ASSEMBLY OF LIPIDS INTO MEMBRANES IN ACANTHAMOEBA PALESTINENSIS : I. Observations on the Specificity and Stability of Choline-14C and Glycerol-3H as Labels for Membrane Phospholipids

In order to determine the feasibility of using radioactive precursors as markers for membrane phospholipids in Acanthamoeba palestinensis, the characteristics of phospholipids labeled with choline-¹⁴C and glycerol-³H were examined. Choline-¹⁴C was found to be a specific label for phosphatidyl choline. There was a turnover of the radioactive moiety of phosphatidyl choline at a rate that varied with the concentration of nonradioactive choline added to the growth medium. Radioactivity was lost from labeled phosphatidyl choline into the acid-soluble intracellular pool and from the pool into the extracellular medium. This loss of radioactivity from cells leveled off and an equilibrium was reached between the label in the cells and in the medium. Radioactive choline was incorporated into phosphatidyl choline by cell-free microsomal suspensions. This incorporation leveled off with the attainment of an equilibrium between the choline-¹⁴C in the reaction mixture and the choline-¹⁴C moiety of phosphatidyl choline in the microsomal membranes. Therefore, a choline exchange reaction may occur in cell-free membranes, as well as living A. palestinensis. In contrast to choline-¹⁴C, the apparent turnover of glycerol-³H-labeled phospholipids was not affected by large concentrations of nonradioactive choline or glycerol in the medium. The radioactivity in lipids labeled with glycerol-³H consisted of 33% neutral lipids and 67% phospholipids. Phospholipids labeled with glycerol-³H turned over slowly, with a concomitant increase in the percentage of label in neutral lipids, indicating a conversion of phospholipids to neutral lipids. Because most (~96%) of the glycerol-³H recovered from microsomal membranes was in phospholipids, whereas only a minor component (~2%) of the glycerol-³H was in the phospholipids isolated from nonmembrane lipids, glycerol-³H was judged to be a specific marker for membrane phospholipids.     

Phospholipids of the Thiobacilli

Phosphatidyl glycerol, disphosphatidyl glycerol, and phosphatidyl ethanolamine were found in all of the Thiobacillus species studied. T. thioparus possessed only these phospholipids. T. intermedius, T. neapolitanus, and T. thiooxidans contained phosphatidyl-N-monomethylethanolamine, and T. novellus lipids contained phosphatidyl-N-monomethylethanolamine, phosphatidyl-N-N-dimethylethanolamine, and phosphatidyl choline, in addition to the three phospholipids common to all of the thiobacilli. Methionine was found to act as a methyl donor in the biosynthesis of the methylated forms of phosphatidyl ethanolamine. Phosphatidyl inositol was not detected in any of the organisms. Changing the nutrient medium did not result in a qualitative change in the phospholipid spectrum of the cultures.     

Effect of Feeding Defatted Millet (Sorghum vulgarie) Flour at Different Protein Levels on Phospholipids of Rat Liver

Effect of feeding defatted millet flour at 5, 10 and 14.5 percent protein levels respectively to rats for 6 weeks has been studied on their liver phospholipids. Feeding of millet at these protein levels significantly increased liver total lipids and total glycerides as compared to control rats fed casein at 10 percent level. Liver phospholipids and phosphatidyl choline were significantly increased in rats fed millet at 10 and 14.5 percent protein levels whereas phosphatidyl ethanolamine was increased in all millet protein fed groups. Incorporation of (counts/liver/100 g body weight) into liver total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine was significantly increased in M–5 and M–15% groups and it was decreased in phosphatidyl choline in rats of M–10% groups as compared to control. The effects of feeding millet on liver phospholipids are due to the quantity as well as quality of the millet protein.     

Serum of normal subjects contains IgG with antibody-like specificity for phospholipids

Using counterimmunoelectrophoresis, sera from normal subjects display precipitin activity against liposomes made by phosphatidic acid, phosphatidyl ethanolamine, phosphatidyl serine and cardiolipin. No activity is detected by using liposomes made with phosphatidyl choline, sphingomyelin and phosphatidyl inositol. The same pattern of precipitation is obtained when pure IgG from normal subjects is tested against phospholipids. No precipitin activity is found by using pure IgM. The precipitin reaction is prevented by preincubating liposomes with Fab obtained from normal IgG.     

The molecular organization of nerve membranes

Summary The lipid content and composition from an axolemma-rich preparation isolated from squid retinal axons was analyzed.The lipids, which accounted for 45.5% of the dry weight of this membrane, were composed of 22% cholesterol, 66.7% phospholipids and 5.2% free fatty acids. The negatively charged species phosphatidyl ethanolamine (37%), phosphatidyl serine (10%) and lysophosphatidyl ethanolamine (4%) made up 51% of the phospholipids. The amphoteric phosphatidyl choline and sphingomyelin accounted for 39% and 4%, respectively.The relative distribution of fatty acids in each of the isolated phospholipids was studied. The most remarkable feature of these phospholipids was the large proportion of long-chain polyunsaturated fatty acids. The 226 acyl chain accounted for 37% in phosphatidyl ethanolamine, 21.7% in phosphatidyl choline, 17.5% on phosphatidyl serine and 20.3% in sphingomyelin (all expressed as area %).The molar fraction of unsaturated fatty acids reached 65% in phosphatidyl ethanolamine and 42.0 and 44.8% in phosphatidyl choline and phosphatidyl serine, respectively. The double bond index in these species varied between 1.0 and 2.6.The lipid composition of the axolemma-rich preparation isolated from squid retinal axons appears to be similar to other excitable plasma membranes in two important features: (a) a low cholesterol/phospholipid molar ratio of 0.61; and (b) the polyunsaturated nature of the fatty acid of their phospholipids.This particular chemical composition may contribute a great deal to the molecular unstability of excitable membranes.The preceding papers of this series were published inArchives of Biochemistry and Biophysics.     

The manipulation of polar head group composition of phospholipids in the wheat Miranovskaja 808 affects frost tolerance

Caryopses of the frost-resistant cultivar of the wheat Triticum aestivum L., Miranovskaja 808, were germinated and grown in the presence of various concentrations of choline chloride. Changes in the composition of leaf total phospholipids and leaf total fatty acids at two extreme temperatures (25°C and 2°C) as well as changes in frost resistance were followed. A choline chloride concentration-dependent accumulation of phosphatidyl choline was observed in the leaves. Seedlings grown at 2°C accumulated more phosphatidyl choline at each choline chloride concentration than those grown at 25°C. There was an inverse relationship between the contents of phosphatidyl choline and phosphatidic acid in the leaves. Neither the temperature nor choline chloride seemed to affect fatty-acid composition. Modification of polar-head group composition of phospholipids affected frost tolerance: Seedlings grown in the presence of 15 mM choline chloride at 25°C exhibited a freezing resistance equal to that of hardened controls. The data indicate that the polar-head group composition of membrane phospholipids in plants can be easily manipulated and point to the importance of phosphatidyl choline in cold adaptation processes.     

Lipids of ocular tissues: VII. Positional distribution of the fatty acids in the phospholipids of bovine retina rod outer segments

The positional distribution of the fatty acids in the major phospholipids of bovine retina rod outer segments was determined. Phosphatidyl ethanolamine and phosphatidyl serine have mostly saturated acids in the 1-position and docosahexaenoic acid in position 2. These phospholipids contain 94 and 79%, respectively, of polyun-saturated acids in the 2-position. Phosphatidyl choline contains mostly saturated acids in the 1-position, but has significant quantities of palmitic in the 2-position along with docosahexaenoic acid. The levels of docosahexaenoic acid in rod outer segment phospholipids are among the highest yet reported for membrane phospholipids, amounting to 23% in phosphatidyl choline, 39% in phosphatidyl ethanolamine, and 45% in phosphatidyl serine.     

Incorporation of32P-orthophosphate into phospholipids by a toxigenic and a nontoxigenic strain of Aspergillus flavus

The incorporation of³²P-orthophosphate into phospholipids by a toxigenic and a nontoxigenic strain ofAspergillus flavus was compared on a glucose-salts medium (AM medium) and a sucrose-yeast extract medium (YES medium). AM medium gave higher incorporation of³²P than YES medium. In AM medium the highest specific activities were found in phosphatidyl choline and phosphatidyl ethanolamine but in YES medium phosphatidyl inositol and phosphatidyl serine also had specific activities of the same order as phosphatidyl choline and phosphatidyl ethanolamine. Mycelia of the toxigenic strain grown and resuspended in AM medium yielded 1.4 times specific activities given by nontoxigenic strain. The two strains did not differ very much in the incorporation obtained in the other media combination tried. These results are in contrast to the large differences obtained in the incorporation of¹⁴C-acetate in earlier studies. The significance of these findings are discussed.These results were presented at the Symposium on the Chemistry and Metabolism of Lipids and Related Subjects held at Vallabhbhai Patel Chest Institute, Delhi on October 3–5, 1969.     

The occurrence of phosphatidyl choline exchange protein in leaves

The transfer of phosphatidyl choline between liposomes was stimulated by the protein fractions from spinach leaves, etiolated and greening leaves of $\text{Avena}$ seedlings. This is confirmed by the transfer of [¹⁴C]phosphatidyl choline or spin-labeled phosphatidyl choline between donor and acceptor liposomes. ESR spectrum changes also indicated that no spin-labeled phosphatidyl choline was released from donor liposomes by spinach leaf protein unless acceptor liposomes were present. [¹⁴C]phospholipids were transferred from liposomes to both spinach chloroplasts and $\text{Avena}$ etiochloroplasts by phosphatidyl choline exchange protein from germinated castor bean endosperms and further from liposomes to spinach chloroplasts by spinach leaf protein. These results support the view that phosphatidyl choline in the plastid is supplied from the synthesis site, the endoplasmic reticulum, by phospholipid exchange protein.     

Effect of phospholipids on the activity of highly-purified liver microsome cytochrome P-450 in a reaction of aniline and naphthalene hydroxylation by hydroperoxides

The effect of different phospholipids on the functional activity of highly purified cytochrome P-450 used as a co-substrate of cumene hydroperoxide was examined. At the molar ratio of phospholipids to cytochrome P-450 that was equal to 30, phosphatidyl serine, phosphatidyl inositol, the total fraction of microsomal phospholipids, and lysophosphatidyl choline increased the hydroxylation rate of aniline and naphthalene. The effect of the above phospholipids on the rate of naphthalene oxidation was much more pronounced. Phosphatidyl choline and sphingomyelin in similar large quantities did not exert a stimulating effect on the reactions studied. The kinetic parameters of aniline oxidation in the systems containing phospholipids that produced an activating effect were investigated.     

Phospholipid-enzyme interactions of chitin synthase of Coprinus cinereus

Abstract The effect of phospholipids on chitin synthase activity has been studied with digitonin-solubilized and partially purified preparations from Coprinus cinereus . When cholate was used as detergent, it inhibited enzyme activity, but this inhibition was reversed by increasing concentrations of phospholipids. Preincubation with cholate and phospholipid caused irreversible loss of activity. When sonicated with solubilized enzyme preparation, dimyristoyl phosphatidyl choline strongly stimulated activity, while dioleoyl phosphatidyl choline was inhibitory. The Arrhenius plot of the effect of temperature on enzyme activity contained breaks, characteristic of a membrane-bound enzyme. It is suggested that chitin synthase requires an annulus of phospholipids for activity.     

Dual effect of tannic acid on the preservation and ultrastructure of phosphatidyl choline vesicles

Summary The use of tannic acid has been proposed to improve the preservation of phospholipids in tissues. We investigated the effects of tannic acid on the preservation of small unilamellar vesicles, prepared from sonicated aqueous suspensions of phospholipids.With cryo-electron microscopy it is demonstrated that small unilamellar vesicles are formed after sonication of the phospholipid suspensions. Fixation of vesicles without tannic acid results in extraction of the phospholipids during dehydration and embedding. Fixation of vesicles containing phosphatidyl choline with tannic acid, with or without glutaraldehyde, results in a fast (within a second) aggregation of the vesicles and the resulting sediment can be dehydrated and embedded when a postfixation in osmium tetroxide is carried out. Small unilamellar vesicles fixed in this way are retrieved in thin sections as multilamellar vesicles with a periodicity of about 5 nm for dimyristoylphosphatidyl choline and about 6 nm for dioleoylphosphatidyl choline.By using ¹³C-phosphatidyl choline it was also demonstrated that tannic acid prevents to a large extend the extraction of phosphatidyl choline during fixation, dehydration and embedding. This dual effect of tannic acid on phosphatidyl choline, aggregation and fixation, should be considered when using tannic acid in tissue preparation.     

Hepatic Subcellular Fractions Lipids in Rats Fed Millet (Sorghum vulgarie) at Different Protein Levels

Effect of feeding defatted millet (Sorghum vulgarie) flour at 5, 10 and 14.5% protein levels respectively for six weeks has been studied on rat liver mitochondrial, microsomal and supernatant fractions total lipids, cholesterol, triglycerides, total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine. The results have been compared with rats fed casein at 10% level for the same period. The metabolism of liver subcellular fractions lipids of millet diet and casein diet fed rats has been studied by the incorporation of acetate-1-¹⁴C and . A significant increase in mitochondrial triglycerides of rats fed millet diet at 5 and 10% protein level, in microsomes of rats fed millet diet at 5, 10 and 15% protein levels and in supernatant fractions of rats fed millet diet at 5 and 15% protein levels was observed. A significant increase in total cholesterol in mitochondria and microsomes and a significant decrease in supernatant fraction of rats fed millet diet at 10% protein level was observed. A significant increase in mitochondrial total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine in rats fed millet diet at 10% protein level and a decrease in these in rats fed millet diet at 5 per cent protein level was observed. In microsomes total phospholipids were increased in rats millet diet at 10% protein level and phosphatidyl choline was increased in rats fed millet diet at 15% protein level. Total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine were significantly reduced in the supernatant fraction of rats fed millet at 10% protein level.

Incorporation of acetate-1-¹⁴C into nonsaponifiable fraction of mitochondria, microsomes and supernatant fractions of rats fed millet diet at 5 and 15 % protein levels was significantly greater, and in saponifiable fractions of the above subcellular fractions was greater in rats fed millet diet at 5 per cent protein level. The specific activity (counts/min/mg) of free cholesterol in mitochondria, microsomes and supernatant fractions of millet diet fed rats was significantly greater, whereas the specific activity of triglycerides was not significantly different from the controls. The acetate-1-¹⁴C specific activity of phosphatidyl choline and phosphatidyl ethanolamine was significantly greater in all the above subcellular fractions of millet diet fed rats (except of phosphatidyl choline in rats fed millet diet at 5 % protein level). The specific activities of phosphatidyl choline were significantly greater in mitochondria of rats fed millet diet at 5 % protein level and of phosphatidyl choline and phosphatidyl ethanolamine in microsomes and supernatant fractions of rats fed millet diet at 5 and 15% protein levels. The specific activities of phosphatidyl choline were significantly decreased in mitochondria and microsomes of rats fed millet diet at 10% protein level. The total acetate-1-¹⁴C activities (counts/min/g equivalent wet liver) of free and esterified cholesterol triglycerides, phosphatidyl choline and phosphatidyl ethanolamine showed that their synthesis from acetate-1-¹⁴C was either enhanced in millet diet fed rats or was comparable to the controls. The total activity of (counts/min/g equivalent wet liver) into phosphatidyl choline and phosphatidyl ethanolamine showed that their synthesis was decreased in microsomes of rats fed millet diet at 10% protein level, increased in rats fed millet diet at 5 and 15% protein levels.     

Coupled Na+ -K+ transport in vesicles containing a purified (NaK)-ATPase and only phosphatidyl choline.

Endogenous phospholipids of a purified (NaK)-ATPase were displaced by exogenous phosphatidyl choline. If vesicles were made from phosphatidyl choline and enzyme containing only phosphatidyl choline, coupled Na⁺K⁺ transport could be demonstrated. This transport was inhibitable by ouabain. Therefore, the number of components necessary for Na⁺K⁺ transport has been reduced to the purified (NaK)-ATPase and one phospholipid.     

Incorporation of bovine thyroid peroxidase in liposomes

Bovine thyroid peroxidase (TPO), an enzyme requiring lipids for demonstrating catalytic activity, was incorporated in liposomes made of pure phospholipids. The enzyme did not show high differences in activity when bilayer thickness was changed, but dipalmitoyl phosphatidyl choline (DPPC) seemed to be more appropiate for activity. The perturbation caused on lipid fluidity by enzyme incorporation was studied by differential scanning calorimetry (DSC) and fluorescence polarization of the apolar probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The complexes of TPO with dimyristoyl phosphatidyl choline (DMPC), DPPC, and distearoyl phosphatidyl choline (DSPC) bilayers showed transition temperatures (T_c) which were lower than the characteristic ones shown by liposomes with the respective phospholipids alone. The microsomal fraction from which TPO was extracted was in the fluid state at 37°C, the temperature at which thyroid peroxidase works ‘in vivo’. Since the effect of the protein in lowering the transition temperature of the phospholipids was so low, the contribution of phospholipids containing unsaturated fatty acids has to be essential for obtaining a fluid bilayer at body temperature.     

Characterization of gastric mucosal membranes: lipid composition of purified gastric microsomes from pig, rabbit, and frog

The lipid compositions of the gradient-purified gastric microsomal membranes from the fundic mucosa of pig, rabbit, and frog were determined. The total lipid content varied widely. Compared to the rabbit (21.6 ± 0.6 mg/100 mg protein), the pig had about twice as much and the frog about three times as much lipid. The levels of cholesterol were higher in both mammalian species (about 32% of the lipid) compared to frog (23%). Phospholipids accounted for about 45, 54, and 52% of the total microsomal lipids from pig, rabbit, and frog and the molar ratios of cholesterol to phospholipid in the three species were 1.95, 1.6, and 1.17, respectively. Phosphatidyl choline and phosphatidyl ethanolamine together constituted about 75% of the total phospholipids in pig and frog and 93% in rabbit gastric microsomes. Sphingomyelin comprised 19.3, 3.2, and 1.5% in pig, rabbit, and frog, respectively. Phosphatidyl inositol constituted 5, 2.7, and 23.6% in pig, rabbit, and frog, respectively. The ratios of phosphatidyl ethanolamine to phosphatidyl choline were 1.17, 1.1, and 0.85 in pig, rabbit, and frog, respectively. The saturated fatty acids 16:0 and 18:0 and the unsaturated fatty acid 18:1 and 18:2 were the predominant fatty acids in all phospholipids. The ratios of saturated to unsaturated fatty acids were between 0.8 and 0.9 in phosphatidyl choline and 0.27 and 0.5 in phosphatidyl ethanolamine from all three species. The contributions by saturated fatty acids were much more in phosphatidyl inositol and sphingomyelin than in phosphatidyl choline and phosphatidyl ethanolamine from all species. Position 1 of phosphatidyl choline had 63% saturated and 37% unsaturated fatty acids; while the reverse was true for position 2. Phosphatidyl ethanolamine, however, had 85% saturated fatty acids in position 1 compared to only 25% in position 2. Arachidonic acid (20:4) was present in significant amounts in all species located exclusively at position 2 of both phosphatidyl choline and phosphatidyl ethanolamine.     

Control of the potassium ion-stimulated adenosine triphosphatase of pig gastric microsomes: effects of lipid environment and the endogenous activator

The K⁺-stimulated ATPase activity associated with the purified gastric microsomes from the pig gastric mucosa can be completely inactivated by treatment with 15% ethanol for 60 s at 37 °C but not at 25 °C. Sequential exposure of the microsomes to 15% ethanol at 25 and 37 °C caused the release of 2.9 and 4.3% of the total membrane phospholipids, respectively, consisting entirely of phosphatidyl choline and phosphatidyl ethanolamine. The ethanol-treated (37 °C) membrane had high basal (with Mg²⁺ as the only cation in the assay mixture) activity, which was further enhanced during reconstitution with phosphatidyl choline or phosphatidyl ethanolamine. The high basal activities could be reduced to the normal control level by assaying the enzyme in presence of the “activator protein,” partially purified from the soluble supernatant of the pig gastric cells. Phosphatidyl choline was somewhat more effective than phosphatidyl ethanolamine in the restoration of the activity of the ethanol-treated enzyme while phosphatidyl serine, phosphatidyl inositol, and sphingomyelin were without any effect. Synthetic phosphatidyl choline with various fatty acid substitutions were tested for their effectiveness in the restoration of the ethanol-inactivated enzyme. The distearoyl (18:0), dioleoyl (18:1), and dilinoleoyl (18:2) derivatives of phosphatidyl choline were almost equally effective while dipalmitoyl (16:0) phosphatidyl choline was somewhat less effective in the reconstitution process. Cholesterol appeared to interfere with phosphatidyl choline in the restoration of the activity of ethanol-treated enzyme. The fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine extracted by 15% ethanol at 37 °C was clearly different than those of the total microsome. Our data suggest that the phospholipids extracted by 15% ethanol at 37 °C are derived primarily from the immediate lipid environment of the enzyme and ATP together with Mg²⁺ and K⁺ help the partially delipidated enzyme to retain the appropriate conformation for the subsequent reconstitution. Furthermore, ethanol appears to either release or inactivate the membrane-associated activator protein, demonstrated to be essential for the K⁺-stimulated activity of the pig gastric ATPase.     

Differential regulation of the N-methyl transferases responsible for phosphatidylcholine synthesis in Saccharomyces cerevisiae

The enzymes catalyzing the conversion of phosphatidylethanolamine to phosphatidylcholine were assayed by measuring the incorporation of label from [¹⁴C-CH₃]-S-adenosyl-methionine into the endogenous phospholipids of particulate, cell-free preparations from S. cerevisiae grown in the presence of N-methylethanolamine, N,N-dimethylethanolamine, or choline. The results indicate that each base in the growth medium results in reduced levels of all the N-methyltransferase activity involved in the formation of the phosphatidyl ester of the given base. By following the conversion of exogenous [³²P]-phosphatidyldimethylethanolamine to [³²P]-phosphatidylcholine it has been shown that the activity of the third methyl transfer is 90% lower in particles prepared from choline grown cells than in particles prepared from cells grown without choline. The results suggest that there are at least two enzymes involved in the conversion of phosphatidylethanolamine to phosphatidylcholine and that their levels can be regulated individually.Supplementing the growth medium with any of the three methylated aminoethanols results in markedly increased cellular levels of their corresponding phosphatidyl esters and decreased levels of the precursor phosphatidyl esters. The fatty acid composition of phosphatidylcholine also changes when the medium is supplemented with choline suggesting that the proportions of the molecular species of this phosphatide depends on whether synthesis is via methylation of phosphatidylethanolamino or from the supplemented aminoethanol.     

N-Methyl Groups in Bacterial Lipids III. Phospholipids of Hyphomicrobia

The phospholipids of Hyphomicrobium vulgare NQ-521 have been separated by preparative thin-layer chromatography and analyzed by paper chromotography of the water-soluble products of acid and mild alkaline hydrolysis. The principal phospholipids are phosphatidyl ethanolamine (23%), phosphatidyl N,N'-dimethylethanolamine (36%), lecithin (29%), and phosphatidyl glycerol (10%). Three other strains of Hyphomicrobium were found to have similar phospholipid compositions. Growing cells incorporated the methyl group of methionine into lipid-bound N,N'-dimethylethanolamine and choline. Experiments with sonic extracts of H. vulgare NQ-521 and (14)C (methyl) S-adenosylmethionine demonstrated the formation of phosphatidyl N-monomethylethanolamine in addition to the dimethylethanolamine and choline phosphatides.     

The Induction of a Phospholipid Requirement and Morphological Abnormalities in Tetrahymena pyriformis by Growth at Supraoptimal Temperatures*†

SYNOPSIS. Supplementation of a chemically-defined medium that supported excellent axenic growth of Tetrahymena pyriformis, mating type II, variety 1, at 35 C, with nucleic acid derivatives allowed full growth at 37, and further addition of phospholipids permitted equivalent growth at 39 and 1/3 of that at 40. No other nutrients tested were active at 40. Population growth could be sustained continuously by serial sub-culture every 48 hr at 40 only if the medium contained synthetic phospholipids or phospholipids isolated from natural sources. The requirement was specific for phosphatidyl choline and phosphatidyl ethanolamine. Phospholipids which contained only saturated fatty acids, and ones which contained unsaturated acids were active. Phospholipids and neutral lipids isolated from 35-grown T. pyriformis were also effective. Phospholipid precursors, sterols, neutral lipids and fatty acids were not. A 48–72 hr exposure to 40 in either the unsupplemented medium or the nucleic acid derivatives-phospholipid-supplemented medium caused extreme variation in size and shape, fractured and displaced kineties, and abnormalities of karyokinesis. The frequency and degree of teratology was greater in the unsupplemented medium, but not markedly so. A possible metabolic basis for the temperature-induced phospholipid requirement and the morphological abnormalities is discussed.