Human osteoarthritis synovium contains an alternatively spliced transcript of ADAMTS4

Purpose: The characterization of an alternatively spliced transcript of the ADAMTS4 aggrecanase. Methods: In human OA synovial cell cultures, RT-PCR was performed using oligonucleotide primers designed to amplify across the exon 8/9 region of human ADAMTS4. The PCR products were purified using a QIAquick purification kit (Qiagen) and sequenced using in house facilities. A pCEP4 (Invitrogen) mammalian expression vector containing ADAMTS4 plus a FLAG epitope was mutated using the QuikChange II site directed mutagenesis kit (Stratagene) to contain the ADAMTS4 splice variant plus a FLAG epitope. The recombinant proteins were purified from HEK293 transfected cells using Anti-FLAG M2 affinity gel (Sigma). Polyclonal antibodies were raised against synthetic peptides representing sequences within the C-terminal region of the splice variant of ADAMTS4 and the raised antibodies were characterized using the recombinant splice variant of ADAMTS4. The antibodies were used in immunohistochemical analysis of human osteoarthritic synovium. The proteolysis of aggrecan and other proteoglycans by the recombinant spice variant of ADAMTS4 was investigated. Results: The degradation of aggrecan is mainly mediated by the aggrecanases, of which ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2) are the best known. We here characterize an alternative splice variant of ADAMTS4. RT-PCR performed as described above resulted in the amplification of normal ADAMTS4, and also a smaller product missing 161 base pairs from the 5’ end of exon 9, the result of alternative splicing in which exon 8 joins to a cryptic 3’ splice site within exon 9. The protein produced by this alternative splicing would lack the spacer domain and have a C-terminus lacking any homologies with the normal ADAMTS4 spacer domain. The alternatively spliced transcript of ADAMTS4 was found in cultured OA synovial cells and in freshly digested OA synovium, but not in human brain, cervix or lung, or in normal bovine synovium. The protein synthesized from this alternatively spliced transcript of ADAMTS4 would lose functions dependent on its spacer domain, like substrate and matrix binding, and inhibition through fibronectin. Removal of the spacer domain from ADAMTS4 has been reported to increase its ability to cleave aggrecan at the Glu373-Ala374 bond, and it may well be that the alternatively spliced transcript produces a protein that is secreted in a more active form. HEK293 cells transfected with a pCEP4 vector containing the cDNA sequence of the splice variant of ADAMTS4 produced the corresponding protein in both the pro and active form. This protein could be found in the media, but mostly associated with the cells, as confirmed using antibodies specific for the splice variant that were produced using synthetic peptides. Immunohistochemical analysis of osteoarthritic synovium using these antibodies showed staining of cells within the synovium. Proteins purified by immunoprecipitation by Anti-FLAG M2 affinity gel from transfected and untransfected HEK293 cells were analysed using the ANASpec SensoLyte 520 Aggrecanase I assay kit. The splice variant had aggrecanase activity comparable to a commercially available ADAMTS4. The splice variant cleaved aggrecan at the G1u373-A1a374 site, as assessed by the neoepitope monoclonal antibody BC3, with activity comparable to ADAMTS4. Conclusions: ADAMTS4 is regulated at multiple levels through control of gene expression, mRNA splicing and protein processing, as well as the expression of naturally occurring inhibitors. We here describe the characteristics of the first known splice variant of ADAMTS4. This alternative splice transcript of ADAMTS4 is expressed as a protein in vivo and can be found in the synovium. It can be speculated that the changes in the C-terminal domain of the protein resulting from this alternatively spliced transcript would have changes in its substrate specificity. The protein produced by the alternative spliced transcript of ADAMTS4 has aggrecanase activity, and the release of low levels of this fully active variant of ADAMTS4 might be a factor in the slow process of superficial zone aggrecan loss in osteoarthritis

Neuroimaging in bulimia nervosa and binge eating disorder: a systematic review.

OBJECTIVE: In recent decades there has been growing interest in the use of neuroimaging techniques to explore the structural and functional brain changes that take place in those with eating disorders. However, to date, the majority of research has focused on patients with anorexia nervosa. This systematic review addresses a gap in the literature by providing an examination of the published literature on the neurobiology of individuals who binge eat; specifically, individuals with bulimia nervosa (BN) and binge eating disorder (BED). METHODS: A systematic review was conducted in accordance with PRISMA guidelines using PubMed, PsycInfo, Medline and Web of Science, and additional hand searches through reference lists. 1,003 papers were identified in the database search. Published studies were included if they were an original research paper written in English; studied humans only; used samples of participants with a diagnosed eating disorder characterised by recurrent binge eating; included a healthy control sample; and reported group comparisons between clinical groups and healthy control groups. RESULTS: Thirty-two papers were included in the systematic review. Significant heterogeneity in the methods used in the included papers coupled with small sample sizes impeded the interpretation of results. Twenty-one papers utilised functional Magnetic Resonance Imaging (fMRI); seven papers utilized Magnetic Resonance Imaging (MRI) with one of these using both MRI and Positron Emission Technology (PET); three studies used Single-Photon Emission Computed Tomography (SPECT) and one study used PET only. A small number of consistent findings emerged in individuals in the acute phase of illness with BN or BED including: volume reduction and increases across a range of areas; hypoactivity in the frontostriatal circuits; and aberrant responses in the insula, amygdala, middle frontal gyrus and occipital cortex to a range of different stimuli or tasks; a link between illness severity in BN and neural changes; diminished attentional capacity and early learning; and in SPECT studies, increased rCBF in relation to disorder-related stimuli. CONCLUSIONS: Studies included in this review are heterogenous, preventing many robust conclusions from being drawn. The precise neurobiology of BN and BED remains unclear and ongoing, large-scale investigations are required. One clear finding is that illness severity, exclusively defined as the frequency of binge eating or bulimic episodes, is related to greater neural changes. The results of this review indicate additional research is required, particularly extending findings of reduced cortical volumes and diminished activity in regions associated with self-regulation (frontostriatal circuits) and further exploring responses to disorder-related stimuli in people with BN and BED

Mast cells produce a unique chondroitin sulfate epitope

The granules of mast cells contain a myriad of mediators that are stored and protected by the sulfated glycosaminoglycan (GAG) chains that decorate proteoglycans. Whereas heparin is the GAG predominantly associated with mast cells, mast cell proteoglycans are also decorated with heparan sulfate and chondroitin sulfate (CS). This study investigated a unique CS structure produced by mast cells that was detected with the antibody clone 2B6 in the absence of chondroitinase ABC digestion. Mast cells in rodent tissue sections were characterized using toluidine blue, Leder stain and the presence of mast cell tryptase. The novel CS epitope was identified in rodent tissue sections and localized to cells that were morphologically similar to cells chemically identified as mast cells. The rodent mast cell-like line RBL-2H3 was also shown to express the novel CS epitope. This epitope co-localized with multiple CS proteoglycans in both rodent tissue and RBL-2H3 cultured cells. These findings suggest that the novel CS epitope that decorates mast cell proteoglycans may play a role in the way these chains are structured in mast cells

Contaminants in Commercial Preparations of ‘Purified’ Small Leucine-Rich Proteoglycans May Distort Mechanistic Studies

This paper reports the perplexing results that came about because of seriously impure commercially available reagents. Commercial reagents and chemicals are routinely ordered by scientists and are expected to have been rigorously assessed for their purity. Unfortunately, we found this assumption to be risky. Extensive work was carried out within our laboratory using commercially-sourced preparations of the small leucine-rich proteoglycans, decorin and biglycan, to investigate their influence on nerve cell growth. Unusual results compelled us to analyse the composition and purity of both preparations of these proteoglycans using both mass spectrometry and Western blotting, with and without various enzymatic deglycosylations. Commercial ‘decorin’ and ‘biglycan’ were found to contain a mixture of proteoglycans including not only both decorin and biglycan but also fibromodulin and aggrecan. The unexpected effects of ‘decorin’ and ‘biglycan’ on nerve cell growth could be explained by these impurities. Decorin and biglycan contain either chondroitin or dermatan sulphate glycosaminoglycan chains whilst fibromodulin only contains keratan sulphate and the large (>2,500 kDa), highly glycosylated aggrecan, contains both keratan and chondroitin sulphate. The different structure, molecular weights and composition of these impurities significantly affected our work and any conclusions that could be made. These findings beg the question as to whether scientists need to verify the purity of each commercially obtained reagent used in their experiments. The implications of these findings are vast, since the effects of these impurities may already have led to inaccurate conclusions and reports in the literature with concomitant loss of researchers’ funds and time

Articular cartilage metabolism in patients with Kashin–Beck Disease: an endemic osteoarthropathy in China

SummaryObjectiveThe objective of this study was to investigate CD44 and proteoglycan metabolism in patients suffering from Kashin–Beck Disease (KBD), an endemic osteoarthropathy that affects 2.5 million of 30 million people living in the KBD regions of China.MethodsImmunohistochemical analyses of cluster of differentiation-44 (CD44), BC-13 and 3-B-3(−) expression were performed in cartilage sections harvested from KBD and normal patients. In addition, the serum levels of soluble CD44 (sCD44), interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and matrix metalloproteinase-1 were determined using a sandwich enzyme linked immunosorbent assay.ResultsHematoxylin & eosin and toluidine blue staining indicated that there was cell necrosis and proteoglycan loss in cartilage from both KBD children and adult cartilage. Strong immunohistochemical staining for CD44, BC-13 and 3-B-3(−) occurred in the majority of adult KBD patients and most KBD children. Furthermore, statistically significant elevated levels of sCD44, IL-1β and TNF-α were found in the sera of both adult and child KBD patients when compared to the levels of normal adult and child controls. Interestingly, IL-1β and TNF-α serum levels were all high in normal children from KBD regions when compared to normal children from non-KBD regions suggesting that unidentified factors (e.g., a genetic predisposition) may protect some people from KBD pathology.ConclusionOur results demonstrate that altered CD44, IL-1β and TNF-α metabolism occurs in the pathogenesis of KBD and there is an increased aggrecanase-generated proteoglycan loss from KBD adult and child cartilage. These primary metabolic changes are likely to be significant contributing factor causing pathological joint formation and instability that leads to secondary osteoarthritis in KBD patients

Fragmentation of decorin, biglycan, lumican and keratocan is elevated in degenerate human meniscus, knee and hip articular cartilages compared with age-matched macroscopically normal and control tissues

Introduction: The small leucine-rich proteoglycans (SLRPs) modulate tissue organization, cellular proliferation, matrix adhesion, growth factor and cytokine responses, and sterically protect the surface of collagen type I and II fibrils from proteolysis. Catabolism of SLRPs has important consequences for the integrity of articular cartilage and meniscus by interfering with their tissue homeostatic functions. Methods: SLRPs were dissociatively extracted from articular cartilage from total knee and hip replacements, menisci from total knee replacements, macroscopically normal and fibrillated knee articular cartilage from mature age-matched donors, and normal young articular cartilage. The tissue extracts were digested with chondroitinase ABC and keratanase-I before identification of SLRP core protein species by Western blotting using antibodies to the carboxyl-termini of the SLRPs. Results: Multiple core-protein species were detected for all of the SLRPs (except fibromodulin) in the degenerate osteoarthritic articular cartilage and menisci. Fibromodulin had markedly less fragments detected with the carboxyl-terminal antibody compared with other SLRPs. There were fewer SLRP catabolites in osteoarthritic hip than in knee articular cartilage. Fragmentation of all SLRPs in normal age-matched, nonfibrillated knee articular cartilage was less than in fibrillated articular cartilage from the same knee joint or total knee replacement articular cartilage specimens of similar age. There was little fragmentation of SLRPs in normal control knee articular cartilage. Only decorin exhibited a consistent increase in fragmentation in menisci in association with osteoarthritis. There were no fragments of decorin, biglycan, lumican, or keratocan that were unique to any tissue. A single fibromodulin fragment was detected in osteoarthritic articular cartilage but not meniscus. All SLRPs showed a modest age-related increase in fragmentation in knee articular and meniscal cartilage but not in other tissues. Conclusion: Enhanced fragmentation of SLRPs is evident in degenerate articular cartilage and meniscus. Specific decorin and fibromodulin core protein fragments in degenerate meniscus and/or human articular cartilage may be of value as biomarkers of disease. Once the enzymes responsible for their generation have been identified, further research may identify them as therapeutic targets

Recommendations of the 2007 Healthy Lifestyle Forum to Help Combat Childhood Obesity

Senator Guy Barnett has held eight Healthy Lifestyle Forums to Help Combat Childhood Obesity since entering the Senate in 2002. The most recent forum, held on 20 June 2007 at Australian Parliament House Canberra, brought together approximately 60 concerned members of health care, academia, industry and public health to develop useful interventions and ideas for fighting childhood obesity. This report details the recommendations of the groups convened. They were asked to consider actions in the key areas of: clinical/health care system monitoring/benchmarking infant and early childhood schools and the wider community, and industry and private sector solutions

Neural response to low energy and high energy foods in Bulimia nervosa and binge eating disorder : a functional MRI Study

Objective: Bulimia nervosa (BN) and binge eating disorder (BED) are eating disorders (EDs) characterized by recurrent binge eating (BE) episodes. Overlap exists between ED diagnostic groups, with BE episodes presenting one clinical feature that occurs transdiagnostically. Neuroimaging of the responses of those with BN and BED to disorder-specific stimuli, such as food, is not extensively investigated. Furthermore, to our knowledge, there have been no previous published studies examining the neural response of individuals currently experiencing binge eating, to low energy foods. Our objective was to examine the neural responses to both low energy and high energy food images in three emotive categories (disgust; fear; and happy) in BN and BED participants. Methods: Nineteen females with BN (n = 14) or BED (n = 5), comprising the binge eating group (BEG; N = 19), and 19 age-matched healthy control (HC)’s completed thorough clinical assessment prior to functional MRI (fMRI). Neural response to low energy and high energy foods and non-food images was compared between groups using whole-brain exploratory analyses, from which six regions of interest (ROI) were then selected: frontal, occipital, temporal, and parietal lobes; insula and cingulate. Results: In response to low energy food images, the BEG demonstrated differential neural responses to all three low energy foods categories (disgust; fear; and happy) compared to HCs. Correlational analyses found a significant association between frequency of binge episodes and diminished temporal lobe and greater occipital lobe response. In response to high energy food images, compared to HC’s, the BEG demonstrated significantly decreased neural activity in response to all high energy food images. The HC’s had significantly greater neural activity in the limbic system, occipital lobe, temporal lobe, frontal lobe, and limbic system in response to high energy food images. Conclusion: Results in the low energy food condition indicate that binge frequency may be related to increased aberrant neural responding. Furthermore, differences were found between groups in all ROI’s except the insula. The neural response seen in the BEG to disgust food images may indicate disengagement with this particular stimuli. In the high energy food condition, results demonstrate that neural activity in BN and BED patients may decrease in response to high energy foods, suggesting disengagement with foods that may be more consistent with those consumed during a binge eating episode

Testing Protein Leverage in Lean Humans: A Randomised Controlled Experimental Study

A significant contributor to the rising rates of human obesity is an increase in energy intake. The ‘protein leverage hypothesis’ proposes that a dominant appetite for protein in conjunction with a decline in the ratio of protein to fat and carbohydrate in the diet drives excess energy intake and could therefore promote the development of obesity. Our aim was to test the ‘protein leverage hypothesis’ in lean humans by disguising the macronutrient composition of foods offered to subjects under ad libitum feeding conditions. Energy intakes and hunger ratings were measured for 22 lean subjects studied over three 4-day periods of in-house dietary manipulation. Subjects were restricted to fixed menus in random order comprising 28 foods designed to be similar in palatability, availability, variety and sensory quality and providing 10%, 15% or 25% energy as protein. Nutrient and energy intake was calculated as the product of the amount of each food eaten and its composition. Lowering the percent protein of the diet from 15% to 10% resulted in higher (+12±4.5%, p = 0.02) total energy intake, predominantly from savoury-flavoured foods available between meals. This increased energy intake was not sufficient to maintain protein intake constant, indicating that protein leverage is incomplete. Urinary urea on the 10% and 15% protein diets did not differ statistically, nor did they differ from habitual values prior to the study. In contrast, increasing protein from 15% to 25% did not alter energy intake. On the fourth day of the trial, however, there was a greater increase in the hunger score between 1–2 h after the 10% protein breakfast versus the 25% protein breakfast (1.6±0.4 vs 25%: 0.5±0.3, p = 0.005). In our study population a change in the nutritional environment that dilutes dietary protein with carbohydrate and fat promotes overconsumption, enhancing the risk for potential weight gain