Roger H. Lumb

A unique phosphatidylcholine exchange protein isolated from sheep lung

It has been established that disaturated phosphatidylchohne is the major surface-active agent in mammalian lung surfactant which lowers surface tension and prevents alveolar collapse at end-expiration [1,2]. Investigations of the synthesis, packaging and release of lung phosphatidylcholine (PC) have demonstrated that the endoplasmic reticulum of Type II pneumocytes is the major site of PC synthesis [3] and that lamellar bodies of these cells make up the primary intracellular storage organelle [4]. The mechanisms by which PC is transferred from the site of synthesis to the storage organelle or onto the alveolar surface are unknown. Phosphatidylcholine exchange proteins which catalyze the exchange of PC between membr~e structures in vitro [5,6], are widely distributed f7] and have been well characterized in bovine liver [8,9], heart [lO,ll] , brain [12] and in rat liver 113,141 andintestine [15]. We report here the purification of a novel phosphatidylcholine exchange protein from sheep lung with pI 7.1 and mol. wt 21 000 as determined by Sephadex G 75 chromatography and SDS-polyacrylamide gel electrophoresis. A second phosphatidylcholine exchange protein also was isolated from sheep lung which is very similar to that purified from bovine liver [S] ; pI 5.8, mol. wt 22 000. Both exchange proteins have been purified over 1 SO-fold.

Polyunsaturated fatty acids enhance the heat induced stress response in rainbow trout (Oncorhynchus mykiss) leukocytes.

The heat shock response has been studied extensively, yet the molecular signals that trigger the response remain elusive. The dogma of the heat shock response contends that denatured proteins initiate the response, but evidence is accumulating to point to a more complex system in which at least more than one signal is involved in this process. Thermal stress initiates changes in cellular phospholipid membrane physical state, which when acted upon by phospholipases may release lipid mediators that could serve as triggering signals during the heat shock response. We have examined the heat shock response in freshly isolated leukocytes from the pronephros of rainbow trout (Oncorhynchus mykiss). In this study, we show that leukocytes isolated from rainbow trout acclimated to 5 or 19 degrees C express elevated levels of heat shock protein 70 (hsp70) mRNA when heat shocked at 5 degrees C above their respective acclimation temperature and supplementation with exogenous docosahexaenoic acid or arachidonic acid followed by heat shock enhanced levels of hsp70 mRNA. The time course for docosahexaenoic acid induced enhancement of hsp70 mRNA was accelerated compared with heat shock alone, and staurosporine inhibited the docosahexaenoic acid induced increase of hsp70 mRNA. We also provide evidence that phospholipase A2 is involved in the heat shock response.

Evidence for protein-catalyzed transfer of platelet activating factor by macrophage cytosol

Platelet activating factor (PAF) is a potent, proinflammatory lipid. PAF is synthesized in response to stimuli and is rapidly destroyed by specific acetylhydrolases. In order to express its biological activity, PAF and its metabolites are transported among subcellular membranes by as yet unexplained mechanisms. We report here an assay system using methylcarbamyl-PAF (CPAF, 1-O-hexadecyl-2-O-(N-methylcarbamyl)-sn-glycero-3-phosphocholine) and a vesicle-extrusion technique for examining protein-catalyzed intermembrane transfer of CPAF, and demonstrate the presence of proteins catalyzing the separate transfer of CPAF and diacyl phosphatidylcholine in macrophage cytosol. The CPAF transfer activity is heat- and trypsin-sensitive and elutes from gel-filtration columns well separated from proteins catalyzing the transfer of phosphatidylcholine.

Rat eustachian tube synthesizes disaturated phosphatidylcholine

Otitis media results when the eustachian tube fails to adequately ventilate the middle ear. A surface tension-lowering substance may be required for normal tube opening, especially in young children with poorly developed naso-pharyngeal musculature. We report here that rat eustachian tube epithelium synthesizes disaturated phosphatidylcholine, which is recognized as the surface tension-lowering substance of pulmonary surfactant.

Spontaneous and protein-catalyzed transfer of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) between phospholipid bilayers

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor or alkylacetyl-GPC), a bioactive phospholipid that possesses hypotensive, platelet-aggregating and inflammatory properties, is known to be secreted by a variety of cell types. The biological activity of alkylacetyl-GPC is related to a precise chemical structure that implicates interaction with proteins. We have studied the spontaneous and protein-catalyzed transfer of alkylacetyl-GPC between phospholipid vesicles and have demonstrated the following: 1. There are at least two transferable pools of alkylacetyl-GPC in sonicated phospholipid vesicles. 2. These two pools differ in the rate at which they dissociate from the vesicles; one pool equilibrates between donor and acceptor vesicles instantaneously while the other pool is transferred much more slowly. 3. Dialysis of alkylacetyl-GPC between phospholipid vesicles through the aqueous phase is slow. 4. A protein fraction derived from rat lung cytosol catalyzes the transfer of the nonequilibrating pool of alkylacetyl-GPC between phospholipid vesicles; this transfer is superimposed on the spontaneous transfer and is unchanged in experiments using vesicles from which the rapidly equilibrating pool has been removed.

Synthesis of platelet activating factor by tissues from the rainbow trout, Salmo gairdneri

In mammals, platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a lipid mediator with biological activity at concentrations in the subnanomolar range. Although PAF is known to have many activities in mammals, little is known about its synthesis and importance in other vertebrate groups. We demonstrate here the synthesis of PAF from [3H]acetate by slices of trout gill, kidney, liver and spleen. PAF synthesis was stimulated by the calcium ionophore A23187 and was time-dependent. The radiolabeled PAF produced was characterized by TLC, HPLC, derivatization and by saponification and phospholipase A2 hydrolysis. These findings suggest that PAF may be an important mediator in fish.

Lung phosphatidylcholine transfer in six vertebrate species: Correlations with surfactant parameters

We have examined phosphatidylcholine transfer activity in lung-soluble fractions from six vertebrate species. There is a significant correlation between the amount of phosphatidylcholine transfer activity and both the alveolar surface area and surface active material. This suggested that phosphatidylcholine exchange proteins have a role in the lung surfactant system.

Transfer of phospholipids by a protein fraction obtained from canine pulmonary lavage

Surfactant phospholipid exists in multicompartment pools within the subphase of the lung. Movement among these pools and back into type II alveolar cells may be catalyzed by a phospholipid transfer protein resident in the subphase. We demonstrate here that a protein fraction obtained from canine lung lavage catalyzes the intermembrane transfer of all the major surfactant phospholipids. The protein is probably not derived from serum and is unrelated to surfactant proteins that have already been described.

sInsect lipids: Carbon number distribution of triglycerides in five species

Abstract 1. 1. Triglycerides were isolated from several species of insects at various developmental stages by thin-layer chromatography. 2. 2. The carbon number distribution of the triglycerides and their fatty acid composition were determined by gas-liquid chromatography. 3. 3. A distinct triglyceride carbon number distribution was obtained for each species. 4. 4. Changes in the carbon number distribution and fatty acid composition of the triglycerides corresponding to metamorphosis were observed. 5. 5. Comparison of percentages of carbon number determiend for triglyceride with calculated randomly distributed values suggests that in insects a non-random distribution of fatty acids occurs biosynthesis of triglyceride.

Effects of Increased Dietary Marine Fish Oil on the Omega-3 Fatty Acid Content of Rainbow Trout Fillets

Abstract We tested the hypothesis that, under commercial production conditions, rainbow trout (Oncorhynchus mykiss) will accumulate omega-3 (n-3) fatty acids in their edible flesh if their diet contains sufficient quantities of marine fish oil. Our results indicate that rainbow trout muscle will accumulate levels of n-3 fatty acids comparable to those reported for marine salmonids after 4 weeks of being fed a diet enhanced with n-3 fatty acids without adversely affecting growth rate.