432 research outputs found
Detection of age-related changes in the distributions of keratan sulfates and chondroitin sulfates in developing chick limbs: an immunocytochemical study
Immunochemical and biochemical comparisons between embryonic chick bone marrow and epiphyseal cartilage chondroitin/dermatan sulphate proteoglycans
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
Distribution of proteoglycans antigenically related to corneal keratan sulfate proteoglycan
Three antibodies reacting with corneal keratan sulfate proteoglycan were used to detect antigenically related molecules in 11 bovine and 13 embryonic chick tissues. Two monoclonal antibodies recognized sulfated epitopes on the keratan sulfate chain and a polyclonal antibody bound antigenic sites on the core protein of corneal keratan sulfate proteoglycan. Competitive immunoassay detected core protein and keratan sulfate antigens in guanidine HCl extracts of most tissues. Keratan sulfate antigens of most bovine tissues were only partially extracted with guanidine HCl, but the remainder could be solubilized by CNBr treatment of the guanidine-extracted residue. Keratan sulfate and core protein antigens co-eluted with purified corneal keratan sulfate proteoglycan on ion exchange high-performance liquid chromatography (HPLC). Endo-beta-galactosidase digestion of the HPLC-purified keratan sulfate antigens eliminated the binding of monoclonal anti-keratan sulfate antibodies in enzyme-linked immunosorbent assay. Extracts of all 11 bovine tissues, except those from brain and cartilage, could bind both anti-keratan sulfate monoclonal antibodies and anti-core protein polyclonal antibody simultaneously. Binding was sensitive to competition with keratan sulfate and to digestion with endo-beta-galactosidase. These results suggest widespread occurrence of a proteoglycan or sulfated glycoprotein bearing keratan sulfate-like carbohydrate and a core protein resembling that of corneal keratan sulfate proteoglycan
Altered Expression Of Chondroitin Sulfate Proteoglycan In The Stroma Of Human Colon Carcinoma. Hypomethylation Of Pg-40 Gene Correlates With Increased Pg-40 Content And Mrna Levels.
Secretory granules of heparin-containing rat serosal mast cells also possess highly sulfated chondroitin sulfate proteoglycans.
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
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