Joseph J. Batenburg

Biosynthesis of lipids in golgi complex and other subcellular fractions from rat liver

Abstract 1. 1. Golgi complex, rough and smooth microsomes, plasma membranes, mitochondria and nuclei from rat liver were isolated and their purity assessed using specific marker enzymes. 2. 2. The various subcellular fractions were assayed for the following processes: biosynthesis of sphingomyelin, CDPdiglycerides, phosphatidylinositol, phosphatidylserine, the conversion of phosphatidylserine into phosphatidylethanolamine, the formation of lecithin via N-methylation, and the activation of palmitic and octanoic acids. 3. 3. None of these processes were found to be present in Golgi complex. 4. 4. The endoplasmic reticulum appears to be the principal site in the cell for the synthesis of sphingomyelin, CDPdiglycerides, phosphatidylinositol, phosphatidylserine and the formation of lecithin. Interestingly, the biosynthesis of phosphatidylserine appears to be four times more active in rough than in smooth microsomes, which might suggest a ribosomal localization of this process. 5. 5. Except for CDPdiglyceride synthesis, mitochondria do not contain any of the synthesizing activities described in 4. Mitochondria are, however, the only site in the cell where phosphatidylserine is decarboxylated. This activity appears to be localized in the inner membrane. 6. 6. The activation of palmitate is localized predominantly in endoplasmic reticulum and mitochondria, though some activity was detected in plasma membranes as well. All other cell organelles, including Golgi and probably nuclei, did not contain significant palmitoyl-CoA synthetase activity. The subcellular distribution of octanoyl-CoA synthetase resembled that of palmitoyl-CoA synthetase except that the former enzyme is more active in mitochondria than in microsomes.

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.

Lipids in host-pathogen interactions: pathogens exploit the complexity of the host cell lipidome.

Abstract Lipids were long believed to have a structural role in biomembranes and a role in energy storage utilizing cellular lipid droplets and plasma lipoproteins. Research over the last decades has identified an additional role of lipids in cellular signaling, membrane microdomain organization and dynamics, and membrane trafficking. These properties make lipids an attractive target for pathogens to modulate host cell processes in order to allow their survival and replication. In this review we will summarize the often ingenious strategies of pathogens to modify the lipid homeostasis of host cells, allowing them to divert cellular processes. To this end pathogens take full advantage of the complexity of the lipidome. The examples are categorized in generalized and emerging principles describing the involvement of lipids in host–pathogen interactions. Several pathogens are described that simultaneously induce multiple changes in the host cell signaling and trafficking mechanisms. Elucidation of these pathogen-induced changes may have important implications for drug development. The emergence of high-throughput lipidomic techniques will allow the description of changes of the host cell lipidome at the level of individual molecular lipid species and the identification of lipid biomarkers.

Characteristics of Surfactant Protein A and D Binding to Lipoteichoic Acid and Peptidoglycan, 2 Major Cell Wall Components of Gram-Positive Bacteria

Infection with gram-positive bacteria is a major cause of pneumonia. Surfactant proteins A (SP-A) and D (SP-D) are thought to play an important role in the innate immunity of the lung. Both proteins can bind to gram-positive bacteria. Until now, it was not known with which surface component(s) of gram-positive bacteria SP-A and SP-D interact. Lipoteichoic acid (LTA) and peptidoglycan (PepG) are components of the cell wall of gram-positive bacteria. By use of a solid phase-based binding assay, LTA of Bacillus subtilis was shown to be bound by SP-D but not by SP-A. Unmodified PepG of Staphylococcus aureus was bound by SP-D. SP-D binding to both LTA and PepG was calcium dependent and carbohydrate inhibitable. These results indicate that SP-D interacts with gram-positive bacteria via binding to the cell wall components LTA and PepG and that the carbohydrate recognition domain is responsible for this binding.

Mechanisms involved in the synthesis of disaturated phosphatidylcholine by alveolar type II cells isolated from adult rat lung.

1. Alveolar type II cells isolated from adult rat lung incorporated radioactively labelled palmitate predominantly into the 2-position of disaturated phosphatidylcholine. In disaturated diacylglycerol, however, the radioactivity was almost equally distributed between the 1- and 2-positions. 2. Exposure of isolated type II cells to the phospholipase A2 inhibitors 4-bromophenacylbromide or quinacrine dihydrochloride led to a decreased synthesis of total phosphatidylcholines from various labelled precursors. Interestingly, it also led to an increased degree of unsaturation of the phosphatidylcholines synthesized by the cells. 3. Incubation of type II cell sonicates with radioactively labelled CDPcholine resulted in the formation of labelled phosphatidylcholine; 56% of this phosphatidylcholine appeared to be disaturated. In similar experiments with homogenates from whole lung, 20% of the synthesized phosphatidylcholine was disaturated. 4. These results suggest that both direct synthesis de novo and remodeling of 1-saturated-2-unsaturated phosphatidylcholines contribute to the biosynthesis of disaturated phosphatidylcholine in isolated alveolar type II cells.

Isolation of alveolar type II cells from fetal rat lung by differential adherence in monolayer culture.

Type II alveolar epithelial cells were isolated from fetal rat lung by differential adherence in monolayer culture. The preparation had a high degree of purity, as assessed by phase contrast microscopy and immunocytochemistry. Purity, based on reactivity with specific anti-adult lung serum (SAALS), which recognizes only type II cells, was 91% for cells isolated from 19-day fetal lungs and 79% for cells isolated from 21-day fetal lungs. The lower purity of type II cells in cultures derived from 1-day postnatal rat lungs (51% cells reactive with SAALS) is probably due to a lower tendency of the type II cells from neonatal rats to adhere to culture dishes than of type II cells from fetal rats. Type II cells isolated from 21-day fetal lungs contained a higher percentage phosphatidylglycerol and incorporated [Me-3H]choline faster into phosphatidylcholine (PC) than type II cells isolated from 19-day fetal lungs. Moreover, in cell preparations derived from lungs at fetal day 21, a higher percentage of epithelial cells contained lamellar bodies than in preparations derived from lungs at fetal day 19. The observation of these differences in the stage of maturation indicates that these differences, which are typical features of the original material, are not obliterated by differentiation during the culture. Type II cells isolated according to the present procedure were capable of synthesizing PC with a high percentage of the disaturated species. This method for the isolation of fetal type II cells may be a useful tool in studies concerning surfactant synthesis and its regulation in the fetal lung.

The rate-limiting step in the biosynthesis of phosphatidylcholine by alveolar type ii cells from adult rat lung

1. The rate-limiting reaction in the biosynthesis of surfactant phosphatidylcholine by type II cells isolated from adult rat lung was examined. 2. Studies on the uptake of [Me-14C]choline and its incorporation into its metabolites over a 5 h period indicated that in these cells the cholinephosphate pool was much larger than both the choline and CDPcholine pool. This is consistent with the idea that the rate-limiting reaction is that catalyzed by cholinephosphate cytidylyltransferase. 3. Evidence that cholinephosphate cytidylyltransferase is the slowest of the three enzymes incorporating choline into phosphatidylcholine was also obtained from pulse-chase experiments. [Me-14C]Choline taken up by the cells was rapidly converted into cholinephosphate during the pulse period. As the radioactivity disappeared from cholinephosphate during the chase period, the label was incorporated immediately into phosphatidylcholine, without much change in the labelling of CDPcholine. This indicates that the cholinephosphotransferase is at least as fast as the cholinephosphate cytidylyltransferase. 4. Inclusion of palmitate in the chase medium accelerated the conversion of labelled cholinephosphate into phosphatidylcholine and decreased the radioactivity associated with CDPcholine. This indicates that under these conditions the cholinephosphate cytidylyltransferase reaction cannot keep up with increased utilization of the CDPcholine in the terminal step of the CDPcholine pathway.

Dimeric N-Terminal Segment of Human Surfactant Protein B (dSP-B1–25) Has Enhanced Surface Properties Compared to Monomeric SP-B1–25

Surfactant protein B (SP-B) is a 17-kDa dimeric protein produced by alveolar type II cells. Its main function is to lower the surface tension by inserting lipids into the air/liquid interface of the lung. SP-Bs function can be mimicked by a 25-amino acid peptide, SP-B(1-25), which is based on the N-terminal sequence of SP-B. We synthesized a dimeric version of this peptide, dSP-B(1-25), and the two peptides were tested for their surface activity. Both SP-B(1-25) and dSP-B(1-25) showed good lipid mixing and adsorption activities. The dimeric peptide showed activity comparable to that of native SP-B in the pressure-driven captive bubble surfactometer. Spread surface films led to stable near-zero minimum surface tensions during cycling while protein free, and films containing SP-B(1-25) lost material from the interface during compression. We propose that dimerization of the peptide is required to create a lipid reservoir attached to the monolayer from which new material can enter the surface film upon expansion of the air/liquid interface. The dimeric state of SP-B can fulfill the same function in vivo.

The Role of Surfactant Proteins in DPPC Enrichment of Surface Films

A pressure-driven captive bubble surfactometer was used to determine the role of surfactant proteins in refinement of the surface film. The advantage of this apparatus is that surface films can be spread at the interface of an air bubble with a different lipid/protein composition than the subphase vesicles. Using different combinations of subphase vesicles and spread surface films a clear correlation between dipalmitoylphosphatidylcholine (DPPC) content and minimum surface tension was observed. Spread phospholipid films containing 50% DPPC over a subphase containing 50% DPPC vesicles did not form stable surface films with a low minimum surface tension. Addition of surfactant protein B (SP-B) to the surface film led to a progressive decrease in minimum surface tension toward 1 mN/m upon cycling, indicating an enrichment in DPPC. Surfactant protein C (SP-C) had no such detectable refining effect on the film. Surfactant protein A (SP-A) had a positive effect on refinement when it was present in the subphase. However, this effect was only observed when SP-A was combined with SP-B and incubated with subphase vesicles before addition to the air bubble containing sample chamber. Comparison of spread films with adsorbed films indicated that refinement induced by SP-B occurs by selective removal of non-DPPC lipids upon cycling. SP-A, combined with SP-B, induces a selective adsorption of DPPC from subphase vesicles into the surface film. This is achieved by formation of large lipid structures which might resemble tubular myelin.

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.