Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.     

Influence of methods of preparation on the infectivity, agglutination, activity, and ultrastructure of bloodstream trypanosomes

Contrary to the reports of others, the surface coat of Trypanosoma brucei brucei was not removed by extensive washing in the various media investigated; in fact, other than a deformed profile when washed in saline, ultrastructure was little affected by column separation and washing in any of these media. Washing in saline increased the agglutinability but reduced the activity and infectivity of the organisms. Washing in bicine-buffered saline-glucose did not impair activity or infectivity but increased agglutinability, albeit to a lesser extent than after washing in phosphate-buffered saline-glucose. The inclusion of 0.1% serum or plasma in the washing medium (phosphate-buffered saline-glucose or bicine-buffered saline-glucose) increased the activity of the T. b. brucei and did not increase agglutinability or impair infectivity. T. congolense and T. vivax were more susceptible to ionic strength changes than were T. b. brucei or T. lewisi, in that not only was their activity impaired, but they began to aggregate on standing in lower ionic strength buffers.