Lycopene in association with alpha-tocopherol or tomato lipophilic extracts enhances acyl-platelet-activating factor biosynthesis in endothelial cells during oxidative stress

Lipophilic compounds contained in tomato can prevent cardiovascular diseases by modulating the atherogenic processes in vascular endothelium mediated by oxidized low-density lipoproteins (LDLs). We investigated the effects of lycopene on the metabolism of platelet-activating factor (PAF) and its much less biologically active acyl analog, acyl-PAF, known to prevent LDL oxidation. Lycopene, or lycopene in association with alpha-tocopherol, or whole tomato lipophilic extracts (containing more than 80% lycopene) were used in experiments in which endothelial cells (ECs) are known to synthesize PAF following H(2)O(2)-induced oxidative stress. The results indicated that in each case H(2)O(2)-stimulated PAF biosynthesis in ECs, which is catalyzed by acetyl-CoA acetyltransferase (AT), appeared strongly inhibited. However, acyl-PAF biosynthesis, which also occurs through the PAF-dependent transacetylase (TA), was significantly increased by lycopene only when it was in association with alpha-tocopherol or with the minor compounds present in the whole lipophilic tomato extract. These findings suggest that alpha-tocopherol or lipophilic compounds present in tomato juice potentiate the effects of lycopene on the modulation of PAF and acyl-PAF biosynthesis in ECs during oxidative stress.     

Regulation of the biosynthesis of acyl analogs of platelet-activating factor by purinergic agonist in endothlial cells

We have previously shown that platelet-activating factor (PAF)-dependent transacetylase (TA) contains three catalytic activities, namely PAF: lysophospholipid TA (TAL), PAF: sphingosine TA (TAs) and PAF acetylhydrolase. It serves as a modifier of PAF actions by producing different lipid signal molecules. The TAL activity is involved in the biosynthesis of acyl analogs of PAF (acyl-PAF, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine, acylacetyl-GPC) in agonist-stimulated endothelial cells. In the present investigation, we have studied the mechanism(s) by which the TA activity is regulated in ATP-treated endothelial cells. We have demonstrated that ATP, and thiol-modifying agents with ATP, specifically regulate only the TAL part of the TA activities.     

Acyl and plasmalogen analogs of platelet activating factor--new lipid cellular bioregulators]

Recent data concerning two structural platelet-activating factor (PAF) analogs-1-O-acyl-2-acetyl-sn-glycero-3-phosphocholine (acyl-PAF) and 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine (vinyl-PAF) identified in some cells and tissues are reviewed. Isolation, identification, biosynthesis, and metabolism of acyl-PAF and vinyl-PAF are considered. The activity of acyl-PAF and vinyl-PAF towards platelets, leukocytes, isolated myocardium, and ileum as well as its in vivo activity are discussed. The influence of acyl-PAF and vinyl-PAF on PAF platelet interaction, Ca2+ mobilization, and platelet adenylate cyclase activity is considered. It is concluded that similar to PAF, acyl-PAF and vinyl-PAF should be regarded as a family of PAF lipid bioregulators.     

Determination of 1-O-acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine,platelet-activating factor and related phospholipids in biological samples by high-performance liquid chromatography-tandem mass spectrometry

Combining normal-phase HPLC separation and tandem mass spectrometric detection, using an ion-spray HPLC-MS interface, a quantitative method for acyl-platelet activating factor (acyl-PAF), platelet-activating factor (PAF) and related phospholipids was developed. Mass spectra, positive ions, showed intense [M+H]⁺ ions; collision-induced dissociation of protonated molecular ions gave characteristic daughter ions corresponding to the polar head. Detection limits of 0.1–0.3 ng injected were obtained by multiple reaction monitoring. Samples of human endothelial cells treated with compounds modulating the levels of acyl-PAF and PAF have been analyzed by the present technique, proving that this approach is suitable for biochemical studies.     

Differential responsiveness of human neutrophils to the autocrine actions of 1-O-alkyl-homologs and 1-acyl analogs of platelet-activating factor.

The phlogistic actions of six molecular species of platelet-activating factor (PAF) (1-O-alkyl-PAF homologs, 16:0-, 18:0- and 18:1-alkyl-PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and their respective 1-acyl-PAF analog counterparts, 16:0-, 18:0- and 18:1-acyl-PAF, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (AGPC)) were assessed relative to five human neutrophilic polymorphonuclear leukocyte (PMN) functional responses: 1) lysosomal enzyme secretion; 2) specific desensitization to 16:0-AGEPC-induced lysosomal enzyme secretion; 3) O2- production; 4) chemotaxis; and 5) priming for enhanced O2- production. With respect to inducing lysozyme secretion, 18:0-AGEPC was 30- and 75-fold less potent than 16:0-AGEPC and 18:1-AGEPC, respectively, and was 25- and 40-fold less potent for inducing beta-glucuronidase secretion. 18:0-AGEPC was also 10-fold less active than 18:1- and 16:0-AGEPC for inducing O2- production. Thus, the rank order of potency of the alkyl-PAF homologs for inducing both lysosomal enzyme secretion and O2- production was 18:1- greater than or equal to 16:0- much greater than 18:0-AGEPC. In contrast, these three alkyl-PAF homologs had the same potency for desensitizing PMN to subsequent 16:0-AGEPC-induced lysosomal enzyme secretion and for priming PMN for augmented O2- production in response to FMLP or human recombinant C5a. Paradoxically, however, the rank order of potency of the alkyl-PAF homologs for effecting PMN chemotaxis was 18:0- greater than 18:1- much greater than 16:0-AGEPC. At concentrations as high as 1.0 microM, the acyl-PAF analogs did not initiate PMN lysosomal enzyme secretion, O2- production, or chemotaxis. However, the acyl-PAF analogs induced partial PMN desensitization to 16:0-AGEPC. A novel finding of potential (patho)-physiologic significance was the ability of acyl-PAF at nM concentrations to prime PMN for significantly enhanced O2- production after stimulation with FMLP or human recombinant C5a. The priming action of acyl-PAF was due to an increase in the rate as opposed to a prolongation of O2- production. The differing rank orders of potency of the alkyl-PAF homologs and acyl-PAF analogs for stimulating several physiologic responses of the same target cell, the human PMN, support the premise that there may be more than one PAF receptor subtype on the PMN and/or that differences in the biophysical properties of the various molecular species of PAF modulate their interaction with PAF receptor(s) linked to stimulus-response coupling.     

Polyphenol tannic acid inhibits hydroxyl radical formation from Fenton reaction by complexing ferrous ions

Tannic acid (TA), a plant polyphenol, has been described as having antimutagenic, anticarcinogenic and antioxidant activities. Since it is a potent chelator of iron ions, we decided to examine if the antioxidant activity of TA is related to its ability to chelate iron ions. The degradation of 2-deoxyribose induced by 6 microM Fe(II) plus 100 microM H2O2 was inhibited by TA, with an I50 value of 13 microM. Tannic acid was over three orders of magnitude more efficient in protecting against 2-deoxyribose degradation than classical *OH scavengers. The antioxidant potency of TA was inversely proportional to Fe(II) concentration, demonstrating a competition between H2O2 and AT for reaction with Fe(II). On the other hand, the efficiency of TA was nearly unchanged with increasing concentrations of the *OH detector molecule, 2-deoxyribose. These results indicate that the antioxidant activity of TA is mainly due to iron chelation rather than *OH scavenging. TA also inhibited 2-deoxyribose degradation mediated by Fe(III)-EDTA (iron = 50 microM) plus ascorbate. The protective action of TA was significantly higher with 50 microM EDTA than with 500 microM EDTA, suggesting that TA removes Fe(III) from EDTA and forms a complex with iron that cannot induce *OH formation. We also provided evidence that TA forms a stable complex with Fe(II), since excess ferrozine (14 mM) recovered 95-96% of the Fe(II) from 10 microM TA even after a 30-min exposure to 100-500 microM H2O2. Addition of Fe(III) to samples containing TA caused the formation of Fe(II)n-TA, complexes, as determined by ferrozine assays, indicating that TA is also capable of reducing Fe(III) ions. We propose that when Fe(II) is complexed to TA, it is unable to participate in Fenton reactions and mediate *OH formation. The antimutagenic and anticarcinogenic activity of TA, described elsewhere, may be explained (at least in part) by its capacity to prevent Fenton reactions.     

Human endothelial cells are target for platelet-activating factor. II. Platelet-activating factor induces platelet-activating factor synthesis in human umbilical vein endothelial cells.

Platelet-activating factor (PAF), a phospholipid mediator with broad and potent biologic activities, is synthesized by several inflammatory cells including endothelial cells (EC). PAF is also an effective stimulating agent for EC leading to increased cell permeability and adhesivity. We examined the synthesis of PAF in human umbilical cord vein EC after stimulation of EC with PAF or with its nonmetabolizable analog 1-O-alkyl-2-N-methyl-carbamyl-sn-glycero-3-phosphocholine (C-PAF). PAF (1 to 100 nM) induced a dose- and time-dependent increase of PAF synthesis as detected by [3H]acetate incorporation into PAF fraction. Stimulation of PAF synthesis occurred via activation of the "remodeling pathway" as the 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase was dose-dependently increased after PAF treatment. The de novo pathway of PAF synthesis was not activated under these conditions. C-PAF was able to mimic the effect of authentic PAF on [3H] acetate incorporation. The inactive metabolite lyso-PAF (100 nM) had no influence on PAF synthesis in EC. CV-3988, BN 52021, and WEB 2086, potent and specific antagonists of PAF suppressed PAF effects on the remodeling pathway completely. The PAF- and C-PAF-induced [3H]PAF remained 93% cell-associated and was not degraded up to 10 min after stimulation. Characterization of the [3H]acetate-labeled material co-migrating with authentic PAF revealed that a significant proportion (approximately 57%) was actually 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. PAF-induced PAF synthesis might be an important mechanism for amplifying original PAF signals and potentiating adhesive interactions of circulating cells with the endothelium.     

Protein kinase C and cyclic AMP modulate thrombin-induced platelet-activating factor synthesis in human endothelial cells.

Stimulation of human endothelial cells (EC) by thrombin elicits a rapid increase of intracellular free Ca2+ [(Ca2+]i), platelet-activating factor (PAF) production and 1-O-alkyl-2-lyso-sn-glycero-3- phosphocholine (lyso-PAF): acetyl-CoA acetyltransferase (EC 2.3.1.67) activity. The treatment of EC with thrombin leads to a 90% decrease in the cytosolic protein kinase C (PKC) activity; this dramatic decline is accompanied by an increase of the enzymatic activity in the particulate fraction. The role of PKC in thrombin-mediated PAF synthesis has been assessed: (1) by the blockade of PKC activity with partially selective inhibitors (palmitoyl-carnitine, sphingosine and H-7); (2) by chronic exposure of EC to phorbol 12-myristate 13-acetate (PMA), which results in down-regulation of PKC. In both cases, a strong inhibition of thrombin-induced PAF production is observed, suggesting obligatory requirement of PKC activity for PAF synthesis. It is suggested that PKC regulates EC phospholipase A2 (PLA2) activity as thrombin-induced arachidonic acid (AA) release is 90% inhibited in PKC-depleted cells. Brief exposure of EC to PMA strongly inhibits thrombin-induced [Ca2+]i rise, acetyltransferase activation and PAF production, suggesting that, in addition to the positive forward action, PKC provides a negative feedback control over membrane signalling pathways involved in the thrombin effect on EC. Forskolin and iloprost, two agents that increase the level of cellular cAMP in EC, are very effective in inhibiting thrombin-evoked cytosolic Ca2+ rise, acetyltransferase activation and PAF production; this suggests that endogenously generated prostacyclin (PGI2) may modulate the synthesis of PAF in human endothelial cells.     

Stimulation of the de novo pathway for the biosynthesis of platelet-activating factor (PAF) via cytidylyltransferase activation in cells with minimal endogenous PAF production

Treatment of Ehrlich ascites cells with 2 mM oleic acid causes a greater than 10-fold increase in the formation of platelet-activating factor (PAF; 1-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine) from the de novo precursor of PAF, 1-[3H]alkyl-2-acetyl-sn-glycerol. Under these conditions, CTP:phosphocholine cytidylyltransferase activity, which is known to catalyze the rate-limiting step in phosphatidylcholine biosynthesis, was stimulated 32% (p less than 0.001) over control cells. Surprisingly, the dithiothreitol-insensitive choline-phosphotransferase activity, which catalyzes the final step in PAF biosynthesis, was reduced approximately 95% in membranes isolated from cells that were pre-treated with 2 mM oleic acid. However, calculations of product formation at this reduced cholinephosphotransferase activity revealed that it was still sufficient to accommodate the increased synthesis of PAF observed in the intact oleic acid-treated cells. Kinetic studies and experiments done with cells treated with phenylmethylsulfonyl fluoride (an acetylhydrolase inhibitor) indicate the various metabolic products formed are derived through the following sequence of reactions: 1-alkyl-2-acetyl-sn-glycerol----1-alkyl-2-acetyl-sn-glycero-3- phosphocholine----1-alkyl-2-lyso-sn-glycero-3-phosphocholine----1-alkyl- 2(long-chain) acyl-sn-glycero-3-phosphocholine. These results indicate PAF is the source of alkylacylglycerophosphocholine through the action of an acetylhydrolase and a transacylase as shown in other cell systems. The relative amounts of PAF, lyso-PAF, and alkylacylglycerophosphocholine produced after treatment of the cells with oleic acid in the absence of the phenylmethylsulfonyl fluoride inhibitor indicate that the acylation rate for lyso-PAF is considerably slower (i.e. rate-limiting) than the deacetylation of PAF by acetylhydrolase. We further conclude that the final step in the de novo pathway for PAF biosynthesis is under the direct control of CTP:phosphocholine cytidylyltransferase, which emphasizes the importance of this regulatory (rate-limiting) step in the biosynthesis of both phosphatidylcholine and PAF.     

Flavonoid biosynthesis in barley primary leaves requires the presence of the vacuole and controls the activity of vacuolar flavonoid transport

Barley (Hordeum vulgare) primary leaves synthesize saponarin, a 2-fold glucosylated flavone (apigenin 6-C-glucosyl-7-O-glucoside), which is efficiently accumulated in vacuoles via a transport mechanism driven by the proton gradient. Vacuoles isolated from mesophyll protoplasts of the plant line anthocyanin-less310 (ant310), which contains a mutation in the chalcone isomerase (CHI) gene that largely inhibits flavonoid biosynthesis, exhibit strongly reduced transport activity for saponarin and its precursor isovitexin (apigenin 6-C-glucoside). Incubation of ant310 primary leaf segments or isolated mesophyll protoplasts with naringenin, the product of the CHI reaction, restores saponarin biosynthesis almost completely, up to levels of the wild-type Ca33787. During reconstitution, saponarin accumulates to more than 90% in the vacuole. The capacity to synthesize saponarin from naringenin is strongly reduced in ant310 miniprotoplasts containing no central vacuole. Leaf segments and protoplasts from ant310 treated with naringenin showed strong reactivation of saponarin or isovitexin uptake by vacuoles, while the activity of the UDP-glucose:isovitexin 7-O-glucosyltransferase was not changed by this treatment. Our results demonstrate that efficient vacuolar flavonoid transport is linked to intact flavonoid biosynthesis in barley. Intact flavonoid biosynthesis exerts control over the activity of the vacuolar flavonoid/H(+)-antiporter. Thus, the barley ant310 mutant represents a novel model system to study the interplay between flavonoid biosynthesis and the vacuolar storage mechanism.     

Effect of short-term vs. long-term elevation of dietary protein intake on responsiveness of rat thick ascending limbs to peptide hormones

We compared the renal responses of rats on three diet regimens. Rats received either 8% protein food (low-protein, LP) for 10 weeks following weaning, 8% protein for 9 weeks followed by 1 week on 30% protein (short-term high-protein, SHP), or 30% protein for 10 weeks (high-protein, HP). Kidneys from HP rats were enlarged by approximately 50%, or 20% when corrected for body mass. Most of this hypertrophy resulted from enlargement of the inner stripe of the outer medulla, site of the thick ascending limbs (TAL), and TAL from HP rats were larger in diameter. SHP rats had TAL diameters similar to HP rats, but changes in renal mass or height of renal zones did not reach statistical significance. The activity of adenylyl cyclase (AC) in TAL, measured from the accumulation of cAMP in isolated tubules, increased with dose of both arginine vasopressin (AVP) and glucagon in all rats. However, HP rats had significantly higher hormone-induced AC activity than LP or SHP rats, which were not different from each other. Our results suggest that tubule hypertrophy may precede up-regulation of hormone-sensitive AC activity during the progression of renal response to elevated dietary protein.     

5-hydroxytryptamine, glucose uptake, glycogen utilization and carbon dioxide fixation in Hymenolepis microstoma (Cestoda)

1. In vitro glucose uptake and glycogen utilization by Hymenolepis microstoma decreased under high oxygen concentrations. 2. 5-Hydroxytryptamine did not stimulate in vitro glucose uptake but did increase glycogen utilizations by H. microstoma. 3. The reduced glucose uptake under high oxygen concentrations (21 and 95%) resulted in a reduction in excretory products. 4. 14CO2-incorporation studies confirmed that, under both 95% O2:5% CO2 and air-minus-CO2 (identical to 21% O2). CO2-fixation by phosphoenolpyruvate carboxykinase (EC 4.1.1.32) was inhibited. 5. The specific activity of hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) was not stimulated by 5-HT. 6. The concentration of ATP required for optimal stimulation of phosphofructokinase activity was 0.67 mM. Activity was further significantly increased by the addition of cAMP and even greater by AMP.     

Platelet-activating factor and prostaglandin E2 impair esophageal ACh release in experimental esophagitis

ACh is a neurotransmitter in cat esophageal circular muscle, as atropine nearly abolishes contraction of in vitro circular muscle strips in response to electric field stimulation (EFS) (5, 12). Experimental esophagitis reduced EFS- but not ACh-induced contraction of esophageal circular muscle, suggesting that esophagitis impairs neurotransmitter release. Because IL-1beta and IL-6 are produced in esophagitis and reproduce these changes in normal esophageal muscle (12), we examined the role of IL-1beta and IL-6 in this motor dysfunction. IL-1beta, IL-6 (12), H2O2, PGE2, and platelet-activating factor (PAF) were elevated in esophagitis specimens. Normal muscle incubated (2 h) in IL-1beta and IL-6 had increases in H2O2, PGE2, and PAF levels. H2O2 contributed to increased PGE2 and PAF, as the increase was partially (60-80%) reversed by the H2O2 scavenger catalase. EFS-induced [3H]ACh release from muscle strips significantly (42%) decreased in esophagitis and after 2 h incubation in PGE2 and in PAF C-16. Similarly, EFS-induced but not ACh-induced muscle contraction decreased in esophagitis and after incubation in PGE2 and PAF C-16. Finally, in normal muscle strips treated with IL-1beta electrical field stimulation (EFS)-induced contraction was partially restored by indomethacin or by the PAF antagonist CV3988 and was completely restored by the combination of CV3988 and indomethacin, whereas in strips treated with IL-6, EFS-induced contraction was partially restored by the PAF antagonist CV3988 and not affected by indomethacin. We conclude that IL-1beta-induced production of H2O2 causes formation of PGE2 and PAF that inhibit ACh release from esophageal cholinergic neurons without affecting ACh-induced contraction of esophageal circular muscle. IL-6 causes production of H2O2, PAF, and other unidentified inflammatory mediators.     

Novel flavonol 2-oxoglutarate dependent dioxygenase: affinity purification, characterization, and kinetic properties

A 2-oxoglutarate-dependent dioxygenase [EC 1.14.11-] that catalyzes the 6-hydroxylation of partially methylated flavonols has been purified to near homogeneity from Chrysosplenium americanum. Enzyme purification was achieved by fast protein liquid chromatography on Superose 12 and Mono Q columns as well as by affinity chromatography on 2-oxoglutarate-Sepharose and immunoaffinity columns. The specific activity of the 6-hydroxylase eluted from Mono Q (97.1 pkat/mg) was enriched 538-fold, with a 0.63% recovery. Both affinity chromatography steps resulted in the elimination of most contaminating proteins, but not without loss of enzyme activity and stability. The molecular mass of both the native and denatured enzyme was found to be 42 and 45 kDa, respectively, suggesting a monomeric protein. The enzyme exhibits strict specificity for position 6 of partially methylated flavonols possessing a 7-methoxyl group, indicating its involvement in the biosynthesis of polymethylated flavonols in this plant. The cofactor dependence of the enzyme is similar to that of other plant dioxygenases, particularly its dependence on ferrous ions for catalytic activity and reactivation. Internal amino acid sequence information indicated its relatedness to other plant flavonoid dioxygenases. The results of substrate interaction kinetics and product inhibition studies suggest an ordered, sequential reaction mechanism (TerTer), where 2-oxoglutarate is the first substrate to bind, followed by O2 and the flavonol substrate. Product release occurs in the reverse order where the hydroxylated flavonol is the first to be released, followed by CO2 and succinate. To our knowledge, this is the first reported 2-oxoglutarate-dependent dioxygenase that catalyzes the aromatic hydroxylation of a flavonoid compound.     

Effects of preparation method, age, and plating technique of thin agar layer media on recovery of Escherichia coli O157:H7 injured by sodium chloride

The thin agar layer (TAL) method was experimentally tested to determine its ability to recover Escherichia coli O157:H7 injured by sodium chloride (NaCl). Cells grown in Brain Heart Infusion broth with 0%, 5%, or 7.5% (w/v) NaCl were spread and spiral plated onto Tryptic Soy agar (TSA), MacConkey Sorbitol agar (MSA), and TSA/MSA TAL combinations. Generally, TSA recovered more injured cells than TAL (p < or =0.05), and TAL recovered more cells than MSA (p < or =0.05). Preparation mode (two vs. three layers) and age (0, 1, or 7 days) of TAL had negligible effect on resuscitation of injured cells (p > 0.05). TAL, which is conventionally used to recover heat, cold, and acid-injured foodborne pathogens, may be used to recover NaCl-injured E. coli O157:H7.     

The involvement of catalase in alcohol metabolism in Drosophila melanogaster larvae

The involvement of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) in the metabolism of alcohols was investigated by comparing Drosophila melanogaster larvae in which catalase was inhibited by dietary 3-amino-1,2,4-triazole (3AT) to larvae fed a diet without 3AT. 3AT inhibited up to 80% of the catalase activity with concordant small increases in the in vitro activities of sn-glycerol-3-phosphate dehydrogenase, fumarase, and malic enzyme, but with a 16% reduction in the in vivo incorporation of label from [14C]glucose into lipid. When the catalase activity was inhibited to different degrees in ADH-null larvae, there was a simple linear correlation between the catalase activity and flux from [14C]ethanol into lipid. By feeding alcohols simultaneously with 3AT, ethanol and methanol were shown to react efficiently with catalase in wild-type larvae at moderately low dietary concentrations. Drosophila catalase did not react with other longer chain alcohols. Catalase apparently represents a minor pathway for ethanol degradation in D. melanogaster larvae, but it may be an important route for methanol elimination from D. melanogaster larvae.     

Hydrogen peroxide detoxification in the midgut of the blood-sucking insect, Rhodnius prolixus

Here we investigated H2O2 production and detoxification in the hematophagous hemiptera, Rhodnius prolixus. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radical (O2-). This reaction produces hydrogen peroxide, which is scavenged by antioxidant enzymes such as catalase (CAT). SOD and CAT activities were found in all tissues studied, being highest in the midgut. CAT was dose-dependently inhibited in vivo by injections of 3-amino-1,2,4-triazole (AT). Insects treated with AT showed a twofold increase in H2O2 levels. Injection of DL-buthionine-[S, R]-sulfoximine (BSO), an inhibitor of glutathione synthesis, also resulted in a fourfold increase in H2O2, together with stimulation of CAT activity. Simultaneous administration of both AT and BSO had a synergistic effect on midgut H2O2 content. Taken all together, our results suggest that CAT and glutathione-dependent mechanisms cooperate to control H2O2 concentration in the midgut cell and prevent hydroxyl radical generation by Fenton reaction in this tissue.     

H(2)O(2) and PARS mediate lung P-selectin upregulation in acute pancreatitis

P-selectin and circulating xanthine oxidase are involved in the process of neutrophil infiltration into the lung associated with acute pancreatitis. This study investigated the mediators that trigger the upregulation of P-selectin in this process. Pancreatitis was induced in rats by intraductal administration of 5% sodium taurocholate. P-selectin expression was measured using radiolabeled antibodies. Neutrophil infiltration and PAF levels were also evaluated. The role of superoxide radical, H(2)O(2), or the enzyme poly (ADP-ribose) synthetase (PARS) on these processes was determined in groups of animals treated with the corresponding inhibitors. Pancreatitis was associated with an increase in P-selectin expression in the lung. Inhibition of PARS or H(2)O(2) abrogated P-selectin upregulation, PAF generation, and neutrophil recruitment. Superoxide dismutation prevented neutrophil recruitment and PAF generation, but had no effect on P-selectin expression. We conclude that during acute pancreatitis, upregulation of P-selectin in the pulmonary endothelium is triggered by H(2)O(2) and PARS activity.     

Transaldolase deficiency influences the pentose phosphate pathway, mitochondrial homoeostasis and apoptosis signal processing

TAL (transaldolase) was originally described in the yeast as an enzyme of the PPP (pentose phosphate pathway). However, certain organisms and mammalian tissues lack TAL, and the overall reason for its existence is unclear. Recently, deletion of Ser(171) (TALDeltaS171) was found in five patients causing inactivation, proteasome-mediated degradation and complete deficiency of TAL. In the present study, microarray and follow-up Western-blot, enzyme-activity and metabolic studies of TALDeltaS171 TD (TAL-deficient) lymphoblasts revealed co-ordinated changes in the expression of genes involved in the PPP, mitochondrial biogenesis, oxidative stress, and Ca(2+) fluxing. Sedoheptulose 7-phosphate was accumulated, whereas G6P (glucose 6-phosphate) was depleted, indicating a failure to recycle G6P for the oxidative branch of the PPP. Nucleotide analysis showed depletion of NADPH and NAD(+) and accumulation of ADP-ribose. TD cells have diminished Deltapsi(m) (mitochondrial transmembrane potential) and increased mitochondrial mass associated with increased production of nitric oxide and ATP. TAL deficiency resulted in enhanced spontaneous and H(2)O(2)-induced apoptosis. TD lymphoblasts showed increased expression of CD38, which hydrolyses NAD(+) into ADP-ribose, a trigger of Ca(2+) release from the endoplasmic reticulum that, in turn, facilitated CD20-induced apoptosis. By contrast, TD cells were resistant to CD95/Fas-induced apoptosis, owing to a dependence of caspase activity on redox-sensitive cysteine residues. Normalization of TAL activity by adeno-associated-virus-mediated gene transfer reversed the elevated CD38 expression, ATP and Ca(2+) levels, suppressed H(2)O(2)- and CD20-induced apoptosis and enhanced Fas-induced cell death. The present study identified the TAL deficiency as a modulator of mitochondrial homoeostasis, Ca(2+) fluxing and apoptosis.