Biomedical subjects

G Dallner

Publications and source records attributed to G Dallner.

At least 91 records · Page 5Linked to original sources

PubMed1991

Characterization of the lipid and protein contents of myelin bodies isolated from the renal cortex of gentamicin-treated rats.

Myelin bodies were isolated from the renal cortex of gentamicin-treated rats (100 mg/kg body weight, twice daily for 3 days, i.p.) employing an initial pelleting by differential centrifugation and subsequent flotation on a discontinuous sucrose gradient. These structures were found to contain almost twice as much protein as phospholipid and SDS-polyacrylamide gel electrophoresis revealed the presence of many different polypeptides. All the major phospholipids are present, although myelin bodies contain a considerably higher proportion of phosphatidylinositol, somewhat more phosphatidylcholine and considerably lower percentages of phosphatidylserine and sphingomyelin than do normal renal phospholipids. The fatty acids of myelin body phospholipids are highly saturated (67.3-87.9%) and a striking feature is the occurrence of relatively large amounts of 22:1, presumably erucic acid, especially in sphingomyelin. Myelin bodies contain small amounts of unesterified cholesterol, unesterified dolichol and coenzymes Q9 and Q10.

Animals↗

PubMed1991

The mevalonate pathway in the bloodstream form of Trypanosoma brucei. Identification of dolichols containing 11 and 12 isoprene residues.

The major surface antigen of the bloodstream form of Trypanosoma brucei, the variant surface glycoprotein, is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. The biosynthesis of the glycosylphosphatidylinositol anchor, as well as the assembly of the asparagine-linked oligosaccharide chains found on the variant surface glycoproteins, involves polyisoprenoid lipids that act as sugar carriers. Preliminary observations (Menon, A.K., Schwarz, R.T., Mayor, and Cross, G.A.M. (1990) J. Biol. Chem. 265, 9033-9042) suggested that the sugar carriers in T. brucei were short-chain polyisoprenoids containing substantially fewer isoprene residues than polyisoprenols in mammalian cells. In this paper we describe metabolic labeling experiments with [3H]mevalonate, as well as chromatographic and mass spectrometric analyses of products of the mevalonate pathway in T. brucei. We report that cells of the bloodstream form of T. brucei contain a limited spectrum of short chain dolichols and dolichol phosphates (11 and 12 isoprene residues). The total dolichol content was estimated to be 0.28 nmol/10(9) cells; the dolichyl phosphate content was 0.07 nmol/10(9) cells. The same spectrum of dolichol chain lengths was also found in a polar lipid that could be labeled with [3H]mevalonate, [3H]glucosamine, and [3H]mannose, and which was characterized as Man5GlcNAc2-PP-dolichol. The most abundant product of the mevalonate pathway identified in T. brucei was cholesterol (140 nmol/10(9) cells). Ubiquinone (0.09 nmol/10(9) cells) with a solanesol side chain was also identified.

Animals↗

PubMed1991

Lipid compositions of intracellular membranes isolated from rat liver nodules in Wistar rats.

The mevalonate pathway gives rise to important end products for the regulation of growth and resistance to oxidative stress and is, consequently, of importance in carcinogenesis. In this study liver nodules were produced in Wistar rats by intermittent feeding with dietary 2-acetylaminofluorene, and the lipid compositions of isolated microsomes, mitochondria, and lysosomes were examined. The phospholipid compositions of these subfractions were unchanged compared to normal hepatic tissue, but the fatty acid patterns were altered, particularly in microsomes. An increase in the content of palmitic acid and a decrease in that of stearic acid were noted. The pattern of fatty acyl moieties on carbon atoms 1 and 2 of the glycerol backbone of phospholipids was unchanged in nodular tissue compared to normal liver. The amount of dolichol was significantly higher in microsomes and mitochondria, but not in lysosomes, and the relative amounts of longer polyisoprenoid compounds were increased in the liver nodules. The relative concentration of esterified dolichol was decreased and an enrichment in saturated fatty acids in this fraction could be observed. The cholesterol concentration was found to be lower in microsomes, but was unchanged in mitochondria and lysosomes, and the normally low concentration of cholesteryl esters was elevated somewhat in microsomes and lysosomes. The ubiquinone content of liver nodular mitochondria was unchanged, but increased 7-fold in microsomes and 2-fold in lysosomes. The alterations found in the lipid composition of liver nodules are significant and have functional implications in many cellular processes of proposed importance for the carcinogenic process, i.e., protein glycosylation cholesterogenesis, regulation of the mevalonate pathway, cellular oxidation-reduction state, and resistance to oxidative stress.

Animals↗

PubMed1991

Inhibition of lipid peroxidation by ubiquinol in submitochondrial particles in the absence of vitamin E.

The relationship between the antioxidant effects of reduced coenzyme Q10 (ubiquinol, UQH2) and vitamin E (alpha-tocopherol) was investigated in beef heart submitochondrial particles in which lipid peroxidation was initiated by incubation with ascorbate + ADP-Fe3+. These effects were examined after extraction of coenzyme Q10 (UQ-10) and vitamin E from the particles and reincorporation of the same components alone or in combination. The results show that UQH2 efficiently inhibits lipid peroxidation even when vitamin E is absent. It is concluded that UQH2 can inhibit lipid peroxidation directly, without the mediation of vitamin E.

Adenosine Diphosphate↗

PubMed1991

Involvement of sterol carrier protein-2 in dolichol biosynthesis.

The effect of sterol carrier protein-2 (SCP-2) on dolichol biosynthesis by rat liver microsomes was investigated. cis-Prenyltransferase activity was stimulated 7-fold in the presence of 5 micrograms of purified SCP-2/mg of microsomal protein, which was similar to the increase obtained by adding detergent. The polyisoprenoid pattern obtained in the presence of SCP-2 was the same as that present in rat liver, in contrast to the pattern appearing upon incubation of microsomes with detergent, which gave shorter polyisoprenoids. Like SCP-2, the cytosolic fraction from rat liver also stimulated cis-prenyltransferase. Incubation with cytosol pretreated with anti-SCP-2 showed no stimulatory effect and led to the accumulation of shorter polyisoprenoids. SCP-2 had no appreciable effect on polyprenol alpha-saturase, dolichol kinase, dolichyl phosphate phosphatase, or acyl-CoA:dolichol acyltransferase. The results demonstrate that SCP-2 greatly stimulates and may regulate the condensation reactions mediated by cis-prenyltransferase in the process of dolichol biosynthesis and permits polymerization of the polyisoprenoid to its natural chain length.

Albumins↗

PubMed1991

Covalent binding of dolichyl phosphate to proteins in rat liver.

Rats were injected via the portal vein with (RS)-[5-3H]-mevalonolactone and the lipids were extracted. From fractions of liver homogenate, all labeled dolichol, cholesterol and ubiquinone could be extracted, but about 40% of microsomal and lysosomal dolichyl phosphate was only released after alkaline hydrolysis. Only a small amount of the non-extractable radioactivity was found to be associated with alpha-unsaturated polyprenyl phosphate. There was no difference in the polyisoprenoid pattern when the two pools of dolichyl phosphate were compared. On the other hand, the specific activity of the bound lipid was only half that of the extractable form. After phenyl-Sepharose chromatography, a peak of protein was isolated exhibiting a 25-fold enrichment in bound radioactive dolichyl phosphate. Treatment with a non-specific protease, followed by chromatography, gave polypeptide fragments associated with bound lipids. On SDS/PAGE a major protein band at 23 kDa and some minor bands with higher molecular masses were found to be associated with this lipid. The results indicate the presence of covalently bound dolichyl phosphate in rat liver.

Animals↗

PubMed1991

Effect of dietary fat on rat liver microsomal and mitochondrial/lysosomal dolichol, phospholipid and cholesterol.

The influence of different fat diets on liver phospholipid, cholesterol and dolichol was studied. Rats were separated into four groups and fed standard laboratory chow (control), a diet containing linolenic acid, a coconut oil diet, or a corn oil-containing diet. After five weeks, microsomes and mitochondrial/lysosomal fractions were prepared from the liver, and lipid compositions were analyzed. No changes in phospholipid content were observed. In control animals, the fatty acid compositions of phosphatidylcholine and phosphatidylethanolamine in the two subfractions were similar. However, these two phospholipids showed different fatty acid patterns, which were altered independently upon dietary treatment. The dietary treatments resulted, in most cases, in decreased cholesterol and dolichol contents and, especially in microsomes, in a decreased level of esterification of both lipids. The fatty acid compositions of cholesteryl esters in the two subfractions showed significant differences and cholesterol was esterified to a large extent with linolenic acid when this fatty acid was supplied in the diet. The same dietary treatment exerted different effects on the cholesterol localized in the two different intracellular compartments. This difference was most pronounced in rats fed the corn oil-containing diet; microsomal cholesteryl esters exhibited increased saturation, whereas cholesteryl esters exhibited increased saturation, whereas cholesteryl esters in the mitochondrial/lysosomal fraction displayed decreased saturation. Dolichyl esters in the two cellular compartments had different fatty acyl compositions, with a considerably higher degree of saturation in microsomes. The various diets influenced the nature of the fatty acid moieties present in the isolated fractions and the effects on the two subfractions were opposite.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Fatty acid composition of brain phospholipids in aging and in Alzheimer's disease.

The two major phospholipid classes, namely, phosphatidylethanolamines (PE) and phosphatidylcholines (PC), were studied in four different regions of human brain, i.e., in frontal gray matter, frontal white matter, hippocampus and in pons. The fatty acid (FA) compositions of these phospholipids were found to be specific for the different regions. PC contains mostly saturated and 18:1 FA, while PE is rich in polyunsaturated FA. Aging has no influence on the FA compositions, while in Alzheimer's disease (AD) PE is modified in all four regions, particularly in frontal gray matter and in hippocampus. The abundance of the major monounsaturated FA of PE, 18:1, is not significantly altered in Alzheimer's disease, but there is a substantial increase in the relative amounts of the saturated components 14:0, 16:0 and 18:0. This is paralleled by a decrease in the polyunsaturated FA 20:4, 22:4 and 22:6. It is not clear whether the changes observed are specific for AD. Changes in saturated/polyunsaturated FA ratio are likely to influence cellular function, which in turn may cause certain neural deficiencies. The findings do not support the hypothesis that AD reflects an accelerated aging process.

Adult↗

PubMed1991

Effects of pravastatin and cholestyramine on products of the mevalonate pathway in familial hypercholesterolemia.

Patients with heterozygous familial hypercholesterolemia (n = 12) were treated either with pravastatin, a specific inhibitor of HMG-CoA reductase, or cholestyramine, followed by a period of combined treatment with both drugs. Initially, these patients had increased serum levels of low density lipoprotein (LDL) cholesterol (8.77 +/- 0.48 mmol/l; SEM), lathosterol (5.32 +/- 0.60 mg/l), and ubiquinone (0.76 +/- 0.09 mg/l), while the serum dolichol concentration was in the normal range. Cholestyramine treatment (n = 6) decreased the levels of LDL cholesterol (-32%) and increased lathosterol (+125%), but did not change dolichol or ubiquinone levels in a significant manner. Pravastatin treatment (n = 6) decreased LDL cholesterol (-27%), lathosterol (-46%), and ubiquinone (-29%). In this case, the amount of dolichol in serum also showed a small but statistically insignificant decrease (-16%) after 12 weeks of treatment. Combined treatment with cholestyramine and pravastatin (n = 6) resulted in changes that were similar to, but less pronounced than, those observed during pravastatin treatment alone. In no case was the ratio between ubiquinone and LDL cholesterol reduced. Possible effects on hepatic cholesterol, ubiquinone, and dolichol concentrations were studied in untreated (n = 2), cholestyramine-treated (n = 2), and pravastatin-treated (n = 4) gallstone patients and no consistent changes could be observed. The results indicate that treatment with pravastatin in familial hypercholesterolemia decreases serum ubiquinone levels in proportion to the reduction in LDL cholesterol.

Adult↗

PubMed1990

Presence of individual enzymes of cholesterol biosynthesis in rat liver peroxisomes.

Cholesterol biosynthesis by isolated rat liver peroxisomes was examined. Labeling of cholesterol from [3H]-mevalonate in the presence of peroxisomes required the addition of cytosol, since peroxisomes, like microsomes, apparently possess only those enzymes of cholesterol biosynthesis subsequent to the steps involving farnesyl-PP. Under the conditions employed the amounts of 4,4-dimethyl and desmethyl sterols generated by peroxisomes were equal to or exceeded those produced by the microsomes. In addition, marker enzyme analysis demonstrated minimal microsomal contamination in the peroxisomal fraction. The metabolite patterns observed by HPLC after incubation of these two fractions with [3H]mevalonate were different. Dihydrolanosterol oxidase, steroid-14-reductase, steroid-8-isomerase, and steroid-3-ketoreductase activities were present in peroxisomes. Separation of peroxisomes into membranes and contents revealed that all the synthesizing activities are associated with the membrane fraction. 7 alpha-Hydroxylase, which catalyzes the first step in the biosynthesis of bile acids, was also present in peroxisomes, but it remains to be clarified to what extent peroxisomal cholesterol is a substrate for bile acid synthesis.

Animals↗

PubMed1990

Metabolism of fatty acids and their incorporation into phospholipids of the mitochondria and endoplasmic reticulum in isolated hepatocytes determined by isolation of fluorescence derivatives.

Isolated hepatocytes were incubated in the presence of [14C]palmitic, [14C]linoleic or [14C]linolenic acid and the time-courses of incorporation of radioactivity into phosphatidylcholine and phosphatidylethanolamine of microsomes and mitochondria were followed. For this purpose a procedure was developed for HPLC separation of 9-diazomethylanthracene (ADAM) derivatives of fatty acids. When [14C]palmitic acid was used, the major product of elongation and desaturation was octadecadienoic acid, which accounted for 35-65% of the total radioactivity. Labeled palmitoleic, stearic and oleic acids could also be isolated. In fatty acids which do not participate to any large extent in deacylation-reacylation reactions, the pattern of incorporation was characteristic: a high rate of incorporation into microsomal and a low rate of incorporation into mitochondrial phospholipids during the first 40 min, followed by a decrease in the former and an increase in mitochondrial labeling. This pattern is consistent with the fact that de novo synthesis of these two phospholipids occurs in the endoplasmic reticulum in vivo. When cells were incubated in the presence of [14C]linoleic acid, 70-90% of the radioactivity recovered in phospholipids was in this same form, whereas the remaining label was mainly in arachidonic acid and, to some extent, in eicosatrienoic acid. When hepatocytes were incubated in the presence of [14C]linolenic acid, 70-85% of the radioactivity in isolated phospholipids was associated with linolenic acid. As much as 20% of the label was recovered in docosahexanoic acid and 5-10% in arachidonic acid. In the case of the two latter labeled substrates the exchange reactions seem to dominate over de novo synthesis. For phospholipids synthesized de novo the transfer from the endoplasmic reticulum to mitochondria requires about 3 h.

Animals↗

PubMed1990

Rates of cholesterol, ubiquinone, dolichol and dolichyl-P biosynthesis in rat brain slices.

Slices from the brain and liver of rats were prepared and upon incubation exhibited a continuous and high capacity for incorporation of radioactive precursors into proteins and lipids. Using [3H]mevalonate as precursor, the rates of biosynthesis of cholesterol, ubiquinone, dolichol and dolichyl-P in brain slices were determined and found to be 5.5, 0.25, 0.0093 and 0.0091 nmol/h/g, respectively. Dolichol and dolichyl-P accumulate to a limited extent, but almost all of these lipids in the brain originate from de novo synthesis. The calculated half-lives for cholesterol, ubiquinone, dolichol and dolichyl-P were 4076, 90, 1006 and 171 h, respectively. The results indicate that lipids formed via the mevalonate pathway in the brain have an active and independently regulated biosynthesis.

Animals↗

PubMed1990

Nonaprenyl-4-hydroxybenzoate transferase, an enzyme involved in ubiquinone biosynthesis, in the endoplasmic reticulum-Golgi system of rat liver.

The properties and distribution of nonaprenyl-4-hydroxybenzoate transferase in rat liver were investigated with subcellular fractions, liver perfusion, and in vivo labeling with [3H]solanesyl-PP. In addition to some ubiquinone-9, only one labeled intermediate, i.e. nonaprenyl-4-hydroxybenzoate, was obtained. In the total microsomal fraction, the enzyme had a pH optimum of 7.5 and was completely inhibited by Triton X-100 and deoxycholate, but not by taurodeoxycholate and beta-octyl glucoside. Liver, kidney, and spleen demonstrated the highest activities of nonaprenyl-4-hydroxybenzoate transferase. Upon subcellular fractionation, high specific activities were found in smooth II microsomes and Golgi III vesicles. The enzyme was also found in lysosomes and plasma membranes, but only at low levels in rough and smooth I microsomes and mitochondria and not at all in peroxisomes and cytosol. When the product of the transferase reaction was used as a substrate in vitro and in a perfusion system, the only product obtained was end product ubiquinone-9. Although the transferase reaction was associated with the inner, luminal surface of microsomal vesicles, the terminal reaction(s) for ubiquinone-9 synthesis are found at the outer cytoplasmic surface. The results suggest that the major site for ubiquinone synthesis is the endoplasmic reticulum-Golgi system, which also participates in the distribution of ubiquinone-9 to other cellular membranes.

Alkyl and Aryl Transferases↗

PubMed1990

Uptake and metabolism of dolichol and cholesterol in perfused rat liver.

The uptake of dolichol and cholesterol by perfused rat liver was studied. When these radioactive lipids were incorporated into egg phosphatidylcholine liposomes, both dolichol and cholesterol appeared initially in the supernatant and in the microsomal fraction and, later on, in the mitochondrial-lysosomal fraction. The lipids taken up were esterified to some extent, but no phosphorylation of dolichol occurred. Incorporation of dolichol and cholesterol into lipoproteins increased the efficiency of uptake, which was receptor-mediated in this case. Accumulation of these lipids occurred in lysosomes followed by a transport to the endoplasmic reticulum (ER). Both labeled dolichol and cholesterol appeared in the bile. In the case of dolichol, the majority of this radioactivity was not associated with the original substance itself, and probably represented lipid-soluble catabolites. In the case of cholesterol, most of the radioactivity was associated with bile acids. It appears that, in contrast to the receptor-mediated uptake of lipoproteins from the perfusate, the uptake of liposomal lipids involves a different mechanism. After association with the plasma membrane, the lipids enter into the cytoplasm and are transported to the ER and later to the lysosomes.

Animals↗

PubMed1990

The effects of inducers of the endoplasmic reticulum, peroxisomes and mitochondria on the amounts and synthesis of ubiquinone in rat liver subcellular membranes.

Rats were treated with inducers of peroxisomes, mitochondria and the endoplasmic reticulum, as well as receiving diets and drug known to influence the mevalonate pathway. Treatment with clofibrate and 2-diethylhexylphthalate (DEHP) increased microsomal and mitochondrial ubiquinone contents, but a decrease was observed in lysosomes. In vivo labeling of this lipid with [3H]mevalonate was also elevated. The amount of cholesterol did not change upon exposure to these inducers of peroxisomes and mitochondria, but its rate of labeling was decreased. The concentration of dolichol increased only after treatment with DEHP and only in lysosomes. The inducers of the endoplasmic reticulum phenobarbital, 3-methylcholanthrene and N-nitrosodiethylamine enhanced the rate of ubiquinone synthesis and exposure to the latter two substances also elevated the amount of this lipid in microsomes. A cholesterol-rich diet increased the labeling of ubiquinone and decreased cholesterol labeling, while cholestyramine treatment had opposite effects on lipid labeling in both microsomes and mitochondria. The results demonstrate that the ubiquinone contents of the various membranes of hepatocytes change in a characteristic manner under the influence of inducers and dietary factors. Clearly, the level of ubiquinone and its biosynthesis are regulated separately from those of the other products of the mevalonate pathway, cholesterol and dolichol.

Animals↗

PubMed1990

Lipid compositions of different regions of the human brain during aging.

The neutral and phospholipid compositions of various regions of the human brain were analyzed using autopsy material covering the life period between 33 and 92 years of age. The protein content was also measured and, on a weight basis, this content is unchanged in the cerebellum, pons, and medulla oblongata, whereas in the 90-year-old group it decreases in the hippocampus, gray matter, and nucleus caudatus. In white matter, the protein content decreases continuously with age. The phospholipid composition is characteristic of the region investigated, but remains unchanged during aging. The total phospholipid content exhibits only a 5-10% decrease in the oldest age group. The content of dolichol and its polyisoprenoid pattern are also characteristic of the region analyzed. Between 33 and 92 years of age, the amount of dolichol in all portions of the brain increases three- to fourfold, but the isoprenoid pattern remains constant. The level of dolichyl-P varies between different regions, but only a moderate increase is seen with age. Ubiquinone content is highest in the nucleus caudatus, gray matter, and hippocampus, and in all areas this content is decreased to a great extent in the oldest age groups. All regions of the human brain are rich in cholesterol, but alterations in the amount of this lipid are highly variable during aging, ranging from no change to a 40% decrease.

Adult↗

PubMed1990

Effects of prolonged treatment with phthalate ester on rat liver.

Rats were fed a diet containing 2%, 0.2% or 0.02% di(2-ethylhexyl)phthalate (DEHP) for a period of 102 weeks. Only the 2% diet caused a substantial decrease in body weight. Both peroxisomal palmitoyl-CoA dehydrogenase and mitochondrial carnitine acetyltransferase activities were greatly induced by exposure to the highest dose of phthalate ester, reaching maximal plateau values after about 20 weeks of treatment. The diet containing 0.2% DEHP increased both activities slowly, but continuously, and at the end of the two-year period these increases were almost comparable to those obtained with the highest dose. Even the lowest dose gave a slowly increase in these activities. Both microsomal NADPH-cytochrome c reductase activity and the level of cytochrome P-450 were increased initially by exposure to 2% DEHP, but returned almost or completely to the control level after about 30 weeks of exposure. Depending on the dose of DEHP in the diet, peroxisomal catalase activity was elevated above the control level during the first year of treatment but was about the same as in the control animals during the second year. A substantial decrease in peroxisomal urate oxidase activity was observed throughout the entire experimental period. When treatment was ceased after one year, all activities returned to the control values within 2-3 weeks. These results demonstrate the complex nature of the effects caused by prolonged treatment with DEHP with cumulative increases at low doses.

Animals↗

PubMed1989

Uptake and modification of dietary polyprenols and dolichols in rat liver.

Short and long dolichols and polyprenols in free form or esterified with fatty acids were incorporated into liposomes and administered to rats through a gastric tube. The free alcohols were taken up by the liver to different extents. While uptake in other organs was less, it also involved the fatty acid esters. The use of systems other than liposomes did not increase the efficiency of uptake. Most of the administered lipids were recovered in the lysosomes. Exogenous dolichols and polyprenols were both partly esterified in the liver and, to some extent, also phosphorylated; a portion of the polyprenols was also alpha-saturated. These results indicate that various polyisoprenes are taken up, to a small extent, from the diet by tissues under normal conditions and in liver these dietary lipids undergo terminal modifications.

Animals↗