Showing papers by "Richard Bucala published in 1984"

Modification of DNA by reducing sugars: a possible mechanism for nucleic acid aging and age-related dysfunction in gene expression.

Abstract: Reducing sugars react nonenzymatically with protein amino groups to initiate a process called nonenzymatic browning. Long-lived proteins, such as collagen and the lens crystallins, accumulate sufficient modification in vivo that they acquire many of the chemical properties characteristic of aged proteins. We have obtained evidence that nucleic acids also can undergo nonenzymatic modification by sugars. Incubation of DNA or nucleotides with glucose 6-phosphate (Glc-6-P) produces spectral changes similar to those described for nonenzymatic browning proteins. The occurrence of chemical modification was verified by measuring the transfection efficiency of viral DNA after incubation with glucose and Glc-6-P. A loss of transfection potential occurred that was first order with respect to time and sugar concentration. The rate of inactivation by Glc-6-P was 25 times that of glucose; 8 days of incubation with 150 mM Glc-6-P decreased transfection by 4 orders of magnitude. Glc-6-P also produced strand scission in a time- and concentration-dependent manner. We conclude that glucose, Glc-6-P, and possibly other sugars can react with DNA to produce significant structural and biological alterations. Since nucleic acids are long-lived molecules in the resting cell, the accumulation of these addition products might be a mechanism for the decreased genetic viability characteristic of the aged organism.

Nonenzymatic addition of glucocorticoids to lens proteins in steroid-induced cataracts.

Abstract: A frequent manifestation of long-term glucocorticoid administration is the occurrence of posterior subcapsular cataracts. The molecular basis for this effect has not yet been elucidated. The addition of prednisolone to the rat lens in culture results in a time- and concentration-dependent lens opacification that correlates with the formation of covalent prednisolone-lens protein adducts. Prednisolone adduct formation was analyzed by [3H]prednisolone incorporation and by immunoprecipitation with antiserum specific for proteins modified by the nonenzymatic addition of prednisolone. In the rat lens, these adducts were localized in both the water-soluble and urea-soluble lens protein fractions. Gel electrophoresis and fluorography revealed that the most extensively modified proteins were two crystallins subunits. Lens proteins from 33 normal and cataractous human lenses were fractionated and analyzed for the presence of prednisolone-protein adducts by competitive radioimmunoassay. Adducts were detected only in those samples derived from glucocorticoid-induced cataractous lenses. We conclude that elevated glucocorticoid levels lead to the formation of glucocorticoid-lens protein adducts both in vitro and in vivo. Lens protein modification by glucocorticoids may lead to sufficient biochemical or structural alterations so as to result in cataract formation. The ability of glucocorticoids to form adducts with proteins in vivo also may play a role in some of the other toxic manifestations of long-term glucocorticoid therapy.

The reaction of 16α‐hydroxyestrone with erythrocytes in vitro and in vivo

Abstract: 16 alpha-Hydroxyestrone (16 alpha OHE ) has been shown previously to react with albumin, forming stable covalent adducts with lysine residues. The incubation of [3H]16 alpha OHE with whole blood also results in the incorporation of tritium into cells, with the rate of incorporation paralleling that of 16 alpha OHE -protein adduct formation. 32% of erythrocyte acid-precipitable radioactivity was found to be present within membrane proteins and electrophoresis demonstrated that several of these proteins are modified by [3H]16 alpha OHE . Membrane proteins from the red cells of ten individuals were reduced with sodium borohydride, acid hydrolyzed, and the 16 alpha OHE -lysine adducts purified by hydrophobic chromatography and reverse-phase high-pressure liquid chromatography. The amount of these adducts was quantitated by radioimmunoassay and found to be five times higher than the plasma levels of free 16 alpha OHE . This reflects most likely the accumulation of 16 alpha OHE -protein adducts during the course of the red cell life. Quantitation of 16 alpha OHE -protein adducts with other cellular proteins may contribute to our understanding of systemic lupus erythematosus, an autoimmune disease in which elevated levels of 16 alpha OHE occur.