Biophysical and enzymatic properties of aminoglycoside adenylyltransferase AadA6 from Pseudomonas aeruginosa

AbstractThe gene coding for the aminoglycoside adenylyltransferase (aadA6) from a clinical isolate of Pseudomonas aeruginosa was cloned and expressed in Escherichia coli strain BL21(DE3)pLysS. The overexpressed enzyme (AadA6, 281 amino-acid residues) and a carboxy-terminal truncated variant molecule ([1-264]AadA6) were purified to near homogeneity and characterized. Light scattering experiments conducted under low ionic strength supported equilibrium between monomeric and homodimeric arrangements of the enzyme subunits. Circular Dichroism spectropolarimetry indicated a close structural relation to adenylate kinases. Both forms modified covalently the aminoglycosides streptomycin and spectinomycin. The enzyme required at least 5mM MgCl2 for normal Michaelis–Menten kinetics. Streptomycin exhibited a strong substrate inhibition effect at 1mM MgCl2. The truncated 17 residues at the C-terminus have little influence on protein folding, whereas they have a positive effect on the enzymic activity and stabilize dimers at high protein concentrations (>100μM). Homology modelling and docking based on known crystal structures yielded models of the central ternary complex of monomeric AadA6 with ATP and streptomycin or spectinomycin

Drug-free remission, functioning and radiographic damage after 4 years of response-driven treatment in patients with recent-onset rheumatoid arthritis

0.05, group 4 versus groups 1 and 2, group 3 versus group 2). Conclusions: In patients with recent-onset active RA, drug-free remission was achieved in up to 18% of patients. DAS-driven treatment maintained clinical and functional improvement, independent of the treatment strategy. Joint damage progression remained significantly lower after initial combination therapy compared with initial monotherap

Fluorogenic MMP activity assay for plasma including MMPs complexed to α2-macroglobulin

Elevated MMP activities are implicated in tissue degradation in, e.g., arthritis and cancer. The present study was designed to measure MMP enzyme activity in plasma. Free active MMP is unlikely to be present in plasma: upon entering the circulation, active MMP is expected to be captured by the proteinase inhibitor α2-macroglobulin (α2M). Reconstituted MMP-13/ α2M complex was unable to degrade collagen (MW 300,000) in contrast to the low-molecular-weight fluorogenic substrate (MW < 1500). Limited access of high-MW substrates to the active site of MMPs captured by α2M presents the most likely explanation. Consistently, the high-MW inhibitor TIMP (MW ~ 28,000) was unable to inhibit MMP/α2M enzyme activity, whereas the low-MW inhibitor BB94 (MW ~ 500) effectively suppressed enzyme activity. By using fluorogenic substrates with Dabcyl/Fluorescein as quencher/fluorophore combin-ation, sensitive MMP-activity assays in plasma were achieved. Spiking of active MMP-13 and MMP-13/α2M complex, and inhibitor studies with TIMP-1 and BB94, indicated that active MMPs are efficiently captured by (α2M in plasma. MMP activity was even detected in control plasma, and was significantly increased in plasma from rheumatoid arthritis patients

Difference in expression of the plasminogen activation system in synovial tissue of patients with rheumatoid arthritis and osteoarthritis

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Manual therapy in osteoarthritis of the hip: outcome in subgroups of patients

OBJECTIVE: To investigate whether manual therapy has particular benefit in subgroups of patients defined on the basis of hip function, range of joint motion, pain and radiological deterioration. METHODS: The study was performed in the out-patient clinic of physical therapy of a large hospital. Data on 109 patients with OA of the hip (clinical ACR criteria) participating in a randomized clinical trial on the effects of manual therapy were used. The outcomes for hip function (Harris hip score), range of joint motion (ROM) and pain (VAS) were compared for specific subgroups. Subgroups were assigned by the median split method. The interaction effect between subgroup and treatment was tested using multiple regression analysis. RESULTS: No differences were observed in the effect of manual therapy in specific subgroups of patients defined on the basis of baseline levels of hip function, pain and ROM. On the basis of radiological grading of osteoarthritis (OA), we found that patients with severe radiological grading of OA had significantly worse outcome on ROM as a result of manual therapy than patients with mild or moderate radiological grading of OA. CONCLUSION: A significant interaction effect was found for only 1 out of 12 hypotheses investigated. Therefore, we conclude that there is no evidence for the particular benefit of manual therapy in subgroups of patient