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Biomedical subjects

G Dallner

Publications and source records attributed to G Dallner.

At least 109 records · Page 6Linked to original sources
PubMed1989

Hydrolysis of dolichyl esters by rat liver lysosomes.

Dolichyl ester hydrolase activity is broadly distributed among the organs of the rat. The highest activity was found in spleen, brain, lung, and thyroid tissues, whereas this activity is very low in stomach and intestine. The esterase involved is localized to the lumen of lysosomes and, to some extent, in the plasma membranes. Hydrolysis occurs with both alpha-saturated and alpha-unsaturated polyisoprenes esterified with different fatty acids, but the rate of hydrolysis is strongly dependent on the nature of the substrate. The enzyme involved is inhibited by divalent cations, EDTA and EGTA and also by one of the products, dolichol. The esterase is activated by 3-[(3-cholamidopropyl) dimethylammonio]-1-propranesulfonic acid and taurodeoxycholate and inhibited by Triton X-100. Dolichyl esterase activity is completely inhibited by alpha- and beta-naphthyl acetate, phenylmethylsulfonyl fluoride, and beta-chloromethylmercurisulfate. These inhibitors, as well as the pH optimum for dolichyl ester hydrolysis, clearly differentiate the enzyme involved from cholesteryl esterase and triglyceride lipase. Microsomal phospholipase A hydrolyzes dolichyl esters at a slow rate only. In vivo labeling experiments with [3H]mevalonate demonstrated that newly synthesized dolichol is transported in esterified form to the lysosomes, where this lipid is slowly hydrolyzed by the esterase. The possibility is raised that the role of the fatty acyl moiety may be to target dolichol to its final location in the cell.

Animals↗
PubMed1989

Conditions for quantitation of dolichyl phosphate, dolichol, ubiquinone and cholesterol by HPLC.

Conditions for the isolation and quantitation of dolichyl phosphate, dolichol, cholesterol, and ubiquinone by reversed phase high performance liquid chromatography were investigated. A simple and fast sample preparation procedure using prepacked mini columns was employed. The UV spectra of the fractions obtained were examined and, in the case of dolichol compounds, the maximum absorbance around 205 nm was shown to be linearly dependent on the number of double bonds present in the isoprenolog. The analytical procedure described shows a very broad range of linearity (five orders of magnitude) and detects single dolichyl phosphate isoprenologs in amounts as small as 0.1 ng. The lowest overall recovery, that for dolichyl phosphate, is 77%. Use of isoprenolog 23 and ergosterol as internal standards reduced the variation in the method to 2.5, 4.0 and 5.5% for cholesterol, dolichyl phosphate and dolichol, respectively. The method described was employed to study the lipid composition of rat organs and biological variations in these compositions.

Animals↗
PubMed1989

Age-related changes in the lipid compositions of rat and human tissues.

The levels of cholesterol, ubiquinone, dolichol, dolichyl-P, and total phospholipids in human lung, heart, spleen, liver, kidney, pancreas, and adrenal from individuals from one-day-old to 81 years of age were investigated and compared with the corresponding organs from 2 to 300 day-old rats. The amount of cholesterol in human tissues did not change significantly during aging, but the level of this lipid in the rat was moderately elevated in the organs of the oldest animals. In human pancreas and adrenal the ubiquinone content was highest at one year of age, whereas in other organs the corresponding peak value was at 20 years of age, and was followed by a continuous decrease upon further aging. A similar pattern was observed in the rats, with the highest concentration of ubiquinone being observed at 30 days of age. Dolichol levels in human tissues increase with aging, but they increase to very different extents. In the lungs this increase is seven-fold, and in the pancreas it is 150-fold. The elevation in the dolichol contents of rat tissues ranges from 20 to 30-fold in our material. In contrast, the levels of the phosphorylated derivative of dolichol increased to a more limited extent, i.e., 2 to 6-fold in human tissues and even less in the rat. These results demonstrate that the levels of a number of lipids in human and rat organs are modified in a characteristic manner during the life-span. This is in contrast to phospholipids, which constitute the bulk of the cellular lipid mass.

Aging↗
PubMed1989

Discharge of newly-synthesized dolichol and ubiquinone with lipoproteins to rat liver perfusate and to the bile.

An effective system for perfusing rat liver using complete tissue culture medium and washed calf erythrocytes as oxygen carriers was devised. Infusion of taurocholate and glucose proved necessary to maintain stable metabolic activity and bile secretion during a 6-hr period. Perfusate pO2, pCO2 and pH values were monitored continuously and found to be stable. Electron microscopic examination revealed the maintenance of normal hepatic structure, even after 6 hr. Normal rates of protein and urea synthesis, no leakage of cytoplasmic enzymes, and continuous bile acid production demonstrated the functional integrity of this system. Using [3H]mevalonic acid as precursor, dolichol, dolichyl phosphate, ubiquinone and cholesterol were found to be continuously synthesized in this perfused liver system. All these lipids appeared in the perfusate, indicating discharge through the ER-Golgi system. The lipoproteins of the perfusate were isolated by ultracentrifugation and characterized with respect to size distribution and lipid composition. Dolichol was found in VLDL, LDL and HDL fractions, with the highest concentration present in the latter. In rat and human blood plasma this lipid was mainly associated with HDL. The ubiquinone in the perfusate was primarily associated with the VLDL fraction, while in rat plasma it was found more evenly distributed among all the three lipoprotein fractions studied. Dolichol, ubiquinone and cholesterol were also discharged to the bile, whereas dolichyl phosphate was not. Thus, newly-synthesized dolichol and ubiquinone are transported out of the hepatocyte to the blood and to the bile.

Animals↗
PubMed1989

The influence of di(2-ethylhexyl)phthalate on protein turnover in rat liver.

Treatment of rats with the plasticizer di(2-ethylhexyl)phthalate increases liver weight and leads to proliferation of mitochondria and peroxisomes. Using in vivo labelling with [3H]leucine, an increased rate of incorporation into the total protein of mitochondria and microsomes was observed. The half-lives of proteins in subcellular fractions were determined using [35S]methionine labelling. The half-lives for the total protein of mitochondria, microsomes, and supernatant were increased from 6 to 25 days, from 3.5 to 5.5 days and from 2.5 to 5 days upon treatment with phthalate esters. Experiments with [14C]guanidino-L-arginine indicated that some reutilization of [35S]methionine occurred, but this did not influence the results substantially. It appears that phthalate esters increase protein synthesis and decrease protein breakdown, the former effect being of greater importance.

Amino Acids↗
PubMed1989

Fatty acid oxidation in hepatic peroxisomes and mitochondria after treatment of rats with di(2-ethylhexyl)phthalate.

Rats were fed a diet containing di(2-ethylhexyl)-phthalate, which increases the number of peroxisomes and mitochondria in the liver. This proliferation does not change the ratio of phospholipid to protein in mitochondria or microsomes, but causes certain changes in the fatty acid composition of the phospholipids. The highest rates of peroxisomal and mitochondrial beta-oxidation are obtained with 12:0 and 16:0 fatty acids as substrates, respectively. A 3-4 fold increase in the rate of beta-oxidation by both organelles is caused by DEHP treatment, but there are no qualitative changes in the relative rates of oxidation of individual fatty acids. Short- and medium-chain carnitine acyltransferases in peroxisomes, microsomes and mitochondria, as well as the mitochondrial long-chain carnitine acyltransferase are induced to various extents. These results indicate that the increased beta-oxidation of fatty acids caused by phthalate treatment involves the same peroxisomal and mitochondrial pathways which operate under normal conditions.

Animals↗
PubMed1988

Esterification of dolichol in rat liver.

In the various subcellular fractions of rat liver 45-75% of the total dolichol was esterified with a fatty acid. The esterification reaction was localized exclusively in the microsomes, and the transferase activity is 3-fold higher in the cation-insensitive smooth microsomes than in other microsomal subfractions. Although fatty acyl-CoAs tested served as substrates, palmitoyl-CoA was the most rapidly utilized. None of the phosphatidylcholine or phosphatidylethanolamine species tested could be utilized to esterify dolichol with a fatty acid, indicating the absence of transacylation. alpha-Saturated dolichols were esterified at a higher rate than their alpha-unsaturated counterparts. Albumin and low concentrations of Triton X-100 activated the esterification reaction, which was not dependent on mono- or divalent cations, ATP, or CoA. The sensitivity of the transferase activity to trypsin indicates localization of the enzyme(s) involved on the outer surface of microsomes (i.e. the cytoplasmic surface of the endoplasmic reticulum), as is also the case for enzymes of dolichol biosynthesis. Transferase activity was detected in all tissues examined but at a much lower level than in liver and testis. The patterns of fatty acids in dolichol esters of different organelles exhibited some specificity. Labeling in vivo indicated that esterification of dolichol may play a role in targeting this lipid from the endoplasmic reticulum to lysosomes.

Acyltransferases↗
PubMed1988

The half-lives of dolichol and dolichyl phosphate in rat liver.

Rat liver dolichol and dolichyl-P were labeled by injection of [3H]mevalonate into the portal vein and their rates of synthesis and breakdown determined. In the initial phase the radioactivity appeared in alpha-unsaturated polyprenols. Subsequent saturation required 90 min. The half-lives of dolichols in microsomes were between 80 and 118 h, and shorter dolichols had shorter values of T1/2. The half-lives of dolichols in lysosomes were between 115 and 137 h, while microsomal dolichyl-P exhibited a T1/2 of 32 h. Injected dolichol was recovered in the lysosomes of hepatocytes and exhibited a rate of breakdown which was slower than that of the endogenous compound. These results indicate differences in the catabolism of dolichol at different subcellular locations, as well as differences between the catabolism of dolichol and dolichyl-P.

Animals↗
PubMed1988

Influence of cyclosporine A on protein synthesis in rat liver.

The influence of the immunosuppressive agent cyclosporine A on protein synthesis was investigated in rat liver in vivo and in vitro. Incorporation of [14C]leucine into total protein by microsomes was inhibited 36% in the presence of cyclosporine A. Treatment of rats with cyclosporine A also resulted in decreased protein synthesis by microsomes isolated from the same animals. This inhibition was dependent on the dose administered and the duration of treatment. The inhibitory factor in this latter case appeared to be associated with the supernatant fraction. Decreased in vivo incorporation of [35S]methionine into proteins of microsomal membranes and peroxisomes, but not into microsomal luminal proteins was also observed after treatment of rats with cyclosporine A. On SDS-polyacrylamide gel electrophoresis certain of the microsomal membrane and peroxisomal proteins displayed decreased labeling. Furthermore, cyclosporine A treatment decreased the inductive effects of phenobarbital, clofibrate, and phthalate ester on microsomal and peroxisomal enzymes. It is suggested that certain of the toxic effects of cyclosporine A are exerted through inhibition of cellular protein synthesis.

Animals↗
PubMed1988

Glutathione transferase in the urine: a marker for post-transplant tubular lesions.

Basic glutathione transferase released from the proximal tubular epithelium in the kidney was monitored in the urine of 69 recipients of renal allografts. The enzyme was isolated from human liver and the urinary analysis performed with radioimmunoassay. Patients receiving cyclosporine A without toxicity or rejection did not excrete this enzyme in their urine; whereas the urine of patients with cyclosporine A-induced nephrotoxicity contained significant amounts of the transferase (P less than 0.001). Patients with allograft rejection also showed increased urinary concentrations of the basic glutathione transferase, but had significantly lower values than patients with cyclosporine induced nephrotoxicity (P less than 0.001). During aminoglycoside and co-trimoxazole treatment, the urinary concentration of this transferase also increased. Patients with renal infarction showed a sudden increase in urinary transferase to very high levels. The results indicate that quantitative analysis of the basic glutathione transferase in urine is useful for monitoring renal tubular lesions present in various complications following transplantation, such as cyclosporine and antibiotic induced nephrotoxicity and renal infarction.

Adolescent↗
PubMed1988

Tissue and subcellular localizations of 3H-cyclosporine A in mice.

The tissue and subcellular localizations of 3H-cyclosporine A after administration to mice were determined with whole-body autoradiography and scintillation counting of lipid extracts of tissues and subcellular fractions. The radioactivity was widely distributed in the body and the pattern of distribution after oral or parenteral administration was the same, except that tissue levels were generally lower after oral administration. Pretreatment of the animals with a diet containing cyclosporine A for 30 days before the injection of radioactive cyclosporine A did not change the pattern of distribution substantially. No significant radioactivity was found in the central nervous system, except for the choroidal plexus and the area postrema region of the brain. In pregnant mice no passage of radioactivity from the placentas to fetuses was observed after a single injection. 3H-cyclosporine A and/or its metabolites showed a high affinity for the lympho-myeloid tissues, with a marked long-term retention in bone marrow and lymph nodes. There was massive excretion in the intestinal tract after parenteral administration, and the liver, bile, pancreas and salivary glands contained high levels of radioactivity. In the kidney radioactivity was confined to the outer zone of the outer kidney medulla. In liver homogenates no quantitatively significant binding of 3H-cyclosporine A and/or its metabolites to cellular molecules such as proteins, DNA, phospho- or neutral lipids was found. After lipid extraction with organic solvents, almost all radioactivity was recovered in the organic phase.

Animals↗
PubMed1988

Presence of dolichol and its derivatives in human blood.

Optimal conditions for the quantitative estimation of dolichol in human plasma were determined. Because of the large amounts of other lipids present in the blood, the extraction procedure, the procedure for hydrolysis, and the HPLC procedure are of decisive importance. Human plasma contains dolichol, dolichyl esters, and dolichyl phosphate at concentrations of 41, 102, and 55 ng/g, respectively. These polyisoprenoid lipids are associated with the high density lipoprotein fraction. The relative amounts and compositions of dolichyl esters in the plasma are similar to those observed in isolated human liver microsomes and Golgi vesicles. Sixty percent of the fatty acids present are saturated and almost no long-chain polyunsaturated components are present. There is no correlation between blood dolichol content and weight, sex, dietary state, or plasma cholesterol level, but there is an inverse relationship to plasma triglyceride content. A linear increase in the total plasma dolichol content with increasing age was found. In a few pathological conditions where the level of blood cholesterol was increased, the total blood dolichol content was not affected. Apparently, dolichol is a stable lipid component of human high density lipoprotein.

Animals↗