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The distribution of tyramine and its methylated derivatives, N-methyltyramine and N,N-dimethyltyramine, was investigated in tissue parts (leaves and fruits) of several plants of Citrus genus by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In the course of our study we discovered the occurrence of N,N,N-trimethyltyramine in all citrus plants examined. This quaternary ammonium compound, known to act in animals as a neurotoxin, was recognized and characterized by mass spectrometric analysis. The substance, never described before in the Citrus genus, is also known as candicine or maltoxin. Results indicate that N,N,N-trimethyltyramine is consistently expressed in leaves of clementine, bitter orange, and lemon. Conversely, low levels were found in the leaves of orange, mandarin, chinotto (Citrus myrtifolia), bergamot, citron, and pomelo. In the edible part of the fruits, N,N,N-trimethyltyramine was found at trace levels.

Different O/W nanoemulsion-based delivery systems were developed in order to optimize the bioavailability of encapsulated resveratrol for potential oral administration. Blank formulations without resveratrol had no negative effect on cell viability or the cytoskeleton structure of Caco-2 cells (XTT viability assay and confocal microscopy). All nanoemulsions were then evaluated based on permeability tests on Caco-2 cells. As a result, the most efficient formulations were lecithin-based nanoemulsions which were able to transport resveratrol through cell monolayers in characteristically shorter times (1-6h) than those required for their metabolization (3-12h), allowing for better preservation of the integrity of the emulsion droplets, thus better protecting the resveratrol molecule. Fluorescence spectroscopy studies confirmed that resveratrol was encapsulated in the inner core of the nanoemulsions, which provides protection against chemical degradation. Furthermore, the developed systems also demonstrated the capability of nanoemulsions in sustained release of resveratrol from dialysis bags compared to the unencapsulated compound., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

N(ε)-trimethyllysine (TML) is a non-protein amino acid which takes part in the biosynthesis of carnitine. In mammals, the breakdown of endogenous proteins containing TML residues is recognized as starting point for the carnitine biosynthesis. Here, we document that one of the main sources of TML could be the vegetables which represent an important part of daily alimentation for most mammals. A HPLC-ESI-MS/MS method, which we previously developed for the analysis of N(G)-methylarginines, was utilized to quantitate TML in numerous vegetables. We report that TML, believed to be rather rare in plants as free amino acid, is, instead, ubiquitous in them and at not negligible levels. The occurrence of TML has been also confirmed in some vegetables by a HPLC method with fluorescence detection. Our results establish that TML can be introduced as free amino acid in conspicuous amounts from vegetables. The current opinion is that mammals utilize the breakdown of their endogenous proteins containing TML residues as starting point for carnitine biosynthesis. However, our finding raises the question of whether a tortuous and energy expensive route as the one of TML formation from the breakdown of endogenous proteins is really preferred when the substance is so easily available in vegetable foods. On the basis of this result, it must be taken into account that in mammals TML might be mainly introduced by diet. However, when the alimentary intake becomes insufficient, as during starvation, it might be supplied by endogenous protein breakdown.

Hyperglycaemia, a characteristic feature of diabetes mellitus, induces endothelial dysfunction and vascular complications by accelerating endothelial cell (EC) senescence and limiting the proliferative potential of these cells. Here we aimed to investigate the effect of stachydrine, a proline betaine present in considerable quantities in juices from fruits of the Citrus genus, on EC under high-glucose stimulation, and its underlying mechanism. The senescence model of EC was set up by treating cells with high-glucose (30 mM) for different times. Dose-dependent (0.001-1 mM) evaluation of cell viability revealed that stachydrine does not affect cell proliferation with a similar trend up to 72 h. Noticeable, stachydrine (0.1 mM) significantly attenuated the high-glucose induced EC growth arrest and senescence. Indeed, co-treatment with high-glucose and stachydrine for 48 h kept the percentage of EC in the G0 /G1 cell cycle phase near to control values and significantly reduced cell senescence. Western blot analysis and confocal-laser scanning microscopy revealed that stachydrine also blocked the high-glucose induced upregulation of p16(INK4A) and downregulation of SIRT1 expression and enzyme activity. Taken together, results here presented are the first evidence that stachydrine, a naturally occurring compound abundant in citrus fruit juices, inhibits the deleterious effect of high-glucose on EC and acts through the modulation of SIRT1 pathway. These results may open new prospective in the identification of stachydrine as an important component of healthier eating patterns in prevention of cardiovascular diseases., (© 2013 Wiley Periodicals, Inc.)

N(G),N(G)-dimethyl-L-arginine (ADMA) and N(G)-methyl-L-arginine (NMMA) are endogenous inhibitors of nitric oxide synthase (NOS). In contrast, N(G),N'(G)-dimethyl-L-arginine (SDMA) possesses only a weak inhibitory potency towards neuronal NOS and it is known to limit nitric oxide (NO) production by competing with L-arginine for cellular uptake. The inhibition of NOS is associated with endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. L-homoarginine (HArg), a structural analog of L-arginine (Arg), is an alternative but less efficient substrate for NOS. Besides, it inhibits arginase, leading to an increased availability of L-arginine for NOS to produce NO. However, its relation with cardiovascular disease remains unclear. To date, several analytical methods for the quantitative determination of Arg, HArg, NMMA, AMDA, and SDMA in biological samples have been described. Here, we present a simple, fast, and accurate HPLC-ESI-MS/MS method which allows both the simultaneous determination and quantification of these compounds without needing derivatization, and the possibility to easily modulate the chromatographic separation between HArg and NMMA (or between SDMA and ADMA). Data on biological samples revealed the feasibility of the method, the minimal sample preparation, and the fast run time which make this method very suitable and accurate for analysis in the basic and clinical settings.

Air pollution exposure in industrialized cities is associated with an increased risk of morbidity and mortality attributed to cardiovascular diseases. Combustion exhausts emitted from motor vehicles and industries represent a major source of nanoparticles in the atmosphere. Flame-generated organic carbon nanoparticles (OC NPs) provide interesting model nanoparticles that simulate fresh combustion emissions near roadways or combustion sources. These model nanoparticles can be produced by controlling flame operating conditions and used to test possible toxicological mechanisms responsible for the observed health effects. OC NPs were used to investigate their possible effect on endothelial cells (EC) growth and production of proinflammatory lipid mediators. Results indicated a dose and time-dependent reduction in cell viability following incubation of EC with OC NPs for 24 and 48h. Fluorescence-activated cell sorting revealed that EC treated with OC NPs showed a cell proliferation index significantly lower than that of control cells and an increased apoptotic cell death. The annexin assay confirmed the increased apoptotic cell death. Moreover, OC NPs also induced a time-dependent increase of proinflammatory lysophospholipid production. These results, establishing that OC NPs induce EC proinflammatory lysophosholipid production and apoptotic cell death, provide the first evidence of the detrimental effect of OC NPs on EC., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The occurrence and distribution in Citrus genus plants of N-methylated derivatives of tryptamine and their 5-hydroxylated forms are reported. Tryptamine, N-methyltryptamine, N,N-dimethyltryptamine, N,N,N-trimethyltryptamine, 5-hydroxytryptamine (serotonin), 5-hydroxy-N-methyltryptamine, 5-hydroxy-N,N-dimethyltryptamine (bufotenine), and 5-hydroxy-N,N,N-trimethyltryptamine (bufotenidine) were quantitated by LC-ESI-MS/MS. Leaves of all citrus plants examined contained N,N,N-trimethyltryptamine, a compound that we first discovered in the bergamot plant. Interestingly, we also found out that all plants examined contained 5-hydroxy-N,N-dimethyltryptamine and 5-hydroxy-N,N,N-trimethyltryptamine, compounds never described so far in the Citrus genus. As N,N,N-trimethyltryptamine and 5-hydroxy-N,N,N-trimethyltryptamine possess nicotine-like activity by exerting their action on acetylcholine receptors, it is conceivable that both represent the arrival point of a biosynthetic pathway aimed to provide Citrus plants with chemical defense against aggressors. This hypothesis is supported by our finding that leaves and seeds, which are more frequently attacked by biotic agents, are the parts of the plant where the highest levels of those compounds were found.

Endogenous methylarginines, N(G),N(G)-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), N(G)-N('G)-dimethyl-L-arginine (symmetric dimethylarginine; SDMA), and N(G)-monomethyl-L-arginine (monomethylarginine; NMMA) are supposed to be produced in human body through the methylation of protein arginine residues by protein arginine methyltransferases (PRMT) and released during proteolysis of the methylated proteins. Micromolar concentration of ADMA and NMMA can compete with arginine for nitric oxide synthase (NOS) reducing nitric oxide (NO) formation, whereas SDMA does not. Indeed, increased ADMA and SDMA plasma levels or a decreased arginine/ADMA ratio is related with risk factors for chronic kidney disease and cardiovascular disease. To the best of our knowledge the exogenous presence of methylarginines, like that in fruits and vegetables, has never been described so far. Here, we report the finding that methylarginines are ubiquitous in vegetables which represent an important part of human daily diet. Some of these vegetables contain discrete amounts of ADMA, SDMA, and NMMA. Specifically, among the vegetables examined, soybean, rye, sweet pepper, broad bean, and potato contain the highest ADMA and NMMA mean levels. Our results establish that the three methylarginines, in addition to being produced endogenously, can also be taken daily through the diet in conspicuous amounts. We propose that the contribution of the methylarginines contained in the vegetables of daily diet should be taken into account when the association between vegetable assumption and their levels is evaluated in clinical studies. Furthermore, a comprehensive understanding on the role of the digestive breakdown process and intestinal absorption grade of the methylarginines contained in vegetables is now needed., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The occurrence of N-methylated tryptamine derivatives in bergamot plant (Citrus bergamia Risso et Poit) is reported for the first time. Interestingly, the most abundant of these substances is N,N,N-trimethyltryptamine, which has not been previously identified in any citrus plant. The N-methylated tryptamine derivatives were identified and quantitated in leaves, peel, juice, and seeds by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. N,N,N-Trimethyltryptamine was confirmed by MS(3) and comparison with the synthesized authentic standard. In addition, the study of the distribution of tryptophan, tryptamine, N-methyltryptamine, N,N-dimethyltryptamine, and N,N,N-trimethyltryptamine indicated that these compounds are differently expressed in the various tissues of the bergamot plant. Intriguingly, chemically synthesized N,N,N-trimethyltryptamine was reported to possess nicotine-like activity being a stimulant of parasympathetic ganglia by exerting its action on acetylcholine receptors. On this basis, the identification of N,N,N-trimethyltryptamine at a relatively high level in leaves suggests a possible role in a physiological mechanism of plant defense.

The presence of pipecolic acid and pipecolic acid betaine, also known as homostachydrine, is herein reported for the first time in Citrus genus plants. Homostachydrine was found in fruits, seeds, and leaves of orange, lemon, and bergamot (Citrus bergamia Risso et Poit). As homostachydrine was not commercially available, as a comparative source, extracts of alfalfa leaves ( Medicago sativa L.) were used, in which homostachydrine is present at high concentration. Then, the results where confirmed by comparison with an authentic standard synthesized and purified starting from pipecolic acid. The synthesized standard was characterized by a ESI-MS/MS study using a 3D ion-trap mass spectrometer. When subjected to MS/MS fragmentation in positive ion mode, homostachydrine, unlike its lower homologue proline betaine (also known as stachydrine), showed a pattern of numerous ionic fragments that allowed unambiguous identification of the compound. For the quantitation in the plant sources, high sensitivity and specificity were achieved by monitoring the transition (158 → 72), which is absent in the fragmentation patterns of other major osmolytes commonly used as markers for studies of abiotic stress. As for the metabolic origin of homostachydrine, the occurrence in citrus plants of pipecolic acid leads to the hypothesis that it could act as a homostachydrine precursor through direct methylation.

Numerous compounds, many of them osmolytes, were quantified in natural juices and in frozen concentrate juices from fruits of plants of the Citrus genus. L-proline, N-methyl-L-proline (hygric acid), N,N-dimethyl-L-proline (stachydrine), 4-hydroxy-L-prolinebetaine (betonicine), 4-hydroxy-L-proline, γ-aminobutyric acid (Gaba), 3-carboxypropyltrimethylammonium (GabaBet), N-methylnicotinic acid (trigonelline), and choline in the fruit juices of yellow orange, blood orange, lemon, mandarin, bitter orange (Citrus aurantium), chinotto (Citrus myrtifolia), and grapefruit were analyzed by sensitive HPLC-ESI-tandem mass spectrometry procedure. It was found that the most represented osmolytes in the juices, that is, L-proline, stachydrine, and betonicine, can be quantified with minimal sample preparation and short analysis time (about 1 min) also by flow injection analysis (FIA) ESI-MS/MS with the same results as obtained by HPLC ESI-MS/MS. In all of the juices, discrete amounts of choline and trigonelline were present. Conversely, GabaBet was always below detection limits. Notably, N-methyl-L-proline and 4-hydroxy-L-prolinebetaine, which were discovered for the first time in the juice of bergamot (Citrus bergamia Risso et Poit), are also present in all of the citrus juices examined.

The content of proline and various compounds deriving from its metabolism (4-hydroxy-L-proline, N-methyl-L-proline, N,N-dimethylproline, and 4-hydroxy-L-prolinebetaine) was determined in fruits and seeds of Bergamot (Citrus bergamia Risso et Poit), growing in the Calabria region (South Italy). A HPLC-ESI-tandem mass spectrometry method, which allowed rapid determination of L-proline, 4-hydroxy-L-proline, N-methyl-L-proline, N,N-dimethylproline, and 4-hydroxy-L-prolinebetaine in juice and extracts of bergamot fruit with minimum sample preparation and short analysis time (about 10 min), is presented. Proline and 4-hydroxy-L-proline levels in the samples were also determined by HPLC analysis with fluorescence detection and the results compared to those obtained with HPLC-ESI-tandem mass spectrometry. For the first time, the presence of N-methyl-L-proline and 4-hydroxy-L-prolinebetaine in the fruits of a plant of the Citrus genus is reported.

Exposure of human endothelial progenitor cells (EPCs) to tumor necrosis factor-α (TNF-α) reduced their number and biological activity. Yet, signal transduction events linked to TNF-α action are still poorly understood. To address this issue, we examined the possible effect of fasudil and Y27632, two inhibitors of Rho kinase pathway, which is involved in endothelial dysfunction, atherosclerosis, and in- flammation. Results demonstrated that incubation with fasudil starting from 50 μM but not Y27632 determined a dose-dependent improvement of EPC number during exposure to TNF-α (P < 0.05 vs. TNF-α alone). Analysis of the signal transduction pathway activated by TNF-α revealed that the increased expression of p-p38 was not significantly altered by fasudil. Instead, fasudil blocked the TNF-α induced phosphorylation of Erk1/2 (P < 0.05 vs. TNF-α) as well as the inhibitor of Erk1/2-specific phosphorylated form, i.e., PD98059 (P < 0.05 vs. TNF-α). These results were confirmed by analysis of these kinases by confocal microscopy. Finally, 2D-DIGE and MALDI-TOF/TOF analysis of EPCs treated with fasudil revealed increased expression levels of an actin-related protein and an adenylyl cyclase associated protein and decreased expression levels of proteins related to radical scavenger and nucleotide metabolism. These findings suggest that fasudil positively affects EPC number and that other major signals might take part to this complex pathway.

Endothelial progenitor cell (EPC) therapy is a promising approach to promote angiogenesis and endothelial repair in patients with cardiovascular diseases (CVD). However, their release of proinflammatory mediators may compromise the therapeutic efficacy. Little is known about the role of Platelet-Activating Factor (PAF) in EPC functional response. Here, we investigated the expression of PAF receptor (PAF-R) in early EPC and the release of PAF under stimulation with factors involved in endothelial dysfunction. Results indicated that early EPC express the PAF-R and respond to PAF signaling via a transient increase of cytoplasmic Ca(2+) concentration. EPC release PAF in a time dependent manner upon stimulation with tumor necrosis factor-alpha (TNF-alpha) or high-glucose concentration with a peak at 30 min and 10 min (p<0.01 vs. control), respectively. PAF, starting at concentration of 50 ng/ml, exerted a detrimental effect on EPC number with a concomitant increase of p38 activity. Furthermore, both the reduction of early EPC number and the enhanced p38 activity induced by PAF were abolished by CV3988, a PAF receptor antagonist. These novel findings, revealing that early EPC respond to PAF signaling, unveil an inflammatory pathway that may play a crucial role in the outcome of cardiovascular cell therapy with EPC., (Copyright @ 2010 Elsevier B.V. All rights reserved.)

Objective: Peripheral arterial disease (PAD) is a major health problem especially when associated to concomitant diabetes and hypercholesterolemia. Hyperglycemia with an overwhelming generation of oxygen radicals and formation of glycation end-products exacerbates oxidation-sensitive mechanisms activated by tissue ischemia. Administration of autologous bone marrow cells (BMC) is an increasing notable intervention to induce therapeutic angiogenesis, ameliorated by metabolic intervention (MT). Recently, hemangioblasts (HS) with functional properties were isolated., Methods: The effects of integrate regimen with intravenous BMC, HS, and MT (1.0% vitamin E, 0.05% vitamin C, and 6% l-arginine) were examined in the ischemic hindlimb of ApoE(-/-) diabetic and non-diabetic. Blood flow ratio was monitored by use of a laser Doppler blood flowmeter. Capillary density was determined in sections of the adductor and semimembranous muscles with antibody against CD31., Results: BMC or HS alone, and BMC plus HS increased blood flow and capillary densities and decreased interstitial fibrosis. These effects were amplified by additional MT, at least in part, through the nitric oxide pathway, reduction of systemic oxidative stress and macrophage infiltration. Investigation of molecular mechanisms in bone marrow (BM)-derived progenitor cells from mice revealed that BMC therapy and, more consistently, in combination with MT ameliorated functional activity via decreased cellular senescence and increased telomerase and chemokine CXCR4 activities. Telomerase activity was also increased by HS alone or HS+MT and, more consistently, by BMC+HS alone or in combination with MT., Conclusions/interpretation: Intravenous autologous BMC and HS intervention together with MT increased therapeutic angiogenesis in the ApoE(-/-) diabetic mouse hindlimb., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

The chemical composition of 30 samples of juices obtained from bergamot (Citrus bergamia Risso and Poit.) fruits is reported and compared to the genuineness parameters adopted by Association of the Industry of Juice and Nectars (AIJN) for lemon juice. It was found that the compositional differences between the two juices are distinguishable, although with difficulty. However, these differences are not strong enough to detect the fraudulent addition of bergamot juice to lemon juice. Instead, we found the high-performance liquid chromatography (HPLC) analysis of the flavanones naringin, neohesperidin, and neoeriocitrin, which are present in bergamot juice and practically absent in the lemon juice, is a convenient way to detect and quantify the fraudulent addition of bergamot juice. The method has been validated by calculating the detection and quantification limits according to Eurachem procedures. Employing neoeriocitrin (detection limit = 0.7 mg/L) and naringin (detection limit = 1 mg/L) as markers, it is possible to detect the addition of bergamot juice to lemon juice at the 1% level. When using neohesperidin as a marker (detection limit = 1 mg/L), the minimal percentage of detectable addition of bergamot juice was about 2%. Finally, it is reported that the pattern of flavonoid content of the bergamot juice is similar to those of chinotto (Citrus myrtifolia Raf) and bitter orange (Citrus aurantium L.) juices and that it is possible to distinguish the three kinds of juices by HPLC analysis.

Increasing evidence indicates that mammalian SIRT1 mediates calorie restriction and influences lifespan regulating a number of biological molecules such as FoxO1. SIRT1 controls the angiogenic activity of endothelial cells via deacetylation of FoxO1. Endothelial dysfunction and reduced new blood vessel growth in diabetes involve a decreased bioactivity of endothelial progenitor cells (EPCs) via repression of FoxO1 transcriptional activity. The relative contribution of SIRT1 with respect to the direct effects of high glucose on EPC number is poorly understood. We report that treatment of EPCs with high glucose for 3 days determined a consistent downregulation of EPC positive to DiLDL/lectin staining and, interestingly, this was associated with reduced SIRT1 expression levels and enzyme activity, and increased acetyl-FoxO1 expression levels. Moreover, EPCs responded to high glucose with major changes in the expression levels of cell metabolism-, cell cycle-, and oxidative stress-related genes or proteins. Proteomic analysis shows increased expression of nicotinamide phosphoribosyl transferase and mitochondrial superoxide dismutase whereas a glucose-related heat shock protein is reduced. These findings show that SIRT1 is a critical modulator of EPCs dysfunction during alteration of glucose metabolism.

Moderate wine intake is associated with a reduced risk of morbidity and mortality from cardiovascular disease. Atherosclerosis is enhanced in arterial segments exposed to disturbed flow. Perturbed shear stress increases also the endothelial expression of oxidation-sensitive responsive genes (such as ELK-1 and p-JUN). This study evaluates the effects of chronic consumption of red wine on perturbed shear stress-induced atherogenesis. Results indicated that chronic treatment with red wine significantly attenuated the activation of redox-sensitive genes (ELK-1 and p-JUN) and increased endothelial nitric oxide synthase (eNOS) expression (which was decreased by perturbed shear stress) in cultured human coronary endothelial cells (EC) and in atherosclerosis-prone areas of hypercholesterolemic mice. Oral administration of red wine to hypercholesterolemic mice reduced significantly the progression of atherosclerosis. Moreover, short-term supplementation with red wine to C57BL/6J mice significantly increased upregulation of aortic eNOS and SIRT1 expression induced by physical training. These findings establish that administration of low doses of red wine can attenuate the proatherogenic effects induced by perturbed shear stress in vitro and in vivo. This evidence may have implications for the prevention of atherosclerotic lesion progression and its clinical manifestations.

Circulating endothelial progenitor cells (EPCs) play a significant role in neovascularization of ischaemic tissues and in re-endothelization of injured blood vessels. Identification of compounds able to enhance EPC levels and improve their functional activity, noticeably compromised by risk factors for coronary heart disease, is of clinical interest. This study evaluates the effects of red wine on EPCs. After being isolated from total peripheral blood mononuclear cells, EPC phenotype was confirmed by the presence of double positive cells for DiLDL uptake and lectin binding and by expression of CD34, CD133 and VE-cadherin cell surface markers. Long-term culture in the presence of red wine (1 microl/ml), containing resveratrol (Resv) at physiological concentration (nM), determined a time-dependent amelioration of cell number (P < 0.05). The presence of red wine prevented the TNF-alpha-induced reduction of EPC number (P < 0.05) and this effect was accompanied by reduced p38-phosphorylation expression levels (P < 0.05) and increased NOx levels (P < 0.05) Indeed, pure Resv alone significantly improved the TNF-alpha reduced EPC number (P < 0.05). This evidence indicates novel beneficial effects of red wine and Resv in the positive modulation of EPCs levels.

The transacetylase (TA), reported to be identical to platelet-activating factor (PAF) acetylhydrolase (II), is a multifunctional enzyme with three catalytic activities: lysophospholipid transacetylase (TA(L)), sphingosine transacetylase (TA(S)), and acetylhydrolase (AH). We report that TA(L) activity participates in the control of PAF levels in monocytes and macrophages and that its regulation differs in these two types of cells. In monocytes, LPS or granulocyte-macrophage colony-stimulating factor (GM-CSF) specifically increased the TA(L) activity. Western blot analysis and enzyme assays on immunoprecipitates revealed that the increased activity can be ascribed to PAF-AH (II) and that both translocation from cytosol to membranes and p38/ERKs-mediated phosphorylation regulate the enzyme activation. Instead, in macrophages differentiated in vitro from monocytes by incubation with FCS, an increase of both TA(L) and AH activities was observed. Moreover, activation of ERKs and p38 MAP kinase was not required for the up-regulation of PAF-AH (II) in differentiated macrophages. The differences observed in macrophages as compared to monocytes can be explained by 1) p38/ERKs-independent phosphorylation of PAF-AH (II) and 2) appearance of plasma PAF-AH in the course of macrophage differentiation.

The molecular model of Lycopersicon esculentum (tomato) pectin methylesterase (PME) was built by using the X-ray crystal structure of PME from the phytopathogenic bacterium Erwinia chrysanthemi as a template. The overall structure and the position of catalytically important residues (Asp132, Asp 153, and Arg 221, located at the bottom of the active site cleft) are conserved. Instead, loop regions forming the walls of the catalytic site are much shorter and form a less deep cleft, as already revealed by the carrot PME crystal structure. The protein inhibitor of pectin methylesterase (PMEI) isolated from kiwi fruit binds tomato PME with high affinity. Conversely, no complex formation between the inhibitor and PME from E. chrysanthemi is observed, and the activity of this enzyme is unaffected by the presence of the inhibitor. Fluorescence quenching experiments on tomato PME and on PME-PMEI complex suggest that tryptophanyl residues present in the active site region are involved in the interaction and that the inhibitor interacts with plant PME at the level of the active site. We also suggest that the more open active site cleft of tomato PME allows the interaction with the inhibitor. Conversely, the narrow and deep cleft of the active site of E. chrysanthemi PME hinders this interaction. The pH-dependent changes in fluorescence emission intensity observed in tomato PME could arise as the result of protonation of an Asp residue with unusually high pKa, thus supporting the hypothesis that Asp132 acts as acid/base in the catalytic cycle., (Copyright 2003 Wiley-Liss, Inc.)

PAF-dependent transacetylase (TA) modifies the functions of platelet-activating factor (PAF), a potent inflammatory lipid, either by transferring the acetyl group from PAF to lysophospholipids (TAL activity), or to sphingosine (TAS activity) or by hydrolyzing PAF (acetylhydrolase activity). In stimulated endothelial cells (EC), TAL activity contributes to the synthesis of acyl-PAF, an acyl analog of PAF, that antagonizes PAF functions and is regulated by the cellular redox state. In this study, we investigated the possible involvement of TA in the flavonoid antioxidant mechanism(s) during oxidative stress in EC induced by hydrogen peroxide. The treatment of EC with H2O2 resulted in 4-fold increase of the acetyl-CoA acetyltransferase activity (AT), that is responsible for PAF biosynthesis, while the TAL activity increased only by 53%. However, the preincubation of H2O2-treated EC with the flavonoids hesperedin, naringin, and quercetin strongly inhibited AT activity and activated TAL by 290%, 340%, and 250%, respectively. The induction of TAL activity resulted in enhanced biosynthesis of 1-acyl-2-[3H]acetyl-PAF in intact EC and was related to the flavonoid structure. These findings suggest that TAL is involved in the flavonoid anti-inflammatory action by enhancing the production of acyl-PAF.

Release of FFA from membrane phospholipids was observed after incubation of umbilical cord vein-derived endothelial cells (HUVEC) with vascular endothelial growth factor (VEGF). In particular, we found an increase of arachidonate, stearate, and palmitate in a time-dependent manner with a peak at 30 min. The maximum increase was reached by arachidonate (4.4-fold), followed by stearate (2.2-fold) and palmitate (1.3-fold). The arachidonate increase can be ascribed to the activation of phospholipase A2 (PLA2). In fact, cells preincubated with arachidonyl trifluoromethyl ketone, a PLA2 inhibitor, showed a marked reduction in arachidonate mobilization. The role of Ca2+ in PLA2 activation was also investigated. Cells incubated with VEGF in the presence of EGTA showed a marked decrease in arachidonate mobilization, whereas incubation with the calcium ionophore A23187 alone produced an increase in arachidonate, although to a lesser extent compared with the VEGF stimulation. Incubation with A23187 in association with PMA produced the same increase in arachidonate as the VEGF treatment. Mitogen-activated protein kinase (MAPK) activity was also found to increase as a consequence of VEGF stimulation. Taken together, these results suggest that the VEGF-mediated activation of PLA2 in HUVEC is dependent on both MAPK-mediated phosphorylation and Ca2+ increase. Furthermore, the increase in stearate and palmitate likely is brought about by the activation of a pathway involving phospholipase D, phosphatidate phosphohydrolase (PAP), and DAG lipase. In fact, the increase in those FFA was prevented when HUVEC were stimulated with VEGF in the presence of ethanol (which inhibits the formation of phosphatidate), propranolol (a specific inhibitor of PAP), or RHC-80267 (a specific inhibitor of DAG lipase).

The effect of phosphate, its analogues, and other substrates on structural features of recombinant 5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was investigated. Phosphate was found to exert a significant stabilizing effect on the protein against the inactivation caused by temperature, sodium dodecyl sulfate (SDS), urea, and proteolytic enzymes. In the presence of 100 mM phosphate: (i) the apparent transition temperature (Tm) of recombinant SsMTAP increased from 111 degrees to 118 degrees C; and (ii) the enzyme still retained 40% and 30% activity, respectively, after 30 min of incubation at 90 degrees C with 2% SDS or 8 M urea. The structure modification of SsMTAP by phosphate binding was probed by limited proteolysis with subtilisin and proteinase K and analysis of polypeptide fragments by SDS-PAGE. The binding of the phosphate substrate protected SsMTAP against protease inactivation, as proven by the disappearance of a previously accessible proteolytic cleavage site that was localized in the N-terminal region of the enzyme. The conformational changes of SsMTAP induced by phosphate and ribose-1-phosphate were analyzed by fluorescence spectroscopy, and modifications of the protein intrinsic fluorophore exposure, as a consequence of substrate binding, were evidenced.

A protein acting as a powerful inhibitor of plant pectin methylesterase was isolated from kiwi (Actinidia chinensis) fruit. The complete amino-acid sequence of the pectin methylesterase inhibitor (PMEI) was determined by direct protein analysis. The sequence comprises 152 amino-acid residues, accounting for a molecular mass of 16 277 Da. The far-UV CD spectrum indicated a predominant alpha-helix conformation in the secondary structure. The protein has five cysteine residues but neither tryptophan nor methionine. Analysis of fragments obtained after digestion of the protein alkylated without previous reduction identified two disulfide bridges connecting Cys9 with Cys18, and Cys74 with Cys114; Cys140 bears a free thiol group. A database search pointed out a similarity between PMEI and plant invertase inhibitors. In particular, the four Cys residues, which in PMEI are involved in the disulfide bridges, are conserved. This allows us to infer that also in the homologous proteins, whose primary structure was deduced only by cDNA sequencing, those cysteine residues are engaged in two disulfide bridges, and constitute a common structural motif. The comparison of the sequence of these inhibitors confirms the existence of a novel class of proteins with moderate but significant sequence conservation, comprising plant proteins acting as inhibitors of sugar metabolism enzymes, and probably involved in various steps of plant development.

A new extraction and chromatographic procedure to quantify free and esterified ergosterol in tomato products was devised. The extraction solution was composed of a dichloromethane/methanol mixture in a 2:1 (v/v) ratio. This extraction solvent allowed for higher ergosterol recovery from tomato products (an average of 25% more) compared to hexane, which is frequently employed for ergosterol extraction. Both free and esterified ergosterol were determined by HPLC reverse-phase chromatography employing a Nova-Pak C-18 column (300 x 3.9 mm), filled with 4 mm average particle size and a guard column of the same material. The elution was performed at a flow rate of 1 mL. min(-1) with a linear gradient of solvent A (methanol/water, 80:20, v/v) and solvent B (dichloromethane). The gradient, starting at sample injection, was from 0 to 50% B for 20 min for the free ergosterol analysis and additional 15 min at 50% B to analyze the ergosterol esters. This technique has proven to be more sensitive for ergosterol determination than other reported chromatographic procedures. Moreover, ergosterol esters, extracted from various fungal sources, separated well and were easily quantified.

A method for the determination of a low level of pectin methylesterase activity from vegetable products is described. The method is based on an affinity chromatography technique that employs a resin-bound pectin methylesterase inhibitor, purified from kiwi fruit, which selectively binds the pectin methylesterase. The resin has the capacity to concentrate the enzyme, allowing measurement of enzyme activities too low to be determined by commonly employed techniques and commensurate with those found in pasteurized food products. The enzyme is eluted from the resin at alkaline pH (9.5) and assayed by a pH-stat method. Depulped orange juices containing different amounts of pectin methylesterase were prepared and used to determine enzyme recovery. The results show a recovery of 90% with a standard deviation of 6.8%.

The pectin methylesterase inhibitor from kiwi fruit (Actinidia chinensis) was purified by a single-step procedure based on affinity chromatography. Partially purified tomato pectin methylesterase was covalently bound to Sepharose. The affinity resin strongly and selectively binds the inhibitor, which could be eluted in high yield as a single, homogeneous and sharp peak by high salt concentration at pH 9.5 without loss of inhibitory activity. The purified protein possesses a molecular mass of 18 kDa, as estimated by SDS/PAGE, whereas by gel filtration under native conditions, its molecular mass appears to be 25 kDa. The inhibitor interacts with pectin methylesterase, forming a 1:1 complex, as demonstrated by gel-filtration experiments. The inhibitor was glycosylated. Its glycidic portion can be removed by digestion with N-glycosidase F after protein denaturation and, to a minor extent, by digestion with N-glycosidase H. No glycidic residue could be removed by digesting the native protein with those N-glycosidases. Antibodies against pectin methylesterase inhibitor were raised in rabbits and used to evidence protein expression during fruit ripening. The results showed that the inhibitor is present in the unripe fruit as an inactive precursor with a higher molecular mass (30 kDa) and is transformed into the active protein, most likely by proteinase action, during the course of the ripening process.

A fluorescent analogue of GDP, the 3'-O-anthraniloyl-GDP (anl-GDP) was demonstrated to bind to the elongation factor Tu (EF-Tu) with an affinity even higher than that of the parent nucleotide. As a consequence of the binding, an increase in fluorescence anisotropy and an emission band arising from non-radiative energy transfer among the protein intrinsic fluorophores and the labelled nucleotide were observed. Therefore, it was possible to study the exchange kinetics and the equilibrium between the protein-bound labelled GDP and the natural nucleotide through modifications, occurring during the course of the reaction, of fluorescence anisotropy and non-radiative energy transfer. In this way, it was also easily proven that, in the presence of aurodox (N-methylkirromycin), an antibiotic impairing EF-Tu biological function, the exchange kinetics between the protein-bound labeled GDP and the natural nucleotide was faster. Moreover, it was also found that the labelled nucleotide is recognized as a substrate by pyruvate kinase, being converted by this enzyme, in the presence of phosphoenolpyruvate, into anl-GTP. Pyruvate kinase is also able to convert, in the presence of phosphoenolpyruvate, the complex EF-Tu.anl-GDP into the complex EF-Tu.anl-GTP. The fluorescence properties of the 3'-O-anthraniloyl-labeled guanyl nucleotides and their feature as excellent acceptors of fluorescence arising from protein intrinsic fluorophores, may make these compounds useful for structural and binding studies on guanosine-nucleotide-binding proteins.

A fluorescent tRNA derivative labeled at 3'-O position of the ultimate adenosine residue by reaction, under mild conditions, of tRNA with isatoic anhydride [3,1-benzoxazine-2,4(1H)-dione] was obtained. The labeling selectivity was determined by several criteria: digestion with RNase, followed by HPLC of the digest, produces only one labeled nucleoside, identified as 3'-O-anthraniloyladenosine; the ratio of the absorbance at 260 nm to 332 nm also suggests a 1:1 molar ratio between the nucleic acid and the fluorophore; finally, the incapacity of the labeled tRNA to be charged by the specific aminoacyltransferase further demonstrates the engagement of the 3'-O position. Although the 3'-O-anthraniloyl-labeled tRNA does not seem to be functionally active, as far as the aminoacyl charging activity is concerned, surprisingly we found that it is able to form the ternary complex with elongation factor Tu (EF-Tu) and GTP with an affinity consistently higher than uncharged tRNA. From fluorescence anisotropy measurements the ternary complex dissociation constant was estimated as 73 nM for Escherichia coli and 140 nM for yeast anthraniloyl-tRNA(Phe). These results may be interpreted in terms of the particular structure of the anthraniloyl group that makes the labeled tRNA similar to an aminoacyl-tRNA.

Porins, a family of hydrophobic proteins located in the outer membrane of the cell wall of gram-negative bacteria and lipopolysaccharide (LPS), were shown to stimulate the synthesis of platelet activating factor (PAF), a phospholipid mediator of inflammation and endotoxic shock, by cultured human glomerular mesangial cells (MC). The synthesis of PAF induced by porins was rapid (peak at 20 min) and independent either from contamination by LPS or from generation of an endotoxin-induced cytokine such as tumor necrosis factor (TNF) since it was not prevented by cycloheximide, an inhibitor of protein synthesis or anti-TNF blocking antibodies. LPS also stimulated PAF synthesis by MC. However, the kinetic of PAF synthesis induced by LPS was biphasic with an early and transient peak at 10 minutes and a second and sustained peak at three to six hours. This second peak required an intact protein synthesis and was prevented by anti-TNF antibodies, suggesting the dependency on LPS-induced synthesis of TNF. Experiments with labeled precursors demonstrated that in MC, either after stimulation with porins or LPS, PAF was synthesized via the remodeling pathway that involves acetylation of 1-0-alkyl-sn-glyceryl-3-phosphorylcholine (2-lyso-PAF) generated from 1-0-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity. Porins and LPS, indeed, induced PLA2-dependent mobilization of [14C]-arachidonic acid that was inhibited by p-bromodiphenacylbromide (PBDB). PBDB, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)

The finding of a powerful inhibitor of pectin methylesterase in ripe kiwi fruit is reported. The inhibitor was revealed to be a glycoprotein. It was purified to homogeneity and found to have a molecular mass of about 28 kDa, as estimated by gel filtration chromatography, SDS/PAGE and analytical ultracentrifugation. The sugar portion is composed of galactose, arabinose and rhamnose, the latter being much less represented. The amino acid composition showed a very high content of acidic residues compared to basic ones, which is the reason for the very low isoelectric point of the protein (less than 3.5). The kind of inhibition on kiwi pectin methylesterase was found to be competitive with an apparent Ki of 0.22 microM, using citrus pectin as a substrate. Moreover, the inhibitor is effective in inhibiting pectin methylesterase in the pH range 3.5-7.5. Kiwi inhibitor appears to be specific for pectin methylesterase, inasmuch as it was found to be ineffective against other polysaccharide-degrading enzymes, such as polygalacturonase and amylase. Conversely, it appears to be completely aspecific as far as the pectin methylesterase source is concerned. In fact, it was found to inhibit this enzyme effectively from all the sources we assayed, i.e. orange, tomato, apple, banana, potato.

The heart ventricle myoglobin of bluefin tuna has been purified to an apparent homogeneity. The amino acid analysis has revealed only a limited number of substitutions between the myoglobins of yellowfin and bluefin tuna. The alpha-helix content of tuna myoglobin has been found considerably lower than that of mammalian myoglobin. No correlation has been discovered between the conformational stability and alpha-helix content. Denaturation experiments have shown that the whole structure of tuna myoglobin results from the interaction of two structural units which represent the product of independent folding processes. The structure of tuna myoglobin has been found more open and disorganized than that of sperm whale. This result has been related to the low content of electrostatic interactions and explained in terms of evolutive adaptations.

The mutual interference between the second-derivative bands of tyrosine and tryptophan in proteins has been evaluated in terms of the ratio r between two peak to peak distances. The r values have been found to be not only related to the tyrosine/tryptophan ratio but also dependent on the polarity of the medium in which tyrosyl residues are embedded. The results obtained on purified proteins have been found consistent with the available X-ray information and with the existing solvent perturbation data.

1. The heart ventricle myoglobin of Atlantic bluefin tuna has been purified and its amino acid composition has been determined. 2. The perturbing effect of guanidine hydrochloride on the molecular structure of tuna ferrimyoglobin and its corresponding apoprotein has been investigated by Soret absorbance and ultraviolet fluorescence. 3. The conformation-free energy of unfolding delta G0 has been calculated by thermodynamic treatments of the data concerning guanidine unfolding. 4. The results have been compared with other known myoglobins, particularly those of yellowfin tuna.

The mixed interaction between human apolipoproteins C-I and A-II, each of which self-associate is aqueous solution, has been evaluated by sedimentation equilibrium measurements. In order to simplify data analysis apoC-I and apoA-II were modified by treatment with 2-nitrophenylsulfenyl chloride and tetranitromethane respectively. The molecular properties of the resulting derivatives, S-apoC-I and N-apo-A-II, each of which appreciable extinction coefficients above 350 nm, were indistinguishable from the corresponding unmodified species. Sedimentation equilibrium data were obtained with mixtures of S-apoC-I and native apoA-II, N-apoA-II and native apoC-I, and native apoC-I and native apoA-II. Mixed complex formation was detected readily with all mixtures investigated. The combined results were most consistent with a single mixed oligomer containing 2 molecules of apoA-II and 4 molecules of apoC-I. The corresponding equilibrium constant was 31248 +/- 890 (l/gm)5.

Second derivative spectroscopy has been used to resolve the complex protein spectrum in the near-ultraviolet region and the contributions of the three aromatic chromophores have been evaluated. A method for the direct quantitative determination of phenylalanine and tryptophan in proteins has been carried out. Phenylalanine determination has been carried out in the spectral region between 250 and 265 nm, where there are no significant contributions from other aromatic chromophores. Tryptophan determination has been performed in the 290-295-nm region and the experimental values have been corrected for the presence of tyrosine. The results obtained on 10 highly purified proteins have been found in good agreement with those obtained from sequence analysis.

The heme and cysteine microenvironments of bluefin tuna apomyoglobin have been investigated by examining the fluorescence properties of two extrinsic chromophores, i.e., ANS and 1,5-AEDANS. 1,5-AEDANS was covalently bound to the single cysteine residue found in the primary structure of tuna apomyoglobin. Recombination experiments with hemin showed that tuna apomyoglobin does not bind 1,5-AEDANS in the same binding site of the heme, although the fluorescence properties of the covalently bound 1,5-AEDANS strongly suggest that the dye is embedded in a rather nonpolar microenvironment. ANS was selected because of its ability to bind the apomyoglobin in the same nonpolar moiety of the heme. Acidification of apoMb--AEDANS to pH 3.0 produced an increase of 1,5-AEDANS fluorescence intensity and a shift of its emission maximum from 475 to 470 nm. In the same pH range apomyoglobin lost its ability to bind ANS. Two independent transitions were observed with increasing concentrations of guanidine. Low guanidine concentration (less than 1.0 M) unfolded the heme binding site as indicated by the disappearance of ANS fluorescence, whereas higher denaturant concentration was required to produce full normalization of 1,5-AEDANS emission spectrum.

High protein dietary content stimulates urea formation in ureotelic animals but does not exert almost any effect on ammonia production from L-amino acids in vitro. L-histidine and L-threonine are the only amino acids which are most actively deaminated by ureotelic animals fed on a high protein diet. All the steps of L-histidine metabolism have been studied: it has been found that both the histidine transaminase pathway and the histidase pathway are stimulated. Glutamic acid is also a product of histidine catabolism through the histidase pathway, but its catabolism is unaffected by the dietary protein content. These data suggest the existence of independent mechanism controlling the catabolism of the two amino acids.

The individual fluorescence of the two tryptophan residues (Trp-7 and Trp-14) of mammalian apomyoglobins has been resolved by comparing the fluorescence properties of these proteins to those of bluefin tuna apomyoglobin, which contains only Trp-14. The two tryptophan residues have been found to have different emission maxima, i.e., 321 for Trp-14 and 333 for Trp-7. The fluorescence of Trp-14 depends on the protonation of a sterically related histidyl residue in the pH range between 8.3 and 5.6, where no conformational change was detected. This residue has been identified as His-119. At pH 8.3 the quantum yield of Trp-7 is lower than that of Trp-14. An increase of the fluorescence intensity of Trp-7 occurs when the heme binding site of apomyoglobin is destroyed by acid or a low concentration of guanidine hydrochloride. An independent unfolding of the N-terminal district of the apomyoglobin molecular occurs on increasing the guanidine concentration. The two distinct structural transitions have been discussed in terms of two domains of tertiary structure.