleave aggrecan at certain glutamyl bonds and are considered to be largely responsible for cartilage aggrecan catabolism observed during the development of arthritis. We have previously reported that certain catechins, polyphenolic compounds found in highest concentration in green tea (Camellia sinensis), are capable of inhibiting cartilage aggrecan breakdown in an in vitro model of cartilage degradation. We have now cloned and expressed recombinant human ADAMTS-1, -4 and -5 and report here that the catechin gallate esters found in green tea potently inhibit the aggrecan-degrading activity of these enzymes, with submicromolar IC50 values. Moreover, the concentration needed for total inhibition of these members of the ADAMTS group is approximately two orders of magnitude lower than that which is needed to partially inhibit collagenase or ADAM-10 activity. Catechin gallate esters therefore provide selective inhibition of certain members of the ADAMTS group of enzymes and could constitute an important nutritional aid in the prevention of arthritis as well as being part of an effective therapy in the treatment of joint disease and other pathologies involving the action of these enzymes.European Journal of Biochemistry 07/2003; 270(11):2394-403. · 3.58 Impact Factor -
Article: Selective inhibition of ADAMTS‐1, ‐4 and ‐5 by catechin gallate esters
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ABSTRACT: Three mammalian ADAMTS enzymes, ADAMTS-1, -4 and -5, are known to cleave aggrecan at certain glutamyl bonds and are considered to be largely responsible for cartilage aggrecan catabolism observed during the development of arthritis. We have previously reported that certain catechins, polyphenolic compounds found in highest concentration in green tea (Camellia sinensis), are capable of inhibiting cartilage aggrecan breakdown in an in vitro model of cartilage degradation. We have now cloned and expressed recombinant human ADAMTS-1, -4 and -5 and report here that the catechin gallate esters found in green tea potently inhibit the aggrecan-degrading activity of these enzymes, with submicromolar IC50 values. Moreover, the concentration needed for total inhibition of these members of the ADAMTS group is approximately two orders of magnitude lower than that which is needed to partially inhibit collagenase or ADAM-10 activity. Catechin gallate esters therefore provide selective inhibition of certain members of the ADAMTS group of enzymes and could constitute an important nutritional aid in the prevention of arthritis as well as being part of an effective therapy in the treatment of joint disease and other pathologies involving the action of these enzymes.European Journal of Biochemistry. 05/2003; 270(11):2394 - 2403.
Top co-authors
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Institutions
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2008–2010
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University of East Anglia
- • School of Biological Sciences
- • Biomedical Research Centre
Norwich, ENG, United Kingdom
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2005–2008
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Addenbrooke's Hospital
Cambridge, ENG, United Kingdom
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