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Biomedical subjects

L M van Golde

Publications and source records attributed to L M van Golde.

At least 127 records · Page 7Linked to original sources
PubMed1984

Culture of rat cerebral oligodendrocytes in a serum-free, chemically defined medium.

Oligodendrocytes were isolated from the cerebra of young rats (5-10 days old) by trypsinization of the tissue followed by cell separation on Percoll gradients. The isolation was carried out in physiological, isotonic media. The cell yield was 2-4 X 10(6) cells per brain; the plating efficiency was greater than or equal to 70%. Isolated cells were seeded on poly-L-lysine-coated culture dishes and maintained in a serum-free, chemically defined medium for at least 30 days. After 10 days in culture 67 +/- 10% of the surviving cells were oligodendrocytes, as judged by immunocytochemical and morphological criteria, whereas most of the other cells reacted positively with antiserum against glial fibrillary acidic protein. The expression of typical oligodendrocyte markers (2':3'-cyclic-nucleotide 3'-phosphodiesterase, galactocerebrosides and myelin basic protein) was greatly enhanced under these serum-free conditions as compared with cultures in serum-containing medium. The antigenic markers (galactocerebrosides, myelin basic protein) were absent in the freshly isolated cells but could be detected after 3 days in culture by immunocytochemistry. The activity of 2':3'-cyclic-nucleotide 3'-phosphodiesterase increased from 75 nmol min-1 mg-1 protein on day 4 to 400 nmol min-1 mg-1 protein on day 14 in culture.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1982

Lamellar bodies isolated from adult human lung tissue.

Lamellar bodies, the intracellular storage form of pulmonary surfactant, were isolated from adult human lung tissue. As shown by electron microscopy, the isolated human lamellar bodies resembled the lamellar bodies isolated from experimental animals. Chemical analysis revealed that the lamellar bodies consisted largely of lipids, particularly phospholipids (85%). The major phospholipid was phosphatidylcholine, which accounted for 71% of the total phospholipids. Phosphatidylglycerol and phosphatidylethanolamine were 10 and 8%, respectively, of the lamellar body phospholipid. Phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, and sphingomyelin were minor components. Cholesterol was found to represent 60% of the neutral lipids or 9% of the total lipids. Phosphatidylcholine contained largely saturated fatty acids, of which palmitic acid was the most abundant. Disaturated phosphatidylcholines comprised 67% of the total phosphatidylcholines. Phosphatidylglycerol and phosphatidylethanolamine contained considerably less saturated fatty acids. Only 24% of phosphatidylglycerol was disaturated. The chemical composition of adult human lamellar bodies was very similar to that of lamellar bodies derived from experimental animals. The similarities in morphology and chemical composition of lamellar bodies suggest that surfactant metabolism in human lung may be similar to that of other mammals.

Aged↗
PubMed1981

Regulation of triacylglycerol synthesis in the liver: a decrease in diacylglycerol acyltransferase activity after treatment of isolated rat hepatocytes with glucagon.

Isolated rat hepatocytes were used to investigate the possibility of a short-term effect of glucagon on the synthesis of triacylglycerols in the liver. Incubation of hepatocytes in the presence of glucagon, followed by homogenization in a buffer containing F- (50 mM) and EDTA (2.5 mM), resulted in a 53% decrease in activity of microsomal diacylglycerol acyltransferase (EC 2.3.1.20), the only enzyme that is exclusively involved in the synthesis of triacylglycerols. The activity of cholinephosphotransferase (EC 2.7.8.2), which also uses diacylglycerols as substrate, was not decreased after exposure of the hepatocytes to glucagon. This may imply that triacylglycerol synthesis can be regulated independently of phosphatidylcholine synthesis. The activity of diacylglycerol acyltransferase in microsomes isolated from a homogenate of whole liver could be reduced by preincubating the microsomes with Mg2+ (5 mM), ATP (1 mM) and 105 000 X g supernatant. The enzyme could be reactivated by incubation of the washed microsomes with a 105 000 X g supernatant in the presence of dithiothreitol (5 mM). Fluoride (50 mM) inhibited this reactivation. It is concluded that the activity of diacylglycerol acyltransferase is subject to hormonal short-term control, possibly via a phosphorylation-dephosphorylation mechanism.

Acyltransferases↗
PubMed1980

Phospholipid-transfer activity in type II cells isolated from adult rat lung.

The soluble fraction from adult rat lung type II cells stimulated the transfer of various phospholipids between either liposomes or rat lung microsomes and rat lung mitochondria. Compared to whole lung, type II cells are highly enriched in transfer activity suggesting that phospholipid-transfer proteins play a role in the transport of surfactant phospholipids. Sephadex chromatography of pH 5.1 supernatant from type II cells yielded only one fraction catalysing the transfer of phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine, while chromatography of pH 5.1 supernatant from whole lung yielded in addition a specific phosphatidylcholine-transfer and a specific phosphatidylglycerol-transfer protein.

Animals↗
PubMed1980

Phospholipid transfer proteins in rat lung. Identification of a protein specific for phosphatidylglycerol.

From the soluble fraction of a rat lung homogenate, two proteins were partially purified which catalyzed the transfer of phosphatidylglycerol between liposomes and rat lung mitochondria. One protein was specific for phosphatidylglycerol, the other also catalyzed the transfer of phosphatidylcholine and phosphatidylethanolamine. The soluble fraction from rat liver also contained the nonspecific, but not the specific phosphatidylglycerol-transfer protein. It is suggested that the specific phosphatidylglycerol-transfer protein, as well as the phosphatidylcholine-transfer protein, which is also present in rat lung, contribute in the lung to the biogenesis of lamellar bodies, the organelles where surfactant phosphatidylcholine and phosphatidylglycerol are stored prior to secretion onto the alveolar surface.

Animals↗
PubMed1980

Effects of cortisol and thyroxine on phosphatidylcholine and phosphatidylglycerol synthesis by adult rat lung alveolar type II cells in primary culture.

1. The effect of cortisol and thyroxine on the formation of phosphatidylcholines and phosphatidylglycerols was studied in adult rat lung type II cells in primary culture. 2. Addition of cortisol enhanced the incorporation of [Me-14C]choline, [1-14C]acetate, [1-14C]palmitate, D-[U-14C]glucose, and [1(3)-3H]glycerol into total and disaturated phosphatidylcholines. 3. Cortisol also stimulated the formation of phosphatidylglycerols from labelled acetate, palmitate, glucose, and glycerol, but did not affect the formation of phosphatidylethanolamines. 4. Thyroxine alone did not significantly affect the formation of total and disaturated phosphatidylcholines nor that of phosphatidylglycerols or phosphatidylethanolamines. 5. Exposure of the cells to a combination of cortisol and thyroxine caused increases in the rates of synthesis of total and disaturated phosphatidylcholines from labelled choline, palmitate, and glycerol and in that of phosphatidyl-glycerols from labelled glycerol. These increases were about the same as those brought about by cortisol alone. In contrast to cortisol alone, the combination of cortisol and thyroxine did not significantly affect the entry of labelled acetate and glucose into phosphatidylcholines and phosphatidylglycerols. 6. The present results suggest that direct effects of glucocorticosteroids on the alveolar type II cell may play a role in the regulation of the synthesis of surfactant lipids in the adult lung.

Animals↗
PubMed1979

Lysolecithin acyltransferase and lysolecithin: lysolecithin acyltransferase in adult rat lung alveolar type II epithelial cells.

1. The specific activity of lysolecithin acyltransferase (EC 2.3.1.23) in sonicated adult rat lung alveolar type II epithelial cells, measured either alone or in combination with acyl-CoA synthetase (EC 6.2.1.3), was found to be an order of magnitude greater than that of lysolecithin:lysolecithin acyltransferase. 2. Lysolecithin acyltransferase in type II cells was found to prefer palmitoyl-CoA over oleoyl-CoA as substrate. The combination of lysolecithin acyltransferase and acyl-CoA synthetase was found to prefer palmitate over oleate for incorporation into phosphatidylcholine. 3. Compared to whole lung homogenate, sonicated adult rat type II cells are highly enriched in lysolecithin acyltransferase but not in lysolecithin:lysolecithin acyltransferase. 4. These observations indicate that in normal adult rat type II cells the deacylation-reacylation cycle is more important for the formation of dipalmitoyl phosphatidylcholine than the deacylation-transacylation process.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1978

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture.

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.

Acetates↗
PubMed1977

Role of lamellar bodies in the biosynthesis of phosphatidylcholine in mouse lung.

1. A lamellar body-enriched fraction was isolated from whole lung homogenates of mouse lung and its contamination with microsomes, mitochondria, and cytosol protein assessed by marker enzyme analyses. 2. By measuring the activity of cholinephosphotransferase (EC 2.7.8.2) in varying amounts of microsomes in the presence and absence of a fixed quantity of lamellar bodies, it could be demonstrated unequivocally that lamellar bodies of mouse lung lack the capacity to synthesize phosphatidylcholine de novo. 3. A similar approach allowed the conclusion that lamellar bodies of mouse lung do not contain lysophosphatidylcholine acyltransferase (EC 2.3.1.23) and lysophosphatidylcholine:lysophosphatidylcholine acyltransferase (EC 2.3.1.--), enzymes which play a putative role in the formation of pulmonary 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine. The activities of these enzymes observed in lamellar body fractions could be attributed completely to contaminating microsomes and cytosol respectively. 4. Lamellar bodies contributed to the activity of microsomal lysophosphatidylcholine acyltransferase by a cooperative effect. The possible role of this cooperation in the biosynthesis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine is discussed.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1977

Effect of fasting and feeding a high-sucrose, fat-free diet on the synthesis of hepatic glycerolipids in vivo and in isolated hepatocytes.

1. The synthesis of glycerolipids from a number of radioactive precursors, such as [2-3H] glycerol, [32P]phosphate, [Me-14C]choline and [1,2-14C2]ethanolamine proceeds in enzymatically isolated hepatocytes with a specificity that agrees very well with that observed in the liver in vivo. 2. The nutritional state of the rat has a profound influence on the glycerolipid metabolism of isolated hepatocytes. Fasting strongly decreased the incorporation of glycerol via sn-glycerol 3-phosphate into triacylglycerols whereas the formation of phosphatidylcholine and, particularly, phosphatidylethanolamine was much less affected by food deprivation. Refeeding a high-sucrose, fat-free diet caused a tremendous increase in the synthesis of diacylglycerols and triacylglycerols to values exceeding those found in hepatocytes from normally fed animals. The formation of phosphatidylcholine increased more slowly and the synthesis of phosphatidylethanolamine was even depressed by refeeding a high-sucrose, fat-free diet. 3. Alteration of the nutritional state resulted in similar changes in the amounts and metabolism of hepatic glycerolipids in vivo. The formation and, consequently, the amount of diacyglycerol and triacylglycerol were decreased by fasting and increased to values above normal in rats refed a high-sucrose, fat-free diet. The formation and the amounts of phospholipids in the liver decreased slightly by fasting which was mainly due to a decrease in phosphatidylcholine. Refeeding caused a significant increase in the formation and amount of phosphatidylcholine. The amount of phosphatidylethanolamine was even further diminished by feeding a high-sucrose, fat-free diet to 48-h-fasted rats. 4. These results show that the alterations, induced in the in vivo metabolism of hepatic glycerolipids, by changes of the dietary state, are also reflected in the isolated hepatocytes. This finding strengthens the potential significance of isolated hepatocytes in studies on the regulation of hepatic lipid metabolism.

Animals↗