Robert A. Greenwald

Human cartilage lysozyme

The lysozyme content of human cartilage was measured by incubation of lyophilized, powdered cartilage in a variety of buffers and salt solutions, and the factors controlling the binding of lysozyme within cartilage were studied. Lysozyme was extracted from hyaline cartilage by buffers of pH greater than 9.0 by solutions 1 M in monovalent cations, and by solutions 0.12-0.40 M in divalent cations. The ability of cations to extract lysozyme from cartilage agreed with their known affinities for binding to chondroitin sulfate. The total extractable lysozyme content of five samples of human costal cartilage ranged from 1.45 to 3.36 mug lysozyme per mg of cartilage; for five samples of hyaline cartilage from peripheral joints the range was 0.80-3.03 mug lysozyme per mg of cartilage. Cartilage incubated in excess exogenous lysozyme could bind 0.053 equivalents of lysozyme per equivalent of chondroitin sulfate. Fibrocartilage and synovium from knee joints yielded no detectable lysozyme, despite the fact that synovium, a tissue rich in lysosomes, contained measurable quantities of beta-glucuronidase. Lysozyme extraction from cartilage was not augmented by incubation with streptolysin S. When incubation was carried out with mild extraction techniques, lysozyme extraction from cartilage tended to parallel uronic acid release, both as a function of time and from one specimen to another. The active material as lysozyme. Lysozyme occurs in human hyaline cartilage as a counterion to polyanionic glycosaminoglycans. Carextracted from cartilage met five criteria for identification tilage lysozyme appears to be extracellular and nonlysosomal. Degradation of cartilage may contribute to the increased serum and synovial fluid lysozyme levels often present in patients with rheumatoid arthritis.

Effect of agarose variability on the measurement of lysozyme activity

Lysozyme assays are often performed by a diffusion technique utilizing agarose gels impregnated with substrate organisms (lysoplates), but the results differ greatly from those obtained with spectrophotometric or immunologic techniques. We have investigated the effect of agarose composition on the lysoplate assay utilizing 10 different gels varying in ionic parameters. Standard curves generated with purified human lysozyme solutions were parallel, but the diameters of the zones of lysis varied inversely with gel sulfate content. The different agaroses had variable effects on determinations of normal serum lysozyme, and the results obtained on any given gel agreed with neither those found on other gels nor with independent assay in another system. The lysoplate assay should be utilized only in those laboratories that can obtain uniform agarose preparations and extensively calibrate normal ranges for their gels.

Complex formation between lysozyme and cartilage proteoglycans

Abstract Density-gradient centrifugation of dissociative extracts of cartilage yields a proteoglycan which forms an ionic complex with lysozyme, a natural component of cartilage whose function in that tissue is unknown. The progress of the reaction is monitored by assay of the supernatant hexuronate content after addition of the precipitant. The lysozyme-proteoglycan complex is solubilized by addition of salts or by alkalinization, but the salt concentration required is greater than that needed to disperse a complex of lysozyme and chondroitin sulfate. Since it appears that the chondroitin sulfate portion of the proteoglycan molecule participates in complex formation with lysozyme, the charge distribution in the proteoglycan probably differs from that in the linear glycosaminoglycan. Cartilage lysozyme could exist in vivo as part of an ionic complex involving proteoglycans, but on a stoichiometric basis, it is unlikely that lysozyme plays a major role in maintenance of the structural integrity of the tissue.

General Principles of IRB Review

In the preceding chapters, we have discussed the historical, legal, and ethical background that led to the formation of the IRB system, and we have outlined how such committees should be constituted and financed. In Section II, we discuss in greater detail the principles and mechanisms of IRB review, the preparation and evaluation of informed consent procedures and forms, the procedures applicable to new drugs and medical devices, and the continuing review of approved research. These chapters are directed to a diverse audience—lay members of IRB committees, nonscientific professionals, and administrative staff—and, thus, the discussions will cover a broad range of issues as befits the multidisciplinary nature of IRB review.