Serum release of hepatic calcium-binding protein regucalcin by liver injury with galactosamine administration in rats

The incorporation and 9 desaturation of exogenous [¹⁴C]stearic acid were studied in HTC 7288c cells in suspension. We examined the uptake of the acid over a wide range of concentrations (0–160 M) after incubating the cells for 6 h in a chemically-defined medium. Under this experimental condition, the uptake of the labeled acid was more extensive than that obtained from static cultures or from monolayer of isolated hepatocytes of rats. At an external concentration of 160 Mca. 52 nmoles of acid per mg of cellular protein was taken up. The production of oleic acid from [¹⁴C]stearate (9 desaturation) correlated well with the uptake curve between 0–80 M concentration. For higher stearate concentrations, the biosynthesis of oleic acid declined substantially and a plateau of 22 nmoles/mg cellular protein was reached. The incorporation and desaturation of an initial exogeneous concentration of [¹⁴C]stearic acid (80 M) was also studied from 0–6 h. The results obtained demonstrated that the uptake of the substrate into cellular lipids was fast and non saturable. Quantitative gas-liquid chromatography of total cellular lipids under the different experimental conditions demonstrated a negative correlation between the decrease in the palmitic and palmitoleic acids and the increase in the intracellular levels of stearic and oleic acids. These analytical modifications took place with no changes in the saturated/monoenoic fatty acid ratio. This work also demonstrated a significant contribution of the stearoyl-CoA desaturase system to the high levels of oleic acid present in this kind of hepatoma cells.Abbreviations FAME Fatty Acid Methyl esters - GLC gas-liquid chromatography - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanosulfonic acid - HTC Hepatoma Tissue Culture - IMEM-Zo Improved Minimal Essential Medium-zinc optional     

Inhibition of arachidonate biosynthesis in hepatoma tissue culture cells by 11-deoxycorticosterone-induced factor

In this work it was demonstrated that the incubation of hepatoma cultured cells (HTC 7288 c) with 11-deoxycorticosterone (DOC) ranging from 0 to 10^–4M concentration provoked a dose-dependent inhibition in the conversion of [1^–14C] eicosatrienoic acid to arachidonic acid. This steroid also produced an increase in the uptake of exogenous 20: 3 (n-6) acid. The depressive effect evoked by DOC on 5 desaturating activity was reflected on the fatty acid composition changes of the hepatoma cells. The 5 desaturase activity was inhibited by a soluble factor that would be induced by the hormone and that was present in the cytosol fraction from DOC-treated cells, corresponding to a low molecular mass below 25 kDa. Presently we report that an 11--OH group on the steroid molecule is not an essential requirement for the production of a 5 desaturase inhibitory factor.Members of the Carrera del Investigador Científico, CONICET, Argentina     

Δ5 Desaturase activity in rat kidney microsomes

Rat kidney microsomal fraction is able to catalyze the enzymatic desaturation of eicosatrienoic acid (20:3n-6) to arachidonic acid (20:4n-6) by the 5 desaturase pathway, in the presence of reduced nicotinamide adenine dinucleotide (NADH), adenosinetriphosphate (ATP) and coenzyme A (CoA). The substrate of the reaction [1-¹⁴C]eicosa-8,11,14trienoic acid (20:3n-6), was separated from the product [1-¹⁴C]eicosa-5,8,11,14-tetraenoic acid (20:4n-6) by reverse phase high-pressure liquid chromatography (RP-HPLC). These fatty acids were individually collected by monitoring the eluent at 205 nm and their radioactivity was measured by liquid scintillation counting. The 5 desaturase activity in kidney microsomes increased linearly with the substrate concentration up to 20 M. Enzymatic activity was sensitive to pH with the maximum at 7.0 and was proportional with incubation time up to 10 min. The apparent Km and Vmax of 5 desaturase were 56 M and 60 pmoles·min^–1·mg^–1 microsomal protein, respectively. Neither the cytosolic renal fraction nor the cytosolic liver fraction enhanced the 5 desaturase activity. Contrary to a report but in accordance to others, the present results suggest that rat kidneys can synthesize arachidonic acid at least to satisfy partially their needs for eicosanoid production.     

Characterization of a structurally and functionally diverged acyl-acyl carrier protein desaturase from milkweed seed

A cDNA for a structurally variant acyl-acyl carrier protein (ACP) desaturase was isolated from milkweed (Asclepias syriaca) seed, a tissue enriched in palmitoleic (16:1⁹)* and cis-vaccenic (18:1¹¹) acids. Extracts of Escherichia coli that express the milkweed cDNA catalyzed ⁹ desaturation of acyl-ACP substrates, and the recombinant enzyme exhibited seven- to ten-fold greater specificity for palmitoyl (16:0)-ACP and 30-fold greater specificity for myristoyl (14:0)-ACP than did known ⁹-stearoyl (18:0)-ACP desaturases. Like other variant acyl-ACP desaturases reported to date, the milkweed enzyme contains fewer amino acids near its N-terminus compared to previously characterized ⁹-18:0-ACP desaturases. Based on the activity of an N-terminal deletion mutant of a⁹ -18:0-ACP desaturase, this structural feature likely does not account for differences in substrate specificities.     

Fatty acid Δ5 desaturation in rat liver cell nuclei

Activity of one of the key enzymes involved in arachidonic acid (20:4 n–6) biosynthesis, the 5 desaturase, was found in rat liver cell nuclei. Up to now, it has been shown that the fatty acid desaturases are located exclusively in the endoplasmic reticulum. Similarly to what happens with microsomal enzyme the nuclear 5 desaturase enzyme was only fully active in the presence of a cytosolic factor. In this condition it reached a specific activity of 50 pmol 20:4 n–6 formed/min/mg of protein. This fact would imply that purified nuclei like purified microsomes lack a soluble cytosol factor necessary for the total desaturation reaction expression. Besides the nuclear 5 desaturase has an optimal pH of 7.6 and is inhibited by 1 or 10 mM KCN. Low long chain acyl-CoA synthetase activity that catalyzes the formation of 20:3 n–6-CoA, was also found in liver nuclei. This step would be essential in nuclear desaturation since when ATP and/or CoA (necessary for the acylation reaction) are omitted from the incubation mixture, the desaturation reaction does not take place.Abbreviation PMSF phenylmethylsulfonyl fluoride     

Effects of cannabinoids on the activities of mouse brain lipases

Cannabinoids were found to augment phospholipase activities and modify lipid levels of mouse brain synaptosomes, myelin and mitochondria. Delta-1-tetrahydrocannabinol (¹-THC) and several of its metabolites induced a dose-dependent (0.32–16 M) stimulation of phospholipase A₂ (PLA₂) activity resulting in the increased release of free arachidonic acid from exogenous [1-¹⁴C]phosphatidylcholine (PC). The potencies of the cannabinoids in modulating PLA₂ activity were approximately of the order: 7-OH-¹-THC > ¹-THC > 7-oxo-¹-THC > ¹-THC-7oic acid = 6 OH-¹-THC 6-OH-¹-THC. The hydrolysis of phosphatidylinositol (PI) by synaptosomal phospholipase C (PLC) was enhanced significantly by ¹-THC and promoted diacylglyceride levels by greater than 100 percent compared to control values. In contrast, arachidonate was the major product resulting from phospholipase activities of a 20,000g pellet. Synaptosomal diacylglyceride lipase activity was inhibited by ¹-THC. [1-¹⁴C]Arachidonic acid was readily incorporated into subcellular membrane phospholipids and after exposure to cannabinoids led to diminished phosphoglyceride levels and concomitant increases in released neutral lipid products. These data suggest that cannabinoids control phospholipid turnover and metabolism in mouse brain preparations by the activation of phospholipases and, through this mechanism, may exert some of their effects.     

Role of fatty acids of (n-3) and (n-6) series on α-linolenic acid desaturation and chain elongation in HTC cells

Summary The desaturation and chain elongation of [1-¹⁴C] -linolenic acid were studied in HTC cells preincubated for 24 h in the presence of different unlabeled fatty acids of (n-3) and (n-6) series. After 24 h in the presence of [1-¹⁴C] -18:3, cells transformed this acid into labeled 20:5 and 22:5(n-3) through the desaturation-elongation pathway and into 20:3 and 22:3(n-3) by the elongation reactions. The preincubation of HTC cells with (n-3) fatty acids (-18:3, 20:5 and 22:6) produced an increase in the amount of [1-¹⁴C] -18:3 that remained in the cells without being metabolized and consequently, a decrease in the last product formed, the 22:5(n-3) was observed. Simultaneously, the desaturation-elongation products decreased significantly and those of the elongation pathway were not modified, except when the cells were pre-incubated with the last fatty acid of this family (22:6) which increased this metabolic route. Fatty acids of (n-6) series (-18:3, 20:3, 20:4 and 22:4) decreased the desaturation-elongation pathway and increased the elongation route from [1-¹⁴C] -18:3. From these results, it can be concluded that fatty acids of (n-3) family and intermediates of (n-6) series would impair the [1-¹⁴C] -18:3 metabolism at the 6 desaturation step. The fatty acid composition of the cells was also modified by the preincubation with (n-3) and (n-6) acids showing a decrease on 9 desaturation activity.     

Effect of different carbon sources on the biosynthesis of polyunsaturated fatty acids ofα-linolenic acid family in culture of minimal deviation hepatoma 7288 c cells

Summary The effect of three different carbon sources on the biosynthesis of polyunsaturated fatty acids of the-linolenic acid series was investigated in hepatoma tissue culture (HTC) cells. Alpha linolenic acid was converted to higher homologs by a desaturating route that synthetized mainly 18:4 (6, 9, 12, 15), 20:4 (8, 11, 14, 17) and 20:5 (5, 8, 11, 14, 17) and an elongating route that produced 20:3 (11, 14, 17) and 20:4 (5, 11, 14, 17) acids. Fasting decreased both biosynthetic routes whereas glucose reactivated only the elongating pathway. Lactalbumin hydrolysate enhanced significantly only the desaturating route whereas glycerol was inactive. Glucose and aminoacids increased similarly the incorporation of labeled linolenic acid in the cells. The results are independent of hormonal effects.Members of the Carrera del Investigador Científico of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

Effect of benzyl viologen on the phospholipid fatty acid composition and some properties in hepatic microsomal membrane of rats

The effect of benzyl viologen (a stimulator of free radical production in cells) on lipid composition, fluidity and enzymes involved in both polyunsaturated fatty acid biosynthesis and cholesterol metabolism was studied in liver microsomal membrane of adult rats. In viologen-treated animals, a significant decrease in the levels of free cholesterol and cholesteryl esters, accompanied to a decrease at the free cholesterol/phospholipid ratio, were observed. The levels of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acyl-coenzyme A : cholesterol acyltransferase (ACAT) were also lower in viologen-treated rats than in controls. Linoleic and arachidonic acids were both severely lower while docosatetraenoic, docosapentaenoic and docosahexaenoic acids were significantly higher as compared with controls. Furthermore, a decrease in monounsaturated/saturated ratio was found. In addition, the treatment evoked a depression in the fatty acid desaturation complex, with a diminish of ⁹, ⁹, and ⁵ desaturase activities in microsomal membrane.It was concluded that changes in phospholipid microsomal fatty acid and cholesterol content could be directly responsible for changes in membrane fluidity and function, and that extensive yield of docosahexaenoic acid may serve to maintain the physical characteristics of particular domains against oxidative stress caused by benzyl viologen treatment.     

Effect of fatty acids of ω6 series on the biosynthesis of arachidonic acid in HTC cells

Summary The influence of the preincubation of HTC cells with fatty acids of 6 series and columbinic acid (St, 9c, 12c 18:3) on the biosynthesis of arachidonic acid was studied. The cells were incubated on a chemically defined medium with or without the addition of unlabeled linoleic, -linolenic, eicosatrienoic, arachidonic, docosatetraenoic, docosapentaenoic and columbinic acids. After 24 hr of preincubation in the presence of the aforementioned fatty acids, [1-¹⁴C]eicosa-8,11,14-trienoic acid was added to the culture medium as the only lipidic source. Twenty-four hours later the synthesis of arachidonic acid and the fatty acid composition of the cells were determined. At 20 MM concentration the 6 fatty acids studied except docosapentaenoic acid produced an increase on the biosynthesis of arachidonic acid compared to the cells incubated in the absence of unlabeled fatty acids in the medium. The fatty acids added to the culture medium were incorporated into the cells and modified their fatty acid composition. Columbinic acid, with a similar structure to linoleic acid, also produced a significant increase on the conversion of eicosatrienoic acid to arachidonic acid. These results would suggest that the effect of both, linoleic and columbinic acids, may be adscribed to their configuration and not necessarily to their transformation in higher homologs, since columbinic acid is unable to be desaturated.All authors are members of the Carrera del Investigador Cientifico of the Consejo Nacional de Investigaciones Cientifícas y Técnicas, Argentina.     

Variation in leaf carbon isotope discrimination in Encelia farinosa: implications for growth,competition, and drought survival

Population-level variation in the leaf carbon isotope discrimination () values was examined in Encelia farinosa, a common Sonoran Desert shrub. There was approximately a 2 range in values among different plants. These differences in values among neighboring plants were maintained through time, both under conditions when neighbors were present and after neighbors had been removed. Individuals with high values were found to have an accelerated growth rate when these plants were released from competition for water. Individuals with low values were better able to persist through long-term drought. These data suggest possible tradeoffs between conditions favoring high- and low--value plants within a natural population. Given the temporal variability in precipitation between years and spatial variability in microhabitat quality in the Sonoran Desert, variation in values among E. farinosa plants will be maintained within a population.     

Regulatory effect of various steroid hormones on the incorporation and metabolism of [14C]stearate in rat hepatoma cells in culture

We have examined the incorporation and metabolism of [¹⁴C] stearic acid within the total lipids of HTC rat-hepatoma cells in suspension culture in presence and in absence of steroidal hormone stimulation. Both, glucocorticoids (dexamethasone, cortisol and corticosterone) and mineralocorticoids (deoxycorticosterone and aldosterone) as well as the estrogen -estradiol and the androgen testosterone enhanced the extent of 9 desaturation to oleic acid of the saturated precursors, whereas only the two mineralocorticoids affected the incorporation rate of the exogenous acid into total cellular lipids, thus promoting a little stimulation. Furthermore, all the hormones tested increased the radiolabelling of the total cellular phospholipids except deoxycorticosterone and testosterone, the former having no effect and the latter exerting a moderate inhibition. On the other hand, the incorporation of¹⁴C into neutral lipids was stimulated by testosterone, in contrast to the inhibition of this parameter observed exclusively with either the mineralocorticoids or the estrogen. Within the phospholipid subclasses, the radiolabelling of phosphatidylcholine was augmented by means of all the steroids tested save deoxycorticosterone and testosterone, whereas phosphatidylethanolamine exhibited a decrease only in the presence of testosterone. In a similar fashion, within the neutral lipids, the predominating triglyceride fraction was preferentially labelled—at the expense of other subclasses of lesser abundance—upon treatment with the steroids except aldosterone, which exerted no effect. The results obtained were correlated with those changes observed in the mass distribution of the different lipid subclasses either with or without prior hormonal stimulation.Abbreviations ALD aldosterone - ANOVAR analyses of variance - CE cholesterol esters - Cl cortisol - CLN cardiolipin - Cst corticosterone - CH cholesterol - DG diacylglycerides - DOC 11-deoxycorticosterone - Dx dexamethasone phosphate salt - ßE 17--estradiol - FAME fatty acid methyl esters - GLC gas-lipid chromatography - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanosulfonic acid - HTC hepatoma tissue culture - IMEM-Zo improved minimal essential medium-Zinc option - LPC lysophosphatidyl-choline - LPL lysophospholipids - MG monoacylglycerides - NEFA free fatty acids - PC phosphatidyl-choline - PE phosphatidyl-ethanolamine - PG phosphatidyl-glycerol - PI phosphadyl-inositol - PS phosphadidyl-serine - SM sphingomyelin - TG triacylglycerides - TLC thin-layer chromatography - Tst testosterone Members of the Carrera del Investigador Cientifico, CONICET, Argentina.     

Effect of L-triiodothyronine on liver microsomal Δ6 and Δ5 desaturase activity of male rats

The effect of different doses of L-triiodothyronine (T₃) on the activity of 6 and 5 desaturases and lipid fatty acid composition was studied in liver microsomes of male rats. The activity of 6 and 5 desaturases was decreased 24 and 28%, respectively, in animals administered a daily intraperitoneal dose of 1000g T₃/100g body wt. for 5 days, whereas with 500g T₃/100g body wt. only 6 desaturase activity was decreased. On the other hand, no enzyme activity changed at a shorter period of hormone treatment. Changes in microsomal fatty acid composition did not seem to be a direct consequence of desaturation activity, since after 1 and 5 days of T₃ treatment, the concentrations of 18:2 (n-6) and 20:3 (n-6) decreased and only after 1 day that of 20:4 (n-6) increased in spite of unchanged or decreased 6 and 5 desaturase activities. Other factors than desaturation activity must be involved in fatty acid composition of thyroid hormonetreated rats, such as diet, membrane lipid synthesis and degradation, fatty acid turn-over and oxidation. (Mol Cell Biochem121: 149–153, 1993)     

Mitochondrial Adaptation to in vivo Polyunsaturated Fatty Acid Deficiency: Increase in Phosphorylation Efficiency

Polyunsaturated fatty acid (PUFA) deficiency affects respiratory rate both in isolated mitochondria and in hepatocytes, an effect that is normally ascribed to major changes in membrane composition causing, in turn, protonophoriclike effects. In this study, we have compared the properties of hepatocytes isolated from PUFA-deficient rats with those from control animals treated with concentrations of the protonophoric uncoupler 2,4-dinitrophenol (DNP). Despite identical respiratory rate and in situ mitochondrial membrane potential (), mitochondrial and cytosolic ATP/ADP–P_i ratios were significantly higher in PUFA-deficient cells than in control cells treated with DNP. We show that PUFA-deficient cells display an increase of phosphorylation efficiency, a higher mitochondrial ATP/ADP–P_i ratio being maintained despite the lower . This is achieved by (1) decreasing mitochondrial P_i accumulation, (2) increasing ATP synthase activity, and (3) by increasing the flux control coefficient of adenine nucleotide translocation. As a consequence, oxidative phosphorylation efficiency was only slightly affected in PUFA-deficient animals as compared to protonophoric uncoupling (DNP). Thus, the energy waste induced by PUFA deficiency on the processes that generate the proton motive force (pmf) is compensated in vivo by powerful adaptive mechanisms that act on the processes that use the pmf to synthesize ATP.     

Expression of fungal desaturase genes in cultured mammalian cells

Long-chain polyunsaturated fatty acids (LC-PUFA) are important components of cellular structure and function. Most of LC-PUFA are derived from linoleic acid and a-linolenic acid. In plants and fungi, these two acids can be synthesized from oleic acid via the action of two enzymes, 12 and 15-desaturases. Due to lack of these enzymatic activities and the ability to synthesize these two essential fatty acids, animals must obtain them from the diet. In this report, we demonstrated the expression of a fungal 12-desaturase gene in mouse L cells incubated in serum-free medium. The results showed a significant increase in the amount of linoleic acid with a concomitant decrease of oleic acid in cellular lipids. Most of the newly formed linoleic acid was incorporated into cellular phospholipids, particularly phosphatidylcholine. The increase of linoleic acid provided the substrate for the endogenous synthesis of (n-6) LC-PUFA, such as eicosadienoic acid (EDA), dihomo--linoleic acid (DGLA) and arachidonic acid (AA). Prolonged incubation further increased the levels of linoleic acid derived from oleic acid by the action of 12-desaturase, and the levels of 20:2n-6 produced from linoleic acid by the action of the endogenous elongase. However, prolonged incubation suppressed significantly the formation of DGLA and AA. In a separate study, a fungal 6-desaturase gene has also been expressed in the mouse L cells incubated in serum-containing medium. The result shows a significant increase in levels of 20:3n-6 and 20:4n-6. These findings demonstrate that through genetic modification, it is possible to (1) generate cell lines which no longer require dietary 'essential' fatty acids and (2) alter the endogenous fatty acid metabolism to enhance the production of LC-PUFA and their derivatives.     

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen,Streptococcus mutans Ingbritt

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (³H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference () dissipator, KSCN. The membrane ATPase inhibitor, N,N¹-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (pH) and . ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to pH.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N¹-dicyclohyxyl carbodiimide - p protonmotive force - pH transmembrane pH gradient - transmembrane electrical potential difference     

Measurements of electrical potential differences across yeast plasma membranes with microelectrodes are consistent with values from steady-state distribution of tetraphenylphosphonium inPichia humboldtii

Summary Electrical potential differences across the plasma membrane () of the yeastPichia humboldtii were measured with microelectrodes (filled with 0.1m KCl) inserted into cells immobilized in microfunnels. The registered signals were reproducible and stable for several minutes. On attainment of stable reading for the specific membrane resistanceR _sp was determined by applying square-current pulses to the preparation. Both andR _sp were pH dependent and displayed equal but opposite deflection, reaching its maximal value of –88±9 mV (n=13) andR _sp its minimal value of 10 k·cm² (maximal conductance) at pH 6.5. Uncouplers and the polyene antibiotic nystatin depolarized the cells, decreasing to –21±15 mV (n=10) with concomitant decrease ofR _sp. Comparison of values from microelectrode measurements with those calculated from the steady-state distribution of tetraphenylphosphonium ions agreed within 10 mV under all physiological conditions tested, except at pH values above 7.0. During microelectrode insertion transient voltage signals (a few msec long) were detected by means of an oscilloscope. These voltage signals were superimposed on the stable recordings described above. These short voltage signals disappeared in uncoupled cells. The closely related values obtained by two independent methods (direct measurements with microelectrodes and calculation from steady-state distribution of a lipophilic cation) provide evidence that these values reffect the true membrane potential of intact cells.     

Incorporation and metabolic conversion of saturated and unsaturated fatty acids in SK-Hep1 human hepatoma cells in culture

We report here a study of the incorporation and metabolism of various long chain fatty acids in SK-Hep-1 cultured hepatoma cells. Medium supplementation with radiolabelled palmitic, stearic, linoleic, -linolenic and eicosa-8, 11,14-trienoic acids (1 µM, 24 H) resulted in an active uptake of each of these precursors by the cultures. Subsequent analysis of the cellular lipids indicated that they exhibit almost all the enzymic activities of polyunsaturated fatty acid metabolism that are characteristic of normal hepatic cells. With respect to the desaturation capacities of this cell line, although -linolenic acid reacted more extensively than did linoleic acid and the conversion of 8,11,14-eicosatrienoic acid by the 5 specific enzyme was more avid than had been previously seen in normal rat or human liver: the saturated fatty acids constituted relatively poor substrates, being preferentially chain-elongated rather than (mono) desaturated at the 9 position. Analysis of the fatty acid profiles of total cellular lipids and of various lipid subclasses, however, revealed a relative paucity of essential fatty acids when compared with the abundance of endogenous monoenoic acids (particularly oleic). Of the total cellular fatty acids, 58% were present in the form of phospholipids; with 33% of the remaining 42% (i.e., the neutral lipids) being associated with triacylglycerol fraction. Within the total lipids, phosphatidyl-choline and phosphatidyl-ethanolamine were the major sites for the incorporation of all metabolic products derived from the incubated radiolabelled 16- and 18-carbon fatty acid precursors, whereas the phosphatidyl-inositol fraction was the predominat recipient of nascent arachidonic acid when the eicosatrienoate was the substrate. The express purpose of this investigation was to characterize the biochemical routes involved in the anabolism of various essential fatty acids in the human hepatocyte, through the use of cultured human hepatoma cells as an experimental model system. In view of the similarities between certain aspects of the polyunsaturated fatty acid metabolism of these cells and the corresponding properties of other mammalian hepatic or liver-derived tissues, the data presented here would thus constitute a significant beginning alone those lines. Moreover, considering the extreme difficulty in obtaining for such investigation relevant tissue samples from normal human sources, we regard these results — and the availability for use of this particular human hepatoma cell line — as important new developments in the effort to characterize a useful experimental model both for gaining immediate information and for designing future experiments.     

On the nature of the uncoupling effect of fatty acids

The effect of palmitic acid on the electrical potential difference across the inner mitochondrial membrane appears to depend on the medium in which mitochondria are incubated. In medium A (cf. Luvisettoet al. (1987),Biochemistry,26, 7332–7338) decreases much more than in medium B (cf. Rottenberg and Hashimoto (1986),Biochemistry,25, 1747–1755) at concentrations of fatty acid which equally stimulate the rate of respiration in state 4. Valinomycin and NaCl were both present in medium B and absent in medium A. However, in both media the pattern of the P/O ratio as a function of antimycin in the presence of a constant amount of palmitic acid or of FCCP shows similar behaviour. We conclude that in both media palmitic acid increases the membrane conductance to protons, but for unclear reasons the assay fails to measure the decline of in medium B. However, the increase in membrane conductance induced by palmitic acid does not quantitatively account for the stimulation of the rate of respiration.     

Negative-ion fast atom bombardment tandem mass spectrometry for characterization of sulfated unsaturated disaccharides from heparin and heparan sulfate

Negative-ion fast atom bombardment tandem mass spectrometry has been used in the characterization of non-, mono-, di- and trisulfated disaccharides from heparin and heparan sulfate. The positional isomers of the sulfate group of monosulfated disaccharides were distinguished from each other by negative-ion fast atom bombardment tandem mass spectra, which provide an easy way of identifying the positional isomers. This fast atom bombardment collision induced dissociation mass spectrometry/mass spectrometry technique was also applied successfully to the characterization of di- and trisulfated disaccharides.Abbreviations FABMS fast atom bombardment mass spectrometry - CID collision induced dissociation - MIKE mass analysed ion kinetic energy - MS/MS mass spectrometry/mass spectrometry - HPLC high performance liquid chromatography - UA d-gluco-4-enepyranosyluronic acid - CS chondroitin sulfate - DS dermatan sulfate - HA hyaluronan - Hep heparin - HS heparan sulfate - UA(14) GlcNAc 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNAc 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcN6S 2-amino-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcN 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcN6S 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcNS 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNS 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(13)GalNAc 2-acetamido-2-deoxy-3-O-(-d-Gluco-4-enepyranosyluronic acid)-d-galatose - UA(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA2S(13)GalNAc 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-galactose - UA2S(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA2S(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA(13)GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose - UA(13)GlcNAc 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose.