Biomedical subjects
E R Schwartz
Publications and source records attributed to E R Schwartz.
Animal models: a means to study the pathogenesis of osteoarthritis.
A number of animal models have been developed to investigate the pathogenesis of osteoarthritis. In general, rabbits, guinea pigs, and dogs have been the animals of choice, although rats, mice, and birds have also been used. The techniques used to induce disease development include: destabilization of a knee joint either by extra- or intraarticular surgical manipulation; immobilization of the knee joint to alter load impact and distribution; or introduction of a foreign substance into the joint space. The success of a particular animal model rests in the ability to parallel, on a temporal basis, the biological changes which characterize osteoarthritic development as seen in the human population. Macroscopic, microscopic, and biochemical evaluations then permit the detailing of the steps which lead to cartilage erosion. In this manner, link proteins have been found to be deficient in osteoarthritic guinea pig cartilage.
The phosphorylation of human link proteins.
Three link proteins of 48,44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain 32P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO4/mole link protein.
Monensin stimulation of arylsulfatase B activity in human chondrocytes.
Secreted and intracellular arylsulfatase B (ASB) activities were measured in normal and osteoarthritic (OA) human chondrocyte cultures in the absence and presence of monensin, ammonium chloride, and chloroquine. Of the three agents added, only monensin produced a significant stimulation of secreted enzyme activity. Osteoarthritic cells consistently exhibited a three-fold higher level of secreted specific ASB activity than did normal cells, with or without monensin. When compared with normal cells, OA cells also consistently exhibited a twofold heightened intracellular specific enzyme activity both in the absence or presence of monensin. With increasing dosage of monensin, secreted and intracellular ASB activity increased for both OA and normal cells. Total enzyme activity of secreted and intracellular ASB was found to be cell density dependent. No inhibition of secreted or intracellular ASB activity was observed for sparsely plated cultures. In contrast to sparse cultures, an inhibition of secreted ASB, with or without monensin, was observed in densely plated cultures. Intracellular total activity was not inhibited by high-density cultures. Secreted ASB activity was found to be time-dependent after passage. Enzyme activity was maximal at 6 h in both OA and normal cells and decreased by the end of 24 h both in serum-free medium and in serum-free medium with monensin. When compared with normal cells, OA cells expressed higher levels of ASB activity under all test conditions. This heightened activity therefore appears to be a property inherent in the OA chondrocyte.
Phosphorylation of proteoglycans. Identification of phosphorylation sites in chondroitin sulfate-rich region of core protein.
Serine residues, which are the sites of phosphorylation in proteoglycans, were demonstrated to be located on chondroitin sulfate-containing peptides. These peptides appeared to be derived primarily from the chondroitin sulfate-rich region of the proteoglycan core protein. The localization of phosphate moieties in the chondroitin sulfate-containing peptides was observed in all experiments. The phosphate moieties were retained on chondroitin sulfate-containing peptides after the protein core was treated with either papain or trypsin. Two phosphopeptide preparations, derived from chondroitin sulfate-containing peptides of proteoglycan subunits, were extensively purified. These 2 phosphopeptide preparations were shown by carbohydrate analysis to be free of keratan sulfate-containing peptides or peptides from the hyaluronic acid binding region of the core protein. One of the phosphopeptide preparations had a phosphate: serine molar ratio of 0.40. This indicated that nearly one-half of the serine residues were phosphorylated rather than glycosylated. Peptides derived from the core protein that contained keratan sulfate had no phosphate moieties.
Characterization of human bone cells in culture.
Cultures of human bone cells were established, maintained, and characterized with respect to several metabolic parameters. These studies were undertaken with a view to using the bone culture system as a means of studying mechanisms of bone metabolism. The donor patients' ages ranged from 1 to 90 years and their disease states included congenital limb anomalies, exostosis, and osteo- and rheumatoid arthritis. Cultures were maintained up to 5 months. The osteoblast-like character of these cells was confirmed with the use of measurements applied to bone cells from other systems. Analyses showed that (a) the cells' appearance resembled that of cultured osteoblasts from other animal sources, b) intracellular cAMP was stimulated by human parathyroid hormone, c) osteocalcin was detected in the medium of all tested bone cell cultures and its production was found to be stimulated by 1,25-dihydroxycholecalciferol, and d) newly synthesized collagen was almost exclusively type I. In contrast, cultures of human fibroblasts, established in one instance from tissue specimens of the same donor patient, grew faster, reached a higher limiting density, and produced a greater proportion of type III collagen than the corresponding bone cells. Furthermore, fibroblasts did not accumulate osteocalcin in their culture medium. The conditions described in this report to maintain human bone cells in culture should provide a suitable test system to study the regulation of human bone metabolism.
The pediatrician's approach to the preschool child with language delay.
A systematic approach to the child with suspected language disorder includes screening of expressive language, receptive language, general development, and hearing. Various screening approaches and tools are discussed. Diagnostic categories and associated referral patterns are recommended.
Biochemical characterizations of human osteoblasts in culture.
Explore the source record for details and available documents.
Phosphorylation of proteoglycans from human articular cartilage.
Previous studies have shown that sulfated proteoglycans from human articular and epiphyseal cartilage were phosphorylated. These macromolecules contribute to the stiffness and resiliency of this tissue. We demonstrate here that the phosphate moieties are an integral part of proteoglycan subunits. Specifically, evidence is presented which indicates that proteoglycan monomers contain 3 to 4 phosphate moieties per core protein and that these appear to exist as phosphoserine residues. Furthermore, the data illustrate that human articular cartilage also contains more than 20 different phosphoproteins, some of which are closely associated with proteoglycan aggregates. Proteoglycan subunits were purified from extracts of articular cartilage or from media fractions which had been used to label tissue specimens with 32P-orthophosphate. Chemical and radiographic analyses revealed that the phosphate concentration with respect to sulfate and uronic acid content remained constant when purified proteoglycan monomers were subjected to equilibrium ultracentrifugation and size-exclusion chromatography. That the phosphate moieties were bound to proteoglycan monomers via monoester linkages was indicated by the release of 32P-orthophosphate from proteoglycan subunits incubated under mild alkaline conditions or reacted with acid or alkaline phosphatases. Identification of serine residues in the core protein as the sites of phosphorylation was made by autoradiography of thin layer plates on which hydrolyzed samples of purified 32P-proteoglycan subunits had been subjected to 2-dimensional electrophoresis/chromatography. Quantification of 3 to 4 phosphate moieties per core protein of 200,000 daltons was made by chemical analysis of inorganic phosphate released from proteoglycans by acid hydrolysis.
Phosphorylation of proteoglycans from human articular cartilage by a cAMP-dependent protein kinase.
Purified proteoglycan subunits from human articular, bovine articular and nasal cartilages, and a rat chondrosarcoma were phosphorylated in vitro by beef heart cAMP-dependent protein kinase in the presence of gamma 32P-ATP. In these experiments, a maximum of 1.7 moles of 32P were incorporated per mole of proteoglycan from human cartilage. Phosphorylation was dependent on the presence of cAMP. Analysis by autoradiography revealed that serine residues in the core protein of the proteoglycan were the sites of phosphorylation. Treatment of proteoglycan subunits with chondroitinase ABC and alkaline phosphatase prior to reaction with cAMP-dependent protein kinase increased the incorporation of 32P by 12-30% when compared with untreated proteoglycans. These data indicate that proteoglycans in cartilage can be phosphorylated by cAMP-dependent protein kinase.
The modulation of osteoarthritic development by vitamins C and E.
Explore the source record for details and available documents.
Characteristics of human chondrocyte cultures in completely defined medium.
Chondrocytes derived from normal human adult articular cartilage were established and maintained for over 5 months in a completely defined medium without the addition of serum or any other growth factors. At the end of 5 months, these cells were still metabolically active. The cells incorporated [3H] thymidine into DNA, incorporated [35S] sulfate into proteoglycans, and exhibited lysosomal enzyme activities. The 35S-labeled proteoglycans isolated from the culture medium had elution profiles on high pressure liquid chromatography (HPLC) similar to those observed for proteoglycans from other mammalian sources. This self-contained growth competence may reflect a need produced by the unusual avascular and alymphatic character of articular cartilage.
Effect of ascorbate on lysosomal enzyme activities in guinea pig cartilage and adrenals.
The effect of ascorbic acid on adrenal and cartilage lysosomal enzyme activity was evaluated in guinea pigs maintained on either 2.4 mg, or 150 mg of ascorbate per day. When compared to adrenal enzyme activities in animals on high dietary levels of ascorbate, the adrenals of animals on 2.4 mg of ascorbate per day expressed 50% increase in the activities of acid phosphatase, arylsulfatase A, and arylsulfatase B (p less than 0.02). In cartilage, the activity of acid phosphatase was significantly elevated in animals on low dietary levels of ascorbate (p less than 0.01), but arylsulfatase A and arylsulfatase B activities were not affected by varying the dietary intake of ascorbate. These data indicate an inverse relationship between tissue vitamin C concentrations and lysosomal enzyme activities.
Experimentally-induced osteoarthritis in guinea pigs: effect of surgical procedure and dietary intake of vitamin C.
The effect of variation in dietary ascorbic acid on surgically induced osteoarthritis was examined in the stifle joints of guniea pigs. Two different surgical procedures were used to induce osteoarthritis in the right stifle joint of these animals. Guinea pigs were maintained either on a high (150 mg/day) or low (2.4 mg/day) dietary intake of vitamin C. Regardless of the surgical procedure used to induce osteoarthritis, the animals maintained on the high level of vitamin C consistently showed severe joint damage than animals on the low level of the vitamin.
Effect of vitamins C and E on sulfated proteoglycan metabolism and sulfatase and phosphatase activities in organ cultures of human cartilage.
Explore the source record for details and available documents.
Phosphorylation of proteoglycans in human articular cartilage.
A study of human articular cartilage indicated that componenet proteoglycans can be phosphorylated. Phosphorylation, also found in a specimen of human epiphysial cartilage, occurred when [gamma-32P]-ATP or 32Pi was included in the in vitro incubation medium. Treatment of the phosphorylated proteoglycans with chondroitinase and chondrosulfatases effectively removed the chondroitin sulfate without dephosphorylating the remaining molecule. Since phosphorylation could be effected in a totally chemically defined medium, it appears that the necessary enzyme systems for this reaction are contained entirely within chondrocytes.
Effect of ascorbic acid on arylsulfatase activities and sulfated proteoglycan metabolism in chondrocyte cultures.
A correlation between increased arylsulfatase activities and decreased sulfated proteoglycan content in human osteoarthritic articular cartilage suggested a possible interrelationship between these parameters. Since we had previously shown that ascorbate caused a decrease in levels of arylsulfatase A and B activities in normal chondrocyte cultures, the validity of the above relationship was examined by measuring the effect of vitamin C on the biosynthesis and distribution of 35S-labeled proteoglycans and arylsulfatase A and B activities in cell extracts of chondrocytes derived from normal and osteoarthritic tissue. Arylsulfatase A and B activities were found to be reduced in the presence of ascorbic acid in all normal and osteoarthritic cell lines examined when measured 3, 6, 10, and 13 days after the introduction of the vitamin in the culture medium. Acid phosphatase activity, on the other hand, was found to be elevated in the presence of ascorbate. The inhibitory effect by ascorbic acid on arylsulfatase activities could be reversed by withdrawing the vitamin from the nutrient medium. Addition of EDTA to the cell extracts before assay also reversed the inhibiton. Sulfated proteoglycan biosynthesis as reflected in 35S-sulfate uptake per milligram of DNA was significantly increased in the presence of ascorbic acid. The distribution of the newly synthesized molecules between the cell layer and medium fractions was altered. In the presence of ascorbate, more deposition into the cell layer of newly synthesized macromolecules occurred. These data suggest an inverse relationship between arylsulfatase activities and the stability of the newly synthesized sulfated proteoglycans in the extracellular matrix.
The effect of salicylate on prostaglandin levels in rabbit knees following inducement of osteoarthritic changes.
Surgical inducement of medial instability in the right knee of rabbits was used to produce joint changes which resemble those observed in human osteoarthritis. Ordinary tap water was supplied to half of the rabbits and tap water plus sodium salicylate to the others. Determinations of prostaglandin were made on the synovial fluid and cartilage from all rabbits five months after surgery. In both groups, the concentration of prostaglandin in synovial fluid was lower in the operated knees, but the total amount of prostaglandin was found to be approximately equal to that in the unoperated knees. The development of degenerative joint changes therefore was not accompanied by increases in prostaglandin content. Salicylate treatment did not alter this observation, however, it did reduce overall prostaglandin levels. These results suggest that prostaglandin interaction is not involved in osteoarthritic joint degeneration.