Phenolic acids protect low density lipoproteins from peroxynitrite-mediated modification in vitro

The role of reactive nitrogen species, such as peroxynitrite, in atherogenesis and the protective effect of dietary phenolic compounds are not yet understood. In this study, we sought firstly to determine whether phenolic acids become nitrated by peroxynitrite and then whether phenolic acid nitration can prevent consumption of gamma-tocopherol and thus enhance the resistance of LDL to oxidation by peroxynitrite. Coumaric acid was found to be readily nitrated by peroxynitrite and it also demonstrated a protective effect on y-tocopherol. Of greater significance was its potent inhibition of lipid peroxidation which was equal to that of caffeic acid.     

Increased nitration and diminished activity of copper/zinc superoxide dismutase in placentas from diabetic rats

Abstract

Nitration-induced protein damage in the placenta leads to impaired blood flow and deficient feto–placental exchange in diabetic pregnancies. This work studied the effect of nitric oxide and peroxynitrite on Cu/Zn SOD activity in rat placentas and evaluated whether Cu/Zn SOD is nitrated in the placenta from diabetic rats at mid-gestation. Protein nitration was evaluated by EIA, Cu/Zn SOD activity by inhibition of the epinephrine auto-oxidation, Cu/Zn SOD expression by western blot and specific nitration by immunoprecipitation. This study found higher levels of protein nitration (p < 0.001), diminished Cu/Zn SOD activity and enhanced protein expression (p < 0.01) in placentas from diabetic rats. Placental Cu/Zn SOD activity was inhibited by peroxynitrite (p < 0.01). Besides, nitration of Cu/Zn SOD was elevated in placentas from diabetic rats (p < 0.01). These results show that rat Cu/Zn SOD can be nitrated, a modification that could lead to the depressed activity of this enzyme found in placentas from diabetic rats.     

Immunological detection of nitrosative stress-mediated modified Tamm–Horsfall glycoprotein (THP) in calcium oxalate stone formers

Abstract

The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in hyperoxaluric condition has been proved experimentally. This may result in the formation of the cytotoxic metabolite peroxynitrite, which is capable of causing lipid peroxidation and protein modification. The presence of nitrotyrosine in proteins has been associated with several pathological conditions. The present study investigated the presence of nitrotyrosine in the stone formers Tamm–Horsfall glycoprotein (THP). In vitro nitration of control THP was carried out using peroxynitrite. New Zealand white rabbits were immunized with peroxynitrated THP at 15-day intervals. Antisera collected following the third immunization were assayed for antibody titres using solid-phase ELISA. Antibodies were purified by affinity chromatography. The carbonyl content of control, stone formers and nitrated THP were determined. Western blotting was carried with control, stone formers and nitrated THPs. Immunodiffusion studies demonstrated cross-reaction with nitrated bovine serum albumin. Significant amounts (p<0.001) of carbonyl content were present in both stone formers and nitrated THPs. Western blot analysis confirmed the presence of nitrated amino acid 3-nitrotyrosine in stone formers, which could bring about structural and functional modifications of THP in hyperoxaluric patients. A cross-reaction with nitrated bovine serum albumin confirms that the raised antibody has certain paratopes similar to the epitope of nitrated protein molecules. Detection of 3-nitrotyrosine in stone formers THP indicates that it is one of the key factors influencing the conversion of THP to a structurally and immunologically altered form during calcium oxalate stone formation.     

Use of a novel double-sandwich enzyme-linked immunosorbent assay method for assaying chondroitin sulfate proteoglycans that bear 3-nitrotyrosine core protein modifications, a previously unrecognized proteoglycan modification in hydrocephalus

3-Nitrotyrosine is a useful marker for nitric oxide-mediated tissue injury. However, which proteins are preferred peroxynitrite modification targets is unclear. Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid of human neonates with hydrocephalus and may be a target for peroxynitrite modification. We examined (1). whether CSPG core protein can be modified by peroxynitrite in vitro; (2). to what degree in comparison to bovine serum albumin (BSA), the most commonly used nitrated protein standard; (3). whether nitrated CSPGs can be measured directly in biological samples; and (4). whether nitrated proteoglycan concentrations in cerebrospinal fluid correlate with disease. In vitro nitration of bovine aggrecan was performed by exposure to different peroxynitrite concentrations, and 3-nitrotyrosine products were measured. Bovine serum albumin (BSA) nitration was also performed in comparison. A larger percentage of tyrosine residues were nitrated in aggrecan than in BSA under all conditions tested. An enzyme-linked immunosorbent assay (ELISA) for 3-nitrotyrosine consistently overestimated aggrecan nitration when nitrated BSA was used as the standard. This is important as most current assays of nitration in biological samples use nitrated BSA as the standard. Therefore, if nitrated CPSGs were a substantial portion of the nitrated proteins in a sample, total nitrated protein content would be overestimated. Aggrecan retained its function of binding hyaluronic acid despite substantial nitration. A double-sandwich ELISA was developed for nitrated CSPGs in biological samples, using nitrated aggrecan as standard. [Nitrated CSPG] was found to be significantly elevated in preterm hydrocephalus cerebrospinal fluid (P<0.02), but correlated poorly with cerebrospinal fluid [nitric oxide] (P>0.069), suggesting that nitrated CSPG and NO levels may be independant markers of tissue injury. Peroxynitrite-mediated protein tyrosine nitration is a previously unrecognized modification of CSPGs, and may reflect level of brain injury in hydrocephalus.     

Oxidation of 2,2,7,8-tetramethyl-6-chromanol,the model compound of γ-tocopherol,by hypochlorous acid

Abstract

Due to its high concentration in soybean oil, γ-tocopherol is probably the most important form of vitamin E in Western diets, whereas, α-tocopherol is by far the most important form of vitamin E as a dietary supplement. Recent studies have shown that γ-tocopherol is an excellent scavenger of nitric oxide and possibly other dangerous electrophiles while α-tocopherol has little if any scavenger activity.¹ The ability of γ-tocopherol to act as a scavenger of electrophiles is due to an absence of substituents in the activated 5-position of the chromanol ring.     

Environmental stresses induce health-promoting phytochemicals in lettuce

Plants typically respond to environmental stresses by inducing antioxidants as a defense mechanism. As a number of these are also phytochemicals with health-promoting qualities in the human diet, we have used mild environmental stresses to enhance the phytochemical content of lettuce, a common leafy vegetable. Five-week-old lettuce (Lactuca sativa L.) plants grown in growth chambers were exposed to mild stresses such as heat shock (40 °C for 10 min), chilling (4 °C for 1 d) or high light intensity (800 μmol m^?2 s^?1 for 1 d). In response to these stresses, there was a two to threefold increase in the total phenolic content and a significant increase in the antioxidant capacity. The concentrations of two major phenolic compounds in lettuce, chicoric acid and chlorogenic acid, increased significantly in response to all the stresses. Quercetin-3-O-glucoside and luteolin-7-O-glucoside were not detected in the control plants, but showed marked accumulations following the stress treatments. The results suggest that certain phenolic compounds can be induced in lettuce by environmental stresses. Of all the stress treatments, high light produced the greatest accumulation of phenolic compounds, especially following the stress treatments during the recovery. In addition, key genes such as phenylalanine ammonia-lyase (PAL), l-galactose dehydrogenase (l-GalDH), and γ-tocopherol methyltransferase (γ-TMT) involved in the biosynthesis of phenolic compounds, ascorbic acid, and α-tocopherol, respectively, were rapidly activated by chilling stress while heat shock and high light did not appear to have an effect on the expression of PAL and γ-TMT. However, l-GalDH was consistently activated in response to all the stresses. The results also show that these mild environmental stresses had no adverse effects on the overall growth of lettuce, suggesting that it is possible to use mild environmental stresses to successfully improve the phytochemical content and hence the health-promoting quality of lettuce with little or no adverse effect on its growth or yield.     

The two faces of α- and γ-tocopherols: an in vitro and ex vivo investigation into VLDL, LDL and HDL oxidation

BackgroundVitamin E and its derivatives, namely, the tocopherols, are known antioxidants, and numerous clinical trials have investigated their role in preventing cardiovascular disease; however, evidence to date remains inconclusive. Much of the in vitro research has focused on tocopherol's effects during low-density lipoprotein (LDL) oxidation, with little attention being paid to very LDL (VLDL) and high-density lipoprotein (HDL). Also, it is now becoming apparent that γ-tocopherol may potentially be more beneficial in relation to cardiovascular health.ObjectivesDo α- and γ-tocopherols become incorporated into VLDL, LDL and HDL and influence their oxidation potential in an in vitro and ex vivo situation?DesignFollowing (i) an in vitro investigation, where plasma was preincubated with increasing concentrations of either α- or γ-tocopherol and (ii) an in vivo 4-week placebo-controlled intervention with α- or γ-tocopherol. Tocopherol incorporation into VLDL, LDL and HDL was measured via high-pressure liquid chromatography, followed by an assessment of their oxidation potential by monitoring conjugated diene formation.ResultsIn vitro: Both tocopherols became incorporated into VLDL, LDL and HDL, which protected VLDL and LDL against oxidation. However and surprisingly, the incorporation into HDL demonstrated pro-oxidant properties. Ex vivo: Both tocopherols were incorporated into all three lipoproteins, protecting VLDL and LDL against oxidation; however, they enhanced the oxidation of HDL.ConclusionsThese results suggest that α- and γ-tocopherols display conflicting oxidant activities dependent on the lipoprotein being oxidized. Their pro-oxidant activity toward HDL may go some way to explain why supplementation studies with vitamin E have not been able to display cardioprotective effects.     

Effects of α-tocopherol on the lipid peroxidation and fluidity of porcine intestinal brush-border membranes

The effect of α-tocopherol on the lipid fluidity of porcine intestinal brush-border membranes was studied using pyrene as a fluorescent probe. Addition of α-tocopherol to the medium decreased fluorescence intensity and lifetime, but increased the fluorescence polarization of pyrene-labeled membranes. β-, γ-, and δ-Tocopherols gave no appreciable effect on the fluorescence intensity and polarization of the complex. The apparent dissociation constant (3.1 ± 0.12 μM) of the interaction of α-tocopherol with the membranes, estimated from the change in the fluorescence intensity with varying concentrations of α-tocopherol, was in good agreement with the concentration required to cause the half-maximal inhibition of lipid peroxidation of the membranes performed by incubation with 100 μM ascorbic acid and 10 μM Fe²⁺. Decrease of the slope in the thermal Perrin plot of the polarization of pyrene-labeled membranes by α-tocopherol suggests that the movement of pyrene molecules in the membranes is restricted by binding of the tocopherol. This interpretation was confirmed by an increased harmonic mean of the rotational relaxation time of the dye molecules in the membranes from 10.9 ± 0.16 to 18.5 ± 0.51 μs after addition of 25 μM α-tocopherol to the medium. The perturbation of lipid phase in the membranes induced by α-tocopherol was also suggested from a decreased quenching rate constant of pyrene fluorescence in the membranes for Tl⁺. Based on these results, the effect of α-tocopherol on the lipid fluidity of the membranes is discussed.     

Dietary α- and γ-tocopherol supplementation attenuates lipopolysaccharide-induced oxidative stress and inflammatory-related responses in an obese mouse model of nonalcoholic steatohepatitis

Oxidative stress contributes towards the development of nonalcoholic steatohepatitis (NASH). Thus, antioxidants may decrease oxidative stress and ameliorate the events contributing to NASH. We hypothesized that α- or γ-tocopherol would protect against lipopolysaccharide (LPS)-triggered NASH in an obese (ob/ob) mouse model. Five-week-old obese mice (n=18/dietary treatment) were provided 15 mg/kg each of α- and γ-tocopherol or 500 mg/kg of α- or γ-tocopherol for 5-weeks. Then, all mice were injected ip once with LPS (250 μg/kg) before being sacrificed at 0, 1.5 or 6 h. Body weight and hepatic steatosis were unaffected by tocopherols and LPS. Hepatic α- and γ-tocopherol increased (P<.05) ~9.8- and 10-fold in respective tocopherol supplemented mice and decreased in response to LPS. LPS increased serum alanine aminotransferase (ALT) by 86% at 6 h and each tocopherol decreased this response by 29–31%. By 6 h, LPS increased hepatic malondialdehyde (MDA) and tumor necrosis factor-α by 81% and 44%, respectively, which were decreased by α- or γ-tocopherol. Serum ALT was correlated (P<.05) to hepatic tumor necrosis factor-α (r=0.585) and MDA (r=0.592), suggesting that inflammation and lipid peroxidation contributed to LPS-triggered hepatic injury. α- and γ-Tocopherol similarly attenuated LPS-triggered increases in serum free fatty acid, and α-tocopherol only maintained the LPS-triggered serum triacylglycerol responses at 6 h. These findings indicate that increasing hepatic α- or γ-tocopherol protected against LPS-induced NASH by decreasing liver damage, lipid peroxidation, and inflammation without affecting body mass or hepatic steatosis. Further study is needed to define the mechanisms by which these tocopherols protected against LPS-triggered NASH.     

Characterization of nitrated phenolic compounds for their anti-oxidant, pro-oxidant, and nitration activities

Coffee is one of the most widely consumed beverages worldwide. Evidence of the health benefits and the important contribution of coffee brew to the intake of anti-oxidants in the diet has increased coffee consumption. Chlorogenic acid (ChA) and caffeic acid (CaA) are the major phenolic compounds in coffee. However, phenolic compounds, which are generally effective anti-oxidants, can become pro-oxidants in the presence of Cu²⁺ to induce DNA damage under certain conditions. On the other hand, sodium nitrite (NaNO₂) is widely used as a food additive to preserve and tinge color on cured meat and fish. It is possible that phenolic compounds react with NaNO₂ under acidic conditions, such as gastric juice. In this study, we identified compounds produced by the reaction between ChA or CaA in coffee and NaNO₂ in artificial gastric juice. The identified phenolic compounds and nitrated phenolic compounds were assessed for their anti-oxidant, pro-oxidant, and nitration activities by performing an in vitro assay. The nitrated phenolic compounds seemed to show increased anti-oxidant activity and decreased pro-oxidant activity. However, one nitrated CaA compound that has a furoxan ring showed the ability to release in the neutral condition.     

Crystal structure of nitrated human manganese superoxide dismutase: mechanism of inactivation

A cellular consequence of the reaction of superoxide and nitric oxide is enhanced peroxynitrite levels. Reaction of peroxynitrite with manganese superoxide dismutase (MnSOD) causes nitration of the active-site residue Tyr34 and nearly complete inhibition of catalysis. We report the crystal structures at 2.4 A resolution of human MnSOD nitrated by peroxynitrite and the unmodified MnSOD. A comparison of these structures showed no significant conformational changes of active-site residues or solvent displacement. The side chain of 3-nitrotyrosine 34 had a single conformation that extended toward the manganese with O1 of the nitro group within hydrogen-bonding distance (3.1 A) of Nepsilon2 of the second-shell ligand Gln143. Also, nitration of Tyr34 caused a weakening, as evidenced by the lengthening, of a hydrogen bond between its phenolic OH and Gln143, part of an extensive hydrogen-bond network in the active site. Inhibition of catalysis can be attributed to a steric effect of 3-nitrotyrosine 34 that impedes substrate access and binding, and alteration of the hydrogen-bond network that supports proton transfer in catalysis. It is also possible that an electrostatic effect of the nitro group has altered the finely tuned redox potential necessary for efficient catalysis, although the redox potential of nitrated MnSOD has not been measured.     

Effects of dietary incorporation of different antioxidant extracts and free-range rearing on fatty acid composition and lipid oxidation of Iberian pig meat

This investigation was designed to evaluate the effects of feeding either free range or in confinement using concentrated diets with the same ingredients and oil source (5.5% of olive oleins) but with different antioxidant supplementation [control diet with a basal level of α-tocopheryl acetate (control); 200 mg/kg synthetic all-rac-α-tocopheryl acetate (Eall-rac); 200 mg/kg natural RRR-α-tocopheryl-acetate (ERRR-); flavonoid extract-enriched diet (AFlav); and phenolic compound-enriched extract (APhen)] on the fatty acid composition and lipid oxidation of Iberian pig muscle longissimus dorsi. The α-tocopherol concentration was significantly higher in muscles from free-range and ERRR- pigs than in muscles from Eall-rac pigs, and γ-tocopherol was only detected in muscles from free-range pigs. Longissimus dorsi muscles from free-range pigs had a significantly lower content of saturated fatty acids and higher content of polyunsaturated fatty acids than muscles from the other five groups of pigs fed in confinement; however, no significant effect on monounsaturated fatty acids was observed. No effect of dietary antioxidant supplementation (synthetic or natural α-tocopherol, flavonoid extract, or phenol extract) on the fatty acid composition of muscles was observed. A significant influence of dietary treatment on lipid oxidation was observed after 3 (P < 0.01), and 7 and 10 (P < 0.001) days of refrigerated storage, respectively. The lowest thiobarbituric acid-reactive substances (TBARS) values were found in pork chops from the free-range and ERRR- groups, intermediate values from the Eall-rac group, followed by AFlav and APhen, while the highest TBARS values corresponded to muscles from pigs fed the control concentrate. The source of α-tocopherol had a significant effect on lipid oxidation (P < 0.05), whereas the AFlav and APhen groups had similar TBARS values.     

The influence of antioxidants in the thiyl radical induced lipid peroxidation and geometrical isomerization in micelles of linoleic acid

Abstract

The biomimetic model of micelles of linoleic acid containing 2-mercaptoethanol and the antioxidant was examined under gamma irradiation up to 400?Gy in aerobic or deoxygenated conditions where thiyl radicals are the main reactive species. Lipid peroxidation was retarded by ascorbic acid and α-tocopherol, whereas this process was strongly inhibited by resveratrol as effectively as the ascorbic acid/α-tocopherol mixture. Furthermore, antioxidants have a much stronger inhibitory effect on the peroxidation in the presence of 2-mercaptoethanol, and at the same time show protective properties of the double bond, decreasing the cis–trans isomerization. Under anaerobic conditions, cis–trans isomerization occurred and antioxidants efficiency increased along the series: resveratrol < α-tocopherol?<?ascorbic acid. This result is explained taking into account the double bond localization in the hydrophobic core of the micelle and the need of co-localization of the antioxidant in order to get an anti-isomerizing activity and protection of the natural lipid geometry.     

Isoprostanes,Prostaglandins and Tocopherols in Pre-eclampsia,Normal Pregnancy and Non-pregnancy

This study is designed to evaluate whether oxidative stress and inflammation are involved in severe pre-eclampsia compared to normal pregnancy and non-pregnancy. We have measured plasma and urinary levels of 8-iso-PGF_2α, a major isoprostane as an indicator of oxidative stress; plasma and urinary 15-keto-dihydro-PGF_2α, a major metabolite of cyclooxygenase-catalysed PGF_2α as an indicator of inflammatory response, and plasma -α-and -γ-tocopherol in 18 pre-eclamptic, 19 normal pregnancy and 20 non-pregnant women. Pregnant women had significantly higher levels of 8-iso-PGF_2α and PGF_2α metabolite as compared to the non-pregnancy. Levels of 8-iso-PGF_2α in the pre-eclamptic women did not differ from the normal pregnancy but PGF_2α metabolite levels were significantly higher in normal pregnancy. On the other hand, γ-tocopherol levels were significantly lower in pre-eclampsia than normal pregnancy. In contrast, the concentration of α-tocopherol was very similar between the groups. α-and γ-tocopherol levels were significantly lower in pregnancy compared to non-pregnancy. Although no direct evidence of oxidative stress and inflammatory response was observed in severe pre-eclampsia, a reduction of γ-tocopherol suggests the possible precedence of oxidative stress in this condition. Higher levels of isoprostanes and prostaglandin metabolite in late pregnancy suggest the importance of both free radicals and cyclooxygenase-catalysed oxidation products in normal biological processes of pregnancy.     

Electron spin resonance and electron nuclear double resonance studies of cation radicals derived from tocopherol model compounds

Electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) measurements were performed for the cation radicals obtained from the model compounds of α-, β-, γ- and δ-tocopherol (vitamin E) by oxidizing the tocopherol precursors in an AlCl₃-CH₂Cl₂ solution. The proton hyperfine coupling constants g-values were precisely determined. The ENDOR spectra of the cation radicals of α-, β-, γ- and δ-tocopherol models in CH₂Cl₂ at ?100°C clearly show 10, 6, 6 and 12 different proton hyperfine couplings, respectively. By varying the temperature, the ESR spectra of the α- and δ-tocopherol model cations exhibit line-width alternation phenomena characteristic of the hindered rotation of the OH group. However, neither the β- nor the γ-tocopherol model cation radical ESR spectra show any sign of an alternative line-width effect. These results are interpreted by assuming that the β- and γ-tocopherol model cations are stabilized in the trans and cis conformations, respectively. On tocopherol model cations are stabilized in the trans and cis conformations, respectively. On the other hand, both the α- and δ-tocopherol model cations exist as cis and trans isomers.     

Vitamin E,glutathione S-transferase and γ-glutamyl transpeptidase activities in cultured hepatocytes of rats treated with carcinogens

• 1.1. The effects of α-tocopherol and γ-tocotrienol on glutathione S-transferase (GST) and γ-glutamyl transpeptidase (γ-GT) activities in cultured hepatocytes prepared from rats treated with diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) were investigated.
• 2.2. Both the α-tocopherol and γ-tocotrienol treated hepatocytes showed significantly higher (P < 0.05) GST activities than untreated hepatocytes prepared from the carcinogen treated rats in the first 3 days of culture. Treatment with α-tocopherol and γ-tocotrienol generally resulted in a tendency to increase the GST activities above that in the untreated hepatocytes.
• 3.3. Treatment with high doses (125–250 μM) of α-tocopherol and low doses (12.5–25 μM) of γ-tocotrienol generally resulted in a significant reduction in γ-GT activities at 1–3 days. γ-GT activities are reduced as the dose of α-tocopherol and γ-tocotrienol are increased.

     

Modulation of lysozyme function and degradation after nitration with peroxynitrite

Background

Peroxynitrite (PN) is formed from superoxide and nitric oxide, both of which are increased during hepatic ethanol metabolism. Peroxynitrite forms adducts with proteins, causing structural and functional alterations. Here, we investigated PN-induced alterations in lysozyme structure and function, and whether they altered the protein's susceptibility to proteasome-catalyzed degradation.

Methods

Hen egg lysozyme was nitrated using varying amounts of either PN or the PN donor, 3-morpholinosydnonimine (SIN-1). The activity, nitration status and the susceptibility of lysozyme to proteasome-catalyzed degradation were assessed.

Results

Lysozyme nitration by PN or SIN-1 caused dose-dependent formation of 3-nitrotyrosine-lysozyme adducts, causing decreased catalytic activity, and enhanced susceptibility to degradation by the 20S proteasome. Kinetic analyses revealed an increased affinity by the 20S proteasome toward nitrated lysozyme compared with the native protein.

Conclusion

Lysozyme nitration enhances the affinity of the modified enzyme for degradation by the proteasome, thereby increasing its susceptibility to proteolysis.

General significance

Increased levels of peroxynitrite have been detected in tissues of ethanol-fed animals. The damaging effects from excessive peroxynitrite in the cell increase hepatotoxicity and cellular death by protein modification due to nitration. Cellular defenses against such changes include enhanced proteolysis by the proteasome in order to maintain protein quality control.     

Immunological detection of nitrosative stress-mediated modified Tamm-Horsfall glycoprotein (THP) in calcium oxalate stone formers.

The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in hyperoxaluric condition has been proved experimentally. This may result in the formation of the cytotoxic metabolite peroxynitrite, which is capable of causing lipid peroxidation and protein modification. The presence of nitrotyrosine in proteins has been associated with several pathological conditions. The present study investigated the presence of nitrotyrosine in the stone formers Tamm-Horsfall glycoprotein (THP). In vitro nitration of control THP was carried out using peroxynitrite. New Zealand white rabbits were immunized with peroxynitrated THP at 15-day intervals. Antisera collected following the third immunization were assayed for antibody titres using solid-phase ELISA. Antibodies were purified by affinity chromatography. The carbonyl content of control, stone formers and nitrated THP were determined. Western blotting was carried with control, stone formers and nitrated THPs. Immunodiffusion studies demonstrated cross-reaction with nitrated bovine serum albumin. Significant amounts (p < 0.001) of carbonyl content were present in both stone formers and nitrated THPs. Western blot analysis confirmed the presence of nitrated amino acid 3-nitrotyrosine in stone formers, which could bring about structural and functional modifications of THP in hyperoxaluric patients. A cross-reaction with nitrated bovine serum albumin confirms that the raised antibody has certain paratopes similar to the epitope of nitrated protein molecules. Detection of 3-nitrotyrosine in stone formers THP indicates that it is one of the key factors influencing the conversion of THP to a structurally and immunologically altered form during calcium oxalate stone formation.     

Peroxynitrite Induces Tyrosine Nitration and Modulates Tyrosine Phosphorylation of Synaptic Proteins

Peroxynitrite, the product of the radical-radical reaction between nitric oxide and superoxide anion, is a potent oxidant involved in tissue damage in neurodegenerative disorders. We investigated the modifications induced by peroxynitrite in tyrosine residues of proteins from synaptosomes. Peroxynitrite treatment (> or =50 microM) induced tyrosine nitration and increased tyrosine phosphorylation. Synaptophysin was identified as one of the major nitrated proteins and pp60src kinase as one of the major phosphorylated substrates. Further fractionation of synaptosomes revealed nitrated synaptophysin in the synaptic vesicles, whereas phosphorylated pp60src was enriched in the postsynaptic density fraction. Tyrosine phosphorylation was increased by treatment with 50-500 microM peroxynitrite and decreased by higher concentrations, suggesting a possible activation/inactivation of kinases. Immunocomplex kinase assay proved that peroxynitrite treatment of synaptosomes modulated the pp60src autophosphorylation activity. The addition of bicarbonate (CO2 1.3 mM) produced a moderate enhancing effect on some nitrated proteins but significantly protected the activity of pp60src against peroxynitrite-mediated inhibition so that at 1 mM peroxynitrite, the kinase was still more active than in untreated synaptosomes. The phosphotyrosine phosphatase activity of synaptosomes was inhibited by peroxynitrite (> or =50 microM) but significantly protected by CO2. Thus, the increase of phosphorylation cannot be attributed to peroxynitrite-mediated inhibition of phosphatases. We suggest that peroxynitrite may regulate the posttranslational modification of tyrosine residues in pre- and postsynaptic proteins. Identification of the major protein targets gives insight into the pathways possibly involved in neuronal degeneration associated with peroxynitrite overproduction.     

Novel vitamin E forms in leaves of Kalanchoe daigremontiana and Phaseolus coccineus

In the present study, we isolated novel tocochromanols from green leaves of Kalanchoe daigremontiana and primary leaves of etiolated seedlings of Phaseolus coccineus that were identified as β-, γ-, and δ-tocomonoenols with unsaturation at the terminal isoprene unit of the side chain. The content of γ-tocomonoenol in leaves of etiolated bean increased gradually with the age of seedlings, reaching 50% of the γ-tocopherol level in 40-day-old plants. The content of this compound in leaves was increased by short illumination of etiolated plants and by addition of homogentisic acid, a biosynthetic precursor of tocopherols. These data indicated that γ-tocomonoenol is synthesized de novo from homogentisic acid and tetrahydro-geranylgeraniol diphosphate, a phytol precursor. Based on these results, a biosynthetic pathway of tocomonoenols is proposed.