Linoleic acid-induced endothelial cell injury: role of membrane-bound enzyme activities and lipid oxidation.

High plasma levels of linoleic acid (18:2) may injure endothelial cells, resulting in decreased barrier function of the vascular endothelium. The effects of linoleic acid on endothelial barrier function (transendothelial movement of albumin), membrane-bound enzyme activities, and possible autooxidation of linoleic acid under experimental conditions were studied. The exposure of endothelial monolayers to 18:2 for 24 hr at 60, 90, and 120 microM fatty acid concentrations caused a significant increase in transendothelial movement of albumin, with maximum albumin transfer at 90 microM. Fatty acid treatment resulted in the increased appearance of cytosolic lipid droplets. Activities of the membrane-bound enzymes, angiotensin-converting enzyme (ACE), and Ca(2+)-ATPase increased steadily with increasing time of cell exposure to 90 microM 18:2, reaching significance at 24 hr. Treatment of endothelial cultures with up to 120 microM 18:2 did not cause cytotoxicity, as evidenced by a nonsignificant change in cellular release of [3H]-adenine. Incubation of 18:2-supplemented serum-containing culture media with 1000 microM 18:2 at 37 degrees C for up to 48 hr did not result in formation of autooxidation products. These results suggest that 18:2 itself, and not its oxidation products, plays a major role in disrupting endothelial barrier function.     

Oxidative lipidomics of gamma-irradiation-induced intestinal injury

Although gamma-irradiation-induced tissue injury has been associated with lipid peroxidation, the individual phospholipid molecular targets have not been identified. We employed oxidative lipidomics to qualitatively and quantitatively characterize phospholipid peroxidation in a radiosensitive tissue, the small intestine, of mice exposed to total body irradiation (TBI) (10 and 15 Gy). Using electrospray ionization mass spectrometry we found that the major classes of intestine phospholipids-phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol-included clusters with highly oxidizable molecular species containing docosahexaenoic fatty acid. Molecular species of cardiolipin were represented by only two major less oxidizable individual molecular species-tetralinoleoylcardiolipin and trilinoleoyl-mono-oleoylcardiolipin. Selective and robust oxidation of two anionic phospholipids-cardiolipin in mitochondria and phosphatidylserine outside of mitochondria-was observed 24 h after gamma-irradiation. MS analysis detected several TBI-induced molecular species of oxidized cardiolipin: (C(18:2))(3)(C(18:2)-OOH), (C(18:2))(2)(C(18:2)-OOH)(2), (C(18:2))(1)(C(18:2)-OOH)(3), and (C(18:2)-OOH)(4). The major molecular species involved in TBI-triggered peroxidation of phosphatidylserine included C(18:0)/C(22:6)-OOH, C(18:0)/C(22:5)-OOH, and C(18:0)/C(22:4)-OOH. More abundant phospholipids-phosphatidylcholine and phosphatidylethanolamine-did not reveal any oxidative stress responses despite the presence of highly oxidizable docosahexaenoic fatty acid residues in their molecular species. A marked activation of caspases 3/7 that was detected in the intestine of gamma-irradiated mice indicates the involvement of apoptotic cell death in the TBI injury. Given that oxidized molecular species of cardiolipin and phosphatidylserine accumulate during apoptosis of different cells in vitro we speculate that cardiolipin and phosphatidylserine oxidation products may be useful as potential biomarkers of gamma-irradiation-induced intestinal apoptosis in vivo and may represent a promising target for the discovery of new radioprotectors and radiosensitizers.     

Linoleic acid-induced endothelial cell injury: Role of membrane-bound enzyme activities and lipid oxidation

High plasma levels of linoleic acid (18:2) may injure endothelial cells, resulting in decreased barrier function of the vascular endothelium. The effects of linoleic acid on endothelial barrier function (transendothelial movement of albumin), membrane-bound enzyme activities, and possible autooxidation of linoleic acid under experimental conditions were studied. The exposure of endothelial monolayers to 18:2 for 24 hr at 60, 90, and 120 μM. fatty acid concentrations caused a significant increase in transendothelial movement of albumin, with maximum albumin transfer at 90 μM. Fatty acid treatment resulted in the increased appearance of cytosolic lipid droplets. Activities of the membrane-bound enzymes, angiotensin-converting enzyme (ACE), and Ca²⁺-ATPase increased steadily with increasing time of cell exposure to 90 μM 18:2, reaching significance at 24 hr. Treatment of endothelial cultures with up to 120 μM 18:2 did not cause cytotoxicity, as evidenced by a nonsignificant change in cellular release of [₃H]-adenine. Incubation of 18:2-supplemented serum-containing culture media with 1000 μM 18:2 at 37°C for up to 48 hr did not result in formation of autooxidation products. These results suggest that 18:2 itself, and not its oxidation products, plays a major role in disrupting endothelial barrier function.     

Effect of dietary alpha-linolenic acid on the activity and gene expression of hepatic fatty acid oxidation enzymes

The activities of hepatic fatty acid oxidation enzymes in rats fed linseed and perilla oils rich in alpha-linolenic acid (alpha-18:3) were compared with those in the animals fed safflower oil rich in linoleic acid (18:2) and saturated fats (coconut or palm oil). Mitochondrial and peroxisomal palmitoyl-CoA (16:0-CoA) oxidation rates in the liver homogenates were significantly higher in rats fed linseed and perilla oils than in those fed saturated fats and safflower oil. The fatty oxidation rates increased as dietary levels of alpha-18:3 increased. Dietary alpha-18:3 also increased the activity of fatty acid oxidation enzymes except for 3-hydroxyacyl-CoA dehydrogenase. Unexpectedly, dietary alpha-18:3 caused great reduction in the activity of 3-hydroxyacyl-CoA dehydrogenase measured with short- and medium-chain substrates but not with long-chain substrate. Dietary alpha-18:3 significantly increased the mRNA levels of hepatic fatty acid oxidation enzymes including carnitine palmitoyltransferase I and II, mitochondrial trifunctional protein, acyl-CoA oxidase, peroxisomal bifunctional protein, mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases, 2, 4-dienoyl-CoA reductase and delta3, delta2-enoyl-CoA isomerase. Fish oil rich in very long-chain n-3 fatty acids caused similar changes in hepatic fatty acid oxidation. Regarding the substrate specificity of beta-oxidation pathway, mitochondrial and peroxisomal beta-oxidation rate of alpha-18:3-CoA, relative to 16:0- and 18:2-CoAs, was higher irrespective of the substrate/albumin ratios in the assay mixture or dietary fat sources. The substrate specificity of carnitine palmitoyltransferase I appeared to be responsible for the differential mitochondrial oxidation rates of these acyl-CoA substrates. Dietary fats rich in alpha-18:3-CoA relative to safflower oil did not affect the hepatic activity of fatty acid synthase and glucose 6-phosphate dehydrogenase. It was suggested that both substrate specificities and alterations in the activities of the enzymes in beta-oxidation pathway play a significant role in the regulation of the serum lipid concentrations in rats fed alpha-18:3.     

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.     

Protein ligand interactions:isoquinoline alkaloids as inhibitors for lactate and malate dehydrogenase

Kinetic analysis has shown that isoquinoline, papaverine and berberine act as reversible competitive inhibitors to muscle lactate dehydrogenase and mitochondrial malate dehydrogenase with respect to the coenzyme NADH. The inhibitor constants Ki vary from 7.5 microM and 12.6 microM berberine interaction with malate dehydrogenase and lactate dehydrogenase respectively to 91.4 microM and 196.4 microM with papaverine action on these two enzymes. Isoquinoline was a poor inhibitor with Ki values of 200 microM (MDH) to 425 microM (LDH). No inhibition was observed for both enzymes in terms of their respective second substrate (oxaloacetic acid - malate dehydrogenase; pyruvate - lactate dehydrogenase). A fluorimetric analysis of the binding of the three alkaloids show that the dissociation constants (Kd) for malate dehydrogenase are 2.8 microM (berberine), 46 microM (papaverine) and 86 microM (isoquinoline); the corresponding values for lactate dehydrogenase are 3.1 microM, 52 microM and 114 microM. In all cases the number of binding sites averaged at 2 (MDH) and 4 (LDH). The binding of the alkaloids takes place at sites close to the coenzyme binding site. No conformational non equivalence of subunits is evident.     

Analyses for phosphatidylcholine hydroperoxides by LC/MS

A new liquid chromatography mass spectrometry (LC/MS) method has been developed for the qualitative and quantitative analyses of phosphatidylcholine hydroperoxides (PC-OOH) in human plasma using a synthetic hydroperoxide (1-stearoyl-2-erucoyl-PC monohydroperoxide, PC 18:0/22:1-OOH) as an internal standard. 1-Stearoyl-2-linoleoyl-PC monohydroperoxide (PC 18:0/18:2-OOH) was identified in plasma by LC/MS by comparison with an authentic standard. The calibration curves obtained for 1-palmitoyl-2-linoleoyl-PC monohydroperoxide, PC 16:0/18:2-OOH and PC 18:0/18:2-OOH were linear throughout the calibration range (0.1–1.0 pmol). The limit of detection (LOD) (S/N = 3:1) was 0.01 pmol, and the limit of quantification (LOQ) (S/N = 6:1) was 0.1 pmol for both PC 16:0/18:2-OOH and PC 18:0/18:2-OOH. Plasma concentrations of PC 16:0/18:2-OOH and PC 18:0/18:2-OOH were 89 and 32 nM, respectively, in a healthy volunteer.     

Linoleate and linolenate desaturation by rat hepatoma cells

Morris 7777 rat hepatoma cells in culture possess high delta 6 and delta 5 desaturase activities over linolenic acid added to the medium as albumin or alpha-fetoprotein complexes. After 2 hours incubation with [1-14C] linolenic acid (7 microM), around 40% of the radioactivity was recovered in other polyene fatty acids, mainly pentaenes. After 24 hours incubation with this substrate the polyene derivatives raised to more than 60%. However, [1-14C] linoleic acid was poorly converted to other polyene fatty acids. Linoleic acid up to 58 microM concentration in the medium do not inhibited linolenic acid desaturation. Long-term supplementation with 50 microM linoleic or linolenic acid, which modified the fatty acid profile of hepatoma lipids, enhanced the desaturase activities against linoleic acid. Desaturase activities were not affected by the fatty acid protein carrier, alpha-fetoprotein or albumin.     

Inhibitory action of polyunsaturated fatty acids on IMP dehydrogenase

We screened the inhibitor of mouse inosine 5'-monophosphate dehydrogenase (IMPDH) type II from natural compounds, and found that a fatty acid, linoleic acid (C18:2), inhibited IMPDH activity. In the C18:2 fatty acid derivatives, all trans-configuration (i.e., linoelaidic acid), ester form, alcohol form, and addition of the hydroxyl group of linoleic acid had no effect on inhibitory activity. Therefore, both parts of a carboxylic acid and an alkyl chain containing cis-type double bonds of fatty acid might be essential for inhibition. Among the various carbon atom lengths and double bonds of fatty acids examined, the strongest inhibitor was C20:2-fatty acid, eicosadienoic acid, and 50% inhibition was observed at a concentration of 16.1 microM. Eicosadienoic acid induced the inhibition of IMPDH activity and was competitive with respect to IMP (K(i)=3.1 microM). For inhibitory effect, the C20-fatty acids ranked as follows: C20:2>C20:3>C20:1> C20:4>C20:5, and C20:0 showed no inhibition. The energy-minimized three-dimensional structures of linear-chain C20-fatty acids were calculated, and it was found that a length of 20.7-22.5A and width of 4.7-7.2A in the fatty acid molecular structure was suggested to be important for IMPDH inhibition. Docking simulation of C20-fatty acids and mouse IMPDH type II, which was homology modeled from human IMPDH type II (PDB code: 1NF7), was performed, and the fatty acid could bind to Cys331, which is a amino acid residue of the active site, competitively with IMP. Based on these results, the IMPDH-inhibitory mechanism of fatty acids is discussed.     

Inhibition of brain prostaglandin D synthetase and prostaglandin D2 dehydrogenase by some saturated and unsaturated fatty acids

The activities of rat brain prostaglandin D synthetase and swine brain prostaglandin D2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC50 values of this acid were 80 microM for prostaglandin D synthetase and 7 microM for prostaglandin D2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC50 values of these derivatives were more than 200 microM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 microM for prostaglandin D synthetase, and 20, 10, 8.5 and 7 microM for prostaglandin D2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC50 values of 40 microM for prostaglandin D synthetase and 3.9 microM for prostaglandin D2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 microM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 microM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 microM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 microM).     

The effect of conjugated linoleic acid on the viability and metabolism of human osteoblast-like cells

Studies in experimental animals and murine osteoblast cells in culture have produced conflicting findings on the effect of conjugated linoleic acid (CLA) on bone formation. The present study investigated the influence of CLA on viability and metabolism of two human osteoblast-like cell lines (SaOS2 and MG63). Both cell lines were exposed to increasing concentrations (0-50 microM) of CLA either as pure cis (c) 9: trans (t) 11 and t10:c12 CLA isomers or a blend of isomers, or linoleic acid (C18:2). Cell cytotoxicity and degree of DNA fragmentation were unaffected by any fatty acid treatment. PGE2 biosynthesis by both cell lines was variably reduced by CLA isomer blend and t10:c12 CLA, but not c9:t11 CLA. Alkaline phosphatase activity was variably increased by all CLA treatments. These results suggest a lack of cytotoxic effect of CLA on human osteoblast-like cells and tentatively suggest a possible beneficial effect on bone formation in humans.     

Differential regulation of calmodulin-dependent and -independent cyclic AMP phosphodiesterases from oviduct by fatty acids.

Regulation of calmodulin-independent and -dependent cAMP phosphodiesterases from quail oviduct by various fatty acids was studied. The calmodulin-independent form was slightly activated by low concentrations (20 microM) of oleic, linoleic and arachidonic acid, higher concentrations were inhibitory. The basal activity of the calmodulin-dependent form was activated by linoleic acid and to a lesser extent by arachidonic acid at low concentrations and inhibited by higher concentrations of the two fatty acids. In contrast, arachidonic acid was a potent reversible inhibitor of calmodulin in the activation of this enzyme (IC50: 20 microM) whereas linoleic acid was inactive from 10 to 150 microM. The present results strongly suggest that the differential regulation of cAMP phosphodiesterases by these fatty acids could profoundly influence the level of cAMP in the oviduct and thus its subsequent effects.     

An improved HPLC assay for phosphatidylcholine hydroperoxides (PCOOH) in human plasma with synthetic PCOOH as internal standard

Quantitative and qualitative analyses of 1-palmitoyl-2-linoleoyl-phosphatidylcholine monohydroperoxide [PC 16:0/18:2-OOH] and 1-stearoyl-2-linoleoyl-phosphatidylcholine monohydroperoxide [PC 18:0/18:2-OOH] in human plasma were improved by chemiluminescence HPLC using synthetic 1-stearoyl-2-erucoyl-phosphatidylcholine monohydroperoxide (PC 18:0/22:1-OOH) as internal standard. The calibration curves of synthetic PC 16:0/18:2-OOH and PC 18:0/18:2-OOH, obtained by their direct injections with the IS into the HPLC system, were linear throughout the calibration range (10-1000 pmol). Within-day and between-day coefficients of variation were below 8%, and the recoveries were between 84% and 101%. Plasma concentrations of PC 16:0/18:2-OOH and PC 18:0/18:2-OOH were 102+/-59 nM (mean+/-SD) and 36+/-20 nM, respectively, in the 33 healthy volunteers. The present method might help understanding incompletely understood pathway of plasma phosphatidylcholine hydroperoxides.     

Synergistic actions of tetradecylthioacetic acid (TTA) and dexamethasone on induction of the peroxisomal beta-oxidation and on growth inhibition of Morris hepatoma cells. Both effects are counteracted by insulin

(1) The relation between the effects of the sulfur-substituted fatty acid analogue, tetradecylthioacetic acid (TTA), dexamethasone and insulin on enzyme induction and growth rate was studied in Morris hepatoma 7800 C1 cells in culture. (2) The activities of the cynanide-insensitive palmitoyl-CoA oxidase and palmitoyl-CoA hydrolase were induced about 2-fold by 50 microM TTA after 72 h of treatment. Catalase was less induced and NADPH-cytochrome-c2 reductase, glucose-6-phosphate dehydrogenase and lactate dehydrogenase were unaffected by the fatty acid analogue. (3) Dexamethasone (250 nM) induced the same enzymes as did TTA, but was a less efficient than 50 microM TTA. However, in combination their effects were more than additive, resulting in 4-7-fold increases. (4) Insulin (400 nM) counteracted the inductive effects of both TTA and dexamethasone on all enzymes except for lactate dehydrogenase, which was induced by the combination of all three compounds. (5) TTA inhibited the growth rate of the cells, and this effect was potentiated by dexamethasone and counteracted by insulin. (6) The enzyme inductions were similar in exponential and plateau phases of growth, indicating that these processes were independently affected by the three compounds.     

The growth inhibitory effect of conjugated linoleic acid on a human hepatoma cell line, HepG2, is induced by a change in fatty acid metabolism, but not the facilitation of lipid peroxidation in the cells

We investigated the growth inhibitory effect of conjugated linoleic acid (CLA) on HepG2 (human hepatoma cell line), exploring whether the inhibitory action occurs via lipid peroxidation in the cells. When the cells were incubated up to 72 h with 5-40 microM of CLA (a mixture of 9c,11t-18:2 and 10t,12c-18:2), cell proliferation was clearly inhibited in a dose and time dependent manner but such an inhibition was not confirmed with linoleic acid (LA). In order to evaluate the possible contribution of lipid peroxidation exerted by CLA to cell growth inhibition, alpha-tocopherol (5-20 microM) and BHT (1-10 microM) as potent antioxidants were added to the medium with CLA (20 microM), which did not restore cell growth at all. Furthermore, after 72 h incubation, the membranous phospholipid hydroperoxide formation in the CLA-supplemented cells was suppressed respectively to 25% and 50% of that in LA-supplemented cells and control cells. No difference was observed by a conventional lipid peroxide assay, the TBA test, between CLA-supplemented cells and LA-supplemented cells. Although the cellular lipid peroxidation was not stimulated, lipid contents (triacylglycerol, total cholesterol and free cholesterol) and fatty acid contents (palmitic acid, palmitoleic acid and stearic acid) markedly increased in CLA-supplemented cells compared with LA-supplemented and control cells. Moreover, supplementation with 20 microM LA and 20 microM arachidonic acid profoundly interfered with the inhibitory effect of CLA in HepG2. These results suggest that the growth inhibitory effect of CLA on HepG2 is due to changes in fatty acid metabolism but not to lipid peroxidation.     

Modulation of DMBA-induced biochemical changes by organoselenium compounds in blood of rats

The protective role of two synthetic organoselenium compounds 1-isopropyl-3-methylbenzimidazole-2-selenone (SeI) and 1, 3-di-p-methoxybenzylpyrimidine-2-selenone (Sell) was examined against the 7,12-dimethylbenz[a]anthracene (DMBA)-induced changes in biochemical parameters in blood of rats. Albino Winstar rats (150-200 g body wt) were treated with single dose of DMBA (50 mg/kg body wt) and organoselenium compounds (25 micromol/kg) for 4 weeks at two days internal. Blood was taken from the anaesthetized rats ventricle from their hearts for biochemical analysis. Administration of DMBA resulted in elevation of urea, uric acid and creatinine levels as well as AST, ALT and LDH activities and decrease in levels of total proteins, albumin and globulin. SeI and SeII caused a significant (p<0.05) decrease in urea, uric acid and creatinine levels and alanine aminotransferase (ALT); aspartate aminotransferase; (AST) and lactate dehydrogenase (LDH) activities and significantly increased the levels of total protein and albumin (p<0.05). These organoselenium compounds are likely to be beneficial in human health.     

Inactivation of soluble 17 beta-hydroxysteroid dehydrogenase of human placenta by fatty acids

The sensitivity of soluble, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) of human placenta to inactivation by fatty acids was examined. Exposure to the unsaturated fatty acids oleic, arachidonic, linoleic and linolenic acid resulted in the loss of activity. Methyl and ethyl esters of oleic acid, the saturated fatty acid, stearic acid and prostaglandins E2 and F2 alpha were without effect. Inactivation by oleic acid required the fatty acid at levels above its critical micelle concentration, 50 microM, as estimated by light-scattering. Steroid substrates and inhibitors did not protect against inactivation. NAD+, NADH, NADP+ and NADPH did protect. The concentrations of NADP+, 50 microM, and NAD, 1.5 mM, necessary for complete protection were significantly greater than their respective Michaelis constants, 0.16 microM and 15.2 microM. The data suggest that soluble 17 beta-HSD can bind to fatty acid micelles and that the binding site(s) on the enzyme are at or near pyridine nucleotide binding sites.     

Application of a continuous bioreactor cascade to study the effect of linoleic acid on hybridoma cell physiology

The aim of the present study is to demonstrate the use of controlled bioreactors for toxicological studies. As a model system the effect of linoleic acid on hybridoma cells is studied in two well-controlled continuously operated bioreactors placed in series. In the first reactor the effect on rapid proliferating cells can be studied, while in the second reactor a special steady state is created, which allows studying the effect on apoptotic cells. Experiments are done at 0, 25, and 50 microM linoleic acid. At the end of the experiment with 50 microM linoleic acid, the concentration of linoleic acid is increased stepwise to determine the cytotoxic level. For rapid proliferating cells exposed to 25 and 50 microM stimulation of growth was observed. At 50 microM there was at the same time an increase in cell death through apoptosis. For stressed apoptotic cells linoleic acid caused partial growth inhibition at 25 and 50 microM and arrest of cell proliferation in the G(2)/M phase at 50 microM. For both, rapid proliferating cells and stressed apoptotic cells, complete growth inhibition occurred at 85 microM, with cells being arrested in the G(2)/M phase and dying mainly through necrosis. Cells in the bioreactor system appeared to be more sensitive towards linoleic acid than cells grown in multi-well plates. (IC(50) = 300 microM; IC(100) = 400 microM). Altogether the results of the present study reveal that the biostat experiments allow detailed analysis of the effect of a bioactive ingredient on cell physiology and behavior.     

Plasmodium falciparum histidine-rich protein-2 (PfHRP2) modulates the redox activity of ferri-protoporphyrin IX (FePPIX): peroxidase-like activity of the PfHRP2-FePPIX complex

Histidine-rich protein-2 from Plasmodium falciparum (PfHRP2) binds up to 50 molecules of ferri-protoporphyrin IX (FePPIX) (Choi, C. Y., Cerda, J. F., Chu, H. A., Babcock, G. T., and Marletta, M. A. (1999) Biochemistry 38, 16916-16924). We reasoned that the PfHRP2-FePPIX complex has antioxidant properties that could be beneficial to the parasite. Therefore, we examined whether binding to PfHRP2 modulated the redox properties of FePPIX. We observed that PfHRP2 completely inhibited the auto-oxidation of ascorbate mediated by free FePPIX. We also investigated the peroxidase activity of PfHRP2-FePPIX using 13-hydroperoxy-9,11-octadienoate (18:2-OOH) as substrate. Reaction of PfHRP2-FePPIX with 18:2-OOH in the presence of added reducing agents gave 13-hydroxy-9,11-octadienoate (18:2-OH) as a major product and 13-keto-9,11-octadienoate (18:2=O) and 9,12,13-trihydroxy-10-octadecaenoate as minor products. Binding of FePPIX to PfHRP2 lowered the rate of decomposition of 18:2-OOH and increased the 18:2-OH to 18:2=O ratio. Similar to other authentic peroxidases, phenols, amines, and biological reductants like ascorbate promoted 18:2-OH production, and NaCN inhibited 18:2-OH production. Thioanisole also acted as a reductant and was converted to thioanisole sulfoxide, suggesting formation of compound I during the reaction. These data show that PfHRP2 modulates the redox activity of FePPIX and that the PfHRP2-FePPIX complex may have previously unrecognized antioxidant properties.