Abnormal high density lipoproteins from patients with liver disease regulate cholesterol metabolism in cultured human skin fibroblasts

Apolipoprotein B (apoB) of plasma low density lipoproteins (LDL) binds to high affinity receptors on many cell types. A minor subclass of high density lipoproteins (HDL), termed HDL1, which contains apoE but lacks apoB, binds to the same receptor. Bound lipoproteins are engulfed, degraded, and regulate intracellular cholesterol metabolism and receptor activity. The HDL of many patients with liver disease is rich in apoE. We tested the hypothesis that such patient HDL would reduce LDL binding and would themselves regulate cellular cholesterol metabolism. Normal HDL had little effect on binding, uptake, and degradation of 125I-labeled LDL by cultured human skin fibroblasts. Patient HDL (d 1.063-1.21 g/ml) inhibited these processes, and in 15 of the 25 samples studied there was more than 50% inhibition at 125I-labeled LDL and HDL protein concentrations of 10 micrograms/ml and 25 micrograms/ml, respectively. There was a significant negative correlation between the percentage of 125I-labeled LDL bound and the apoE content of the competing HDL (r = -0.54, P less than 0.01). Patient 125I-labeled HDL was also taken up and degraded by the fibroblasts, apparently through the LDL-receptor pathway, stimulated cellular cholesterol esterification, increased cell cholesteryl ester content, and suppressed cholesterol synthesis and receptor activity. We conclude that LDL catabolism by the receptor-mediated pathway may be impaired in liver disease and that patient HDL may deliver cholesterol to cells.     

Protection of ribosomal RNA from kethoxal in polyribosomes: Implication of specific sites in ribosome function

In an attempt to probe the topography of 5 S, 16 S and 23 S RNAs in a functionally engaged ribosome, polysomes were probed using the structure-sensitive, guanine-specifie reagent kethoxal. Reactivities of guanine residues at 38 specific ribosomal RNA sites in polysomes were compared with their corresponding reactivities in vacant 70 S ribosomes. No polysome-specific protection was seen for 5 S RNA. In 16 S RNA, positions 530, 693 or 1079, 966, 1338 and 1517 showed protection in polysomes; all of these sites have highly conserved primary and secondary structures, and include several methylated nucleotides. In 23 S RNA, polysome protection is seen at positions 277, 1071, 1475 or 2112, 2116 and 2751. We attribute polysome-specific protection either to direct contact of transfer RNA and/or messenger RNA with the protected sites or to tRNA and/or mRNA-induced changes in ribosome conformation involving the protected sites.     

Cholesterol biosynthesis in transplantable hepatomas: evidence for impairment of uptake and storage of dietary cholesterol

Cholesterol feeding inhibits cholesterol biosynthesis in normal but not in malignant liver tissue. It has been postulated that hepatomas have suffered a specific intracellular deletion of the cholesterol feedback control mechanism, but there is little direct evidence to support this hypothesis. Rats bearing Morris transplantable hepatomas were fed high cholesterol diets for periods of up to 21 days. Cholesterol biosynthesis, as expected, was suppressed in the normal liver but not in hepatomas. The livers accumulated large amounts of cholesteryl ester but the hepatomas showed little or no increase in ester content. Cholesterol-1alpha-(3)H was administered intragastrically to other tumor-bearing rats. Uptake of radioactivity by the tumors was much slower than by normal liver. Comparison of the specific activities of liver and tumor cholesterol with that of the plasma suggested that the liver took up dietary cholesterol selectively from the blood, while the appearance of radioactivity in the tumors could be explained by slow equilibration with plasma cholesterol. Our results suggest that the insensitivity of cholesterol biosynthesis to dietary cholesterol in hepatomas could be explained by an impairment in the uptake and storage of dietary cholesterol and that the concept of an intracellular deletion of the feedback mechanism requires further evidence.     

Effect of Diurnal Temperature Cycles on the Production of Aflatoxin

Exposures to short periods of high temperature (40 to 50 C) in each 24-hr diurnal temperature cycle (average temperature ca. 25 C) reduced growth of Aspergillus parasiticus and production and accumulation of the aflatoxins when compared with cultures held continuously at 25 C. In contrast, diurnal cycles with an average temperature of ca. 25 C but with minima as low as 10 C did not appreciably affect either growth or toxin production. The ratio of production of aflatoxin B to aflatoxin G increased as the maximal temperature was raised but remained essentially unchanged with decreasing minimal temperatures.     

Quantification of propranolol enantiomers in small blood samples from rats by reversed-phase high-performance liquid chromatography after chiral derivatization

A high-performance liquid chromatographic (HPLC) technique is described for quantification of R(+)- and S(−)-propranolol from 100-μl rat blood samples. The procedure involves chiral derivatization with tert.-butoxycarbonyl- -leucine anhydride to form diastereomeric propranolol- -leucine derivatives which are separated on a reversed-phase HPLC column. The method as previously reported has been modified for assaying serial blood microsamples obtained from the rat for pharmacokinetic studies. An internal standard, cyclopentyldesisopropylpropranolol, has been incorporated into the assay and several derivatization parameters have been altered. Standard curves for both enantiomers were linear over a 60-fold concentration range in 100-μl samples of whole rat blood (12.5–750 ng/ml; r=0.9992 for each enantiomer). Inter- and intra-assay variability was less than 12% for each enantiomer at 25 ng/ml. No enantiomeric interference or racemization was observed as a result of the derivatization. No analytical interference was noted from endogenous components in rat blood samples. Preliminary data from two male Sprague-Dawley rats given a 2.0 mg/kg intravenous dose of racemic propranolol revealed differential disposition of the two enantiomers. R(+)-Propranolol achieved higher initial concentration but was eliminated more rapidly than S(−)-propranolol. Terminal half-lives of R(+)- and S(−)-propranolol were 19.23 and 51.95 min, respectively, in one rat, and 14.50 and 52.07 min, respectively, in the other.