Integration of bacteria capture via filtration and in situ lysis for recovery of plasmid DNA under industry-compatible conditions

Combining capture and lysis of the bacteria with partial purification of the plasmid DNA is beneficial for the design of efficient plasmid production processes at larger scale. Such an approach is possible when the bacteria are captured by filtration. Taking industrial requirements into account, however, such a capture requires complex filtration mixtures containing retentive additives such as bentonite and polycations. This makes the straightforward transfer of established lysis protocols to in situ lysis difficult. In this contribution, the different steps of such a protocol are designed for complex filter cakes, including fragilization (by lysozyme), lysis (alkaline pH/acidic pH, 70/37 degrees C, urea/NaCl/Triton), and specific elution (pH, NaCl, CaCl2, guanidinium hydrochloride). Results are compared in regard to plasmid quality (topoisomeric form) and quantity (compared to the yield obtained by a commercial miniprep of a small aliquot of the bacteria suspension from the bioreactor). Best results in these terms were obtained by the Triton lysis protocol performed at 37 degrees C (30 min of contact with a lysis buffer composed of 50 mM Tris pH 8, 1% Triton, 1 g/L lysozyme, and 6 M guanidinium hydrochloride) followed by the specific elution of the plasmid DNA in 50 mM Tris buffer pH 8.     

One-step purification of the recombinant catalytic subunit of pyruvate dehydrogenase phosphatase

A facile one-step affinity chromatographic purification of the recombinant catalytic subunit (PDPc) of bovine pyruvate dehydrogenase phosphatase (PDP) to near homogeneity is described. PDPc binds in the presence of Ca(2+) to the inner lipoyl domain (L2) of the dihydrolipoamide acetyltransferase component (E2) of the mammalian pyruvate dehydrogenase complex. The affinity column consists of a glutathione S-transferase (GST)-L2 fusion protein bound to glutathione-Sepharose 4B beads. An extract of transformed Escherichia coli cells containing 50 mM Tris buffer (pH 7.5), 2 mM CaCl(2), 5 mM MgCl(2,) 150 mM NaCl, 0.5 mM dithiothreitol, 1% Triton X-100, and l M urea was passed through the affinity column, and the column was washed extensively with this buffer mixture. PDPc was eluted with 50 mM Tris buffer (pH 7.5) containing 5 mM MgCl(2), 0.5 mM dithiothreitol, and 1 mM EGTA. Approximately 22 mg of highly purified PDPc was obtained from 10 g (wet weight) of transformed cells. The preparation contained a small amount of a "nicked" form of PDPc. The cleavage is between Arg-394 and Arg-395.     

Purification of penicillin G acylase using immobilized metal affinity membranes

The immobilized metal affinity membrane (IMAM) with modified regeneration cellulose was employed for purification of penicillin G acylase (PGA). For studying PGA adsorption capacity on the IMAM, factors such as chelator surface density, chelating metal, loading temperature, pH, NaCl concentration and elution solutions were investigated. The optimal loading conditions were found at 4 degrees C, 0.5 M NaCl, 32.04 micromol Cu(2+) per disk with 10 mM sodium phosphate buffer, pH 8.5, whereas elution conditions were: 1 M NH(4)Cl with 10 mM sodium phosphate buffer, pH 6.8. By applying these chromatographic conditions to the flow experiments in a cartridge, a 9.11-fold purification in specific activity with 90.25% recovery for PGA purification was obtained. Meanwhile, more than eight-times reusability of the membrane was achieved with the EDTA regeneration solutions.     

Glycosylation-inhibiting factor from human T cell hybridomas constructed from peripheral blood lymphocytes of a bee venom-sensitive allergic patient.

Human T cell hybridomas, which constitutively secrete glycosylation inhibiting factor (GIF), were constructed from PBL of an allergic individual who was sensitive to honey bee venom. PBMC of the patient were stimulated with either denatured or cyanogen bromide-treated bee venom phospholipase A2 (PLA2), and Ag-activated cells were propagated by IL-2 in the presence of human recombinant lipocortin I. T cells obtained in the cultures were fused with a HAT-sensitive mutant of the human lymphoblastoid cell line CEM. Approximately one-third of hybridoma clones constitutively secreted GIF. The GIF-producing hybridomas were CD3+ and bore TCR-alpha beta. GIF formed by unstimulated hybridomas lacked affinity for bee venom PLA2. Upon cross-linking of CD3, however, a majority of the GIF-producing hybridomas formed IgE-binding factors and GIF, the latter of which had affinity for bee venom PLA2. Both nonspecific GIF and Ag-binding GIF from the hybridomas bound to an immunosorbent coupled with the anti-lipomodulin mAb 141-B9. Using an affinity-purified GIF as an immunogen, we established mouse B cell hybridomas that secreted monoclonal anti-human GIF. In order to characterize human nonspecific GIF, one of the GIF-producing hybridomas was adapted to a serum-free medium, and culture supernatant was fractionated by DEAE-Sepharose column chromatography and by gel filtration. The majority of nonspecific GIF in the culture supernatant was recovered from DEAE-Sepharose by elution of the column with 10 mM Tris-HCl buffer, pH 8.0, containing 50 mM NaCl. Affinity-purification of GIF in the DEAE Sepharose fraction by using anti-GIF-coupled Affigel, and analysis of the purified GIF by SDS-PAGE revealed that human GIF is a single polypeptide chain of 14 to 15 kDa. Gel filtration of both crude and affinity-purified GIF preparations confirmed the molecular size of the cytokine.     

Coupling reaction and properties of poly(ethylene glycol)-linked phospholipases A2

Secretory phospholipases A2 (PLA2) from Naja naja naja (cobra snake) venom, from Bothrops neuwiedii (crotalid snake) venom (two isoforms) and from bee venom were modified with tresylated monomethoxy poly(ethylene glycol) (TMPEG). The kinetic and inflammatory properties of the adducts (PEG-PLA2) were measured. As found by gel permeation chromatography, 95-100% of P-1 PLA2 from B. neuwiedii and PLA2 from N. naja naja venom change their chromatographic mobility after TMPEG treatment. By contrast, only 50-60% of both P-3-PLA2 from B. neuwiedii and PLA2 from bee venom modify their elution profile from Superdex 75. All the modified proteins preserved the enzymatic activity toward phospholipid monolayers, but with a reduced specific activity and greater lag times than the unmodified controls. These results suggest that the PEG-PLA2 complexes would have an altered interaction with lipid membranes. The PEG-linked proteins preserve their edema-inducing activity evaluated by the rat hind-paw edema test except for N. naja naja PEG-PLA2 in which inflammatory activity was significatively decreased. Altogether, the results show a partial dissociation of catalytic and inflammatory activities of Group II and III secretory PLA2s after their modification with PEG.     

Inhibitory effect of Ca2+ on formation of Mg2(+)-mediated two-dimensional hexagonal lattice structure by an R-form lipopolysaccharide from Klebsiella pneumoniae

When the R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-:K1-), from which cationic material had been removed by electrodialysis, was suspended in 50 mM Tris buffer at pH 8.5 containing 0.1 mM or higher concentrations of MgCl2, it formed an ordered two-dimensional hexagonal lattice structure and its center-to-center distance (lattice constant) depended upon the concentration of MgCl2 and reached the shortest value (14 nm) at 10 mM. In contrast, in the presence of 0.1 to 10 mM CaCl2 in place of MgCl2, the electrodialyzed LPS did not form such an ordered hexagonal lattice structure but formed an irregular network structure with a center-to-center distance of 19 to 20 nm. We investigated interaction of Mg2+ and Ca2+ in formation of the hexagonal lattice structure by the electrodialyzed LPS suspended in 50 mM Tris buffer at pH 8.5. When 0.1 mM or higher concentrations of CaCl2 were mixed with 1 mM MgCl2 or when 1 mM or higher concentrations of CaCl2 was mixed with 10 mM MgCl2, the electrodialyzed LPS did not form the hexagonal lattice structure of the magnesium salt type but formed the irregular network structure of the calcium salt type. In the coexistence of equimolar or higher concentrations of CaCl2 together with 1 or 10 mM MgCl2, the binding of Mg to the electrodialyzed LPS was significantly inhibited and, conversely, the binding of Ca was enhanced as compared with when MgCl2 or CaCl2 was present alone. However, the coexistence of 10 times less molar concentrations of CaCl2 did not significantly inhibit the binding of Mg to the electrodialyzed LPS. Therefore, the inhibition of formation of the Mg2(+)-mediated hexagonal lattice structure of the electrodialyzed LPS by equimolar or higher concentrations of CaCl2 accompanied the inhibition of binding of Mg but that by 10 times less molar concentrations of CaCl2 did not accompany it.     

Molybdenocene-oligonucleotide binding study at physiological pH using NMR spectroscopy and cyclic voltammetry

The self-complementary oligonucleotide CGCATATATGCG was used as a model to establish the binding interactions of antitumor molybdenocene dichloride and DNA. The free dodecamer was first characterized using ¹H, NOESY, and DQF-COSY NMR experiments, which enable to pinpoint the guanines and adenines as well as the cytosines and thymines signals in the aromatic region. Molybdenocene dichloride was characterized in saline and buffer solutions as function of pH by ¹H NMR spectroscopy. In 10 mM NaCl/D₂O solution at pH of 6.5 and above, Cp₂Mo(OD)(D₂O)⁺ is in equilibrium with its dimeric species, [Cp₂Mo(μ-OH)₂MoCp₂]²⁺. In 25 mM Tris/4 mM NaCl/D₂O at physiological pH, a new stable species is formed, coordinated by the buffer, Tris(hydroxymethyl)aminomethane. The interactions of molybdenocene dichloride species with CGCATATATGCG were studied at different pH. At pH 6.5, in 4 mM NaCl/D₂O solution, ¹H NMR spectra of CGCATATATGCG exhibit downfield shifts in the signals associated mainly to adenines and guanines, upon addition of molybdenocene dichloride. At pH 7.4, in 25 mM Tris/4 mM NaCl/D₂O, molybdenocene species causes broadening and small downfield shifts to the purines and pyrimidine signals, suggesting that molybdenocene dichloride can get engaged in binding interactions with the oligonucleotide in a weak manner. ³¹P NMR spectra of these interactions at pH 7.4 showed no changes associated to Mo(IV)-OP coordination, indicating that molybdenocene–oligonucleotide binding interactions are centered, most likely, on the bases. Cyclic voltammetry titration showed a 4.9% of molybdenocene–oligonucleotide interaction. This implicates that possible binding interactions with DNA are weak.     

Snake venom cardiotoxin induces G-actin polymerization

Snake venom cardiotoxin showed the ability to induce polymerization of G-actin from rabbit skeletal muscle in a low ionic strength buffer composed of 0.2 mM CaCl2/0.2 mM ATP/0.5 mM mercaptoethanol/2.0 mM Tris-HCl, pH 8.0. The activity was enhanced greatly when 0.4 mM MgCl2 was present in the buffer and could be inhibited if G-actin was preincubated with deoxyribonuclease I. Furthermore, the DNAase could also partially depolymerize actin polymer previously formed by the interaction of G-actin with the toxin.     

An amylase from fresh fruiting bodies of the monkey head mushroom Hericium Erinaceum

An amylase with a molecular mass of 55 kDa and an N-terminal sequence exhibiting similarity to enzyme from Bacteroides thetaitaomicron was isolated from fruiting bodies of the monkey head mushroom Hericium erinaceum. The purification scheme included extraction with distilled water, ion exchange chromatography on DEAE-cellulose and SP-sepharose, and gel filtration by FPLC on Superdex 75. The amylase of H. erinaceum was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and eluted with 0.2 M NaCl in the same buffer. The enzyme was subsequently adsorbed on SP-Sepharose in 10 mM ammonium acetate buffer (pH 4.5) and eluted with 0.3 M NaCl in the same buffer. This fraction was subsequently subjected to gel filtration on Superdex 75. The first peak eluted had a molecular mass of 55 kDa in SDS-PAGE. The amylase of H. erinaceum exhibited a pH optimum of 4.6 and a temperature optimum of 40°C. The enzyme activity was enhanced by Mn²⁺ and Fe³⁺ ions, but inhibited by Hg²⁺ ions.     

Calcium binding to intestinal goblet cell mucin.

1. Binding of Ca-2+ to goblet cell mucin of rat small intestine was studied using equilibrium dialysis against 0.01 M Tris/HCl buffer (pH 7.4) and tracer amounts of 45-CaCl2. Binding was found to reach saturation at a Ca free -2+ concentration of 0.1--1.0 mM, to be independent of temperature (4-37 degrees C), and to increase with increasing pH (5.0-8.7). At low concentrations of Ca free -2+ (smaller than 0.03 mM) the binding curve was sigmoidal, suggesting positive cooperativity of binding sites and a possible change in the tertiary structure of the mucin. Binding was markedly reduced, and sigmoidicity abolished, by removal of sialic acid from the mucin, or by adding 0.14 M NaCl to the dialysis medium. This latter finding suggests that, in vivo, other cations would compete for Ca-2+ binding ligands. 2. Under conditions mimicking those used for binding studies, CaCl2 (10- minus 5 M) was found to cause a small increase (0.03 units) in the absorbance of mucin solutions, especially in the ultraviolet region, possibly indicating increased light scattering. No change in the solubility of the mucin was observed after the addition of CaCl2 (10- minus 6-10- minus 4 M). A significant decrease in viscosity of the mucin was noted, however, with the addition of CaCl2 (10- minus 6-10- minus 2 M). Together with the binding data, these findings suggested that during binding, Ca-2+ combines with negative charges on goblet cell mucin (especially those of sialic acid carboxyl groups) and induces contraction or folding of the macromolecule which promotes cooperative cation binding. No evidence was obtained to suggest that CaCl2 caused precipitation, polymerization or gelation of the mucin in 0.01 M Tris/HCl.?     

Isolation of lectins from hemolymph of decapod crustaceans by adsorption on formalinized erythrocytes

Hemolymph of decapod crustaceans contains lectins of important specificity. An isolation procedure, based on adsorption of hemolymph lectins on red blood cells (RBC) fixed with formaldehyde, is described. Hemolymph is let to clot for 3 h at 22-28 degrees C (RT) and for 24 h at 5 degrees C; centrifuged at 13000 g for 30 min; filtered through 5-microm filters; diluted with an equal volume of 50 mM NaCl, 100 mM CaCl(2); supplemented with protease as well as phenoloxidase inhibitors; centrifuged at 13000 g for 20 min. Formalinized RBC (FRBC) are mixed with diluted hemolymph to a suspension of about 20% v/v FRBC. After incubation for 30 min at RT, FRBC are washed five times with 150 mM NaCl, 10 mM CaCl(2). The lectins adsorbed on FRBC are desorbed using either 100-500 mM of carbohydrate solutions in 0.9% NaCl or 50 mM Tris-HCl buffer, pH 8.0 containing 100 mM NaCl and 20 mM entylenediaminetetraacetate (EDTA). The procedure is efficient in isolating the hemolymph lectins of the decapods Liocarcinus depurator and Potamon potamios.     

Flocculation and adsorption of enzymes during growth of a moderate halophile, Micrococcus varians var. Halophilus.

Flocculation of a moderate halophile, Micrococcus varians ATCC 2197, occurred during growth in complex medium containing 3 M NaCl and a concentration of MgSO4 and KH2PO4 greater than 40 and 14 mM, respectively. Extracellular nuclease activity was absent in the flocculated cultures. Repeated washing of flocs by Mg2+-free Tris buffer containing 3 M NaCl, lowering of pH value of floc suspension below 6.3, or addition of ethylenediaminetetraacetic acid resulted in complete dissociation of the flocs and release of Mg2+ ions as well as nuclease and amylase. Inhibition of extracellular enzyme production accompanied by flocculation appeared to be the result of adsorption of enzyme proteins to surfaces of the flocs, but not of inhibition of biosynthesis. Floc formation could also occur in media containing 18 mM CaCl2 and 3.0 mM KH2PO4, but the Ca flocs were not deflocculated by washing with Ca2+-free buffer, suggesting that the affinity of Ca2+ for cell envelopes was stronger than that of Mg2+. It was also observed that most halophilic Planococcus and Micrococcus flocculated in the presence of MgSO4 and phosphate but halophilic Pseudomonas, Acinetobacter, and Bacillus did not.     

重组酶法定量分析吡咯喹啉醌

用ＤＥＡＥ－ｓｅｐｈａｃｅｌ和ＣＭ－ｃｅｌｕｌｏｓｅ柱层析的方法从Ｃｏｍａｍｏｎａｓｔｅｓｔｏｓｔｅｒｏｎｉ菌体中纯化得到一定纯度的脱辅基的乙醇脱氢酶。在含３ｍＭＣａＣｌ２的Ｔｒｉｓ／ＨＣｌ缓冲液（２０ｍＭ,ｐＨ７．０）中,该酶能与ＰＱＱ重组成有活性的全酶,测出的全酶活性大小与外加ＰＱＱ的量成正比,从而定量分析ＰＱＱ。该法专一、灵敏、可靠。     

Analytical grade C-phycocyanin obtained by a single-step purification process

C-phycocyanin (C-PC) is a phycobiliprotein that can be used as a natural blue dye in the food and cosmetic industries, as a biomarker or as an agent in medical treatments, depending on its purity grade. Here we described for the first time a single-step purification process of C-PC extracted from the wet biomass of Spirulina (Arthrospira) platensis LEB-52 using ion exchange chromatography with pH gradient elution. Different conditions varying the elution buffers and volumes, the loading pH and the addition of salt in the elution buffer were studied. The chromatographic condition that resulted in high recovery and purity consisted in equilibration and washing with 0.025 mol/L Tris-HCl buffer pH 6.5 and elution combining a step with 0.08 mol/L NaCl in 0.025 mol/L Tris-HCl buffer pH 6.5 and a pH gradient elution with 0.05 mol/L citrate buffer pH 6.2–3.0. This process resulted in C-PC with purities of 4.2 and 3.5 with recoveries of 32.6 and 49.5 %, respectively, in one purification step.     

Specific adsorption of a platelet membrane glycoprotein by human insoluble collagen

A platelet membrane glycoprotein, 61 kDa, has been identified, which binds specifically to insoluble collagen. The detection of this protein was accomplished by incubating radiolabeled Triton-solubilized platelet supernatant with insoluble collagen, and, after washing the collagen pellet, extracting the 61-kDa glycoprotein from the pellet with sodium dodecyl sulfate buffer. The optimal conditions for specific binding were incubation of 120 micrograms of total platelet supernatant protein with 2 mg of collagen at 4 degrees C for 0.5 h in 0.5 ml of the incubating buffer (20 mM Tris, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, and 0.2% Triton, pH 7.4). The 61-kDa glycoprotein is cleaved by trypsin into a major peptide (44 kDa) and a smaller peptide(s) linked together by disulfide bonds in a molecule which still binds to collagen. When intact platelets are treated first with trypsin and then with dithiothreitol, the 44-kDa peptide is released and was shown to bind to collagen. We conclude that the 61-kDa glycoprotein is a platelet membrane protein which specifically interacts through its extracellular domain with insoluble collagen, and, thus, must be considered as a possible component of the initial platelet-matrix adhesion process which leads to platelet aggregation in vivo.     

A novel ribonuclease with antiproliferative activity from fresh fruiting bodies of the edible mushroom Hypsizigus marmoreus

An 18-kDa ribonuclease (RNase) with a novel N-terminal sequence was purified from fresh fruiting bodies of the mushroom Hypsizigus marmoreus. The purification protocol comprised ion exchange chromatography on DEAE cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose and Q-Sepharose and gel filtration by fast protein liquid chromatography on Superdex 75. The starting buffer was 10 mM Tris-HCl buffer (pH 7.2), 10 mM Tris-HCl buffer (pH 7.2), 10 mM NH(4)OAc buffer (pH 5), 10 mM NH(4)HCO(3) buffer (pH 9.4) and 200 mM NH(4)HCO(3) (pH 8.5), respectively. Absorbed proteins were desorbed using NaCl added to the starting buffer. A 42-fold purification of the enzyme was achieved. The RNase was unadsorbed on DEAE cellulose, Affi-gel blue gel and CM-cellulose but adsorbed on Q-Sepharose. It exhibited maximal RNase activity at pH 5 and 70 degrees C. Some RNase activity was detectable at 100 degrees C. It demonstrated the highest ribonucleolytic activity (196 U/mg) toward poly C, the next highest activity (126 U/mg) toward poly A, and much weaker activity toward poly U (48 U/mg) and poly G (41 U/mg). The RNase inhibited [(3)H-methyl]-thymidine uptake by leukemia L1210 cells with an IC(50) of 60 microM.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Purification and properties of a magnesium-dependent endodeoxyribonuclease endogenous to rat-liver nuclei

An Mg2+-dependent endonuclease has been purified from a 0.6 M NaCl extract of rat-liver nuclei by a series of chromatographic procedures and finally by isoelectric focusing (IEF) electrophoresis. The nuclease fraction prepared by the IEF electrophoresis (IEF fraction) was shown to have a pI value of 7.1 and to migrate as a single band to a molecular-weight position of 36,500 on SDS-polyacrylamide gel. The IEF fraction was subjected to a sedimentation analysis. In a hypotonic buffer (10 mM Tris), the nuclease activity sedimented to have an S value of 4.1 S. However, in an isotonic buffer (0.15 M NaCl), this fraction exhibited two activity peaks of 2.8 and 4.3 S. In a hypertonic buffer (0.3 M NaCl), almost all of the nuclease activity sedimented at 2.7-2.8 S. In this connection, values of 2.8 and 4.3 S were determined to correspond to molecular weights of about 36,000 and 70,000, respectively. Thus, an Mg2+-dependent endonuclease, endogenous to rat-liver nuclei, has been inferred to exist in the reversible form of a monomer/homodimer as its native state. Moreover, the IEF fraction formed single-strand nicks more rapidly than double-strand cuts in pBR322 DNA, and preferentially produced deoxyguanosine 5'-monophosphate termini in the DNA at an early incubation time. In addition, RNAase activity was not detected in this fraction.     

Two latent metalloproteases of human articular cartilage that digest proteoglycan

Human articular cartilage contains very low levels of metalloprotease activity; the activity in 1 g of cartilage is approximately equivalent to the activity of 1 microgram of trypsin. Development of a sensitive assay, based on the digestion of radioactive proteoglycan, has made it possible to study protease activity in 1-2-g specimens of cartilage. Cartilage was extracted with Tris buffer in the cold and with Tris buffer containing 10 mM CaCl2 at 60 degrees C. The extracts were passed through Sepharose 6B; two major and two minor metalloprotease activities were detected. A neutral metalloprotease activity, pH optimum 7.4, was found as a latent form of Mr = 56,000. It could be activated with aminophenylmercuric acetate or trypsin with a resultant decrease of Mr to 40,000. An acid metalloprotease, pH optimum 5.3, also occurred as a latent form of Mr = 50,000. Activation converted this to Mr = 35,000. Removal of calcium ions by dialysis reduced the activity of the neutral enzyme by 80-85% and of the acid enzyme by 100%. Both activities were restored by 10 mM Ca2+. Both enzymes were completely inhibited by 1 mM o-phenanthroline in the presence of excess calcium. This inhibition was overcome by 1 mM Zn2+ and, to a lesser extent, by Co2+. These proteases may be important in the metabolism of the cartilage matrix and in its destruction in osteoarthritis.