Inhibition of selenoprotein synthesis by selenocysteine tRNA[Ser]Sec lacking isopentenyladenosine.

A common posttranscriptional modification of tRNA is the isopentenylation of adenosine at position 37, creating isopentenyladenosine (i(6)A). The role of this modified nucleoside in protein synthesis of higher eukaryotes is not well understood. Selenocysteyl (Sec) tRNA (tRNA([Ser]Sec)) decodes specific UGA codons and contains i(6)A. To address the role of the modified nucleoside in this tRNA, we constructed a site-specific mutation, which eliminates the site of isopentenylation, in the Xenopus tRNA([Ser]Sec) gene. Transfection of the mutant tRNA([Ser]Sec) gene resulted in 80% and 95% reduction in the expression of co-transfected selenoprotein genes encoding type I and II iodothyronine deiodinases, respectively. A similar decrease in type I deiodinase synthesis was observed when transfected cells were treated with lovastatin, an inhibitor of the biosynthesis of the isopentenyl moiety. Neither co-transfection with the mutant tRNA gene nor lovastatin treatment reduced type I deiodinase mRNA levels. Also, mutant tRNA expression did not alter initiation of translation or degradation of the type I deiodinase protein. Furthermore, isopentenylation of tRNA([Ser]Sec) was not required for synthesis of Sec on the tRNA. We conclude that isopentenylation of tRNA([Ser]Sec) is required for efficient translational decoding of UGA and synthesis of selenoproteins.

Identification and sequencing of two isopentenyladenosine-modified transfer RNAs from Chinese hamster ovary cells.

To determine the presence and identity of isopentenyladenosine-containing transfer RNAs (tRNAs) in a mammalian cell line, we adopted a novel method to isolate, clone and sequence these RNAs. This method was based on 3' polyadenylation of the tRNA prior to cDNA synthesis, PCR amplification, cloning and DNA sequencing. Using this unique procedure, we report the cloning and sequencing of the selenocysteine-tRNA and mitochondrial tryptophan-tRNA from Chinese hamster ovary cells which contain this specific tRNA modification. This new method will be useful in the identification of other tRNAs and other small RNAs where the primary sequence is unknown.

Effects of intracellular free cholesterol accumulation on macrophage viability: a model for foam cell death.

This study was designed to identify cellular responses associated with free cholesterol (FC) accumulation in model macrophage foam cells. Mouse peritoneal macrophages (MPMs) or J774 macrophages were loaded with cholesteryl esters using acetylated LDL and FC/phospholipid dispersions and were subsequently exposed to an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor. This treatment produced a rapid accumulation of cellular FC. The FC that accumulated due to ACAT inhibition was more readily available for efflux to 2-hydroxypropyl-beta-cyclodextrin (which removes cholesterol from the plasma membrane) than FC in untreated control cells. After a 3-hour exposure to an ACAT inhibitor, a significant increase in phospholipid synthesis was seen, followed by the leakage of LDH after 12 hours of treatment. We also observed, by electron and fluorescence microscopy, morphological indications of both apoptosis and necrosis in cells treated with an ACAT inhibitor. In addition, inhibition of ACAT for 48 hours resulted in the formation of FC crystals in MPMs but not in J774 cells. If compound 3beta-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A), which modulates intracellular trafficking of cholesterol, was added together with the ACAT inhibitor, each of the metabolic changes elicited by the accumulation of excess FC was either diminished or eliminated. The protective affect of U18666A was not due to a decrease in cellular FC concentrations, because cells treated with an ACAT inhibitor accumulated similar amounts of FC in the presence or absence of U18666A. Thus, treatment with U18666A results in the sequestering of FC in a pool that prevents it from causing various responses to FC deposition in macrophages. The metabolic changes that were produced when these model foam cells were treated with the ACAT inhibitor parallel the pathological events that have been shown to occur in the developing atherosclerotic plaque.

Compartmental isolation of cholesterol participating in the cytoplasmic cholesteryl ester cycle in Chinese hamster ovary 25-RA cells.

Using the Chinese hamster ovary cell line, 25-RA, we have demonstrated that lipoprotein-derived cholesterol and endogenously synthesized cholesterol are selectively differentiated with respect to their cellular locations. These cells lack sterol-mediated regulation, spontaneously storing large amounts of esterified cholesterol, which turns over with a half-time of 7.5 h. When [3H]cholesterol was provided to the cells in serum to trace cellular cholesterol, the specific activities of cellular free and esterified cholesterol (6238 +/- 273 and 5128 +/- 277 cpm/ microg, respectively) failed to equilibrate, indicating that bulk cellular free cholesterol is isolated from that participating in the cholesteryl ester cycle. Using [3H]acetate to trace the fate of endogenously synthesized cholesterol, a failure of equilibration was also observed (specific activities of free and esterified cholesterol = 280 +/- 37 and 458 +/- 8 cpm/ microg, respectively). The lower specific activity of the precursor indicates that endogenously synthesized cholesterol is preferentially esterified. When cells radiolabeled with [3H]acetate were post-incubated in the absence of radiolabel, the specific activity of the esterified cholesterol pool remained significantly higher than that of the free cholesterol, suggesting that cholesterol derived from hydrolysis of esterified cholesterol is preferentially re-esterified.

Cell toxicity induced by inhibition of acyl coenzyme A:cholesterol acyltransferase and accumulation of unesterified cholesterol.

Considerable evidence supports the involvement of acyl-CoA:cholesterol acyltransferase (ACAT) in the maintenance of intracellular cholesterol homeostasis. A number of recently developed ACAT inhibitors may have potential use as pharmacological agents to reduce the development of atherosclerosis. Recently, however, reports arose describing cytotoxic effects following administration of a specific ACAT inhibitor to experimental animals. In order to address the specific intracellular mechanisms involved with the cytotoxic effect, we examined the consequences of ACAT inhibition in cholesterol-enriched mouse peritoneal macrophages. Mouse peritoneal macrophages were cholesterol-enriched by incubation with acetylated low density lipoprotein and free cholesterol:phospholipid dispersions prior to the addition of an ACAT inhibitor, either Sandoz 58-035 or Pfizer CP-113,818. The adenine pool of the macrophages was radiolabeled prior to addition of the ACAT inhibitors, in order to monitor the release of radiolabeled adenine, a technique shown to be a sensitive method to monitor drug-induced toxicity. The ACAT inhibitors were added for up to 48 h and at concentrations up to 2 micrograms/ml. These conditions resulted in an approximately 2-fold increase in adenine release. The increase in cell toxicity paralleled an increase in the cellular free cholesterol content. Reducing the cellular free cholesterol content, by the addition of extracellular acceptors, decreased the cytotoxic effects of the ACAT inhibitors. Addition of an intracellular cholesterol transport inhibitor, either progesterone or U18666A, together with CP-113,818 blocked the toxic effect of CP-113,818. These results suggest that ACAT inhibition of cholesterol-enriched macrophages increases cell toxicity due to the buildup of cellular free cholesterol. Removal of free cholesterol by the addition of extracellular cholesterol acceptors or by blocking intracellular sterol transport relieves the ACAT inhibitor-induced toxicity.

Atherogenic and anti-atherogenic factors in the human diet.

New atherosclerosis causative factors and preventive modalities have been identified. Atherogenic factors include lipid oxidation products, such as cholesterol oxidation products, malonaldehyde and other aldehydes; trans-fatty acids; some saturated fatty acids (lauric, myristic and possibly palmitic acids); and myristic acid plus cholesterol. Lipid oxidation products are well suited to induce arterial damage, based on their known cytotoxic effects; evidence also indicates the possibility of plaque promotion and stimulation of thrombogenesis. Anti-atherogenic factors include antioxidants, fish oils and other polyunsaturates (if protected from oxidation), fibre and trace minerals such as copper, manganese, selenium and zinc. Iron is unique, being considered as both a potential promoter of atherosclerosis (component of ferritin, conceivably inducing lipid oxidation) and a possible anti-atherogenic component (of antioxidant enzyme catalase). It is apparent that an entire new series of research challenges has been uncovered.

Changes in triglyceride fatty acid composition of mammary secretions during involution.

Changes in fatty acid composition were determined for fat triglycerides from mammary secretions of 5 Holstein cows during the first 31 d of the nonlactating period. Proportions of short-chain (4:0 to 8:0) and medium-chain (10:0 to 14:0) fatty acids declined by about 50% during the first 3 d of involution, whereas proportions of stearic (18:0) and oleic acids (18:1) increased during the same period. Little change in proportions of those fatty acids occurred after d 3 of involution. Palmitic acid (16:0) was unchanged in proportion during involution. Fatty acid composition of mammary secretion triglycerides changed rapidly in the early nonlactating period, which may reflect a specific decline in de novo fatty acid synthesis in the involuting gland.