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.     

Preparation and characterization of (9-methyladenine)triammineplatinum(II) and trans-dihydroxo(9-methyladenine)triammineplatinum(IV) complexes

The preparation is reported of [(NH₃)₃Pt(9- MeA)] X₂ (9-MeA = 9-methyladenine) with XCl (1a) and XClO₄ (1b) and of trans-[(OH)₂Pt(NH₃)₃- (9-MeA)]X₂ with XCl (2a) and XClO₄ (2b), and the crystal structure of 1b. [(NH₃)₃Pt(C₆H₇N₅)](ClO₄)₂ crystallizes in space group P2₁/n with a = 20.810(7) Å, b = 7.697(3) Å, c = 10.567(4) Å, β = 91.57(6)°, Z = 4. The structure was refined to R = 0.054, R_w = 0.063. In all four compounds Pt coordination is through N7 of 9-MeA, as is evident from ³J coupling between H8 of the adenine ring and ¹⁹⁵Pt. Pt(II) and Pt(IV) complexes can be differentiated on the basis of different ³J values, larger for Pt(II) than for Pt(IV) by a factor of 1.57 (av). In Me₂SO-d₆, hydrogen bonding occurs between Cl^? and C(8)H of 9-MeA as weil as between Cl^? and the NH₃ groups in the case of the Pt(II) complex 1a. Protonation of the 9-MeA ligands was followed using ¹H NMR spectroscopy and pK_a values for the N1 protonated 9-MeA ligands were determined in D₂O. They are 1.9 for 1a and 1.8 for 2a, which compares with 4.5 for the non-platinated 9-MeA. Possible consequences for hydrogen bonding with the complementary bases thymine or uracil are discussed briefly. Protonation of the OH groups in the Pt(IV) complexes has been shown not to occur above pH 1.     

X-ray structure of a mono(1-methylthyminato) complex of cisplatin,chloro-(1-methylthyminato-N3)-cis-diammineplatinum(II) monohydrate

The crystal structure of chloro-(1-methyltyminato- N3)-cis-diammineplatinum(II) monohydrate, cis- (NH₃)₂Pt(C₆H₇N₂O₂)Cl·H₂O, is reported. The compound crystallizes in space group P$\text{1}$ with a = 6.911(2) Å, b = 8.598(3) Å, c = 11.464(4) Å, α = 100.13(3)°, β = 120.03(3)°, γ = 93.16(3)°, Z = 2. The structure was refined to R = 0.048 and R_w = 0.057. The compound contains the deprotonated 1-methylthymine ligand coordinated to Pt through N3 (1.973(10) Å). This distance represents the shortest Pt-N3(pyrimidine-2.4-dione) bond reported so far. The two PtNH₃ bond lengths differ significantly: PtNH₃ (trans to Cl) is longer (2.052(10) Å) than PtNH₃ (trans to N3 of 1-MeT) (2.002(11) Å). The PtCl distance (2.326(3) Å) is normal, as is the large dihedral angle between the Pt coordination plane and the nucleobase (76.5°).     

Deuterium nuclear magnetic resonance study of the interaction of branched chain compounds (phytanic acid, phytol) with a phospholipid model membrane

Deuterium nuclear magnetic resonance (2H-NMR) spectra have been determined for 50 wt% aqueous dispersions of 1-palmitoyl(stearoyl)-2-[2H31]palmitoyl-sn-glycero-3-phosphocho lin e (PC-d31) containing 20 mol% of the isoprenoid compounds phytol or phytanic acid over the temperature range -5-55 degrees C. Concentration effects of the isoprenoid compounds are also reported. First moments (M1) and order parameters were calculated from the spectra. 20 Mol% of either branched chain compound causes an approximate 9% increase in the mean order parameter SCD. Significant effects are seen on the PC-d31 phase behavior. 20 Mol% of either branched chain compound causes the gel to liquid crystalline onset temperature (Ts) to drop to 28 degrees C from 38 degrees C for PC-d31 alone, as seen from the temperature dependent M1 values. The melting range ([Tl--Ts]) is congruent to 1.5 degrees C for PC-d31 and congruent to 11 degrees C for PC-d31 containing 20 mol% of the branched chain compounds. This is in direct contrast to their straight chain analogues, hexadecanol and palmitic acid, which have been shown to elevate the phase transition temperature. The isoprenoid compounds cause significant disruption of the gel phase, forcing nearest neighbor phospholipid chains apart. Transverse relaxation times (T2e, the time constant for decay of the quandrupolar echo) have been determined over the temperature range -5-50 degrees C. Possible explanation for the effect of the isoprenoid compounds on the dynamic structure of phospholipids in the bilayer are proffered.