Edith R. Schwartz

Delay of UV-induced eye lens protein damage in guinea pigs by dietary ascorbate.

Large accumulations of postsynthetically oxidized proteins are observed in the aged and cataractous eye lens. Ascorbate has previously been used to delay photooxidative damage in vitro. The goals of this study were to confirm that dietary ascorbate can be used to enhance lens ascorbate levels and to determine if lenses with enhanced ascorbate can better withstand photooxidative stress in the form of ultraviolet (UV) light exposure. Guinea pigs were placed on high dietary ascorbate (HDA), 50 mg/day, and low dietary ascorbate (LDA), 2 mg/day, for 21 weeks. Lenses from HDA animals were found to contain 3.3 times more ascorbate than LDA animals. Prior to irradiation, SDS-PAGE protein profiles and exopeptidase activity in HDA and LDA lens soluble proteins were indistinguishable. However upon exposure to UV light, more protein damage (e.g., high-molecular-weight aggregates and enhanced loss of exopeptidase activity) was seen in lens preparations from LDA as compared to HDA animals. These results suggest that ascorbate protects lens components against cataract-like and age-related postsynthetic changes in vivo. As in previous tests on lens preparations, attenuated exopeptidase activity was observed before protein aggregation.

Effect of vitamins C and E on sulfated proteoglycan metabolism and sulfatase and phosphatase activities in organ cultures of human cartilage.

SummaryPrevious studies conducted in cell culture utilizing chondrocytes isolated from human articular cartilage showed that inclusion of ascorbic acid in the nutrient medium resulted in an increase in the net biosynthesis of sulfated proteoglycans concomitant with a reduction in the activities of arylsulfatases A and B. This suggested a possible connection between the stability of the sulfated macromolecules and the activity of these desulfating enzymes. To examine whether this relationship also was operative in a more native milieu, the effect of ascorbate on these parameters was measured in an organ culture system using slices of human articular cartilage where the structural extracellular framework surrounding the chondrocytes is comparable to that present in vivo. In addition, since numerous studies have shown that vitamin E inhibits lysosomal enzymes, the effect of alpha-tocopherol on these parameters was also investigated.Concurrent with an inhibition of arylsulfatase A and B activities, an increase in sulfated proteoglycan biosynthesis per unit of DNA as reflected in35S-sulfate uptake was present in all cartilage specimens examined 6 days after the introduction of vitamin C in the culture fluid. A larger fraction of the newly synthesized sulfated macromolecules was incorporated into the matrix of the tissue in samples maintained in the vitamin-supplemented cultures.Supplementation of the organ culture medium with vitamin E resulted in the inhibition of arylsulfatase A and no change in arylsulfatase B activities. In contrast to the action of vitamin C which stimulated acid phosphatase activity, a potent inhibition of this enzyme was effected by vitamin E.Sulfated proteoglycan content was greatly enhanced by alpha-tocopherol, although the vitamin did not alter the distribution of the newly synthesized molecules between the tissue and medium fractions.Inhibition of acid phosphatase and arylsulfatase A by alpha-tocopherol was evident in tissue lysates and partially purified enzyme preparations. This implies a direct interaction between the enzymes and vitamin E.The actions of alpha-tocopherol and ascorbate on cartilage enzymes and structural components were dissimilar. Both vitamins, however, appeared to enhance the stability of sulfated proteoglycans in the complex structure comprising articular cartilage.

Proteoglycan analysis by high-performance liquid chromatography

Abstract Three different well-characterized preparations of proteoglycan subunits were analyzed by high-performance liquid chromatography on a silica-based material bonded with an amide phase. The biochemical integrity of the proteoglycan subunits was retained during this procedure. The high sensitivity coupled with the increased speed of high-performance liquid chromatography will permit rapid analysis and comparisons of very small specimens.

Characteristics of human chondrocyte cultures in completely defined medium

SummaryChondrocytes 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. The35S-labeled proteoglycans isolated from the culture medium had elution profiles on high pressure liquid chromatography (HPCL) similar to those observed from 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.

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.