Subject(s)
Cytokines/biosynthesis , Extracellular Matrix Proteins , Proteoglycans/immunology , T-Lymphocytes/immunology , Aggrecans , Amino Acid Sequence , Animals , Cartilage , Cattle , Cells, Cultured , Chondroitin Sulfate Proteoglycans/immunology , Concanavalin A , Humans , Interferon-gamma/biosynthesis , Interleukin-10/biosynthesis , Interleukin-2/biosynthesis , Interleukin-4/biosynthesis , Lectins, C-Type , Lymph Nodes/immunology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/immunology , Peptide Fragments/pharmacology , Proteoglycans/chemistry , Proteoglycans/pharmacology , T-Lymphocytes/drug effectsABSTRACT
Aims-To investigate the level of matrix metalloproteinase activity during the time-course of cartilage explant proteoglycan breakdown; to determine the effects of selective small-molecule inhibitors of matrix metalloproteinases on proteoglycan degradation.Methods-The levels of matrix metalloproteinase activity in cartilage explant cultures and conditioned media were monitored by use of a quenched fluorescent substrate. The constants for inhibition of certain matrix metalloproteinases by a series of synthetic inhibitors were determined. Bovine and human cartilage explant cultures were treated with interleukin-1, tumor necrosis factor or retinoic acid and the amount of proteoglycan released into the culture medium in the absence and presence of the inhibitors was quantified. Control experiments, examining the inhibition of other proteinases, and investigating possible toxic or non-specific effects of the inhibitors, were carried out.Results-The profile of inhibition of proteoglycan release suggested the involvement of interstitial collagenase-like, rather than gelatinase- or possibly stromelysin-like, proteinases. No evidence was found for toxic or non-specific mechanisms of inhibition. Very low levels of activity of the known matrix metalloproteinases were present during the time-course of aggrecan breakdown.Conclusions-A novel collagenase-like proteinase(s) may be involved in cartilage proteoglycan breakdown. Gelatinase-type matrix metalloproteinases do not seem to be involved in this process. Specific collagenase inhibitors may be therapeutically efficacious in the treatment of arthritis.
ABSTRACT
Phosphoenolpyruvate carboxylase [PEPC; orthophosphate:oxaloacetate carboxy-lyase (phosphorylating); EC 4.1.1.31] is a major anaplerotic enzyme in the polyketide producer Streptomyces coelicolor A3(2). PEPC was purified from S. coelicolor and the amino-acid sequences of four tryptic peptides were determined. Synthetic oligonucleotides based on the sequences of two of the peptides hybridized to the same bands in various restriction-enzyme digests of S. coelicolor genomic DNA. This hybridization allowed molecular cloning of an 8 kb BamHI fragment of genomic DNA. Partial DNA sequencing of this fragment showed that it could encode amino acid sequences similar to those of PEPC from other microorganisms. A BamHI/PstI fragment was subcloned into the streptomycete high-copy-number plasmid vector pIJ486 and transferred into Streptomyces lividans. The resulting strain over-expressed PEPC activity 21-fold and also over-expressed a protein with a subunit of 100,000 M(r), the same as that of purified S. coelicolor PEPC.
Subject(s)
DNA, Bacterial/chemistry , Phosphoenolpyruvate Carboxylase/isolation & purification , Streptomyces/enzymology , Amino Acid Sequence , Base Sequence , Chromatography, Affinity , Chromatography, High Pressure Liquid , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Molecular Sequence Data , Phosphoenolpyruvate Carboxylase/chemistry , Phosphoenolpyruvate Carboxylase/genetics , Restriction MappingABSTRACT
A method is proposed for the determination of paracetamol in whole undiluted blood, based on the enzymatic hydrolysis of the drug to p-aminophenol, which is then measured by chronoamperometry at a glassy carbon electrode. Hydrolysis of the paracetamol prior to electro-oxidation is shown to alleviate problems that arise from high background currents in the whole blood and so ensures a good linear correlation (r greater than 0.99) between the current and the paracetamol concentration. Recovery experiments and comparison with a reference method based on spectrophotometry suggest that the electrochemical assay only measures that proportion of paracetamol that is not bound to serum albumin.