Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is responsible for the catabolic turnover and loss of whole aggrecan whereas other protease activity is required for C-terminal processing in vivo

2001 ◽  
Vol 358 (3) ◽  
pp. 615-626 ◽  
Author(s):  
John D. SANDY ◽  
Christie VERSCHAREN
Keyword(s):  
Synovial Fluid ◽  
Core Protein ◽  
Globular Domain ◽  
Human Cartilage ◽  
Human Knee ◽  
Synovial Fluids ◽  
Human Joints ◽  
Non Destructive

Studies of aggrecan proteolysis in human joints have implicated both the aggrecanase [ADAMTS, a disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1 motif] and matrix metalloproteinase (MMP) families. We have analysed the aggrecan core protein species present in vivo in both articular cartilage and synovial fluids from normal, acutely injured and osteoarthritic joints. Normal cartilage contains at least seven major G1 domain (the N-terminal globular domain of aggrecan)-bearing species, of which three (full-length core, G1-NITEGE373 and G1-VDIPEN341) have been identified. The C-terminals of the others are unknown but digestion of fetal human aggrecan with MMP-3 and crude aggrecanase suggests that they are products of MMP-like activity in vivo. Normal synovial fluids contain at least 10 species, of which nine result from ADAMTS-dependent cleavage, and this cleavage occurs at all of the five known aggrecanase sites. Aggrecan fragments in the cartilage and synovial fluids of acutely injured joints are generally similar to normal, but all contain a markedly increased ratio of G1-NITEGE to G1-VDIPEN. Aggrecan from the cartilage of late-stage osteoarthritis patients is remarkably similar to normal, whereas the synovial fluid aggrecan is more fragmented than that from normal or injured knees. The analyses suggest that the role of the ADAMTS and these MMP-like activities in human cartilage are distinctly different. Excessive ADAMTS activity in vivo is destructive to cartilage matrix, since the bulk of the glycosaminoglycan (GAG)-bearing products are released from the tissue into the synovial fluid following cleavage of the Glu373–Ala374 bond. In contrast, the MMP-like activity appears to be essentially non-destructive, since much of the GAG-bearing product is retained in the tissue following cleavages that are in the more C-terminal regions of the molecule.

scholarly journals The molecular structure and lubricating activity of lubricin isolated from bovine and human synovial fluids

1985 ◽  
Vol 225 (1) ◽  
pp. 195-201 ◽  
Author(s):  
D A Swann ◽  
F H Silver ◽  
H S Slayter ◽  
W Stafford ◽  
E Shore
Keyword(s):  
Amino Acid ◽  
Synovial Fluid ◽  
Test System ◽  
Sedimentation Equilibrium ◽  
Human Knee ◽  
Synovial Fluids ◽  
Molecular Masses ◽  
Friction Measurements

Lubricin was isolated from bovine ankle, metacarpophalangeal and knee and human knee synovial fluids. The lubricins isolated from the bovine joint fluids had the same amino acid and carbohydrate compositions, but differences were observed in the relative molecular masses. The Mr values of bovine metacarpophalangeal and ankle lubricin determined by light-scattering measurements were about 200 000, whereas values of 132 000 and 143 000 were obtained for the bovine knee lubricin. The human knee lubricin had a similar carbohydrate composition to bovine knee lubricin except for the higher glucosamine content, and the amino acid composition differed slightly. The human sample had a lower glutamic acid content and a leucine/isoleucine ratio of 2:1 compared with 1:1 in the bovine. The Mr value of the human knee lubricin (166 000) was also lower than that of the bovine metacarpophalangeal and ankle samples. The Mr value of the bovine knee lubricin determined by sedimentation-equilibrium measurements was 171 000. The length measurements determined by electron microscopy and also the sedimentation measurements showed considerable polydispersity and indicate that the degree of extension of lubricin molecules can vary. Friction measurements showed that the human knee synovial-fluid lubricin had equivalent lubricating ability in a test system in vitro to that observed for lubricin isolated from normal bovine synovial fluids. The lubricating ability of lubricin was concentration-dependent, and each lubricin sample was able to act as a lubricant in vitro in an equivalent manner to whole synovial fluid at concentrations that are thought to occur in vivo.

Activation Of Extravascular Coagulation In The Inflamed Joints In Rheumatoid Arthritis (RA)

1981 ◽  
Author(s):  
C J W van Ginkel ◽  
J I H Oh ◽  
J A van Mourik ◽  
W G van Aken ◽  
J Vreeken
Keyword(s):  
Synovial Fluid ◽  
Coagulation Factors ◽  
Coagulation Cascade ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Synovial Fluids ◽  
Normal Human ◽  
Coagulation Pathway ◽  
Activation Capacity

Deposition of fibrin-like material on the synovival membrane of inflamed joints is a prominent and persistent feature of RA. To evaluate the involvement of the classical coagulation cascade in the formation of this material, fibrinopeptide A (FPA) and coagulation factors were analyzed in synovial fluid from RA patients. FPA was 1100 ng/ml (range 300-3200; n=l6), compared to 200 ng/ml (60-300;n=13) in synovial fluid from patients with osteoarthritis. No significant change (p> 0.2; n=l4) in FPA immunoreactivity (using R2 anti-FPA serum from Dr Nossel beside the antiserum from our institute) was observed after thrombin treatment of the diffusate of dialyzed synovial fluids. This finding indicates that the high levels of FPA immunoreactivity in joint fluids of RA reflect FPA (Aα 1-16) instead of FPA-like fibrinogen fragments (which can be cleaved from fibrinogen by e.g. granulocytic proteases). In view of the finding that all coagulation factors including fibrinogen were sufficiently present (30-100%; n=l8) - except FV (<5%) - the accumulation of FPA suggests activation of extravascular coagulation in the inflamed joints in RA.To investigate the mechanisms of this activation, normal plasma was incubated with human articular cartilage and activation of FXII was determined by measuring the generation of bradykinin (radioimmunoassay). Cartilage activated FXII whereas its activation capacity was strongly enhanced by pre-exposition to granulocytic enzymes (compare in vivo!). Among its constituents collagen (type II) activated FXII, but proteoglycans were only weak activators. Both substances were isolated from human cartilage, obtained from a normal knee joint at necropsy. With respect to the contribution of the extrinsic coagulation pathway it was shown that synovial fluids from RA patients stimulated the thromboplastin generation by normal human monocytes. Taken together, these findings suggest that both intrinsic and extrinsic coagulation pathways, presumably triggered by the inflammatory process itself, are involved in articular fibrin deposition.

THE RELATION BETWEEN JOINT STIFFNESS UPON EXPOSURE TO COLD AND THE CHARACTERISTICS OF SYNOVIAL FLUID

1952 ◽  
Vol 30 (5) ◽  
pp. 367-377 ◽  
Author(s):  
John Hunter ◽  
E. H. Kerr ◽  
M. G. Whillans
Keyword(s):  
Synovial Fluid ◽  
Joint Stiffness ◽  
In Vivo Studies ◽  
Maximum Speed ◽  
Ambient Temperatures ◽  
Human Knee ◽  
Exposure To Cold ◽  
Human Knee Joint ◽  
Predominant Type

Previous laboratory tests have shown that joint temperatures, on exposure to low ambient temperatures, fall to a greater extent than muscle, rectal, or average skin temperatures. The fall in temperature is accompanied by an increased resistance of joints to movement, and the maximum speed with which the joint can be moved decreases. The predominant type of movement at the human knee joint and interphalangeal joints is a gliding one. The characteristics of synovial fluid explain the increased forces required to move a joint and the loss in speed of movement on exposure to cold. In vivo studies support such predictions.

The role of protein content on the steady and oscillatory shear rheology of model synovial fluids

Soft Matter ◽  
2014 ◽  
Vol 10 (32) ◽  
pp. 5965-5973 ◽  
Author(s):  
Z. Zhang ◽  
S. Barman ◽  
G. F. Christopher
Keyword(s):  
Hyaluronic Acid ◽  
Synovial Fluid ◽  
Acid Solution ◽  
Protein Content ◽  
Shear Viscosity ◽  
Steady Shear ◽  
Interfacial Rheology ◽  
Steady Shear Viscosity ◽  
Synovial Fluids

Model synovial fluid steady shear viscosity to hyaluronic acid solution are identical when interfacial rheology effects are removed.

The histone H1 family: specific members, specific functions?

2008 ◽  
Vol 389 (4) ◽  
pp. 333-343 ◽  
Author(s):  
Annalisa Izzo ◽  
Kinga Kamieniarz ◽  
Robert Schneider
Keyword(s):  
General Structure ◽  
Distinct Species ◽  
Histone H1 ◽  
Globular Domain ◽  
Compensatory Mechanisms ◽  
Biochemical Analyses ◽  
Related Proteins

AbstractThe linker histone H1 binds to the DNA entering and exiting the nucleosomal core particle and has an important role in establishing and maintaining higher order chromatin structures. H1 forms a complex family of related proteins with distinct species, tissue and developmental specificity. In higher eukaryotes all H1 variants have the same general structure, consisting of a central conserved globular domain and less conserved N-terminal and C-terminal tails. These tails are moderately conserved among species, but differ among variants, suggesting a specific function for each H1 variant. Due to compensatory mechanisms and to the lack of proper tools, it has been very difficult to study the biological role of individual variants in chromatin-mediated processes. Our knowledge about H1 variants is indeed limited, andin vitroandin vivoobservations have often been contradictory. Therefore, H1 variants were considered to be functionally redundant. However, recent knockout studies and biochemical analyses in different organisms have revealed exciting new insights into the specificity and mechanisms of actions of the H1 family members. Here, we collect and compare the available literature about H1 variants and discuss possible specific roles that challenge the concept of H1 being a mere structural component of chromatin and a general repressor of transcription.

Essential role of heparan sulfates in axon navigation and targeting in the developing visual system

Development ◽  
1997 ◽  
Vol 124 (12) ◽  
pp. 2421-2430 ◽  
Author(s):  
A. Walz ◽  
S. McFarlane ◽  
Y.G. Brickman ◽  
V. Nurcombe ◽  
P.F. Bartlett ◽  
...  
Keyword(s):  
Visual System ◽  
Core Protein ◽  
Optic Tract ◽  
Axon Growth ◽  
Axon Elongation ◽  
Axon Extension ◽  
Heparan Sulfates

Heparan sulfate (HS) is abundant in the developing brain and is a required co-factor for many types of fibroblast growth factor (FGF) signaling in vitro. We report that some HSs, when added exogenously to the developing Xenopus optic pathway, severely disrupt target recognition causing axons from the retina to bypass their primary target, the optic tectum. Significantly, HS sidechains from a neuroepithelial perlecan variant that preferentially bind FGF-2, HS(FGF-2), cause aberrant targeting, whereas those that preferentially bind FGF-1 do not. Charge-matched fragments of HS(FGF-2) show that the mistargeting activity associates with the FGF-binding fragments. Heparitinase removal of native HSs at the beginning of optic tract formation retards retinal axon elongation; addition of FGF-2 restores axon extension but axons lose directionality. Late HS removal, after axons have extended through the tract, elicits a tectal bypass phenotype indicating a growth promoting and guidance function for native HSs. Our results demonstrate that different HS sidechains from the same core protein differentially affect axon growth in vivo, possibly due to their distinct FGF-binding preferences, and suggest that growth factors and HSs are important partners in regulating axon growth and guidance in the developing visual system.

Host defense role of platelets: engulfment of HIV andStaphylococcus aureus occurs in a specific subcellular compartment and is enhanced by platelet activation

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 4021-4029 ◽  
Author(s):  
Tayebeh Youssefian ◽  
Arnaud Drouin ◽  
Jean-Marc Massé ◽  
Josette Guichard ◽  
Elisabeth M. Cramer
Keyword(s):  
Platelet Activation ◽  
Core Protein ◽  
Morphological Evidence ◽  
Subcellular Compartment ◽  
Virus Particles ◽  
Acquired Immunodeficiency ◽  
Immunodeficiency Syndrome ◽  
Granule Release

Platelets can bind and phagocytose infectious microorganisms and so enable their transport for a prolonged time. To investigate the subcellular events of these interactions, platelets were incubated either with Staphylococcus aureus or with HIV and analyzed by electron microscopy (EM) and immuno-EM. HIV and bacteria internalization occurred exclusively within platelets showing morphological evidence of activation. Platelet activation enhanced the degree of bacterial internalization. Immunolabeling revealed that the engulfing vacuoles and the open canalicular system (OCS) were composed of distinct antigens. The engulfing vacuoles eventually became the site of prominent α-granule release. In platelets incubated with HIV, characteristic endocytic vacuoles were identified close to the plasma membrane, tightly surrounding 1 or 2 HIV particles. Virus particles were also located within the OCS. Immunogold labeling for the viral core protein p24 confirmed the presence of HIV within platelets. Finally, examination of platelets from a patient with acquired immunodeficiency syndrome and high viremia suggested that HIV endocytosis may also occur in vivo.

scholarly journals Maintenance of Dimer Conformation by the Dengue Virus Core Protein α4-α4′ Helix Pair Is Critical for Nucleocapsid Formation and Virus Production

2014 ◽  
Vol 88 (14) ◽  
pp. 7998-8015 ◽  
Author(s):  
Pak-Guan Teoh ◽  
Zhi-Shun Huang ◽  
Wen-Li Pong ◽  
Po-Chiang Chen ◽  
Huey-Nan Wu
Keyword(s):  
Dengue Virus ◽  
Core Protein ◽  
Virus Production ◽  
Hydrophobic Surface ◽  
Pair Interaction ◽  
Virus Core ◽  
Virion Production

ABSTRACTThe virion of dengue virus (DENV) is composed of a viral envelope covering a nucleocapsid formed by a complex of viral genomic RNA and core protein (CP). DENV CP forms a dimer via the internal α2 and α4 helices of each monomer. Pairing of α2-α2′ creates a continuous hydrophobic surface, while the α4-α4′ helix pair joins the homodimer via side-chain interactions of the inner-edge residues. However, the importance of dimer conformation and the α4 helix of DENV CP in relation to its function are poorly understood. Loss of association between CP and lipid droplets (LDs) due to mutation suggests that the CP hydrophobic surface was not exposed, offering a possible explanation for the absence of dimers. Further assays suggest the connection between CP folding and protein stability. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects were detected in virus translation and replication. Thein vitrocharacterization assays further highlighted that the α4-α4′ helix pair conformation is critical in preserving the overall α-helical content, thermostability, and dimer formation ability of CP, features correlated with the efficiency of nucleocapsid formation. Addition of Tween 20 improvesin vitronucleocapsid-like particle formation, suggesting the role of the LD in nucleocapsid formationin vivo. This study provides the first direct link between the α4-α4′ helix pair interaction and the CP dimer conformation that is the basis of CP function, particularly in nucleocapsid formation during virion production.IMPORTANCEStructure-based mutagenesis study of the dengue virus core protein (CP) reveals that the α4-α4′ helix pair is the key to maintaining its dimer conformation, which is the basis of CP function in nucleocapsid formation and virus production. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects in virus translation and replication were detected.In vitroinefficiency and size of nucleocapsid-like particle (NLP) formation offer a possible explanation forin vivovirus production inefficiency upon CP mutation. Further, the transition of NLP morphology from an incomplete state to an intact particle shown by α4-α4′ helix pair mutants in the presence of a nonionic detergent suggests the regulatory role of the intracellular lipid droplet (LD) in CP-LD interaction and in promoting nucleocapsid formation. This study provides the first direct link between the α4-α4′ helix pair interaction and CP dimer conformation that is the fundamental requirement of CP function, particularly in nucleocapsid formation during virion production.

scholarly journals AB0100 PHENOTYPIC AND FUNCTIONAL CHARACTERIZATION OF SYNOVIAL FLUID-DERIVED FIBROBLAST-LIKE SYNOVIOCYTES IN RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1349.1-1349
Author(s):  
D. Køster ◽  
J. H. Egedal ◽  
M. Hvid ◽  
M. R. Jakobsen ◽  
U. Müller-Ladner ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Synovial Tissue ◽  
Mononuclear Cells ◽  
Functional Characterization ◽  
Human Cartilage ◽  
Fibroblast Like Synoviocytes

Background:Fibroblast-like synoviocytes (FLS) are central cellular components in persistent inflammatory joint diseases such as rheumatoid arthritis (RA). Pathological subsets of FLS have been identified from synovial tissue. However, the synovial tissue obtained from arthroplasty procedures is acquired at late disease stages and the cellular yield obtained from synovial tissue biopsies is fairly low. Collectively, challenging the robustness of human RAin vivoandin vitromodels. FLS obtained from the synovial fluid (SF-FLS) are proposed as an alternative source of FLS, but a detailed phenotypical and functional characterization of FLS subsets from the synovial fluid has not been performed.Objectives:The aim of this study was to determine the phenotypical and functional characteristics of synovial fluid-derived fibroblast-like synoviocytes in rheumatoid arthritis.Methods:In the present study, paired peripheral blood mononuclear cells (PBMC) and SF-FLS from patients with RA were obtained (n=7). FLS were isolated from the synovial fluid by a strict trypsinization protocol1and their cellular characteristics and functionality were evaluated at passage 4. Monocultures (SF-FLS) and autologous co-cultures (SF-FLS and PBMC) were established from five patients with RA and subsequently evaluated by flow cytometry, Western blotting and multiplex immunoassays. Human cartilage-sponges (n=3) with SF-FLS and without SF-FLS (n=3) were co-implanted subcutaneously in SCID mice (n=15), mice with only cell-free human cartilage-sponges were used as controls (n=12). After 45 days, the implants were evaluated using stained sections to determine the SF-FLS invasion score based on perichondrocytic cartilage degradation. Data are expressed as median (25-75 percentile). P-values <0.05 were considered statistically significant.Results:The homogeneous subpopulations of FLS, isolated from the synovial fluid, were negative for CD34 and CD45 [98.9%, (97.5-99.7]) and positive for Thy-1 and PDPN [94.6%, (79.9-97.4]). Without stimulation, RA SF-FLS showed high and comparable levels of NFκB related pathway proteins and secreted multiple pro-inflammatory cytokines and chemokines dominated by IL-6 [2648 pg/mL, (1327-6116)] and MCP-1 [2458 pg/mL, (692-8719)]. SF-FLS increased their ICAM-1 and HLA-DR expression after encountering autologous PBMCs (p<0.01), (p<0.05). Further, SF-FLS and PBMC interacted synergistically in a co-culture model of RA and significantly increasing the secretion of several cytokines (IL-1β, IL-2, IL-6, (p<0.01)) and a chemokine (MCP-1, (p<0.01)). The invasion score of the human SF-FLSin vivowas at primary site, [1.6, (1.3-1.7)] and contralateral implantation site [1.5, (1.1-2.2)]. The invasion score of the human SF-FLS-containing implants both at primary and contralateral site were significantly higher compared with cartilage-sponges evaluated from SF-FLS-free control mice (p<0001).Conclusion:This phenotypical and functional characterization of SF-FLS, acquired and activated at the site of pathology, lays a foundation for establishingin vivoandin vitroFLS models. These FLS models will be beneficial in our understanding of the role of this cellular subset in arthritis and for characterization of drugs specifically targeting this pathological RA FLS subset.References:[1]Nielsen M. A. et al. Responses to Cytokine Inhibitors Associated with Cellular Composition in Models of Immune-Mediated Inflammatory Arthritis. ACR Open Rheumatology, 2(1):3-10.http://doi.org/10.1002/acr2.11094Disclosure of Interests:Ditte Køster: None declared, Johanne Hovgaard Egedal: None declared, Malene Hvid: None declared, Martin Roelsgaard Jakobsen: None declared, Ulf Müller-Ladner Speakers bureau: Biogen, Bent Deleuran: None declared, Tue Wenzel Kragstrup Shareholder of: iBio Tech ApS, Consultant of: Bristol-Myers Squibb, Speakers bureau: TWK has engaged in educational activities talking about immunology in rheumatic diseases receiving speaking fees from Pfizer, Bristol-Myers Squibb, Eli Lilly, Novartis, and UCB., Elena Neumann: None declared, Morten Aagaard Nielsen: None declared

scholarly journals Trypanosome Spliced-Leader-Associated RNA (SLA1) Localization and Implications for Spliced-Leader RNA Biogenesis

2008 ◽  
Vol 8 (1) ◽  
pp. 56-68 ◽  
Author(s):  
Avraham Hury ◽  
Hanoch Goldshmidt ◽  
Itai Dov Tkacz ◽  
Shulamit Michaeli
Keyword(s):  
Core Protein ◽  
Rnase H ◽  
Differential Sensitivity ◽  
Minor Effect ◽  
Sm Proteins ◽  
Spliced Leader ◽  
Rna Biogenesis ◽  
Leptomonas Collosoma

ABSTRACT Spliced-leader-associated RNA (SLA1) guides the pseudouridylation at position −12 (relative to the 5′ splice site) of the spliced-leader (SL) RNA in all trypanosomatid species. Nevertheless, the exact role of this RNA is currently unknown. Here, we demonstrate that the absence of pseudouridine on Leptomonas collosoma SL RNA has only a minor effect on the ability of this RNA to function in trans splicing in vivo. To investigate the possible role of SLA1 during SL RNA biogenesis, the structure of the SL RNA was examined in permeable Trypanosoma brucei cells depleted for CBF5, the H/ACA pseudouridine synthase, lacking SLA1. Our results suggest that in the absence of SLA1, the SL RNA secondary structure is changed, as was detected by differential sensitivity to oligonucleotide-directed RNase H cleavage, suggesting that the association of SLA1 maintains the SL RNA in a structural form which is distinct from the structure of the SL RNA in the steady state. In T. brucei cells depleted for the SL RNA core protein SmD1, SL RNA first accumulates in large amounts in the nucleus and then is expelled to the cytoplasm. Here, we demonstrate by in vivo aminomethyltrimethyl UV cross-linking studies that under SmD1 depletion, SLA1 remains bound to SL RNA and escorts the SL RNA to the cytoplasm. In situ hybridization with SLA1 and SL RNA demonstrates colocalization between SLA1 and the SL RNA transcription factor tSNAP42, as well as with Sm proteins, suggesting that SLA1 associates with SL RNA early in its biogenesis. These results demonstrate that SLA1 is a unique chaperonic RNA that functions during the early biogenesis of SL RNA to maintain a structure that is most probably suitable for cap 4 modification.

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