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We conducted a narrative review in six areas of obstetric emergencies: category-1 caesarean section; difficult and failed airway; massive obstetric haemorrhage; hypertensive crisis; emergencies related to neuraxial anaesthesia; and maternal cardiac arrest. These areas represent significant research published within the last five years, with emphasis on large multicentre randomised trials, national or international practice guidelines and recommendations from major professional societies. Key topics discussed: prevention and management of failed neuraxial technique; role of high-flow nasal oxygenation and choice of neuromuscular drug in obstetric patients; prevention of accidental awareness during general anaesthesia; management of the difficult and failed obstetric airway; current perspectives on the use of tranexamic acid, fibrinogen concentrate and cell salvage; guidance on neuraxial placement in a thrombocytopenic obstetric patient; management of neuraxial drug errors, local anaesthetic systemic toxicity and unusually prolonged neuraxial block regression; and extracorporeal membrane oxygenation use in maternal cardiac arrest.

Background: The interplay between cardiovascular disease (CVD) genetic risk indexed by a polygenic risk score (PRS) and diet quality still requires further investigation amongst older adults or those with established or treated CVD. The present study aimed to evaluate the relative contribution of diet quality, measured using the Australian Recommended Food Score (ARFS) and PRS, with respect to explaining variation in plasma lipids CVD outcomes in the Hunter Cohort. Methods: The study comprised a secondary analysis of cross-sectional data from the Hunter Cohort study. Single-nucleotide polymorphisms from previously derived polygenic scores (PGSs) for three lipid classes were obtained: low-density lipoprotein, high-density lipoprotein and triglycerides, as well as PRS for coronary artery disease (CAD) from the PGS catalogue. Regression modelling and odds ratios were used to determine associations between PRS, ARFS and CVD risk. Results: In total, 1703 participants were included: mean ± SD age 66 ± 7.4 years, 51% female, mean ± SD total ARFS 28.1 ± 8 (out of 74). Total diet quality and vegetable subscale were not significantly associated with measured lipids. By contrast, PGS for each lipid demonstrated a markedly strong, statistically significant correlation with its respective measured lipid. There was a significant association between CAD PRS and 5/6 CVD phenotypes (all except atrial fibrillation), with the largest effect size shown with coronary bypass. Adding dietary intake as a covariate did not change this relationship. Conclusions: Lipid PGS explained more variance in measured lipids than diet quality. However, the poor diet quality observed in the current cohort may have limited the ability to observe any beneficial effects. Future research should investigate whether the diet quality of older adults can be improved and also the effect of these improvements on changes in polygenic risk.

Kashin–Beck disease (KBD) is a chronic, deforming endemic osteoarticular disease with altered metabolism of the cartilage matrix. Matrix metalloproteinases (MMPs), aggrecanases (ATAMTSs), and their inhibitors (TIMPs) play important roles in cartilage formation and matrix degradation. This study investigated these proteases and inhibitors in young KBD cartilage. The percentages of chondrocytes staining for MMP-1/-13 and MMP-generated DIPEN neoepitope, aggrecanase-generated ITEGE neoepitope in aggrecan in KBD patients were significantly higher than in controls. However, TIMP-1 was significantly less numerous than in controls in the superficial and middle zones of KBD samples, the percentage of chondrocytes staining for the TIMP-2 was significantly higher than in controls. Staining for MMP-1/-13 and, TIMP-1/-2 in KBD patients was prominent in the superficial zone and the middle zone of articular cartilage. Staining for ITEGE and DIPEN neoepitopes in KBD samples was prominent in the superficial zone and the middle zone of articular cartilage. The strongest staining for the MMP and aggrecanase-generated neoepitopes was adjacent to areas of chondronecrosis. These results indicated that KBD cartilage destruction depends on collagen- and aggrecan-degrading proteases such as collagenases (MMP-1/-13), as well as aggrecanases. Increased TIMP-2 level adjacent to necrotic areas suggest that attempted repair mechanism are also activated. © 2014 Orthopaedic Research Society.

Aim To describe factors people consider important in deciding whether or not to donate their brain for research after death. Background Brain tissue retrieved at post-mortem is needed to further research into neurological conditions such as Parkinson's disease. Previous research has focussed mainly on attitudes to organ donation for transplantation. Design Data were gathered and analysed using a qualitative approach based on grounded theory. Methods Nineteen people who had made a decision about brain donation, five people with Parkinson's and 14 unaffected individuals, were identified through theoretical sampling. Interviews conducted between September 2007–January 2008 were analysed to identify themes representing the concerns of participants, when making a decision. Findings The three main themes identified were views and beliefs about post-mortem, the importance of family and the things people do not talk about. Although participants were more familiar with the concept of organ donation for transplantation, unanimous support was expressed for brain donation for research. However, beliefs about death and post-mortem, influence of family and the difficulty in talking and thinking about things to do with death all posed barriers to consent when actually asked to make a decision. For some, however, being asked had acted as a catalyst, transforming previously held positive attitudes into a decision to consent. Conclusion Guidelines for asking developed from these findings highlight the importance of discussing the issue to raise awareness in potential donors, involving family members, and giving accurate and appropriate information to inform, reassure and to dispel misconceptions.

Reasons for performing study: Recombinant equine growth hormone (reGH) has recently been evaluated for effects on body condition and wound healing. It has the potential to influence articular cartilage via stimulation of IGF-1. Objectives: To investigate effects of administration on synovial joint metabolism. Methods: Six mature horses were given 20 μg/kg bwt reGH daily for 8 weeks by i.m. injection. Three control horses were injected with sterile water. Serum and synovial fluid samples were collected at 6, 8, 11 and 16 weeks for GH and IGF-1 assays. Articular cartilage harvested at week 16 was evaluated by Western analysis using monoclonal antibodies BC-13, BC-4, 8-A-4 and CH-3. Results: Concentrations of IGF-1 in serum and synovial fluid were significantly elevated (P < 0.05) at 6 and 8 weeks in the reGH group. Glycosaminoglycan concentrations in synovial fluid were significantly less than controls at these time points, suggesting that reGH may modulate proteoglycan metabolism in articular cartilage. In the reGH group, there were not any alterations in synovial fluid content of 3B3(-) epitope or aggrecan metabolite, or in aggrecan or link protein catabolites retained within cartilage, that might be expected with development of osteoarthritis. Conclusions: Intramuscular administration of reGH may be a more efficient means of delivery of IGF-1 to joints for cartilage resurfacing initiatives. Potential relevance: We found no alterations in cartilage metabolism indicative of development of osteoarthritis.

Lubricin is a secreted, cytoprotective glycoprotein that contributes to the essential boundary lubrication mechanisms necessary for maintaining low friction levels at articular cartilage surfaces. Diminishment of lubricin function is thereby implicated as an adverse contributing factor in degenerative joint diseases such as osteoarthritis. Lubricin occurs as a soluble component of synovial fluid, and is synthesized and localized in the superficial layer of articular cartilage (and thus has also been described as "superficial zone protein", or SZP); however, defined interactions responsible for lubricin retention at this site are not well characterized. In the current studies, we identified molecular determinants that enable lubricin to effectively bind to articular cartilage surfaces. Efficient and specific binding to the superficial zone was observed for synovial lubricin, as well as for recombinant full-length lubricin and a protein construct comprising the lubricin C-terminal (hemopexin-like) domain (LUB-C, encoded by exons 7-12). A construct representing the N-terminal region of lubricin (LUB-N, encoded by exons 2-5) exhibited no appreciable cartilage-binding ability, but displayed the capacity to dimerize, and thus potentially influence lubricin aggregation. Disulfide bond disruption significantly attenuated recombinant lubricin and LUB-C binding to cartilage surfaces, demonstrating a requirement for protein secondary structure in facilitating the appropriate localization of lubricin at relevant tissue interfaces. These findings help identify additional key attributes contributing to lubricin functionality, which would be expected to be instrumental in maintaining joint homeostasis.

Objective: The major proteases responsible for aggrecan turnover in articular cartilage are the aggrecanases (ADAMTS-4 and ADAMTS-5). Although several studies have demonstrated C-terminal truncation of these aggrecanases, the mechanism and importance of this processing are poorly understood. The objective of this study was to further investigate ADAMTS-4 and ADAMTS-5 C-terminal truncation in a porcine model in vitro culture system. Methods: Chondrocyte–agarose cultures with well-established extracellular matrices were treated with or without interleukin-1 (IL-1), for a variety of different culture time periods. Cultures were analyzed for release of sulfated glycosaminoglycan, aggrecanase-generated interglobular domain (IGD)–aggrecan cleavage, and the presence of ADAMTS-4 and ADAMTS-5 isoforms. Inhibition of aggrecanase activity with monoclonal antibodies, tissue inhibitor of metalloproteinases 3 (TIMP-3), and cycloheximide pretreatment were used to identify ADAMTS isoforms involved in IGD–aggrecan catabolism. Results: Multiple isoforms, including possible zymogens, of ADAMTS-4 and ADAMTS-5 were sequestered within the extracellular matrix formed by 3-week chondrocyte–agarose cultures. IL-1 exposure induced production of a low molecular weight (37 kd) isoform of ADAMTS-4. This isoform was capable of degrading exogenous aggrecan at the IGD–aggrecanase site, was inhibited by TIMP-3, was blocked after preincubation with an antibody to a sequence in the catalytic domain of ADAMTS-4, and required de novo synthesis in the presence of IL-1 for its generation. Conclusion: In porcine chondrocyte–agarose cultures, a 37-kd ADAMTS-4 isoform appears to be the major matrix protease responsible for the IGD–aggrecanase activity detected in response to exposure to IL-1. This conclusion contradicts that of recent studies of transgenic knockout mice and highlights the need to determine the roles of the different aggrecanase(s) in human disease.

The objective of this study was to test the methods for a large study of the incidence and prognosis of Parkinson's disease and other degenerative parkinsonian disorders and provide provisional incidence figures. This was a community-based prospective study to identify patients with newly diagnosed non-drug-induced Parkinsonism (>or=2 of tremor, rigidity, bradykinesia, postural instability) from a population of 148,600 people in Aberdeen, Scotland, over 18 months. Multiple search strategies were used to identify patients, including some population screening. Incident patients and age/sex-matched controls had assessments of impairment, disability, quality of life, mood, and cognition and are being followed up yearly. Two hundred and two people with possible parkinsonian symptoms were assessed, and 82 incident patients were identified, 50 with probable Parkinson's disease. The crude incidences of probable Parkinsonism and probable Parkinson's disease were 31.4/100,000/year (95% CI: 24.5-39.7) and 22.4/100,000/year (95% CI: 16.6-29.6), respectively. The mean age of diagnosis of Parkinson's disease was 76.1 +/- 10.0 years and the incidence was greater in men. The methods were generally successful. Provisionally, we found a higher incidence of Parkinson's disease than other comparable studies, and our patients were considerably older. This may reflect better case ascertainment in the elderly. A larger study is planned.

OBJECTIVE: To determine the effect of cyclosporin A (CSA) on aggrecanase- and matrix metalloproteinase (MMP)-mediated catabolism of proteoglycan (aggrecan) in articular cartilage explants stimulated with interleukin-1 (IL-1) in a culture system that mimics early pathologic processes associated with arthritic disease. METHODS: Proteoglycan (glycosaminoglycan) and lactate quantification, Western immunoblot analyses of aggrecan degradation products, reverse transcription-polymerase chain reaction analyses of aggrecanase-1, aggrecanase-2 (ADAM-TS4, ADAM-TS5, respectively), MMP-1, MMP-3, MMP-13, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, and TIMP-3 messenger RNA (mRNA) expression in articular cartilage explant cultures, and electrophoretic mobility shift assay analysis of nuclear factor of activated T cells (NF-AT) transcription factor activation were used. RESULTS: CSA inhibited, in a dose-dependent and noncytotoxic manner, aggrecanase-mediated proteoglycan catabolism and loss from IL-1-stimulated cartilage explants. There was no evidence of MMP-mediated aggrecan catabolism in this in vitro model. Treatment of articular cartilage explant cultures with 10 ng/ml of IL-1alpha up-regulated the expression of mRNA for ADAM-TS4, ADAM-TS5, MMP-1, MMP-3, and MMP-13. The expression of ADAM-TS4, ADAM-TS5, and MMP-13 was abrogated by the inclusion of 10 microM CSA in the culture medium. NF-AT activation was observed in chondrocytes but could not be inhibited by preincubation with 10 microM CSA. CONCLUSION: CSA can inhibit IL-1-induced aggrecanase-mediated proteoglycan catabolism in articular cartilage explants maintained in culture for 4 days, thus demonstrating molecular mechanisms whereby CSA may be an effective therapy for degenerative joint disease.

We have previously described a large proteoglycan named superficial zone protein that was isolated and purified from culture medium of superficial slices of bovine articular cartilage. Monoclonal antibodies were raised against superficial zone protein and used as probes in Western blot analyses for immunohistochemical studies both to determine precisely which cells within the joint synthesize the proteoglycan and to isolate a cDNA fragment from a bovine chondrocyte lambdagt11 library that encodes part of the proteoglycan. The cDNA fragment that was obtained with use of monoclonal antibody 6-A-1 encodes the 3' end of the sequence for superficial zone protein. On Western blots, monoclonal antibody 3-A-4 recognized an epitope on native, but not reduced, superficial zone protein, whereas monoclonal antibody 6-A-1 reacted with both native and denatured antigen. The proteoglycan was immunolocalized with monoclonal antibody 3-A-4 in chondrocytes predominantly within the superficial zone of fetal and adult articular cartilage and in some cells of the synovial lining. However, the proteoglycan was not detected in chondrocytes deep in articular cartilage, in nasal septal cartilage, or in synovial stromal cells. The only matrix staining positively for superficial zone protein was at the articular surface bordering the synovial cavity in adult, but not fetal, joints. Isolated chondrocytes and synovial cells showed intracellular binding of monoclonal antibody 3-A-4, and flow-cytometric analysis with the antibody gave the following percentages of immunopositive cells: 37.4, 52.5, 3.4, and 7.5 from chondrocytes from the full-thickness, superficial, and deep zones and from synovial cells, respectively. Thus, both chondrocytes and synovial cells bordering the joint cavity synthesize superficial zone protein and substantiate its usefulness as a phenotypic marker of particular cellular species lining the articular cavity.

Introduction Proteoglycan-4 (PRG-4), also known as superficial zone protein/proteoglycan (SZP), is an approximately 345-kDa mucinous proteoglycan that has been detected in a variety of tissues including cartilage, tendon, bone, heart and liver (Ikegawa et al. 2000). In the synovial joint, PRG-4 is specifically synthesized by chondrocytes located in the superficial zone of articular cartilage and by some surface-lining cells of the synovium (Schumacher et al. 1994). Sequence analyses have shown that the N- and C-terminal vitronectin-like domains of PRG-4 may impart interesting functions relevant to synovial joint metabolism (Merberg et al. 1993; Flannery et al. 1999). The objective of this study was to investigate these potential functions, facilitated by the production of PRG-4 N- and C-terminal domains as recombinant proteins. Methods cDNAs for the human N-terminal (exons 2–5) and bovine C-terminal (exons 7–12) domains of PRG-4 were obtained by RT-PCR and cloned into the expression vector pMT-BiP for inducible, secreted expression in Drosophila S2 cells. Proteins were purified using FLAG-M2 antibody affinity chromatography and visualized by SDS-PAGE and Western blotting with PRG-4-specific antibodies. The heparin-binding properties of recombinant proteins were investigated using heparin affinity chromatography. The interactions of recombinant PRG-4 domains with human plasminogen activator-inhibitor (PAI)-1 and bovine type-II collagen were assayed using standard ELISA techniques. Results Stable cell lines have been generated that express human N-terminal and bovine C-terminal PRG-4 domains. In both cases, two proteins have been purified, possibly due to a splice mechanism by the expression system. N-terminal sequence data and Western blotting indicate that the two species in each case could represent full-length and truncated proteins. Analyses of the two PRG-4 N-terminal domain species have confirmed the presence of a predicted heparin-binding domain and indicate that the molecule can bind to PAI-1, with binding activity localized towards its two somatomedin B domains. The somatomedin B domain of vitronectin is known to bind PAI-1 (Seiffert 1997). Analyses of the two PRG-4 C-terminal species have demonstrated self-association under nonreducing conditions and binding to heparin and PAI-1. Discussion The exact role of PRG-4 in the synovial joint is yet to be elucidated. However, these results point towards the interaction of the N- and C-terminal domains of PRG-4 with structural molecules such as type-II collagen and heparin, and functional molecules such as PAI-1, a serpin that is involved in the fibrinolytic cascade and cell adhesion. These properties are in addition to the well-documented boundary lubricating activity of the central mucinous region of PRG-4.

The chondrocytes of articular cartilage synthesise a number of proteinases which are capable of degrading the component molecules of this specialized extracellular matrix. The use of class-specific proteinase inhibitors indicates that major activities responsible for catabolism of proteoglycan (aggrecan) and collagen are attributable to zinc-dependent metalloproteinases. Amino acid sequence analysis of aggrecan catabolites isolated from human cartilage and synovial fluids has identified two major sites of proteolytic cleavage which occur in vivo at Asn341-Phe342 and Glu373-Ala374. Whilst cleavage at the latter site is attributed to an unknown ‘aggrecanase’, the Asn341-Phe342 bond is readily cleaved by many of the known matrix metalloproteinases. While considerable attention has focused in recent years on the expression and activity of MMPs in articular cartilage, it has now been shown (McKie et al. 1997; Patel et al. 1998) that chondrocytes can also express other metalloproteinases (e.g. the ADAM family of disintegrin-metalloproteinases, most of which have a consensus hydrophobic transmembrane sequence at their C-terminus, and many of which possess a consensus zinc-binding catalytic site which is also present in the MMPs). In this study, we have compared and quantified the mRNA expression profiles of two matrix metalloproteinases (MMP-3 and MMP-13) and five disintegrin metalloproteinases (ADAM-10, ADAM-9, ADAM-15, TNF-alpha converting enzyme and decysin) in chondrocytes (human, porcine and bovine) from fresh cartilage and in cartilage explant cultures and isolated cells cultured in monolayer or in agarose gels.

Introduction The sequencing of the genome has provided us with important information regarding the primary structure of many matrix proteins. This in turn has lead to advances in studies of the functions of post-translational modifications on connective tissue proteoglycans (PGs). Changes in GAG structure with ageing and disease have been well documented (Thonar et al. 1986; Brown et al. 1998). However, little is known about the exact sites of and differential substitution of GAGs on the aggrecan core protein and how these substitutions facilitate normal function or the changes seen with disease. The CS : KS ratio of substitution change significantly, with KS levels increasing with age and decreasing with the onset of disease. Objective was to produce monoclonal antibody (MAb) reagents to keratanase (k'ase) generated stub epitopes, that could be used to help identify, characterize and quantify sites of KS substitution on PGs, providing the potential to determine how the arrangement of such substitutions change with development, ageing and pathology. Methods Bovine Nasal Cartilage aggrecan (BNC A1D1) was trypsin digested, generating a range of glycosaminoglycan (GAG) fragments. The sample was then subjected to anion-exchange and size exclusion chromatography to separate KS from CS fragments. Fractions collected were analysed by SDS-PAGE and Western blotting. Fractions positive for KS were pooled and kinase digested to expose the KS stub antigens. Immunization and fusions were carried out as previously described (Nieduszynski et al. 1990). Initial screenings were carried out using ELISA. Briefly, 96-well microtitre plates were coated with the immunizing antigen overnight at 37 °C. The plates were then blocked prior to the addition of hybridoma media for 1–2 h at 37 °C. Binding was detected using an alkaline phosphatase-conjugated secondary antibody for 1 h at 37 °C prior to the addition of the substrate. Positive wells were further screened by ELISA and SDS-PAGE using the immunizing antigen, chondroitinase-digested BNC and an A1D1 BNC preparation to establish the kinase stub specificity of the hybridomas. Further screenings by Western blotting was carried out on positive hybridomas selected. Antigens used included keratanase-digested bovine corneal KS-PGs, keratanase-II-digested KS-PGs and a nonkeratanase-digested corneal KS-PG sample. Results Screening: Screening identified two positive hybridomas, B-KS-I and B-KS-II, which were specific for kinase-generated KS stub. On screening, these antigens showed reactivity specifically for kinase-digested BNC abc core, with no reactivity to the nonkinased linear KS GAG epitopes. Reactivity to kinase-digested corneal KS-PGs indicated that the MAbs generated were indeed to a stub structure in the KS chain and not to some linkage region epitope, amino acid sequence or oligosaccharide present on the core protein. Application: Immunohistochemistry utilizing B-KS-I was used to localize KS in a range of tissues along side anti-KS 5D4. In human articular cartilage engineered grafts, labelling showed B-KS-I and 5D4 to have broadly overlapping labelling patterns for KS; however, label for B-KS-I had a much more restricted and subtle tissue distribution than that of antibody 5D4. Discussion These new KS stub MAbs have potential to be used in many different areas of research. They may be used in analysis of trypsin-digested purified aggrecan from cattle joints of different ages to determine sites of KS substitution, which remain common or change with development and ageing. They may also be used in analysis of cartilage explant culture metabolites to assess KS substitution on the aggrecan fragments generated after stimulation of these cultures with cytokines such as IL-1 or TNF-α. Collectively it will provide important new information on the changing pattern of KS substitution in connective tissue PGs with development, ageing and the onset of pathology.

Introduction A primary event in the destruction of cartilage in arthritic diseases is the loss of aggrecan from the extracellular matrix of articular cartilage. During aggrecan breakdown, cleavage sites are utilized, which reside within the IGD of the aggrecan core protein. The Asn341–Phe342 bond is cleaved by members of the MMP family, whereas the second of the two cleavage sites, the Glu373–Ala374 bond, is cleaved by the aggrecanases which are all members of the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family. Both ADAMTS-4 and -5 have been shown to readily cleave aggrecan at this so-called aggrecanase site (Tortorella et al. 1999; Abbaszade et al. 1999; Sandy et al. 2000). An in vitro model of cartilage degradation has also shown that these enzymes are responsible for the loss of aggrecan from explant cultures of articular cartilage stimulated with IL-1-a or TNF-a (Tortorella et al. 2001). Both ADAMTS-4 and -5 are thought to be synthesized as inactive zymogens that are activated via removal of their propeptide domain by the Golgi enzyme furin. The secreted active ADAMTS-4 and -5 have predicted molecular weights of 67.9 and 73.6 kDa, respectively. The objective of this study was to monitor ADAMTS-4 and -5 secretion and sequestration in the extracellular matrix of chondrocyte-agarose cultures. Methods Porcine articular chondrocytes were isolated and embedded in agarose (Hughes et al. 1997) before preculture in DMEM + 50 µg/ml gent. with 10% FBS and 25 µg/ml Phos.C for 21 days. The plates were washed, then cultured in serum-free DMEM with or without IL-1-α for 96 h. GAG release to the medium was measured using the DMMB assay. Media samples were analysed by Western blotting for aggrecan metabolites using mAb BC-3 to recognize the aggrecanase-generated neoepitope ARGSV. The presence of ADAMTS-4 in the media was analysed using the mAb anti-TS-4N, which recognizes the metalloproteinase domain, and commercially available polyclonal antibodies to the pro- and spacer domains of ADAMTS-4. The presence of ADAMTS-5 was detected using commercially available polyclonal antibodies to the pro- and spacer domains of ADAMTS-5. Agarose plugs were extracted in detergent buffer and analysed by Western blotting for ADAMTS-4 and -5 using the same monoclonal and polyclonal antibodies. Results In control cultures, only 20–30% of the total GAG was released into the medium after 96 h of culture. In contrast, 80–90% of the total GAG was released in cultures exposed to IL-1. Western blot analysis showed aggrecanase-generated aggrecan metabolites in the IL-1-treated cultures but none in the control cultures. Sequestered forms of both ADAMTS-4 and -5 are present in the matrix prior to treatment in serum-free conditions, and following treatment with or without IL-1 for 96 h, there are no differences in the high molecular weight isoforms of the enzymes sequestered in the matrix. Western blots of partially purified media samples showed no differences in the zinc chelator-bound isoforms of either ADAMTS-4 or -5 between control and IL-1-treated cultures. However, the predominant heparin sepharose-bound isoforms of ADAMTS-4 and -5 co-migrate at approximately 37 kDa. Each of the heparin-bound 37-kDa isoforms of ADAMTS-4 and -5 are detected in increased amounts in IL-1α-treated cultures compared to controls. Discussion The increased amounts of the 37-kDa isoforms of both ADAMTS-4 and -5 in the IL-1-treated cultures suggest a role for these smaller isoforms in the increased aggrecanase activity seen in the IL-1-treated cultures compared to controls. This study has identified multiple isoforms of putative aggrecanase activity that could be responsible for increased aggrecan catabolism that leads to cartilage degradation in arthritis.

Introduction Loss of aggrecan metabolites from the cartilage matrix into the surrounding synovial fluid have allowed for identification of two major cleavage sites within aggrecan: Asn341–Phe342 and Glu373–Ala374 peptide bonds. Proteolysis at the Glu373–Ala374 bond has been specifically attributed to ADAMTS-4 and ADAMTS-5. Genetic reporters are commonly used in cell biology to study mechanisms regulating gene expression. Firefly luciferase is widely used as such a reporter for the immediate availability of reporter activity, the sensitivity of the assay, and quickness and ease of use. By making time-controlled cuts in the 5′-noncoding region of ADAMTS-4 and -5 and inserting the deletions into the genetic reporters, which are then transfected into a suitable cell line, activity can be measured. The project aim was to identify the cis and trans acting factors in the 5′-noncoding regions that regulate ADAMTS-5 expression using the Luciferase Assay System (Promega, Madison, WI, USA). Methods A 3-kb fragment containing the 5′-flanking region of ADAMTS-5 was subcloned into pGL3-Basic (Promega), a luciferase reporter vector lacking a eukaryotic promoter. Truncations have also been made to the full-length construct using Erase-a-Base System (Promega) at restriction enzyme sites SacI at 11 bp from the origin and NheI at 21 bp from the origin, resulting in 200–2300 bp deletions on the TS-5 vector. Adherent kidney embryonic fibroblast-like cells (PEAK) were grown in RPMI 1640 media containing supplements (Gibco Invitrogen Corporation) at 37 °C in 5% CO2. Cells were transfected using Fugene-6 Reagent (Roche) as recommended by the manufacturer. 1.5 × 105 cells were plated out in 12-well plates and grown overnight. The next day, serum media was removed and RPMI 1640 media containing only glutamate was added. DNA complexes were prepared by mixing 97 µl of serum-free medium, 3 µl of Fugene-6, and 2 µg of DNA from Control, Basic, or TS-5 full constructs and selected truncations and incubated for 30 min at RT. Hundred microlitres of the complex was pipetted directly into each well. After a 4–5-h incubation, 100 µl of FBS was added to each well and left overnight at 37 °C in 5% CO2. The next morning, the cells were harvested and washed twice in PBS, and resuspended in ×1 Passive Lysis Buffer. About 20 µl of the cell lysate was assayed for luciferase activity as described in the manufacturer's protocol (Promega). Results and discussion Full-length and seven deletion fragments (numbering downstream from the ATG start codon) spanning the 5′-noncoding region were selected from a library of 41 deletions prepared by the Erase-a-Base System: 3b.4 (−2322 bp), 4a.5 (−2110 bp), 5a.2 (1810 bp), 4a.2 (1427 bp), t3a.3 (−718 bp), 2a.1 (−433 bp) and 4a.1 (−278 bp). Firstly, an increase in activity occurs with 256 base pairs deleted from the full-length vector (3b.4). With control considered 100% of activity, the full-length vector produces greater than that amount indicating TS5 vector as having double the activity. A greater increase in activity (×6 that of the control) between the full length and 3b.4 suggests the presence of a suppressor somewhere in the first 256 base pairs of the 5′-noncoding region. Activity then drops between 3b.4 and 4a.5. However, activity is still greater than that produced by the full length. A levelling-off occurs at 5a.2, which continues until a sudden decrease at t3a.3 that is maintained in 2a.1 and 4a.1. Loss of activity between 4a.2 and t3a.3 is linked to loss of a TATA box at 1753–59 bp. There is a total of seven motifs existing in the first few hundred base pairs that could be possible suppressors including an H1 conserved site at 248–254 base pairs. By following the same methods using selected deletion fragments within −2565 to −2322, it will be possible to identify which motif is responsible for suppressing activity in this cell line. Similar experiments have been carried out in isolated articular chondrocytes.