Cellular Phospholipid Content Research Articles

Bioinspired fine-tuning of the mechanical rigidity of SNEDDS for the efficient crossing of multiple gastrointestinal barriers

Nanoproperties, such as size, charge, and rigidity, have been demonstrated to be crucial for nanovehicles to overcome numerous gastrointestinal obstacles. However, the facile approach of modifying the rigidity of nanovehicles remains scarce, limiting understanding of how rigidity impacts their oral delivery. Inspired by the fact that cellular phospholipid content regulates plasma membrane rigidity, the rigidity of self-nanoemulsifiying drug delivery system (SNEDDS) could be fine-tuned via phosphocholine content while their size and zeta potential remain unchanged, using insulin as a model drug. Notably, soft SNEDDS exerted longer gastrointestinal transit time, higher drug release rate, stronger gastrointestinal stability and relatively lower mucus permeation but superior epithelial transcytosis than their hard counterparts in a macropinocytosis-dependent manner. The rigidity-related enhanced transcytosis was attributed to improved endocytosis, lysosome escape capability and exocytosis. Rats with type 1 diabetes exhibited greater oral insulin absorption and blood glucose lowering effect with soft SNEDDS. This study demonstrated the regulatory role of phospholipids in nanovehicle rigidity, which could help develop mechanically optimized nanomedicines in the future.

Putative biomarker for phospholipid accumulation in cultured cells treated with phospholipidosis-inducing drugs: alteration of the phosphatidylinositol composition detected using high-performance liquid chromatography-tandem mass spectrometry.

We developed a high-performance liquid chromatography-tandem mass spectrometric method for phospholipid biomarker discovery and applied it to a cell-based assay system for the screening of phospholipidosis-inducing drugs. We studied the compositions of phospholipid molecules exceeding 100 species in cultured cells and found a characteristic alteration in the composition by treatment with cationic amphiphilic drugs possessing phospholipidosis-inducing potency. The compositions of phosphatidylinositol in RAW264 cells were significantly affected by the drug treatment. Similar alterations were also found in THP-1 cells. These phenomena were not observed when cells were treated with warfarin, which does not have phospholipidosis-inducing potency. Structural analysis of the altered phosphatidylinositols by a product ion scan revealed the presence of certain fatty acyl chains. Based on our findings, we proposed a prediction parameter (PP) for phospholipid accumulation calculated from the relative compositions of phosphatidylinositol species. As the dosage of imipramine (a cationic amphiphilic drug) increased, both the PP and cellular phospholipid content increased. Our results suggest that PP has potency as a biomarker for phospholipid accumulation in cells treated with drugs.

Application of Choline and Glycine Betaine Alters Levels of Total Phosholipids in Olea europaea L. Related to Cold Hardiness

Many plants accumulate cryoprotectant compounds in response to low temperatures such as Glycine betaine (GB). GB is synthesized by a simple two‐step pathway from the primary metabolite choline (Cho). Exogenous applications can improve the growth and survival of numerous species under several abiotic stresses. Although GB and Cho are studied in various plants, only a small number of studies investigated their roles during abiotic stress in the Olea europaea L. In addition, no study has yet investigated effects of GB and Cho application on phospholipid levels. Hence, the aim of the present study was to elucidate whether accumulation and the alterations in the phospholipids were related to GB and Cho applications in cold‐tolerant (Domat) and cold‐sensitive (Manzanilla) cultivars in non‐acclimated stages. For this purpose, 50 mM of GB and Cho solutions were applied as a foliar spray on leaves of two year‐old olive genotypes for three weeks. Leaf samples were collected for evaluations of freezing tolerance and phospholipid analysis. Results revealed that total phospolipids were greater in cv. Domat than those in cv. Manzanilla parallelling to their cold hardiness. In addition, total phospholipid content was greater in GB‐applied samples than that in Cho‐applied samples. Consequently, Cho and GB applications may protect olive leaves by altering cellular phospholipid content.

Hydrostatic pressure affects membrane and storage lipid compositions of the piezotolerant hydrocarbon‐degrading Marinobacter hydrocarbonoclasticus strain #5

A new piezotolerant alkane-degrading bacterium (Marinobacter hydrocarbonoclasticus strain #5) was isolated from deep (3475 m) Mediterranean seawater and grown at atmospheric pressure (0.1 MPa) and at 35 MPa with hexadecane as sole source of carbon and energy. Modification of the hydrostatic pressure influenced neither the growth rate nor the amount of degraded hexadecane (approximately 90%) during 13 days of incubation. However, the lipid composition of the cells sharply differed under both pressure conditions. At 0.1 MPa, M. hydrocarbonoclasticus #5 biosynthesized large amounts ( approximately 62% of the total cellular lipids) of hexadecane-derived wax esters (WEs), which accumulated in the cells under the form of individual lipid bodies. Intracellular WEs were also synthesized at 35 MPa, but their proportion was half that at 0.1 MPa. This lower WE content at high pressure was balanced by an increase in the total cellular phospholipid content. The chemical composition of WEs formed under both pressure conditions also strongly differed. Saturated WEs were preferentially formed at 0.1 MPa whereas diunsaturated WEs dominated at 35 MPa. This increase of the unsaturation ratio of WEs resembled the one classically observed for bacterial membrane lipid homeostasis. Remarkably, the unsaturation ratio of membrane fatty acids of M. hydrocarbonoclasticus grown at 35 MPa was only slightly higher than at 0.1 MPa. Overall, the results suggest that intracellular WEs and phospholipids play complementary roles in the physiological adaptation of strain #5 to different hydrostatic pressures.

Real Time Single Cell Analysis of Bid Cleavage and Bid Translocation during Caspase-dependent and Neuronal Caspase-independent Apoptosis

Bcl-2 homology domain (BH) 3-only proteins couple stress signals to evolutionarily conserved mitochondrial apoptotic pathways. Caspase 8-mediated cleavage of the BH3-only protein Bid into a truncated protein (tBid) and subsequent translocation of tBid to mitochondria has been implicated in death receptor signaling. We utilized a recombinant fluorescence resonance energy transfer (FRET) Bid probe to determine the kinetics of Bid cleavage and tBid translocation during death receptor-induced apoptosis in caspase 3-deficient MCF-7 cells. Cells treated with tumor necrosis factor-alpha (200 ng/ml) showed a rapid cleavage of the Bid-FRET probe occurring 75.4 +/- 12.6 min after onset of the tumor necrosis factor-alpha exposure. Cleavage of the Bid-FRET probe coincided with a translocation of tBid to the mitochondria and a collapse of the mitochondrial membrane potential (DeltaPsim). We next investigated the role of Bid cleavage in a model of caspase-independent, glutamate-induced excitotoxic apoptosis. Rat cerebellar granule neurons were transfected with the Bid-FRET probe and exposed to glutamate for 5 min. In contrast to death receptor-induced apoptosis, neurons showed a translocation of full-length Bid to the mitochondria. This translocation occurred 5.6 +/- 1.7 h after the termination of the glutamate exposure and was also paralleled with a collapse of the DeltaPsim. Proteolytic cleavage of the FRET probe also occurred, however, only 25.2 +/- 3.5 min after its translocation to the mitochondria. Subfractionation experiments confirmed a translocation of full-length Bid from the cytosolic to the mitochondrial fraction during excitotoxic apoptosis. Our data demonstrate that both tBid and full-length Bid have the capacity to translocate to mitochondria during apoptosis.

Metabolic changes during cell growth inhibition by the IRF-1 system

A genetic approach based on the activation of interferon-regulated-factor-1 (IRF-1) has been applied to arrest BHK cell growth in spinner flasks through the presence of estradiol in the culture medium, leading to the activation of the constitutively expressed IRF-1/estrogen receptor fusion protein (IRF-1-hER). Two days after estradiol addition cell proliferation was inhibited; cell concentration being kept for the next 6 days, although a decrease in cell viability was observed. IRF-1 activation alters the cell energetic metabolism, as there is extra metabolic activity with higher glucose, glutamine and oxygen consumption rates. Although the proteolytic activity is higher than in the control cells, the cell protein content increases significantly after IRF-1 activation, suggesting an overall increase in protein synthesis. A significant increase in lactate dehydrogenase activity and a higher reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2–5-diphenyl tetrazolium bromide) were also observed, showing an increase in cell activity during cell growth inhibition. Changes in ATP and in cellular phospholipid content as well in glucose metabolism were detected, in real time by 31P and 13C nuclear magnetic resonance spectroscopy (NMR). The levels of ATP were unaffected but changes in cellular phospholipid content were observed. Although an increase on ADP and AMP content was observed 48 to 72 h after IRF-1 activation, both the ATP content and the energy charge (EC) remained constant. It can be concluded that the IRF-1 activation leads to a significant increase of the cell activity until the cell is no longer able to maintain the same energetic metabolism and starts to lose viability.

Accumulation of cardiolipin and lysocardiolipin in fibroblasts from Tangier disease subjects

Tangier disease (TD) is an inherited disorder of lipid metabolism characterized by very low high density lipoprotein (HDL) plasma levels, cellular cholesteryl ester accumulation and reduced cholesterol excretion in response to HDL apolipoproteins. Molecular defects in the ATP binding cassette transporter 1 (ABCA1) have recently been identified as the cause of TD. ABCA1 plays a key role in the translocation of cholesterol across the plasma membrane, and defective ABCA1 causes cholesterol storage in TD cells. Not only cholesterol efflux, but also phospholipid efflux was shown to be impaired in TD cells. By use of thin layer chromatography, high performance liquid chromatography and time-of-flight secondary ion mass spectrometry, we characterized the cellular phospholipid content in fibroblasts from three homozygous TD patients. The cellular content of the major phospholipids was not found to be significantly altered in TD fibroblasts. However, the two phospholipids cardiolipin and lysocardiolipin, which make up minute amounts in normal cells, were at least 3–5-fold enriched in fibroblasts from TD subjects. A structurally closely related phospholipid (lysobisphosphatidic acid) has recently been shown to be enriched in Niemann–Pick type C, another lipid storage disorder. Altogether these data may indicate that the role of these phospholipids is a regulatory one rather than that of a bulk mediator of cholesterol solubilization in sterol trafficking and efflux.

The PLB2 gene of Saccharomyces cerevisiae confers resistance to lysophosphatidylcholine and encodes a phospholipase B/lysophospholipase.

The PLB1 gene of Saccharomyces cerevisiae encodes a protein that demonstrates phospholipase B, lysophospholipase, and transacylase activities. Several genes with significant homology to PLB1 exist in the S. cerevisiae genome, raising the possibility that other proteins may contribute to the total phospholipase B/lysophospholipase/transacylase activities of the cell. We report the isolation of a previously uncharacterized gene that is highly homologous to PLB1 and that, when overexpressed, confers resistance to 1-palmitoyllysophosphatidylcholine. This gene, which is located adjacent to the PLB1 gene on the left arm of chromosome XIII and which we refer to as PLB2, encodes a phospholipase B/lysophospholipase. Unlike PLB1, this gene product does not contain significant transacylase activity. The PLB2 gene product shows lysophospholipase activity toward lysophosphatidylcholine, lysophosphatidylserine, and lysophosphatidylethanolamine. Whereas deletion of either PLB1 or PLB2 resulted in the loss of 80% of cellular lysophospholipase activity, a plb1/plb2 double deletion mutant is completely devoid of lysophospholipase activity toward the preferred substrate lysophosphatidylcholine. Overexpression of PLB2 was associated with an increase in total cellular phospholipase B/lysophospholipase activity, as well as the appearance of significant lysophospholipase activity in the medium. Moreover, overexpression of PLB2 was associated with saturation at a higher cell density, and an increase in total cellular phospholipid content, but no change in phospholipid composition or fatty acid incorporation into cellular lipids. Deletion of PLB2 was not lethal and did not result in alteration of membrane phospholipid composition or content. PLB2 gene expression was found to be maximal during exponential growth conditions and was decreased in late phase, in a manner similar to other genes involved in phospholipid metabolism.

Changes in cellular and plasma membrane phospholipid composition after lipopolysaccharide stimulation of human neutrophils, studied by 31P NMR.

Lipopolysaccharide (endotoxin, LPS) exerts potent proinflammatory effects on neutrophils which may involve membrane phospholipid metabolism. The cellular and plasma membrane phospholipid composition of resting neutrophils and those stimulated with 50 microg ml(-1) LPS were studied by 31P NMR and chemical analysis. A rapid new method for plasma membrane purification was employed, involving the direct lysis of cytoplasts. Chemical analyses showed that, although total cellular phospholipid content did not change with LPS stimulation, there was twice the amount of phospholipid present in plasma membranes isolated from stimulated cells, resulting in a lowered cholesterol/phospholipid ratio. Since internal membranes have lower cholesterol content this result is consistent with an origin from insertion of these membranes (most probably from the endoplasmic reticulum) into the plasma membrane, thereby increasing its fluidity. The individual phospholipid classes of both cells and membranes were quantified by 31P-NMR spectroscopy after dissolution in sodium cholate without prior extraction of lipids, allowing partial resolution of the major phospholipid classes and ether-linked phospholipids. Ether-linked lipids were distinguished from diacyl phospholipids by hydrolysis of lipid extracts with HCl and phospholipase A1, There was a significant increase in phosphatidylserine in both cells and plasma membranes after stimulation, with a decrease in the phosphatidylethanolamine (diacyl and plasmalogen) content in the cells. Plasma membranes from stimulated cells exhibited a significant decrease in a phospholipid tentatively identified as 2-arachidonoyl-1-alkyl-sn-glycero-3-phosphocholine, a precursor of the lipid inflammatory mediator, platelet-activating factor. This report is the first to elaborate the changes in phospholipid composition in human neutrophils as a whole, and in plasma membranes separated from them, before and after stimulation by the physiological activator, LPS.

Differential Modification of Activities of the High-Affinity and Low-Affinity Insulin Receptors of 3T3-L1 Fibroblasts by Phosphonolipidsin Vivo

The low-affinity and high-affinity forms of the insulin receptor respond differently to modifications of cellular phospholipid content in mouse 3T3-L1 fibroblastsin vivo.When cells are cultured with 2-aminoethylphosphonate the resulting phosphonolipid, which has previously been demonstrated to prevent the insulin-induced differentiation of the fibroblasts into adipocytes [J. D. Smithet al., Biochem. Arch.8,339–344 (1992)] results in alterations in both the affinity for insulin and receptor number of the low-affinity receptor while leaving the high-affinity receptor unaffected. That this phospholipid modification induces a specific change in the cellular insulin effect is demonstrated by the lack of alteration in the mobilization of GLUT-4 and glucose transport in the lipid modified cells. The results suggest that this specific cellular phospholipid modification will be useful in dissecting the specific functions of the two forms of the mammalian insulin receptor.

Correlation of Cellular Phospholipid Content with Nisin Resistance of Listeria monocytogenes Scott A

A nisin-resistant mutant of Listeria monocytogenes Scott A has been characterized by comparing its phospholipid composition with the nisin-sensitive parental strain. The total phospholipids of resistant cells were significantly (P < 0.001) decreased compared to the parental strain. The types of phospholipids isolated from nisin-resistant and sensitive cells were identical, but there was a significant decrease (P < 0.01) in the amount of three individual phospholipids. Nisin-resistant cells were found to bind less nisin and release less phospholipids than sensitive cells when treated with same concentrations of nisin. The cell surface of resistant cells was less hydrophobic compared to sensitive cells, which also may have contributed to the observed nisin resistance. The results suggest that fundamental changes occurred in the membrane structure and function of the resistant mutant as a response to nisin.

High-performance liquid chromatographic study of the regulation of phospholipid metabolism in cultured adrenocortical cells

A rapid high-performance liquid chromatographic (HPLC) method for the separation of phospholipids was developed for minute samples of total lipids ( ca. 200 μg). The method was applied to the study of the phospholipid metabolism in adrenocortical cell cultures. A complete separation of the different cellular phospholipid classes was achieved in 40 min. Good resolution of the phospholipid peaks was obtained, which allowed the collection of each individual class of phospholipids for further analysis of radioactivity and fatty acid composition by gas chromatography. When cells were incubated with [U- 14C]glycerol or [U- 14C]palmitate the bulk of the radioactivity was found in cellular phosphatidylcholines. Exogenous phospholipids were incorporated into cellular lipids to a large extent, however without an increase in the cellular phospholipid content. 12-O-Tetradecanoyl-phorbol-13-acetate induced a 20% increase in the polyunsaturated fatty acid content of the cellular phosphatidylethanolamines, but no change was detected in the cellular phosphatidylcholines. The developed method is well-suited to the study of the phospholipid metabolism in adrenocortical cells where the phospholipid metabolism is closely linked to the specialized functions of the cells.

Cholestasis, metabolism and biliary lipid secretion during perfusion of rat liver with different bile salts

The biological effects of bile acids depend largely upon their molecular structure. When bile acid uptake exceeds the maximal biliary secretory rate (SRm) cholestasis occurs. In order to characterize the influence of bile acid structure on its cholestatic potency we systematically studied SRm, maximal bile flow, maximal and cumulative phospholipid and cholesterol secretion with different taurine-conjugated tri-, di- and keto bile acids (Table I) in the isolated perfused rat liver. Bile acids with a high critical micellar concentration (CMC) promoted the greatest bile flow; a positive non-linear correlation between CMC and maximal bile flow was found. 3α-Hydroxylated bile acids with a hydroxyl group in 6α and/or 7β position and lacking a 12α hydroxy group had a high SRm. SRm was not related to CMC or maximal bile flow, respectively. Phospholipids and cholesterol were secreted in a nearly fixed ratio of 12:1; a strong linear relationship could be observed. Cumulative phospholipid secretion over 48 min was significantly lower for non and poor micelle forming bile acids (TDHC and TUC) than for those with comparatively low CMC values (TUDC, TC, THC, THDC, TCDC) (70–140 vs. 210–450 nmol/g liver). At SRm all bile acids with good micelle forming properties showed a similar cumulative biliary lipid output. However, when biliary lipid output was related to 1 μmol bile acid secreted bile acids with a low SRm induced the highest lipid secretion (TCDC, TC). These data (1) demonstrate that a 6α and/or a 7β hydroxy group on the steroid nucleus reduce cholestatic potency if the 12α hydroxy group is absent, (2) suggest that in the case of micelle forming bile acids the total amount of phospholipids secreted in bile (depletion of cellular phospholipids) is associated with the occurrence of cholestasis whereby bile acids with a low SRm deplete the cellular phospholipid content at much lower bile acid concentrations than those with a higher SRm and (3) imply that bile acids with non and poor micelle forming properties (TDHC, TUC) presumably do not cause cholestasis (solely) by depletion of cellular phospholipids.

Electrical properties of canine subendocardial Purkinje fibers surviving in 1-day-old experimental myocardial infarction.

The passive electrical properties of subendocardial Purkinje fibers surviving in infarcted regions of canine ventricle 24 hours after coronary ligation were studied by using microelectrode techniques and cable theory. In normal hearts, cells within the subendocardial Purkinje fiber strands were found to be well coupled to each other but electrically isolated from neighboring myocardium. Voltage response to intracellular current injection was consistent with one-dimensional cable behavior and yielded estimates of passive electrical properties in general agreement with previous work on free-running Purkinje strands (membrane length constant, 1.2 +/- 0.1 mm; membrane time constant, 7.3 +/- 0.8 msec; input resistance, 67.4 +/- 7.4 K omega; membrane resistance, 8.2 +/- 0.7 K omega.cm; axial resistance, 0.52 +/- 0.06 M omega/cm; membrane capacitance, 960 +/- 102 nF/cm) (n = 21). On the day after coronary ligation, subendocardial Purkinje fiber action potentials were prolonged and slightly depolarized. Significant increases were measured in input resistance (+40.5%), membrane resistance (+43.9%), and axial resistance (+47.5%), whereas membrane capacitance was found to be significantly decreased (-24.3%) (n = 19). Conduction velocity, membrane length constant, membrane time constant, and the time constant and capacitance for the foot of the action potential remained unchanged. These results are consistent with electrical uncoupling between adjacent cells, which will increase internal resistivity, accompanied by changes in cellular phospholipid content, which can increase membrane resistance and alter membrane capacitance. Alternatively, the results can be explained by a simple model in which the apparent electrical structure is altered by changes in electrical coupling alone, with specific electrical properties remaining constant. Although the mechanisms underlying the observed changes remain uncertain, the present study indicates that myocardial infarction is associated with alterations in the passive electrical structure of surviving subendocardial Purkinje fibers, which, together with changes in action potential configuration, may provide a substrate for the generation of ventricular arrhythmias 24 hours after coronary ligation.

Biochemical mechanisms of aminoglycoside cell toxicity. II. Accumulation of phospholipids during myeloid body formation and histological studies on myeloid bodies using twelve aminoglycoside antibiotics.

Cultured human skin fibroblasts take up aminoglycoside antibiotics into lysosomes to form myeloid bodies. Gentamicin (GM), one such antibiotic, was taken up until the cellular concentration reached an estimated 64 mM on the 3rd day when cells were incubated with 2 mM gentamicin. The rate of release of intracellular GM was high on the first day of incubation and gradually slowed down over the next 4 d. About 50% of the GM remained in the cells even on longer incubation in GM-free medium, suggesting it may irreversibly bind to cellular components. With myeloid body formation, the cellular phospholipid content increased 1.5 times. Bis(monoacyl-glyceryl)phosphate, which is known as a marker of lysosomal phospholipid, phosphatidylcholine and phosphatidylserine showed 250, 162, and 153% increases, respectively. Sphingomyelin was not accumulated, while lysosomal sphingomyelinase was dramatically inhibited. Of 12 different aminoglycoside antibiotics, paromomycin is the most prominent myeloid body-forming antibiotic. The myeloid body-formation is not directly correlated to human nephrotoxicity. On the other hand, the number of myeloid bodies is well correlated to the affinity to the brush border membrane, suggesting that such aminoglycoside antibiotics are taken up easily through cellular endocytosis. The cytotoxic effects of aminoglycoside antibiotics may be due to by their binding to cellular organelles other than lysosomes.

Effects of sodium chloride concentration on phospholipid fatty acid composition of yeasts differing in osmotolerance

The effect of growth medium NaCl concentration on the fatty acid composition of phospholipids of 3 strains of Saccharomyces cerevisiae and 6 osmotolerant yeast strains was examined. The S. cerevisiae strains were characterized by a high content of palmitoleic (C16:1) acid and by having no polyunsaturated C18 acids, whereas the osmotolerant strains had a low content of C16:1 and a high proportion of polyenoic C18 acids. An increase of the NaCl concentration from 0% to 8% resulted in a decrease of the cellular phospholipid content on a dry-weight basis, for all strains but one of the osmotolerant strains. For the S. cerevisiae strains increased salinity produced a slight decrease of the proportion of C16 fatty acids with a concomitant increase of C18 acids, whereas the osmotolerant strains showed an increase of the relative content of oleic acid (C18:1) at the expense of the proportion of polyenoic C18 acids.

Thermal regulation of phospholipid fluidity in Corynebacterium glutamicum

AbstractIncreasing temperatures of cultivation caused the cellular phospholipid content of C. glutamicum to decrease significantly. Simultaneously, the melting point of phospholipid fractions, i. e. the temperature of the ordered‐disordered phase transition, increased. A hypothetical connection between the cellular phospholipid composition and the temperature‐depended lysine efflux is discussed.

Kinetic analysis of N-acylphosphatidylserine accumulation and implications for membrane assembly in Rhodopseudomonas sphaeroides

The accumulation of N-acylphosphatidylserine (NAPS) in response to the inclusion of Tris in the growth medium of Rhodopseudomonas sphaeroides strain M29-5 has been examined. In the accompanying paper (Donohue et al., J. Bacteriol. 152:000--000, 1982), we show that in response to Tris, NAPS accumulated to as much as 40% of the total cellular phospholipid content. NAPS accumulation began immediately upon addition of Tris and was reflected as an abrupt 12-fold increase in the apparent rate of NAPS accumulation. We suggest that Tris altered the flow of metabolites through a preexisting and previously unknown metabolic pathway. NAPS accumulation ceased immediately upon the removal of Tris; however, accumulated NAPS remained largely metabolically stable. Importantly, under conditions in which NAPS was not accumulated, the intracytoplasmic membrane was shown to be virtually devoid of newly synthesized NAPS. The significance of this observation is discussed in terms of its physiological implications on phospholipid transfer and membrane biogenesis in R. sphaeroides.

Plasma membrane associated metabolic parameters and the aging of human diploid fibroblasts

Substrate uptake, portions of the cyclic AMP system, membrane fluidity and cellular phospholipid content are some of the parameters which are structurally associated with the plasma membrane and which have been linked to the control of cell proliferation. These parameters were studied with respect to cellular aging of human embryo lung fibroblasts (HELF) in culture. We observed in late passage an increase in the rate of uridine transport and in cellular cyclic AMP levels. These results were examined in relation to the increase in cell volume which occurs in senescing HELF. We also observed an increase in V max of uridine transport, and a decrease in the K m of cyclic AMP phosphodiesterase (PDE) as quiescent, passage 18–25, HELF were stimulated to divide with fresh serum. A similar effect of serum occured in the lated passage (p. 43) cells despite the inability of these late passage cultures to undergo further proliferation. There was no change in cAMP-PDE activity with increasing passage number suggesting that the observed alterations of the cAMP levels, basal and in response to extracellular effectors, were due to alterations in the adenyl cyclase system. We observed no change in senescent HELF in membrane fluidity or phospholipid and neutral fat content.

Relation between Cellular Phospholipids and the Excretion of L-Glutamic Acid by a Glycerol Auxotroph of Corynebacterium alkanolyticum

Relation between cellular phospholipids and L-glutamic acid excretion was investigated using Corynebacterium alkanolyticum GL–21 (a glycerol auxotroph).When strain GL–21 was cultured in glycerol-limited medium which contained n-hexadecane, acetic acid or fructose as carbon source, there occurred the limitation of cellular phospholipid content and the over-accumulation of L-glutamic acid in the broth. Two-dimensional thin-layer chromatograms provided evidence that both the parent and the mutant strains contained the same phospholipids such as cardiolipin, phosphatidylethanolamine, phosphadityl-glycerol, phosphatidylinositol and phosphatidic acid. Limited supply of glycerol to the mutant did not greatly alter the proportions of the individual phospholipids.