Biomedical subjects

D A Leonard

Publications and source records attributed to D A Leonard.

At least 37 records · Page 2Linked to original sources

PubMed1994

Evaluation of a rodent peroxisome proliferator in two species of freshwater fish: rainbow trout (Onchorynchus mykiss) and Japanese medaka (Oryzias latipes)

Rainbow trout (Onchorynchus mykiss) and Japanese medaka (Oryzias latipes) were exposed to the hypolipidemic drug gemfibrozil, a known rodent peroxisome proliferator. Trout were injected (i.p.) daily for 2 weeks at doses of 0, 46, 87, or 152 mg/kg/day. Medaka were exposed to the nominal concentrations of 0, 1.25, 2.5, or 5 ppm in water for 2 weeks in a static-renewal system. Peroxisome proliferation was assessed by measuring fatty acyl-CoA oxidase (FAO) activity, peroxisomal bifunctional enzyme (PBE) quantity, and changes in liver-to-body weight ratios (LWR). Results indicate that a mild peroxisome proliferative response was observed in rainbow trout (significant increases in FAO activity at all dose levels and in LWR at the highest dose level). Medaka demonstrated a significant increase in PBE at the highest dose level, while nonsignificant increases in FAO activity were observed at the mid- and high-dose levels.

Animals↗

PubMed1994

Oxolanosterol oximes: dual-action inhibitors of cholesterol biosynthesis.

A series of oxolanosterol oximes and oxime ethers have been prepared as potential dual-action inhibitors of cholesterol biosynthesis. The synthesis of these oximes along with the evaluation of their ability to inhibit lanosterol 14 alpha-methyl demethylase (P450DM) and to suppress 3-hydroxy-3-methylglutaryl coenzyme. A reductase (HMGR) activity is presented. 3 beta-Hydroxylanost-7-en-15-one 15-oxime XIX was found to be an effective inhibitor of P450DM in rat liver microsomal preparations. In [14C]acetate incorporation studies using Chinese hamster ovary (CHO) cells, compound XIX was found to cause a dramatic reduction in the incorporation of acetate into C27 sterols with a concomitant increase in radiolabeled C30 sterols which is consistent with the inhibition of P450DM. In addition, 15-oxime XIX was shown to suppress HMGR activity in both wild-type CHO and P450DM-deficient (AR45) cells, indicating that suppression of HMGR is independent of any effects of this oxime on P450DM. In both cell lines, parallel declines in HMGR activity and HMGR protein levels were observed suggesting that compound XIX suppresses HMGR activity by regulation of gene expression. These results demonstrate that, as predicted, 15-oxime XIX is indeed a dual-action inhibitor of cholesterol biosynthesis which causes both the inhibition of P450DM and a reduction in HMGR activity.

Animals↗

PubMed1993

Modulation of 3-hydroxy-3-methylglutaryl-CoA reductase by 15 alpha-fluorolanost-7-en-3 beta-ol. A mechanism-based inhibitor of cholesterol biosynthesis.

The chemical synthesis and metabolic characteristics of the lanosterol analogue, 15 alpha-fluorolanost-7-en-3 beta-ol, are described. The 15 alpha-fluorosterol is shown to be a competitive inhibitor of the lanosterol 14 alpha-methyl demethylase (Ki = 315 microM), as well as substrate for the demethylase enzyme. Metabolic studies show that the 15 alpha-fluorosterol is converted to the corresponding 15 alpha-fluoro-3 beta-hydroxylanost-7-en-32-aldehyde by hepatic microsomal lanosterol 14 alpha-methyl demethylase but that further metabolic conversion to cholesterol biosynthetic intermediates is blocked by virtue of the 15 alpha-fluoro substitution. When cultured cells are treated with the fluorinated lanosterol analogue, a decrease in 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity and immunoreactive protein was observed. However, when the lanosterol 14 alpha-methyl demethylase-deficient mutant cell line, AR45, is treated with the fluorosterol, no effect upon HMG-CoA reductase is observed. Thus, metabolic conversion of the sterol to its 32-carboxaldehyde analogue by the lanosterol 14 alpha-methyl demethylase is required for HMG-CoA reductase suppressor activity. Measurement of HMG-CoA reductase mRNA levels in 15 alpha-fluorosterol-treated Chinese hamster ovary (CHO) cells reveals that mRNA levels are not decreased by the sterol as would be expected for a sterol regulator of HMG-CoA reductase activity. The decrease in HMG-CoA reductase protein is due to inhibition of enzyme synthesis, suggesting that the 15 alpha-fluorosterol reduces the translational efficiency of the reductase mRNA. Measurements of the half-life of HMG-CoA reductase show that, in contrast to other oxysterols, the 15 alpha-fluorolanostenol does not increase the rate of degradation of the enzyme. Collectively, these data support the premise that oxylanosterols regulate HMG-CoA reductase expression through a post-transcriptional process which may be distinct from other previously described sterol regulatory mechanisms.

Animals↗

PubMed1993

32-Methyl-32-oxylanosterols: dual-action inhibitors of cholesterol biosynthesis.

Lanosterol 14 alpha-methyl demethylase (P-450DM) is the cytochrome P-450 monooxygenase which oxidatively removes the 14 alpha-methyl group of lanosterol. This demethylation is considered to be a rate-limiting step in the conversion of lanosterol to cholesterol. The intermediates in this transformation are known to bind very tightly to P-450DM and have been implicated in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity, the rate-limiting enzyme in overall cholesterol biosynthesis. Three 32-methylated analogs of the intermediates generated during the removal of the 14 alpha-methyl group by P-450DM, compounds 17a, 17b, and 18, have been prepared and their biochemical activities assessed. All three compounds were found to be direct inhibitors of P-450DM. These compounds were also shown to suppress HMGR activity by reducing the level of enzyme protein.

Animals↗

PubMed1993

Ornithine decarboxylase (ODC) activity in the liver of individual medaka (Oryzias latipes) of both sexes.

Ornithine decarboxylase (ODC) activity has been determined in the liver of individual medaka (N = 48). The results indicate a mean ODC activity of 1.69 +/- 1.77 nmol 14CO2/hr/mg protein. The fish displayed a large interindividual variation of normal ODC activity with a range of 140fold. However, no sex differences were observed. The findings also indicate that the mean ODC activity in this fish model is approximately 100fold greater than observations in the published literature as well as in our laboratory for commonly employed rodent models.

Animals↗

PubMed1993

Effect of hypolipidemic drugs gemfibrozil, ciprofibrate, and clofibric acid on peroxisomal beta-oxidation in primary cultures of rainbow trout hepatocytes.

Primary cultures of hepatocytes were established from sexually mature male rainbow trout (Oncorhyncus mykiss) and treated with the hypolipidemic drugs gemfibrozil (0.25-1.25 mM), clofibric acid (2.25-3.00 mM), or ciprofibrate (0.25-1.00 mM). Significant dose-related increases in peroxisomal fatty acyl-CoA oxidase (FACO) were seen after exposure for 48 hr to clofibric acid (P < 0.01) and ciprofibrate (P < 0.05) but not gemfibrozil (P = 0.08). Strong correlation was obtained between increased acyl-CoA oxidase activity and the relative amount of peroxisomal bifunctional enzyme (PBE), further supporting evidence of a proliferative effect. These preliminary studies demonstrate that peroxisomal beta-oxidation can be induced in vitro in a primary rainbow trout hepatocyte system.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1993

Interactive potential of omega-3 fatty acids with clofibrate or DEHP on hepatic peroxisome proliferation in male Wistar rats.

The interactive potential of three known peroxisome proliferators, omega-3 fatty acids, clofibrate and di(2-ethylhexylphthalate (DEHP), was evaluated in male weanling Wistar rats for the effect on peroxisomal beta-oxidation. Omega-3 fatty acids were supplied by menhaden oil which was fed in six regimens: low fat (5% w/w), low fat and clofibrate (0.3% w/w) or DEHP (0.25% w/w), high fat (20% w/w), high fat and clofibrate or DEHP in the aforementioned concentrations. Induction of peroxisomal beta-oxidation was measured by changes in liver-to-body weight ratio, fatty acyl-CoA oxidase (FAO) activity, and peroxisomal bifunctional enzyme (PBE) quantity. Analysis of transformed data indicated a less than additive response in FAO activity with no deviation from additivity seen with liver-to-body ratios and PBE.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1992

Effects of joint exposures to selected peroxisome proliferators on hepatic acyl-CoA oxidase activity in male B6C3F1 mice.

The interaction potential of peroxisome proliferators of similar and dissimilar structure was examined in B6C3F1 mice. Mice were fed diets containing varying concentrations of ciprofibrate (Cipro), clofibrate (Clof) or di(2-ethylhexyl)phthalate (DEHP), or combinations of Cipro and Clof or Cipro and DEHP for 4 d. Induction of peroxisomal beta-oxidation, measured by increased acyl-CoA oxidase activity, was used as the endpoint for analysis. An additive response occurred following joint exposure to the structurally related compounds Cipro and Clof, whereas a possible synergistic response occurred at low dose combinations of the structurally dissimilar Cipro and DEHP. These findings represent the first report assessing the in-vivo interaction potential of structurally similar and dissimilar peroxisome proliferators and provides insight into the dose-response nature of joint exposures to certain non-genotoxic carcinogens.

Acyl-CoA Oxidase↗

PubMed1989

The making of a smoke-free medical center.

The concept of smoke-free medical facilities is in its formative stages, and such policies have received broad support. Although smoking has been restricted at Mayo Medical Center for many years, in 1986 it was decided that the medical center should become smoke free. This report summarizes the methods used in developing and implementing a smoke-free policy. This experience suggests that with proper planning, the implementation can be smooth. The message being given to patients and staff concerning the health risk of smoking is now more consistent, and there has been wide acceptance of the policy by staff and patients. It is concluded that such a policy can be effectively implemented, if it is well planned and supported.

Adult↗

PubMed1988

An ATP-dependent system specific for degradation of long-lived proteins in permeabilized cells.

We have characterized a digitonin-permeabilized cell system for the ATP-dependent degradation of endogenous long-lived proteins. Proteolysis requires Mg2+ and ATP hydrolysis. Other nucleotide triphosphates (CTP, UTP) can partially replace the ATP requirement. The enhanced rate of degradation of long-lived proteins in response to serum starvation is maintained in the permeabilized cell system and can be partially inhibited by lysosomal inhibitors. The maintenance of intracellular architecture and ease of manipulation of soluble components make the permeabilized cell system ideal for studying the proteolysis of both endogenous and exogenous substrates.

Adenosine Triphosphate↗

PubMed1988

A mammalian mutant cell lacking detectable lanosterol 14 alpha-methyl demethylase activity.

A Chinese hamster ovary cell mutant, AR45, was selected for amphotericin B resistance after treatment with the mutagen ethyl methanesulfonate. The mutant is a cholesterol auxotroph with a deficiency in cholesterol biosynthesis. Whole cell experiments demonstrate that the mutant accumulates the C30 sterols, lanosterol and dihydrolanosterol, under culture conditions which promote active sterol biosynthesis. Metabolic studies show that the C29 sterol demethylation product of lanosterol, but not lanosterol itself, is actively converted to end product cholesterol by whole cells as well as by microsomal preparations derived from the mutant. Detectable amounts of several cytochromes can be observed spectrally in the AR45 demonstrating that it is not a general heme-deficient mutant. Collectively, these results characterize the AR45 mutant cells as being lanosterol 14 alpha-methyl demethylase-deficient. The cell line should prove useful in studying regulation of the demethylase enzyme and the putative endogenous regulatory oxysterol. It should also be a useful tool in the molecular cloning and elucidation of genetic properties of the demethylase.

Amphotericin B↗

PubMed1987

ATP-dependent degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in permeabilized cells.

A system for the assay of 3-hydroxy-3-methyglutaryl (HMG) coenzyme A (CoA) reductase in digitonin-permeabilized Chinese hamster ovary cells is described. Under these conditions, HMG-CoA reductase remained intact and associated with the endoplasmic reticulum, and values for Km (HMG-CoA), Ki (mevinolin), and active/total activity were similar to those seen in sonicated cell preparations. However, the mechanism by which this rapidly turned over (half-life approximately 2 h) enzyme is degraded was disrupted. Addition of ATP at physiological concentrations to digitonin-permeabilized cells resulted in the rapid, irreversible loss of enzyme activity. Immunoblot analysis showed that this loss of activity was followed by cleavage of the intact 97-kilodalton enzyme to a 68-kilodalton fragment which was distinct from the catalytically active fragments generated by nonspecific proteolysis in sonicated cell homogenates. Assay of a lysosomal marker enzyme confirmed that ATP-mediated inactivation and cleavage of reductase was not due to release of lysosomal proteases. The possible role of ATP in phosphorylation, inactivation, and degradation of reductase is discussed.

Adenosine Triphosphate↗

PubMed1987

The effects of vitamin C supplementation on blood and hair levels of cadmium, lead, and mercury.

Fifty-two adult male subjects were randomly assigned to one of three possible treatment groups: supplemental ascorbic acid at one of two levels, 500 mg or 1000 mg, or a placebo. Hair and blood samples were taken before vitamin C or placebo supplementation was started and at monthly intervals thereafter for three months. Samples were analyzed for cadmium, lead, and mercury. Results indicate that vitamin C did not significantly affect levels of these metals in either hair or blood samples.

Adult↗

PubMed1984

Chloroquine inhibits cyclization of squalene oxide to lanosterol in mammalian cells.

Chloroquine inhibits the incorporation of [14C]acetate into sterols at a concentration of 10 microM or more in mouse L cells but has no effect on fatty acid synthesis and CO2 production from the same substrate even at a 10-fold higher concentration of the drug. The site of inhibition is distal to the formation of mevalonate since chloroquine also inhibits [14C]mevalonate metabolism to sterols and does not decrease the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) or the incorporation of [14C]acetate into the total nonsaponifiable lipids. Analyses by thin layer and high pressure liquid chromatography of the nonsaponifiable lipid fraction from cultures incubated with chloroquine show an accumulation of radioactivity in the region of squalene oxide. Identification of the radiolabeled lipid as squalene oxide has been established by: (a) its co-migration with the authentic squalene oxide standard; (b) its conversion into squalene glycol by acid hydrolysis; and (c) its further metabolism to desmosterol when chloroquine is removed from the medium. Addition of chloroquine (12.5-50 microM) to 20,000 X g supernatant fractions of mouse liver homogenates inhibits the incorporation of [14C]mevalonolactone into cholesterol and lanosterol, with corresponding increases of [14C]squalene oxides, in a concentration-dependent manner. It appears, therefore, that chloroquine inhibits the enzymatic step catalyzed by 2,3-oxidosqualene-lanosterol cyclase (EC 5.4.99.7). Incubation of cell cultures with chloroquine (50 microM) arrests cell growth and causes cell death after 1-3 days. However, simultaneous incubation of chloroquine with either cholesterol or lanosterol prevents cell death and permits cell growth. Uptake of chloroquine is not affected by exogenous sterols since intracellular chloroquine concentrations are the same in cells grown with or without added sterols. The cytotoxicity of chloroquine, under our experimental conditions, must, therefore, be due primarily to its inhibition of sterol synthesis. In addition to its well known effect on protein catabolism, chloroquine has been found to inhibit protein synthesis. The significance of these findings concerning the use of chloroquine in studying the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity is discussed.

Acetates↗

PubMed1984

Aphidicolin, a specific inhibitor of DNA polymerase alpha, inhibits conversion of lanosterol to C-27 sterols in mouse L cells.

Aphidicolin, a fungal metabolite which is a specific inhibitor of DNA polymerase alpha, inhibited the incorporation of [14C]acetate into desmosterol in mouse L cells by 50% at a concentration of 8.8 microM. It had no effect on acetate metabolism into fatty acids or CO2. The site of inhibition was determined to be distal to the formation of mevalonic acid since aphidicolin also inhibited the incorporation of [14C]mevalonolactone into desmosterol but had no effect on the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) or the incorporation of [14C]acetate into total nonsaponifiable lipids. High pressure liquid chromotographic analysis of the distribution of radioactivity among the nonsaponifiable lipids formed from [14C]acetate in the presence of aphidicolin indicated an accumulation of lanosterol accompanied by a proportional decrease in radiolabeled desmosterol and two of its precursors, delta 5,7,24-cholestatrienol, and 4 alpha-methyl-delta 8,24-cholestadienol. In cells exposed to aphidicolin, lanosterol accumulation was rapid (15 min) and reversible after a 3-h exposure when cells were rinsed and fresh medium added. It was concluded that aphidicolin inhibits the conversion of lanosterol to C-27 sterols. Although the exact mechanism of this inhibition has not yet been determined, addition of aphidicolin to 20,000 X g supernatant fractions of mouse liver homogenates inhibited the incorporation of [14C]mevalonolactone into cholesterol in a concentration-dependent manner, suggesting that aphidicolin may act directly on one or more of the enzymatic steps involved in lanosterol demethylation. The ubiquitous occurrence of an aphidicolin binding site on eukaryotic DNA alpha polymerases and the inhibitory action of aphidicolin at a proposed secondary regulatory site in sterol biosynthesis (lanosterol metabolism) suggest that a naturally occurring compound may exist which can regulate both DNA replication and cholesterogenesis.

Acetates↗

PubMed1984

The effects of di- and trichloroacetic acid on sheep erythrocytes: an animal model with a glucose-6-phosphate dehydrogenase deficiency.

Erythrocytes of Dorset sheep, an animal model with an erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, responded in a dose-dependent manner to the oxidant stress of di- and trichloroacetic acids (DCA and TCA) as measured by increases in methemoglobin (METHB) and decreases in glutathione (GSH). Given the fact that TCA and DCA are now being found in community drinking water supplies at levels greater than 100 micrograms/liter, there is a need to further investigate their effects on biological systems, including those with a compromised ability to deal with oxidant stress (e.g., G-6-PD-deficient erythrocytes).

Acetates↗

PubMed1984

Phonological process analysis of the speech of mentally retarded adults.

A phonological process analysis was performed on the speech of 20 mentally retarded adults. Results indicated that these subjects exhibited deletion of final consonant, cluster reduction, weak syllable deletion, and stopping. These processes are similar to those previously reported for mentally retarded children and unintelligible nonretarded children. Differences between subjects classified as moderately and severely retarded appear to be quantitative rather than in type of process exhibited.

Adult↗

PubMed1983

The characterization of alpha-glycerophosphate dehydrogenase mutants in Drosophila melanogaster.

Thirty mutants of alpha-glycerophosphate dehydrogenase (alpha GPDH, EC 1.1.1.8) from Drosophila melanogaster were produced with the chemical mutagen ethyl methanesulfonate (EMS). These mutants and nine others previously obtained have been characterized with respect to level of enzymatic activity, viability, flight ability, and presence of cross-reacting material (CRM). The presence of alpha GPDH mRNA in several of the mutans has been tested by in vitro translation. There are strong correlations between the level of enzyme activity, viability and flight ability. Thirteen of the mutants are CRM- by solution immunoprecipitation experiments, but of these, only three are CRM- by a more sensitive 125I-protein A-based radioimmune gel assay. The viability of the three CRM- mutants suggests that the absence of alpha GPDH protein is not a lethal condition. The immunoprecipitated protein of the low activity mutant, alpha GpdhnGL3, has a smaller apparent molecular weight on polyacrylamide-SDS gels than does the protein from wild type. Criteria for the identification of nonsense mutations in Drosophila are discussed.

Animals↗