Cell surface lipids and adhesion. III. The effects on cell adhesion of changes in plasmalemmal lipids.

The two preceding papers of this series suggest that the state of the plasmalemmal lipids affects cell adhesion. Plasmalemmal composition was altered by the experimental incorporation of fatty acids into R1 and R2 positions in the phosphatidyl components of the cell surface. In this paper we report that: (1) If the incorporation is of long chain length fatty acids (saturated) cell adhesion rises. (2) If the incorporation is of unsaturated fatty acids cell adhesion falls as the unsaturation increases. (3) Incorporation has to be extensive to produce a large change in adhesion. (4) Changes in adhesion parallel the plasmalemmal incorporation but do not follow the total cell incorporation. Item (4) argues that it is plasmalemmal and not other membrane lipids that are involved in cell adhesion. Item (3) suggests that bulk membrane properties and not some very specific grouping are involved in the effects of lipids on adhesion. The similar extents of incorporation of the various different fatty acids and the negligible amounts of lysophospholipids in the membranes of cells that have incorporated fatty acids argue that the effects are not due to differential accumulations of these lysolipids when incubations are done with different fatty acids. The changes in adhesion cannot be accounted for by changes in surface charge density since the electrophoretic mobility of the cells is unchanged by these incubations. It is suggested that these effects on adhesion due to changes in plasmalemmal lipids can be explained either in terms of the action of intermembrane van der Waals--London (electrodynamic) forces in cell adhesion or of changes in surface fluidity. These alternatives are discussed.     

The effects of sodium arsenate on the free amino acid levels in erythrocytes and plasma in rats.

The effects of arsenate on the levels of free amino acids in rat plasma and red blood cells have been investigated. The biggest changes occur in the branched-chain amino acids valine, isoleucine and leucine which are markedly decreased. The levels of several other amino-acids also change. The effects on the amino acids are not due to a change in the food intake caused by dosing with arsenate. This has been shown by comparing the results with those on fasting rats (40 h). Branched-chain amino acids in particular are markedly increased in plasma of fasting rats as opposed to a decrease in arsenate-treated rats.     

Lipid regulation of carbohydrate transport system in Mycoplasma]

Cultivation of Acholeplasma laidlawii cells in media containing unsaturated fatty acids results in changes of the physiological state of the membrane lipid bilayer due to preferable incorporation of an unsaturated fatty acid into lipids. The lipids are capable to regulate the transport activity since the transport rates for glucose, 3-O-methyl-C-glucose, glucerol and erythritol change considerably when the cells are cultivated in media containing different unsaturated fatty acids. The transport activity is also affected by the length of the carbon chain, the degree of the fatty acid saturation and the presence of cholesterol. At the same time the activation energy of the transport activity also changes, which suggests that the regulation by lipids (presumably local changes of the physical properties of lipid domen) is involved in the process of the carrier association with the substrate and/or in translocation of this complex through the membrane.     

Amino acid compartmentation in chick blood during the peri-hatching period

Individual amino acid levels and compartmentation in chick blood were measured on day 20 of incubation, at hatching (day 0), or after 1 or 5 days of free life, and compared with those of adult chickens. Blood cell amino acid concentrations were almost one order of magnitude higher than those of plasma, with higher values than those found in mammalian erythrocytes. This difference may be due to the capability for protein synthesis of the nucleated cells coupled with a postulated utilization of amino acids as fuel. The most common pattern of individual plasma amino acid levels was a slight rise at hatching followed by a large decrease, with minimal values for adults. The patterns in the cells did not always coincide with those for plasma. Total blood amino acid levels increased steadily during the period studied due to the increase in intracellular amino acids, giving rise to increasing blood-cell/plasma concentration ratios. These changes showed higher availability of plasma amino acids just after hatching, while the cell concentrations increased steadily to the maximum values in adults. The increase in alanine levels in cells with little changes in plasma can be correlated with the role of this amino acid as the main 2-amino nitrogen carrier in the avian bloodstream. The high amino acid levels in the cells suggest that these cells act as inter-organ transporters and reservoirs of amino acids, they have a different role in their handling and metabolism from those of mammals.     

Evidence for a high proportion of inactive ribosomes in slow-growing yeast cells.

From the protein and RNA content of Saccharomyces cerevisiae growing in different media we calculate that ribosome efficiency is changed: incorporation of amino acids into protein decreases from 8.8 amino acids/s per ribosome in fast-growing cells (0.54 doubling/h) to 5.2 amino acids/s per ribosome in slow-growing cells (0.30 doubling/h). We could not detect significant protein turnover in either fast-or slow-growing cultures, so the lower ribosome efficiency does not seem to be an artifact caused by changes in unstable protein production at different growth rates. Nor is the lower ribosome efficiency due to slower migration of ribosomes along mRNA: the times required to complete polypeptides of known molecular weights are the same in slow-growing cells as those previously determined for fast-growing cells [Waldron, Jund & Lacroute (1974) FEBS Lett. 46, 11-16]. We therefore deduce that ribosome efficiency changes in yeast because the fraction of ribosomes engaged in protein synthesis falls (from 84% in fast-growing cells to 50% in slow-growing cells.     

Glycochenodeoxycholate plays a carcinogenic role in immortalized mouse cholangiocytes via oxidative DNA damage

Bile acids have been suggested to be involved in biliary carcinogenesis, although the underlying mechanisms are yet to be established. The aim of this study was to investigate the carcinogenic effect of bile acids in the biliary tract in relation to oxidative stress. Immortalized mouse cholangiocytes were incubated with various bile acids, followed by measurement of reactive oxygen species (ROS) and the glutathione (GSH) level. As a marker of oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OHdG) expression in cholangiocytes was analyzed by flow cytometry. Then the expression of oxidative DNA repair enzymes in cholangiocytes was examined by real-time PCR. In addition, the long-term effect of bile acid-induced oxidative DNA damage on cholangiocytes was investigated using a mouse oligo DNA microarray. It was found that glycochenodeoxycholate (GCDC) induced the generation of ROS and the depletion of GSH. In contrast, no marked changes were induced by the other bile acids. The percentage of 8-OHdG-positive cells was also increased by GCDC, but the expression of oxidative DNA repair enzymes was not up-regulated. DNA microarray analysis showed marked changes of various genes associated with carcinogenesis (genes related to cell proliferation, angiogenesis, invasion, and metastasis). In conclusion, the long-term effect of oxidative DNA damage due to GCDC may promote carcinogenesis in the biliary tract. Furthermore, accumulation of 8-OHdG due to GCDC might contribute to the dysfunction of oxidative DNA repair enzymes.     

The composition of cell wall skeleton and outermost lipids of Mycobacterium vaccae is modified by ethambutol treatment

Ethambutol (EMB) is a first line drug in tuberculosis treatment inhibiting the biosynthesis of arabinogalactan, which is a component of the mycobacterial cell wall. The growth of Mycobacterium vaccae cells in the presence of EMB increases cell wall permeability, which was monitored by beta-sitosterol biotransformation. GC/MS and GLC/MS (gas chromatography/mass spectrometry) analysis revealed dramatic changes in the content of covalently bound mycolic acids and in molar ratio galactose (Gal) to arabinose (Ara) in the cell envelopes of EMB-treated cells. The detected variations in the compositions of fatty acids indicate that both the cell wall skeleton and outer layer (free lipids) are decomposed due to EMB treatment.     

Effects of global change factors on fatty acids and mycosporine‐like amino acid production in Chroothece richteriana (Rhodophyta)

Under natural conditions, Chroothece richteriana synthesizes a fairly high proportion of fatty acids. However, nothing is known about how environmental changes affect their production, or about the production of protective compounds, when colonies develop under full sunshine with high levels of UV radiation. In this study, wild colonies of C. richteriana were subjected to increasing temperature, conductivity, ammonium concentrations and photosynthetically active radiation (PAR), and UV radiations to assess the potential changes in lipid composition and mycosporine‐like amino acids (MAAs) concentration. The PERMANOVA analysis detected no differences for the whole fatty acid profile among treatments, but the percentages of α‐linolenic acid and total polyunsaturated fatty acids increased at the lowest assayed temperature. The percentages of linoleic and α‐linolenic acids increased with lowering temperature. γ‐linolenic and arachidonic acids decreased with increasing conductivity, and a high arachidonic acid concentration was related with increased conductivity. The samples exposed to UVB radiation showed higher percentages of eicosapentaenoic acid and total monounsaturated fatty acids, at the expense of saturated fatty acids. MAAs accumulation increased but not significantly at the lowest conductivity, and also with the highest PAR and UVR exposure, while ammonium and temperature had no effect. The observed changes are probably related with adaptations of both membrane fluidity to low temperature, and metabolism to protect cells against UV radiation damage. The results suggest the potential to change lipid composition and MAAs concentration in response to environmental stressful conditions due to climate change, and highlight the interest of the species in future research about the biotechnological production of both compound types.     

Changes in adenylate energy charge in Ehrlich ascites tumor cells deprived of serum, glucose, or amino acids.

The adenylate energy charge in Ehrlich ascites tumor cells increases when cells are cultivated in serum-limiting medium and decreases when they are incubated in glucose- or amino acid-limited media. Protein synthetic rates decrease in cells deprived of serum, glucose, or amino acids. Supplementation of deprived cells with respective nutrients restores normal protein synthetic rates and adenylate energy charge values. Serum-deprived cells incubated in depleted serum media do not increase their rates of protein synthesis and their adenylate energy charge remains elevated. These results suggest that serum factors regulate protein synthetic rates by mechanisms other than those regulating the availability in cells of glucose or of amino acids. The increased rates of utilization of glucose and of amino acids following the addition of serum are probably due to increased biosynthetic requirements.     

Effects of the trivalent and hexavalent chromium on the physicochemical properties of mammalian cell nucleic acids and synthetic polynucleotides

The effects of trivalent (chromium chloride) and hexavalent (potassium dichromate) chromium have been studied on the nucleic acids of cultured mammalian cells (BHK hamster fibroblast line), commercial DNA and RNA, and synthetic polynucleotides of known base composition. Modifications of UV absorption spectra and alterations of thermal denaturation and renaturation patterns have been observed by directly treating purified nucleic acids, as well as by examining nucleic acids extracted from cells treated with chromium compounds.Cr(III) interacts with nucleic acid bases, mostly guanine and cytosine, but also with phosphate groups, leading to deprotonation of bases as well as intramolecular cross-links, sandwich complexes between bases and chelation between bases and phosphates. Such interactions destabilize the DNA structure. On the contrary, stabilization of RNA, due to intramolecular metal bonds between nitrogen bases in GC-rich regions, is mainly produced. The kind of interaction of Cr(III) with nucleic acids is not significantly different when intact BHK cells are treated.Cr(VI) interacts similarly with DNA and RNA giving instead different effects when purified nucleic acids or intact cells are treated. Treatment of purified DNA produces breakages in the polynucleotide chain due to the oxidizing power of Cr(VI). In intact cell treatments, changes in the properties of DNA are observed. These could result from the combined action of Cr(III), produced by the intracellular reduction of Cr(VI) and the oxidizing activity of residual Cr(VI).The relevance of Cr(VI) and Cr(III) interactions to the mechanisms of chromium (carcino)genic action is summarized. It is stressed that Cr(VI), if not completely reduced to Cr(III) by extracellular and endoplasmic constituents, can reach the cell nucleus and directly interact with DNA.     

The biochemical composition of the pyloric caeca and its relation to DNA levels in the female starfishAsterias rubens during the reproductive cycle

Summary The biochemical composition of the pyloric caeca of femaleAsterias rubens was studied during the annual reproductive cycle. The amounts of nucleic acids, DNA and RNA were measured in addition to the amounts of lipids, glycogen, other reducing carbohydrates, free amino acids and proteins.A positive correlation of the pyloric caeca index to the biochemical constituents at the cellular level was established. The total DNA content of the pyloric caeca was found to increase with the pyloric caeca index.The relation of the biochemical composition of the pyloric caeca to the size changes of the organ is discussed. The hypothesis that the changes in size of the organ are due to changes both in the number and size of the cells is confirmed. The biochemical composition of the pyloric caeca and its relation to the reproductive cycle is discussed.Abbreviations GI gonad index - CPI pyloric caeca index     

Nature of virally mediated changes in membrane permeability to small molecules.

1. The changes in membrane permeability to small molecules caused by Sendai virus [Pasternak & Micklem (1973) J. Membr. Biol. 14, 293-303] have been further characterized. The uptake of substances that are concentrated within cells is inhibited. Choline and 2-deoxyglucose, which become phosphorylated, and aminoisobutyrate and glycine, which are driven by a Na+-linked mechanism, are examples. The uptake of each compound under conditons where its diffusion across the plasma membrane is rate-limiting is stimulated by virus. Choline, 2-deoxyglucose and amino acids at high concentration, amino acids in Na+-free medium, and most substances at low temperature, are examples. It is concluded that virally mediated decrease of uptake is due to one of two causes. Substances that are accumulated by phosphorylation are not retained because of leakage of the phosphorylated metabolites out of cells. Substances that are accumulated by linkage to a Na+ gradient are no longer accumulated because of collapse of the gradient resulting from an increased permeability to Nat 2. Increased permeability to K+ and Na+ results in (a) membrane depolarization and (b) cell swelling. The latter event leads to haemolysis (for erythrocytes) and can lead to giant-cell (polykaryon) formation (for several cell types). 3. Recovery of cells can be temporarily achieved by the addition of Ca2+; permanent recovery requires incubation for some hours at 37 degrees C. 4. The possible significance of virally mediated permeability changes, with regard to clinical situations and to cell biology, is discussed.     

Aluminum affects membrane physical properties in human neuroblastoma (IMR-32) cells both before and after differentiation

The capacity of Al(3+) to induce changes in the physical properties of plasma membrane from human neuroblastoma cells (IMR-32) was investigated, and the magnitude of the changes was compared with that obtained after cell differentiation to a neuronal phenotype. Similarly to our previous results in liposomes, Al(3+) (10 to 100 microM) caused a significant loss of membrane fluidity, being the differentiated cells more affected than the nondifferentiated cells. Al(3+) also increased the relative content of lipids in gel phase and promoted lipid rearrangement through lateral phase separation, with the magnitude of this effect being similar in nondifferentiated and differentiated cells. Since membrane physical properties depend on bilayer composition, we characterized the content of proteins, phospholipids, cholesterol, and fatty acids in the IMR-32 cells before and after differentiation. Differentiated cells had a significantly higher content of unsaturated fatty acids, creating an environment that favors Al(3+)-mediated effects on the bilayer fluidity. The neurotoxic effects of Al(3+) may be, at least in part, due to alterations of neuronal membrane physical properties, with potential consequences on the normal functioning of membrane-related cellular processes.     

Changes in phospholipid composition and calcium flux in LLC-PK cells cultured at low magnesium concentrations

Monolayers of porcine kidney cells (LLC-PK) were grown in a series of Nu-Serum-supplemented media containing different Mg(2+) concentrations (480, 250, 25, 6.3 or 2.6 microM) to study the effect of Mg(2+) depletion on cellular phospholipid changes and the consequent effect on the membrane permeability to Ca(2+). Cells grown on 6.3 or 2.6 microM Mg(2+) showed a decrease in PE, PS, Sph, PI and an increase of PC. These changes were attributed mainly to the decreased rate of Sph synthesis through the transfer of phosphocholine from PC to ceramide, or due to the increase of PE N-methylation as found in Mg(2+)-deficient cells. The (45)Ca uptake was increased in cells grown on 25.0 microM Mg(2+), while it was decreased in cells grown on 6.3 or 2.6 microM Mg(2+). These changes in Ca(2+) uptake were related to changes of cellular phospholipids and fatty acids which affect adenylate cyclase activity in the membrane, as well as the membrane fluidity.     

Increases in enzyme levels during the formation of phenolic acids in carrot cell cultures

The levels of glutamic acid dehydrogenase (GDH), phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (CAH) and O-methyltransferase (OMT) were measured during the formation of phenolic acids in carrot cells in suspension culture. Caffeic, ferulic and p-hydroxybenzoic acids were always present as the culture proceded. Total content of these acids increased at the early logarithmic and linear phases. GDH showed high activity at the early logarithmic and stationary phases. PAL activity was much enhanced at the linear and stationary phases. CAH activity was found in actively growing cells, especially at the early and late logarithmic phases OMT behaved similarly to PAL. The increases in GDH and CAH might be responsible for the rapid synthesis of phenolic acid at the early logarithmic phase. The increase in phenolic acid at the linear phase would certainly be due to enhancements of both PAL and OMT. On the other hand, the accumulation of vanillic acid was observed in cells which were transferred and cultured on an agar medium, but not in cells in suspension culture. This accumulation is related to increases in OMT levels and also to changes in the degree of β-oxidation.     

Effects of unsaturated fatty acid deprivation on neutral lipid synthesis in Saccharomyces cerevisiae

The effects of unsaturated fatty acid deprivation on lipid synthesis in Saccharomyces cerevisiae strain GL7 were determined by following the incorporation of [14C]acetate. Compared to yeast cells grown with oleic acid, unsaturated fatty acid-deprived cells contained 200 times as much 14C label in squalene, with correspondingly less label in 2,3-oxidosqualene and 2,3;22,23-dioxidosqualene. Cells deprived of either methionine or cholesterol did not accumulate squalene, demonstrating that the effect of unsaturated fatty acid starvation on squalene oxidation was not due to an inhibition of cell growth. Cells deprived of olefinic supplements displayed additional changes in lipid metabolism: (i) an increase in 14C-labeled diacylglycerides, (ii) a decrease in 14C-labeled triacylglycerides, and (iii) increased levels of 14C-labeled decanoic and dodecanoic fatty acids. The changes in squalene oxidation and acylglyceride metabolism in unsaturated fatty acid-deprived cells were readily reversed by adding oleic acid. Pulse-chase studies demonstrated that the [14C]squalene and 14C-labeled diacylglycerides which accumulated during starvation were further metabolized when cells were resupplemented with oleic acid. These results demonstrate that unsaturated fatty acids are essential for normal lipid metabolism in yeasts.     

Evidence that protein length expansion and contraction is partly due to mutational events in premeiotic cells

Studies on the rate of evolution of proteins typically concentrate on rates of change of orthologous amino acids rather than on changes in size (i.e., generation of nonorthologous domains). Recent work has focused attention on Ser/Thr-rich regions in yeast as these tend to undergo size changes rapidly, with size polymorphisms commonly being found, especially in proteins with cell-surface localization. The underlying mechanism generating the indels is presently unclear though, due to a lack of correlation with the location of meiotic double-strand breaks, it has, by exclusion, been conjectured to be replication slippage. Here we provide new evidence to support this possibility. Notably, we show that Ser/Thr-rich repeat regions are more generally associated with the location of Mre11p in premeiotic cells. This is to be expected if the repeats were produced by mutational events in mitotic cells possibly through replication slippage.     

Monoenoic fatty acid requirement in V79 cells

When lipids were removed from the culture medium, growth of V79 cells ceased. Supplementation with cis-octadecenoic acids satisfied the requirement for lipids by V79 cells. After starvation of the exogenous lipids by the shift-down of the medium to lipid-free medium, the content of octadecenoic acid in phospholipids increased more slowly in V79 cells than in V79-R cells, which can grow in the lipid-starved medium. The incorporation of [14C]acetic acid into monoenoic fatty acids and phospholipid molecular species containing monoenoic fatty acids in V79 cells was lower than that in V79-R cells. The reduced formation of monoenoic fatty acids was shown to be due to deficiency in the stimulation of activity of stearoyl-CoA desaturase which is a key enzyme to convert saturated fatty acids to monoenoic fatty acids.     

Fatty acid desaturation and microsomal lipid fatty acid composition in experimental hypothyroidism.

We have studied the influence of experimental hypothyroidism in the rat on the synthesis of unsaturated fatty acids and on liver microsomal lipid fatty acid composition. Hypothyroid rats demonstrated an 80% decrease in delta 9 (stearate) desaturation and a 43% decrease in delta 6 (linoleate) desaturation. Liver microsomal fatty acid composition was altered in the hypothyroid animals with a significantly decreased proportion of arachidonate and increased proportions of linoleate, eicosa-8,11,14-trienoate, eicosapentaenoate and docosahexaenoate. The bulk of these changes occurred in both of the two major phospholipid components, phosphatidylcholine and phosphatidylethanolamine. All of the changes were corrected by treatment of the hypothyroid rat with 25 micrograms of tri-iodothyronine/100 g body wt. twice daily. The diminished delta 9 desaturation did not lead to any changes in fatty acid composition. The increased linoleate and decreased arachidonate levels may be due to the diminished delta 6 desaturase activity, the rate-controlling step in the conversion of linoleate into arachidonate. The increases in the proportions of the other polyunsaturated fatty acid components cannot be explained by changes in the synthesis of unsaturated fatty acids, but are probably due to diminished utilization of these fatty acids.