Continuous culture of rat C6 glioma in serum-free medium

In this communication we describe serum-free culture conditions for the serial propagation of the C6 glioma cell line. The growth rate, saturation density, and morphology of these cells are equivalent to those of their serum-grown counterparts when cultured in a 3:1 mixture of Dulbecco's modified Eagle's medium and Ham's medium F-12 supplemented with trace elements, insulin, transferrin, fibroblast growth factor, linoleic acid complexed to fatty acid-free bovine serum albumin, and a serum-spreading factor (SSF) partially purified from human plasma. The requirement for SSF in the medium can be satisfied by preincubating the tissue culture dishes with SSF. Tissue culture dishes sequentially pretreated with poly-D-lysine and purified cold insouluble globulin will also substitute for this requirement. The fatty acid-free bovine serum albumin/linoleic acid complex increases the growth rate of these cells but has no appreciable effect on their morphology, saturation density, or ability to grow with repeated subculture. The growth stimulation caused by this complex appears to be dependent on the fatty acid, as the fatty acid-free bovine serum albumin alone has no effect on the growth rate. Linoleic acid is cytotoxic in the absence of bovine serum albumin, and the fatty acid-free bovine serum albumin prevents this toxicity. Other fatty acids including oleic, arachidonic, and palmitic only partially substitute for the growth-promoting effect of linoleic acid.     

The growth requirements of SV40 virus transformed Balb/c-3T3 cells in serum-free monolayer culture

The growth requirements of SV40 transformed Balb/c-3T3 cells have been studied in the absence of serum. For growth in serum-free medium, the cells require (i) insulin, (ii) transferrin, and (iii) cis-unsaturated fatty acids added in combination with fatty acid free bovine serum albumin. The growth rate, saturation density, and morphology of cells grown in this serum-free medium are the same as those of cells grown in serum supplemented medium. This mixture also supports the growth of SV40 transformed Swiss-3T3 cells and SV40 transformed primary mouse embryo cells, but does not support the growth of untransformed Balb/c-3T3 cells. The addition of fibronectin to this mixture allows routine subculture, repeated passage, and indefinite propagation of SV40 transformed Balb/c-3T3 cells. Cells grown in this medium for a period of two months retain their ability to induce tumors when injected into athymic nude mice.     

Identification of albumin-bound fatty acids as the major factor in serum-induced lipid accumulation by cultured cells

Factors responsible for the high lipogenic activity of rabbit serum were investigated using an assay procedure based on the gravimetric determination of the 24 hr increase in cell lipid. Cellular synthesis of fatty acids was inhibited by the presence of serum in the assay medium. Approximately 90% of the increase in cell lipid produced by serum fractions was due to triglyceride accumulation. Fractionation of rabbit serum by precipitation with ammonium sulfate or by ultracentrifugation in high density medium, both indicated that three-quarters of its lipogenic activity was associated with albumin. The lipoproteins prepared by ultracentrifugation also exhibited about one-half the activity of whole serum. The lipogenic activity of albumin was confirmed by the high potency of the albumin isolated in a nearly pure form from proteins of d>1.21 by precipitation with trichloroacetic acid and extraction with ethanol. As judged from chemical and isotopic analysis, neither the lipid content nor the lipid composition of the albumin was appreciably altered during its isolation. Of the albumin-bound lipids, only the free fatty acids, as determined by DEAE column chromatography, were present in an amount sufficient to account for the observed increase in cell triglycerides. In control experiments with horse serum of low lipogenic activity, the proteins of d>1.21 also possessed low activity in conjunction with a low content of free fatty acid. However, the albumin isolated from the latter preparation exhibited the high lipogenic activity of rabbit serum albumin. Chemical and isotopic analysis of the recovered horse serum albumin revealed that its free fatty acid content was the same as that of rabbit serum albumin. These results indicated that the isolation of horse serum albumin was attended by a substantial increase in its free fatty acid content. When the rabbit serum and horse serum content of media were adjusted to provide equivalent concentrations of albumin-bound fatty acids, the rabbit liver cells grown on the former media accumulated more lipid than cells grown on the latter media. This difference was shown to be due to the higher concentration of albumin per micro mole of fatty acid in horse serum as compared with rabbit serum. Consequently, the albumin to fatty acid ratio also controls the lipogenic activity of a serum. A linear relationship is presented which relates the cell lipid content to the molar ratio of albumin to free fatty acids and to the absolute concentration of free fatty acids in the medium.     

Characterization of the stimulatory effects of PGF2α on the release of arachidonic acid

Fibroblasts derived from a rat carrageenin granuloma were cultured in the presence of radioactive arachidonic acid, palmitic acid and linoleic acid. More than 90% of each labeled fatty acid was incorporated into a phospholipid fraction by the cells in 18 hrs. Arachidonic acid was evenly incorporated into phosphatidylcholine and phosphatidylethanolamine, while both palmitic acid and linoleic acid were almost entirely incorporated into phosphatidylcholine. The position of phosphatidylcholine where the fatty acids were incorporated was different for each fatty acid. The ratio of the amount of fatty acid incorporated into the 2-position to the amount incorporated into the 1-position of phosphatidylcholine for each fatty acid was >90% for arachidonic acid, 2:1 for palmitic acid and 5:1 for linoleic acid. In the case of phosphatidylethanolamine, most arachidonic acid (>90%) was incorporated into the 2-position. PGF_2^α caused the stimulation of arachidonic acid release but not of palmitic acid and linoleic acid from pre-labeled fibroblasts.The serum in the medium was completely replaceable by bovine serum albumin. The effect of PGF_2^α increased with an increasing concentration of bovine serum albumin, suggesting that serum only acts as a ‘trap’ for released arachidonic acid. The effect of PGF_2^α was greater than bradykinin, and no synergistic effect was seen, although an additive effect was observed.The effect of PGF_2^α depended on the concentration of calcium ions under magnesium-supplemented conditions.     

Free fatty acids as feedback regulators of adenylate cyclase and cyclic 3':5'-AMP accumulation in rat fat cells.

Rat fat cells incubated with lipolytic agents released substances to the medium which acted as feedback regulators of cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. The feedback regulators were not removed by adenosine deaminase. Dialyzed medium that had previously been incubated with fat cells in the presence of norepinephrine markedly inhibited cyclic AMP accumulation by fresh cells, whereas dialyzed medium from control cells did not inhibit cyclic AMP accumulation. The effects of lipolytic agents could be mimicked by adding dialyzed medium previously incubated with fat cells in the presence of oleic acid. This suggested that free fatty acids were the nondialyzable and adenosine deaminase-insensitive inhibitors of cyclic AMP accumulation released to the medium by fat cells incubated with lipolytic agents. The regulatory function of free fatty acids was related to the molar ratio of fatty acid to albumin. Profound inhibition of both lipolysis and cyclic AMP accumulation was seen as the free fatty acid/albumin ratio exceeded 3. The inhibition of cyclic AMP accumulation by oleate was seen as soon as there was a detectable increase in cyclic AMP due to lipolytic agents. Protein kinase activity (in the presence of cyclic AMP) of the infranatant obtained after centrifugation of fat cell homogenates at 48,000 x g was inhibited by medium from cells incubated with lipolytic agents or added oleate. Adenylate cyclase activity of rat fat cell ghosts was also inhibited by dialyzed or nondialyzed medium that previously had been incubated with lipolytic agents or added fatty acids. The direct addition of oleate markedly inhibited adenylate cyclase activity as the free fatty acid/albumin ratio exceeded 2. These data suggest that the prolonged drop in cyclic AMP accumulation seen during the incubation of rat fat cells with lipolytic agents is due to the inhibition of adenylate cyclase. This occurs when the free fatty acid/albumin ratio exceeds 3.     

Effect of phospholipid fatty acid composition of endothelial cells on cholesterol efflux rates.

Human endothelial cells (EA.hy 926 line) were loaded with cationized low density lipoprotein (LDL) and subsequently incubated with fatty acid/bovine serum albumin complexes. The fatty acids were palmitic, oleic, linoleic, arachidonic, and eicosapentaenoic acids. The preincubations resulted in extensively modified fatty acid profiles in cell membrane phospholipids and in cellular cholesteryl esters. The cholesterol efflux from these fatty acid-modified cells was measured using 0.2 mg high density lipoprotein3 (HDL3)/ml medium. The efflux was significantly higher for the palmitic acid-treated cells, compared to all other fatty acid treatments. These differences in efflux rates were not caused by changes in the binding of HDL3 to high affinity receptors on the EA.hy 926 cells. Efflux mediated by dimethyl suberimidate-treated HDL3, which does not interact with high affinity HDL receptors, was similar to efflux induced by native HDL3 after all fatty acid treatments. Our results indicate that high affinity HDL receptors are not important for HDL-mediated efflux of cell cholesterol. The fatty acid composition of the cell membrane phospholipids may be an important determinant.     

The binding of L-tryptophan to serum albumins in the presence of non-esterified fatty acids.

Bovine, human and rat serum albumins were defatted and palmitic acid, oleic acid and lauric acid added in various molar ratios. The binding of L-tryptophan to these albumins was measured at 20 degrees C in a 0.138 M salt solution at pH 7.4, by using an ultrafiltration technique, and analysed in terms of n, the number of available tryptophan-binding sites per albumin molecule, with apparent association constant, k. 2. n and k were 0.90 and 2.3x10(-4)M(minus-1) respectively for defatted bovine serum albumin and 0.87 and 9.7x10(-3)M(-minus-1) for human albumin. Addition of palmitic acid did not decrease n until the molar ratio, fatty acid/bovine albumin, approached and exceeded 2. The decrease in k was small and progressive. In contrast, lauric caused a marked decrease in n and k at ratios as low as 0.5. A similar distinction between the effects on n of palmitic acid and oleic acid and those of lauric acid was seen for human albumin. k for human albumin was not significantly affected by fatty acids under the conditions studied. 3. It is concluded that primary long-chain fatty acid sites interact only weakly with the tryptophan site on albumin and that inhibition of tryptophan binding occurs when secondary long-chain sites are occupied. Primary medium-chain fatty acid sites are distinct from primary long-chain sites but may be grouped with secondary long-chain sites. 4. The relationship between free and bound tryptophan in samples of rat plasma (Stoner et al., 1975) is discussed in terms of a similar but limited study of rat albumin.     

Free amino acids mimic the anabolic but not the proliferative effect of albumin in OK proximal tubular cells

When plasma proteins leak from circulation into the renal tubular lumen in the proteinuric renal diseases, nephrotoxicity of filtered albumin (and/or molecules bound to it) may be important in the subsequent development of tubulo-interstitial damage which contributes to the progression of the disease. When cultured opossum kidney (OK) proximal tubular cells were exposed to bovine serum albumin for 3 days in vitro, increased cell division ([3H]-thymidine incorporation) and cellular hypertrophy (increased protein/DNA ratio) were observed. Both effects were halved if defatted albumin was used. A trivial explanation for the growth responses is that free fatty acids carried on the albumin, and amino acids generated by intracellular degradation of the albumin, are exerting a non-specific growth effect as metabolic fuels which are oxidized to generate ATP. However, the water-soluble free fatty acid octanoate (1 mmol l(-1)) had no significant effect on protein/DNA ratio and a very variable stimulatory effect on [3H]-thymidine incorporation, whereas an essential amino acid mixture or 1 mmol/l(-1) l-Ala or l-Phe only increased the protein/DNA ratio. Furthermore no carnitine was added to the culture medium. This absence would have impaired mitochondrial transport (and hence oxidation) of long-chain free fatty acids derived from the albumin. l-Phe is also a poor substrate for mitochondrial oxidation in kidney. It is therefore concluded that the growth effects of albumin in OK proximal tubular cells are specific effects of the albumin protein and of the free fatty acids and amino acids derived from it, and not a non-specific effect on metabolic fuel supply.     

A kinetic study of glycerophosphate acyltransferase of rat adipocytes in relation to its control by noradrenaline.

Glycerophosphate acyltransferase present in an extract of rat adipocytes is strongly inhibited by excess palmitoyl-CoA. This inhibition is released by serum albumin but an excess of serum albumin is inhibitory, particularly at low palmitoyl-CoA concentrations. An optimal activity is reached when the ratio palmitoyl-CoA/albumin is in the range of 3-6. In the absence of albumin, oleic acid inhibits the activity at all palmitoyl-CoA concentrations. This inhibition is released by albumin and, inversely, oleic acid releases the inhibition by high concentrations of albumin. Another effect of fatty acids is to favour the inactivation of the glycerophosphate acyltransferase in extracts of adipocytes kept at 0 degree C. This inactivation is time-dependent and cannot be reversed by the addition of albumin to the assay mixture. Treatment of adipocytes with noradrenaline had no effect on the activity of the enzyme as long as the cells had been separated from fatty acids and albumin. With extracts of unwashed cells, the effect of noradrenaline on both the activity and stability of glycerophosphate acyltransferase could be explained by the presence of fatty acids in the extract.     

Role of fatty acids in growth-promoting effect of serum albumin on hamster cells in vitro

Dialyzed serum albumin had considerable growth-promoting effect on cultivated hamster cells. This effect was virtually lost on removal of the fatty acids, and it was completely restored by recombination of the fatty acid-free albumin with the isolated and purified fatty acids. The role of albumin itself appeared to be largely that of a carrier of fatty acids, protecting the cells against toxic effects of fatty acids in free solution. This conclusion was based on two observations: Fatty acids in the absence of albumin were growth-inhibitory except in extremely dilute solutions, and beta-lactoglobulin, a protein possessing, like albumin, the ability to bind and release fatty acids, could replace albumin in the presence of fatty acids with similar growth-promoting effect. Examination of individual molecular types of fatty acids showed that all unsaturated acids tested were growth-promoting, whereas the saturated acids were growth-inhibiting, with the exception of stearic acid in low concentrations. Although the possibility of a mitotic triggering effect was not excluded, the fatty acids presumably stimulated growth by providing substrate for cellular metabolism, since there was a direct relationship between the degree of growth stimulation and the duration of exposure of cells to the fatty acids.     

Utilization of free fatty acids complexed to human plasma lipoproteins by mammalian cell suspensions

The purpose of this study was to determine whether lipoprotein-bound free fatty acid could be utilized by isolated mammalian cells. Ehrlich ascites tumor cells were incubated in vitro with radioactive free fatty acids that were bound to human plasma lipoproteins. Under these conditions, lipoprotein-bound free fatty acids were readily taken up by the cells. After 2 min of incubation with free fatty acids bound to low density lipoproteins, most of the radioactivity that was associated with the cells was in the form of free fatty acids. As the incubation continued, increasing amounts of radioactivity were incorporated into CO(2) and cell lipids, particularly phospholipids. Most of the free fatty acid uptake was the result of fatty acid transfer from low density lipoproteins to the cell, not from irreversible incorporation of the intact free fatty acid-low density lipoprotein complex. Fatty acid uptake increased as the ratio of free fatty acid to low density lipoprotein was raised. When albumin was added to the medium, free fatty acid uptake decreased. A large percentage of the newly incorporated cellular radioactivity was released into the medium if the cells were exposed subsequently to a solution containing albumin. Most of the released radioactivity was in the form of free fatty acid. The results with this experimental model suggest that lipoprotein-bound free fatty acid, like albumin-bound free fatty acid, is readily available for uptake by isolated cells. The mechanism of free fatty acid utilization by the Ehrlich cell is similar when either low density lipoprotein or serum albumin serves as the fatty acid carrier.     

The effect of bovine serum albumin on the synthesis of prostaglandin and incorporation of [3H]acetate into platelet-activating factor

The binding of fatty acids by bovine serum albumin (BSA) is well documented. However, the interaction between the synthesis of prostaglandins (PGs) and the trapping of arachidonate released from cellular lipid stores is not as well understood. In this communication, we relate the trapping of fatty acids to the synthesis of PGs and the incorporation of [3H]acetate into platelet-activating factor (PAF). Our results show that, as determined by radioimmunoassay, BSA inhibits bradykinin (BK) (5 ng/ml) and ionophore A23187 (10 microM)-stimulated synthesis of PGs in human embryo lung fibroblasts (IMR-90) in a concentration-dependent manner. Experiments using prelabel with [3H]arachidonate followed by extraction and thin-layer chromatography show that, in the presence of 2 mg/ml BSA, IMR-90 release essentially only fatty acid following stimulation with bradykinin. Little if any prostaglandin and no endoperoxide are detected. In the same experiment, in absence of BSA, about 70% of the released label is detected as prostaglandin. alpha-Cyclodextrin, another trapper of fatty acid, inhibits PG synthesis in much the same way. BSA and alpha-cyclodextrin also inhibit prostacyclin synthesis in endothelial cells derived from the calf pulmonary artery. However, the inhibition of PG synthesis in these cells is not as complete as that in the IMR-90. In contrast to the effect of the trappers on PG synthesis, BSA and alpha-cyclodextrin are observed to potentiate BK- and ionophore-stimulated incorporation of [3H]acetate into PAF in the endothelial cells. The labeled PAF is not released from the cells in either the presence or absence of the trappers, leading us to conclude that BSA causes an increase in acetate-labeled cellular PAF by trapping released fatty acid.     

Release of free fatty acids from Ehrlich ascites tumor cells

Ehrlich ascites tumor cells release free fatty acids (FFA) during in vitro incubation in media that contain albumin. The released FFA are derived by lipolysis from endogenous lipid esters. Addition of glucose to the incubation medium greatly decreases the quantity of fatty acid released by the cells. Cyanide, which inhibits endogenous lipid oxidation but not lipolysis, increases the quantity of fatty acid released to media containing albumin and causes free fatty acid to accumulate in the cells in the absence of exogenous albumin. The release of fatty acid, either preformed or derived by lipolysis during prolonged incubations, occurs under conditions of net fatty acid uptake from the incubation medium. Net release of fatty acid from the cell occurs only when fatty acid-extracted albumin is present in the extracellular medium; extrapolation of the data suggests that net release will not occur under physiological conditions. It is postulated that free fatty acid uptake and release are independent processes, the direction of net fatty acid movement being determined by the relationship between cellular free fatty acid concentration (regulating efflux) and the molar ratio of free fatty acid to albumin in the extracellular medium (regulating uptake).     

Metabolism of n-6 fatty acids by NIH-3T3 cells transfected with the ras oncogene

N-6 fatty acid metabolism was compared in NIH-3T3 cells and DT cells, which differ only in the presence of the v-Ki-ras oncogene. Non-dividing cells were incubated with [1-¹⁴C]-labelled fatty acids (18:2n-6, 18:3n-6, 20:3n-6 and 20:4n-6) at different time intervals (2–24 h) and concentration (0–120 M). In both cells lines, the uptake of different fatty acids from the medium was similar and reached a maximum at 6–8 h. All fatty acids reached the same maximum level in DT cells, whereas, the relative uptake of added fatty acids by NIH-3T3 cells was different: 20:4n-6>20:2n-6>18:2n-6=18:3n-6. Throughout the incubation (2–24 h), desaturation and elongation of n-6 fatty acids was more active in DT cells than in NIH-3T3 cells. However, in both cell lines, incubated with different n-6 fatty acid precursors, the levels of radiolabelled 20:4n-6 were relatively constant. In DT cells, phosphatidylcholine was found to be the major fraction labelled with n-6 fatty acids precursors and those of endogenous synthesis, whereas, in NIH-3T3 cells the neutral lipid fraction, particularly triglycerides, was also strongly labelled. In concentration dependent studies, phospholipid labelling by fatty acids was saturable. At lower concentrations, especially in DT cells, phospholipids were labelled predominantly. As the concentration increased there was an overflow into the triglyceride fraction. Since the differences in fatty acid metabolism between the two cell lines cannot be related to the growth rate, it is suggested that they were a consequence of the expression of the v-Ki-ras oncogene.Abbreviations BSA bovine serum albumin - CE cholesterol ester - DG diglyceride - DMEM Dulbecco's modification of Eagle's medium - EL ether lipids (glyceryl ether diesters) - FAME fatty acid methyl ester - FCS fetal calf serum - FFA free fatty acids - HEPES N-2-(hydroxyethyl)piperazine-N-2-ethanesulphonic acid - MG monoglyceride - NL neutral lipid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PL phospholipid - s.a specific activity - TG triglyceride - TLC thin layer chromatography     

Lipid peroxidation and growth inhibition of human microvascular endothelial cells

Peroxidation products of polyunsaturated fatty acids may cause growth inhibition of cells in culture. This study was carried out to elucidate to what extent peroxidation products may be found in growth media, with and without cells and albumin, using thiobarbituric acid-reactive substances (TBARS) and protein carbonyl groups as measures of peroxidation. The growth of human microvascular endothelial cells was studied as influenced by docosahexaenoic (C22:6, n - 3), arachidonic acid (C20:4. n - 6), and serum albumin. Cell growth was strongly inhibited by the fatty acids, and the inhibition was related to the concentration of TBARS in the medium. Defatted albumin (0.5 g/100 ml) nullified the increase of TBARS in the medium and released the growth inhibition by the fatty acids. With polyunsaturated fatty acids (PUFA) there was a time- and concentration-dependent increase in media TBARS, observed both with and without cells, but the TBARS increase was somewhat greater in the presence of cells. Surprisingly, TBARS in cell-free media also increased somewhat upon increasing the albumin concentration from 0.5 to 5 g/100 ml, and the TBARS increase differed among various preparations of albumin. Unexpectedly, the albumin that had not been defatted gave the lowest TBARS values. The amount of protein carbonyl groups did not differ among various albumin preparations. It is concluded that PUFA may autooxidize in media used for cell cultures, and thereby cause an unspecific growth inhibition, which can be prevented by a low albumin concentration. However, even defatted albumin preparations may contain lipid peroxidation products, the causes and implications of which remain to be elucidated.     

Cell-associated nonesterified fatty acid levels and their alteration during lipolysis in the isolated mouse adipose cell

A rapid and flexible method has been developed for measuring cell-associated, probably intracellular, nonesterified fatty acids (CAFA) in isolated mouse adipose cells. A variety of lipolytic agents as well as various concentrations of epinephrine elevate CAFA levels in rough proportion to their stimulation of glycerol and fatty acid release. Insulin reduces epinephrine-elevated CAFA levels. A detailed, quantitative study of the relationship among lipolytic activity, CAFA levels, and the extracellular molar ratio of fatty acids to albumin has been carried out. Epinephrine-elevated CAFA levels rise linearly with, while epinephrine-stimulated lipolytic activity is independent of, fatty acid to albumin ratios below 2-3. As the ratio increases from 3 to 5, CAFA levels continue to increase, whereas lipolytic activity decreases. Above ratios of 5, fatty acid release almost completely ceases; CAFA levels increase dramatically with residual glycerol release. A temperature-dependent efflux of epinephrine-elevated CAFA can be elicited through blockade of stimulated lipolysis with propranolol, but only in the presence of extracellular fatty acid to albumin ratios below 3. These observations suggest that during stimulated lipolysis, a fatty acid gradient exists between the cell and extracellular serum albumin and that CAFA represent the intracellular component of this gradient. In addition, these observations support the concept that intracellular fatty acids play a role in the feedback regulation of adipose cell function as extracellular fatty acids accumulate during the lipolytic response.     

Effects of chronic exposure to insulin on lipid synthesis in 3T3-L1 fatty fibroblasts

Summary The acute effect of insulin on ³H incorporation into lipid from glucose was measured in 3T3-L1 fatty fibroblasts cultured with and without insulin at 10 µg/ml for 7 days. Basal lipid synthesis did not differ between control cells and cells treated chronically with insulin. There was no insulin stimulation in treated cells while ³H incorporation into lipid in control cells increased from a basal level of 1.39 to 3.85 nmol/dish/90 min with a maximally-stimulating concentration of insulin. This is the first study of 3T3-L1 fatty fibroblasts which describes a lack of acute insulin responsiveness in cells exposed chronically to insulin as compared to control cells.Abbreviations KRP buffer Kreb's Ringer phosphate buffer - BSA bovine serum albumin Dr. Pohl is the recipient of Research Career Development Award AM 00183.     

Inhibition of CD3-induced Ca2+ signals in Jurkat T-cells by myristic acid.

Stimulation of T lymphocytes with antibodies against the T cell receptor/CD3 complex induces within seconds a rise in the concentration of intracellular free Ca2+. Here we show that treatment with 20 microM free myristic acid completely inhibits this Ca2+ signal and the cellular proliferation in Jurkat T cells. Also lauric acid inhibited cell growth while its blocking effect on the Ca2+ signal was weaker than that of myristic acid. Other saturated free fatty acids were inactive. The inhibitory effect of myristic acid could be reversed by the addition of fatty acid free albumin, which will bind the fatty acid. Myristic acid, but not its methyl ester, inhibited both the anti-CD3-induced Ca2+ influx across the cell membrane and Ca2+ release from intracellular stores, but not the formation of inositol phosphates. In contrast, thapsigargin-induced release of Ca2+ from the same intracellular stores was unaffected by myristic acid. Thus, myristic acid specifically blocks T cell antigen receptor-CD3 induced Ca2+ mobilization in T cells.     

Stimulation of anaphylaxis in the mouse footpad by dietary fish oil fatty acids

The effect of fish oil-derived omega-3 (omega-3) fatty acids on anaphylaxis, Arthus and delayed type hypersensitivity reactions in mice has been investigated. Mice on a normal chow diet were fed eicosapentaenoic acid and docosahexaenoic acid at a dose of 500 and 333 mg/kg/day, respectively, by a gastric tube over a period of 61 days. Control groups were given water, safflower oil or oleic acid. Anaphylactic and Arthus type reactions were induced in the mouse footpad using bovine serum albumin as an antigen. Carrageenin was utilized to produce a delayed type hypersensitivity reaction. The animals fed omega-3 fatty acids induced a more anaphylactic foodpad reaction. There was no significant effect of the diet on Arthus and delayed type hypersensitivity responses. There was no effect of the fish oil-supplemented diet on production of antibodies to bovine serum albumin. Synthesis of prostaglandin E2 by peritoneal macrophages was significantly inhibited in the animals fed omega-3 fatty acid-enriched fish oil, while leukotriene B4 production was not affected. These results suggest that a diet enriched in omega-3 fatty acids modulates production of arachidonic acid metabolites and this may influence anaphylaxis, but not Arthus and cellular mediated hypersensitivity responses.     

Dielectric behavior and atomic structure of serum albumin

After 1946, serum albumin was available for studies. Its residue sequence and internal disulfide bonding was developed by 1976. We began to make dielectric dispersion studies and apply Perrin's equations for rotational relaxation times around the two axes of revolution in 1938. These data indicated that albumin should have an elongated shape. In 1992 atomic structure data indicated the molecule was heart-shaped. A similar 1998 study of albumin complexed with fatty acid showed that the molecule was substantially rearranged. We found that the dielectric constant of albumin solutions was sensitive to fatty acid content, making this property an attractive probe in stop–flow kinetic studies. Such studies show that the fatty acid reaction is a two-step process. The fatty acid first binds to exterior sites in a diffusion-limited second order reaction complete in 1 ms. Then a first order rearrangement reaction with 400 ms half-life follows. Thus the highly specialized serum albumin sequence of amino acid residues determines not only the structure of the unligated molecule, but also the distinctive structures of the numerous multiligated molecules.