Howard M. Jenkin

Growth ofChlamydia psittaci strain menigopneumonitis in mouse L cells cultivated in a defined medium in spinner cultures

SummaryL cells were grown in spinner cultures in a defined medium consisting of Waymouth medium MB752/1 (19) supplemented with 2 mg of fatty acid-free bovine serum albumin (BSA) per ml and 5 μg of oleate per ml (WO5 medium). Growth in WO5 medium was comparable to spinner L cell growth in two serum-containing media. The optimal concentration of oleate in the WO medium was 5 to 10 μg per ml. The use of 20 to 80 μg of oleate per ml of medium resulted in lower peak populations and earlier declines in viable cell counts. Cell death occurred rapidly in WO160 medium. Cell growth in WO medium containing 5 to 80 μg of oleate per ml was well above the level of growth observed when no oleate was present in the medium. Since the total lipid and fatty acid compositions of the BSA used in this study have been characterized by the authors, the WO medium may be considered a defined medium. L cells have been continuously maintained in spinner cultures in WO5 medium for over 50 passages with no major variation in the growth pattern. A 1000-fold increase inChlamydia psittaci strain meningopneumonitis, with a peak titer of 9.3×107 plaque-forming units per ml, was observed when the chlamydial agents were grown in spinner L cells in WO5 medium.

Phosphatidylcholine as the choline donor in sphingomyelin synthesis

Sphingomyelin synthesis was studied in cultured Novikoff rat hepatoma cells by following transfer of [14C]choline label into sphingomyelin (SPH). The study was facilitated by the fact that prelabeling of the cells with [Methyl-14C]choline resulted in rapid accumulation of essentially all the label (≈95%) in phosphatidylcholine (PC). The redistribution of PC label during a 15-hr chase was dependent upon the extracellular choline concentration. Under conditions of free choline diffusion (500 μM choline), loss of lable from PC was most pronounced, and the percentage of total radioactivity that became trapped in the extracellular water-soluble choline pool was an order of magnitude greater than in low choline medium (27 μM choline). Despite the significant loss of water-soluble label from the cells in high choline medium, SPH labeling proceeded at essentially the same rate at either choline concentration. During the label chase in 500 μM choline, the specific radioactivity of PC decreased, but the specific radioactivity of SPH continued to increase for 9–12 hr until it reached the specific radioactivity of PC. In the presence of 300 μM neophenoxine (NPO), transfer of label from PC into SPH was stimulated. NPO also decreased the specific radioactivity of PC to about the same extent as that of SPH was increased. Because transfer of choline label from PC to SPH was not affected by loss or dilution of water-soluble precursors, and because the specific radioactivity of PC and SPH, in the absence or presence of NPO, responded in a characteristic precursor product fashion, we conclude that sphingomyelin synthesis in Novikoff cells circumvents the water-soluble choline pool and that phosphatidylcholine serves as the immediate choline source. All our data support the phosphatidylcholine:ceramide cholinephosphotransferase pathway of sphingomyelin synthesis.

Comparison of lipid composition ofAedes aegypti andAedes albopictus cells obstained from logarithmic and stationary phases of growth

The total lipids, total neutral lipids, and total phospholipids fromAedes aegypti andAedes albopictus cells cultivated in vitro in a medium containing fetal calf serum were analyzed. The mosquito cells were harvested in the logarithmic and stationary phases of growth. The fatty acid profiles of the lipids showed differences during the aging of the cells but not between species. There was an increase in chain elongation and unsaturation of the fatty acids in the stationary phase when compared with the logarithmic phase of growth. The major components of the fatty acid profiles of the cells were 16∶1, 16∶1, and 18∶1 fatty acids. Few similarities were found between the lipid analysis of the mosquito cells and the growth medium.

Lipid analysis of Aedes aegypti cells cultivated in vitro

Abstract The constituent lipids of cloned Aedes aegypti cells cultivated in vitro in a medium containing fetal calf serum were analysed. The major phospholipids were phosphatidylcholine (40%) and phosphatidylethanolamine (26%). The minor phosphatides were sphingomyelin (13%), lysophosphatidyl choline (8%), phosphatidylinositol (7%) and cardiolipin (6%). The major neutral lipids were free fatty acids (48%) and triglycerides (20%). The major fatty acid in most lipid classes was 18:1; however, cardiolipin contained 16:1 and the sterol esters contained 18:2 as the major fatty acid. The sterol ester had a fatty acid profile similar to that found in calf serum. The A. aegypti cells contained a small amount of free sterol (2%). Lysophosphatidylcholine contained a high amount of polyunsaturated fatty acids (25%), whereas sphingomyelin had a higher amount of saturated fatty acids (55%) compared to the fatty acid profiles of other classes of lipids. Plasmalogens were not detected in these cells.

Rapid phospholipase A2 stimulation and diacylglycerol cholinephosphotransferase inhibition in baby hamster kidney cells during initiation of dengue virus infection

Abstract Infection of baby hamster kidney (BHK-21) cells by dengue type 2 (DEN-2) virus is accompanied by a rapid but transient stimulation of phospholipase A2 activity which reaches a maximum within 40 min. Thereafter, the phospholipase A2 activity, that is associated with the microsomal fraction, returns to normal levels at an equally rapid pace. In contrast, microsomal phospholipase A1, lysophosphatidylcholine acyltransferase, or lysophospholipase are not altered early in the infection. Microsomal diacylglycerol cholinephosphotransferase was inhibited in phase with phospholipase A2 stimulation. The data obtained on the DEN-2 BHK-21 system suggest that virus-triggered phospholipase A2 activation and ensuing lysophospholipid production may facilitate initial interaction of the virion with the cell plasma membrane and may aid viral penetration.

Alk-1-enyl ether phospholipids (plasmalogens) and glycolipids of Treponema hyodysenteriae: Analysis of acyl and alk-1-enyl moieties

The lipids of Treponema hyodysenteriae B78, the etiologic agent of swine dysentery, comprised 16.4% of the cell dry weight, and consisted of 37.4% glycolipids, 28.6% phospholipids, and 34.0% neutral lipids. Monogalactosyldiacylglycerol, a major lipid in all Treponema except Treponema pallidum, comprised 80% of the glycolipids. An unidentified galactolipid less polar than monogalactosyldiacylglycerol was also detected. Phosphatidylglycerol (19.5% of the total lipids) was the major phospholipid. Phosphatidylcholine, characteristically the major phospholipid of treponemes, comprised 6.1% of the total lipids. Cardiolipin and lysophosphatidylcholine were minor components. The alk-1-enyl ether forms of both the phospholipids (plasmalogens) and glycolipids predominated. The alk-1-enyl ether forms of monogalactosyldiacylglycerol, the unidentified galactolipid, phosphatidylglycerol, cardiolipin, and phosphatidylcholine were 88.3, 96.4, 74.8, 60.6, and 6.3%, respectively. The acyl and alk-1-enyl chains of the organism were qualitatively similar and differed dramatically from those of the medium indicating a capability for fatty acid synthesis that most Treponema do not possess. Saturated C14, C15, and C16 chains comprised more than 95% of the acyl and alk-1-enyl groups. About 25% of the chains were iso-15:0, anteiso-15:0, and other branched moieties.

The effect of heparin on growth of mammalian cells in vitro.

Summary The effect of heparin on the growth of four cell types cultivated in vitro has been investigated. The results suggest that heparin appears to have some growth promoting effects on prepuce cells, while it showed little effect on the growth of Novikoff hepatoma, monkey kidney and baby hamster kidney cells. Heparin reversed the inhibitory effect of prednisolone-21-sodium-succinate on the growth of prepuce and baby hamster kidney cells. The authors thank Gregg Jorgenson and Chris Bjornson for their excellent techincal assistance. The opinions and statements contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department of the Naval Service at large.

Sphingophospholipids of species ofAedes andCulex mosquito cells cultivated in suspension culture from logarithmic and stationary phases of growth

Two sphingophospholipids, sphingomyelin (ceramide phosphorylcholine), and ceramide phosphorylethanolamine, were isolated and purified from cells of four mosquito species obtained from the logarithmic and stationary phases of growth. Quantitation of the two lipids in both phases of growth from cells of each species was made. The fatty acid composition of the two lipids was compared between the cell types and the two phases of growth. There was a tendency for an increase of ceramide phosphorylcholine and a decrease of ceramide phosphorylethanolamine in the stationary phase. Longer chain fatty acids of ceramide phosphorylcholine were observed in the stationary phase than in the logarithmic phase of growth of the mosquito cells. The four species had distinctly different fatty acid patterns in the two complex lipids which might be useful for taxonomic purposes and cell identification.

Phospholipid composition ofCulex quinquefasciatus andCulex tritaeniorhynchus cells in logarithmic and stationary growth phases

Culex quinquefasciatus andCulex tritaeniorhynchus cells were grown in spinner culture and harvested in logarithmic and stationary phases of growth. The phospholipids were extracted from the cells, and the fatty acid profiles of the phospholipid classes were determined and compared. The major components were phosphatidylcholine and phosphatidylethanolamine, constituting≥80% of the phospholipid. The fatty acid profiles of lysophosphatidylcholine, phosphatidylinositol, and cardiolipin showed changes with aging of the Culex cells and between the species. In the lysophosphatidylcholine fraction, there was an increase in saturation of the fatty acids of theC. quinquefasciatus cells, and chain lengthening occurred in both species from the logarithmic to stationary phases of growth. In the phosphatidylinositiol fraction, both Culex species showed a decrease in monoenes and an increase in polyenes, while only theC. tritaeniorhynchus cells showed an increase in fatty acid chain length with aging. TheC. quinquefasciatus cells had an increase in polyenes with aging in the cardiolipin fraction. Differences in the percentage composition of the fatty acids were shown in all the phospholipid fractions between the Culex species in the logarithmic phase of growth and all except the phosphatidylinositiol and cardiolipin fractions in the stationary phase.

The effect of two isomeric octadecenoic acids on the lipid metabolism and growth of Novikoff hepatoma cells

The origin and metabolism of octadecenoic acid (18 : 1) was examined in intact Novikoff rat hepatoma cells by using labeled precursors and two isomeric octadecenoic acids which differed in their abilities to stimulate cell growth in a serum-free medium. The isomers (ci-6-18 : 1 and cis-9-18 : 1) were measured in the cellular lipid by ozonolysis and reduction of the ozonides. The results indicate that the 18 : 1 fatty acid accumulated in the cell lipid by uptake of the preformed acid from the medium. The cis-9-18 : 1 to 16 : 1 and 20 : 1 fatty acids by chain shortening and chain elongation. Both isomers inhibited de novo fatty acid synthesis from acetate by cells suspended in a serum-free medium. The isomers did not exert coordinate control of both fatty acid and cholesterol biosynthesis in the Novikoff cells.