Application of graphite carbon black assisted-laser desorption ionization-mass spectrometry for soy sauce product discrimination.

In a previous study, we developed a novel analytical method to directly and simultaneously detect taste- and odor-active compounds using graphite carbon black (GCB)-assisted laser desorption ionization mass spectrometry (LDI-MS). In this study, we aimed to evaluate food quality using a variety of soy sauces using the method to discriminate each product. Graphite carbon black-laser desorption ionization-mass spectrometry allowed the provision of hundreds of MS peaks derived from soy sauces in both positive and negative modes without any tedious sample pretreatments. Principal component analysis using the obtained MS peaks clearly distinguished three soy sauce products based on the manufacturing countries (Japan, China, and India). Moreover, this method identified distinct MS peaks for discrimination, which significantly correlated with their quantitative amounts in the products. Thus, GCB-LDI-MS analysis was established as a simple and rapid technique for food analysis, illustrating the chemical patterns of food products.

Simultaneous Evaluation of Membrane Permeability and UDP-Glucuronosyltransferase-Mediated Metabolism of Food-Derived Compounds Using Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells.

Pharmacokinetic prediction after oral ingestion is important for quantitative risk assessment of food-derived compounds. To evaluate the utility of human intestinal absorption prediction, we compared the membrane permeability and metabolic activities of human induced pluripotent stem cell-derived small intestinal epithelial cells (hiPSC-SIECs) with Caco-2 cells or human primary enterocytes (hPECs). We found that membrane permeability in hiPSC-SIECs had better predictivity than that in Caco-2 cells against 21 drugs with known human intestinal availability (r = 0.830 and 0.401, respectively). Membrane permeability in hiPSC-SIECs was only 0.019-0.25-fold as compared with that in Caco-2 cells for 7 in 15 food-derived compounds, primarily those that were reported to undergo glucuronidation metabolism. The metabolic rates of the glucuronide conjugate were similar or higher in hiPSC-SIECs as compared with hPECs but lower in Caco-2 cells. Expression levels of UDP-glucuronosyltransferase (UGT) isoform mRNA in hiPSC-SIECs were similar or higher as compared with hPECs. Therefore, hiPSC-SIECs could be a useful tool for predicting human intestinal absorption to simultaneously evaluate membrane permeability and UGT-mediated metabolism. SIGNIFICANCE STATEMENT: Gastrointestinal absorption is an important step for predicting the internal exposure of food-derived compounds. This research revealed that human induced pluripotent stem cell-derived small intestinal cells (hiPSC-SIECs) had better predictivity of intestinal availability than Caco-2 cells; furthermore, the metabolic rates of UDP-glucuronosyltransferase (UGT) substrates of hiPSC-SIECs were closer to those of human primary enterocytes than those of Caco-2 cells. Therefore, hiPSC-SIECs could be a useful tool for predicting human intestinal absorption to simultaneously evaluate membrane permeability and UGT-mediated metabolism.

Novel Approach for Simultaneous Analysis of Peptide Metabolites from Orally Administered Glycinin in Rat Bloodstream by Coumarin-Tagged MALDI-MS.

The lack of an appropriate analytical approach characterizing metabolites from dietary proteins may prevent further studies that could clarify their health benefits. In this study, we attempted to establish a novel analytical assay of peptide metabolites from glycinin using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), in combination with the amine derivatization technique with coumarin (Cou). Cou (30 mmol/L) derivatization of peptides under rapid (30 min) and mild (25 °C, pH 8.5) conditions caused higher MS detection of the peptides as compared to nonderivatized peptides. In addition, an MS shift of the target by Cou derivatization (+202.0 m/z) can help to easily discriminate peptide metabolites in glycinin-administered blood, by comparing the MALDI-MS spectra of Cou-derivatized plasma with those of preadministered blood. After the oral administration of glycinin (100 mg/kg) to Sprague-Dawley rats, 15 di- to tetrapeptides were successfully characterized as glycinin-derived metabolites, demonstrating that the proposed Cou-tagged MALDI-MS is an appropriate characterization technique for peptide metabolites.

In vitro and in silico characterization of adiponectin-receptor agonist dipeptides.

The aim of this study is to develop a dipeptide showing an adiponectin receptor 1 (AdipoR1) agonistic effect in skeletal muscle L6 myotubes. Based on the structure of the AdipoR1 agonist, AdipoRon, 15 synthetic dipeptides were targeted to promote glucose uptake in L6 myotubes. Tyr-Pro showed a significant increase in glucose uptake among the dipeptides, while other dipeptides, including Pro-Tyr, failed to exert this effect. Tyr-Pro induces glucose transporter 4 (Glut4) expression in the plasma membrane, along with adenosine monophosphate-activated protein kinase (AMPK) activation. In AdipoR1-knocked down cells, the promotion by Tyr-Pro was ameliorated, indicating that Tyr-Pro may directly interact with AdipoR1 as an agonist, followed by the activation of AMPK/Glut4 translocation in L6 myotubes. Molecular dynamics simulations revealed that a Tyr-Pro molecule was stably positioned in the two potential binding pockets (sites 1 and 2) of the seven-transmembrane receptor, AdipoR1, anchored in a virtual 1-palmitoyl-2-oleoyl-phosphatidylcholine membrane. In conclusion, we demonstrated the antidiabetic function of the Tyr-Pro dipeptide as a possible AdipoR1 agonist.

Accumulation of Plasma-Derived Lipids in the Lipid Core and Necrotic Core of Human Atheroma: Imaging Mass Spectrometry and Histopathological Analyses.

Objective: To clarify the pathogenesis of human atheroma, the origin of deposited lipids, the developmental mechanism of liponecrotic tissue, and the significance of the oxidation of phospholipids were investigated using mass spectrometry-aided imaging and immunohistochemistry.Atherosclerotic lesions in human coronary arteries were divided into 3 groups: pathologic intimal thickening with lipid pool, atheroma with lipid core, and atheroma with necrotic core. The lipid pool and lipid core were characterized by the deposition of extracellular lipids. The necrotic core comprised extracellular lipids and liponecrotic tissue. The proportion of cholesteryl linoleate in cholesteryl linoleate+cholesteryl oleate fraction in the extracellular lipid and liponecrotic regions differed significantly from that of the macrophage foam cell-dominant region, and the plasma-derived components (apolipoprotein B and fibrinogen) were localized in the regions. The liponecrotic region was devoid of elastic and collagen fibers and accompanied by macrophage infiltration in the surrounding tissue. Non-oxidized phospholipid (Non-OxPL), OxPL, and Mox macrophages were detected in the three lesions. In the atheroma with lipid core and atheroma with necrotic core, non-OxPL tended to localize in the superficial layer, whereas OxPL was distributed evenly. Mox macrophages were colocalized with OxPL epitopes.In human atherosclerosis, plasma-derived lipids accumulate to form the lipid pool of pathologic intimal thickening, lipid core of atheroma with lipid core, and necrotic core of atheroma with necrotic core. The liponecrotic tissue in the necrotic core appears to be developed by the loss of elastic and collagen fibers. Non-OxPL in the accumulated lipids is oxidized to form OxPL, which may contribute to the lesion development through Mox macrophages.

Characteristics of Electrospray-Ionization Detection of Synthetic Di- to Penta-Oligopeptides by Amine Derivatizations.

Chemical derivatizations have been extensively developed for highly sensitive detection of bioactive small peptides; however, their advantages from the viewpoint of longer oligopeptides remain unverified. In this study, electrospray-ionization (ESI)-mass spectrometric (MS) detection of synthetic di- to pentapeptides consisting of glycine and sarcosine were characterized by four amine derivatization methods. It was concluded that the ESI-MS detection of di- to pentapeptides was characterized by the molecular surface area of derivatized peptide moieties with an optimal value of 250 - 300 Å2, regardless of hydrophobicity and derivatization methods.

Optimization of prediction methods for risk assessment of pathogenic germline variants in the Japanese population.

Predicting pathogenic germline variants (PGVs) in breast cancer patients is important for selecting optimal therapeutics and implementing risk reduction strategies. However, PGV risk factors and the performance of prediction methods in the Japanese population remain unclear. We investigated clinicopathological risk factors using the Tyrer-Cuzick (TC) breast cancer risk evaluation tool to predict BRCA PGVs in unselected Japanese breast cancer patients (n = 1,995). Eleven breast cancer susceptibility genes were analyzed using target-capture sequencing in a previous study; the PGV prevalence in BRCA1, BRCA2, and PALB2 was 0.75%, 3.1%, and 0.45%, respectively. Significant associations were found between the presence of BRCA PGVs and early disease onset, number of familial cancer cases (up to third-degree relatives), triple-negative breast cancer patients under the age of 60, and ovarian cancer history (all P < .0001). In total, 816 patients (40.9%) satisfied the National Comprehensive Cancer Network (NCCN) guidelines for recommending multigene testing. The sensitivity and specificity of the NCCN criteria for discriminating PGV carriers from noncarriers were 71.3% and 60.7%, respectively. The TC model showed good discrimination for predicting BRCA PGVs (area under the curve, 0.75; 95% confidence interval, 0.69-0.81). Furthermore, use of the TC model with an optimized cutoff of TC score ≥0.16% in addition to the NCCN guidelines improved the predictive efficiency for high-risk groups (sensitivity, 77.2%; specificity, 54.8%; about 11 genes). Given the influence of ethnic differences on prediction, we consider that further studies are warranted to elucidate the role of environmental and genetic factors for realizing precise prediction.

Matrix-assisted laser desorption/ionization mass spectrometry-guided visualization analysis of intestinal absorption of acylated anthocyanins in Sprague-Dawley rats.

It is unknown whether intestinal absorption of acylated anthocyanins occurs in their intact or metabolized form. In this study, with the aid of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging, intestinal absorption of acylated anthocyanins was visually investigated. Anthocyanin extracts from purple carrots were orally administered to Sprague-Dawley rats. Acylated cyanidins were absorbed into portal and circulating blood systems in their intact form, and aglycon; cyanidin 3-O-(6-O-feruloyl-ß-d-glucopyranosyl)-(1 â†’ 6)-[ß-d-xylopyranosyl-(1 â†’ 2)]-ß-d-galactopyranoside (Cy3XFGG), and showed a high absorption of 39.3 ± 0.1 pmol/mL-plasma at 60 min after administration. MALDI-MS imaging analysis of the rat jejunum membranes showed that an organic anion transporting polypeptide (OATP) transporter was involved in Cy3XFGG transport, while deacylated anthocyanins were incorporated through both the glucose transporter 2 and OATP routes. In conclusion, acylated anthocyanin, Cy3XFGG, can be absorbed in its intact form through intestinal OATP.

Identification of peptides in blood following oral administration of ß-conglycinin to Wistar rats.

In this study, ß-conglycinin (100 mg/kg) was orally administered to Wistar rats in order to identify peptides that may be derived from the protein in the blood. Plasma samples taken from the tail vein up to 8 h after administration were analyzed by matrix-assisted laser desorption/ionization (MALDI) and liquid chromatography-time-of-flight (LC-TOF) mass spectrometry (MS). In total, 126 signals were detected by MALDI-MS. Among the signals, nine oligopeptides (SEL, KGPL, SILGA, DSEL, GDANI, SYFV, CLQSC, GEQPRPF, and LVINEGDA) were successfully identified as ß-conglycinin-derived peptides by LC-TOF/MS at a plasma concentration of 0.75-756 pmol/mL. The results demonstrated that ß-conglycinin could be the dietary source protein for the oligopeptides produced prior to entering the circulating bloodstream of rats.

Identification of characteristic compounds of moderate volatility in breast cancer cell lines.

In this study, we were challenging to identify characteristic compounds in breast cancer cell lines. GC analysis of extracts from the culture media of breast cancer cell lines (MCF-7, SK-BR-3, and YMB-1) using a solid-phase Porapak Q extraction revealed that two compounds of moderate volatility, 1-hexadecanol and 5-(Z)-dodecenoic acid, were detected with markedly higher amount than those in the medium of fibroblast cell line (KMST-6). Furthermore, LC-TOF/MS analysis of the extracts clarified that in addition to the above two fatty acids, the amounts of five unsaturated fatty acids [decenoic acid (C10:1), decadienoic acid (C10:2), 5-(Z)-dodecenoic acid (C12:1), 5-(Z)-tetradecenoic acid (C14:1), and tetradecadienoic acid (C14:2)] in MCF-7 medium were higher than those in medium of KMST-6. Interestingly, H2O2-oxidation of 5-(Z)-dodecenoic acid and 5-(Z)-tetradecenoic acid produced volatile aldehydes that were reported as specific volatiles in breath from various cancer patients, such as heptanal, octanal, nonanal, decanal, 2-(E)-nonenal, and 2-(E)-octenal. Thus, we concluded that these identified compounds over-produced in breast cancer cells in this study could serve as potential precursors producing reported cancer-specific volatiles.

Omental herniation through the esophageal hiatus: A rare cause of gastric outlet obstruction and its CT findings.

A man in his 60s visited the emergency department because of epigastric pain and vomiting. Noncontrast helical abdominal CT revealed a lipomatous mass on the right side of the lower esophagus. Caudal slices showed that the mass appeared to displace the gastric antrum causing gastric outlet obstruction. Further observation revealed a linear structure from the greater curvature of the stomach into the omental vessels. Based on these findings, a diagnosis of intrathoracic omental hernia through the esophageal hiatus was made. Intraoperatively, a defect in the lesser omentum was seen, and the greater omentum herniated through the defect into the esophageal hiatus. Omental herniation through the lesser omentum into the esophageal hiatus can present as gastric outlet obstruction.

Novel in situ visualisation of rat intestinal absorption of polyphenols via matrix-assisted laser desorption/ionisation mass spectrometry imaging.

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is presently used in physiological evaluations for visualisation of targets in organs. In the present study, MALDI-MSI was used as a visualisation technique to investigate the intestinal absorption of polyphenols. Nifedipine/phytic acid-aided MALDI-MSI was performed to visualise theaflavin-3'-O-gallate (TF3'G) and epicatechin-3-O-gallate (ECG) in the rat jejunum for 50-µM, 60-min transport experiments. Non-absorbable TF3'G was successfully visualised at the apical region, whereas absorbable ECG was detected throughout the rat jejunum. MALDI-MSI was also performed to determine the transport routes of the target metabolites. Signals corresponding to TF3'G and ECG in the membranes were diminished following treatment with inhibitors targeting the monocarboxylic acid transporter and organic anion transporting polypeptides. Enhanced visualisation of TF3'G was achieved by inhibiting efflux routes. Our findings demonstrated that the present MALDI-MSI can provide critical spatial informations on intestinal absorption of targets, by which TF3'G and ECG were incorporated into intestinal tissues, followed by efflux back to the apical compartment. In addition, MALDI-MSI analyses suggested that TF3'G was resistant to phase II metabolism during the influx/efflux processes, whereas ECG was susceptible to methylation and sulphation reactions. In conclusion, inhibitor-aided MALDI-MSI could serve as a powerful in situ visualisation technique for verifying intestinal transport routes and investigating the metabolism of penetrants.

[Research of the Cancer-Odor].

Early detection and resection of cancer is the most effective in the treatment of solid cancer. Development of a new cancer detection method is expected to become a breakthrough to solve various problems for early detection. It has been reported that there is the specific odors of cancer by using bio olfaction such as dogs, and it has been recognized that there is the odors of cancer. Cancer cells acquire malignant traits as a result of metabolic changes originating from genetic mutation. The cancer specific odorous substances may be considered to be the end products of their metabolic changes. Omics researches such as genomics, proteomics, and metabolomics have been extensively performed to comprehensively analyze changes in DNA, RNA, protein, metabolism and its products specific to cancer for the purpose of developing a new cancer detection marker. It is thought that the research on the odor of cancer is also on the line of omics research. It is difficult to identify cancer-specific odorants buried in various environmental substances. However, it is expected that human will be able to acquire the technology, from the fact that they can be recognized by biological olfaction. We are continuing the research with the dream that identification of the odorous substances as a new cancer detection marker and sensor development for it will lead to the happiness of colleagues in the world.

Seasoning ingredients in a medium-fat diet regulate lipid metabolism in peripheral tissues via the hypothalamic-pituitary axis in growing rats.

We fed rats noodle (N) -diet containing 30 wt.% instant noodle with a 26% fat-to-energy ratio for 30 days (N-group). Compared with rats that were fed the same amount of nutrients (C-group), the N-group showed lower liver triacylglycerol levels and higher fecal cholesterol levels. We then analyzed transcriptome of the hypothalamic-pituitary (HP), the liver and the white adipose tissue (WAT). Thyroid stimulating hormone (Tshb), and its partner, glycoprotein hormone genes were up-regulated in the HP of N-group. Sterol regulatory element binding transcription factors were activated in the liver of N-group, while an up-regulation of the angiogenic signal occurred in the WAT of N-group. N-group showed higher urine noradrenaline (NA) level suggesting that these tissue signals are regulated by NA and Tshb. The N-diet contains 0.326 wt.% glutamate, 0.00236 wt.% 6-shogaol and Maillard reaction products. Our results suggest that these ingredients may affect lipid homeostasis via the HP axis.

Effect of Aging on the Absorption of Small Peptides in Spontaneously Hypertensive Rats.

In the present study, we aimed to evaluate the effect of aging on the absorption of small peptides in spontaneously hypertensive rats (SHRs). Three kinds of dipeptides, glycyl-sarcosine (Gly-Sar), Trp-His, and captopril (a dipeptidomimetic drug), a Gly-Sar-Sar tripeptide, a Gly-Sar-Sar-Sar tetrapeptide, and a Gly-Sar-Sar-Sar-Sar pentapeptide were administered at doses of 10 mg/kg each to 8- and 40-week-old SHRs. The peptides were all detected in their intact forms in the blood. There was a significantly promoted absorption of di/tripeptides in aged SHRs compared with young SHRs. In contrast, the absorption of tetra/pentapeptides was not affected by aging. PepT1 expression in the mid-jejunum was significantly increased in 40-week-old SHRs compared with 8-week-old SHRs, whereas aging did not alter the expression of claudin-1, a tight junction related protein. Thus, the present results suggest that SHR aging may enhance the absorption of di/tripeptides through the enhanced PepT1 transport route, although oligopeptides may be absorbed in an age-independent manner.

[Validation of Whole Slide Imaging for Primary Diagnosis of Gastrointestinal Pathology].

Whole-slide imaging (WSI) technology enables the primary diagnosis of histopathological slides. This study aimed to determine the diagnostic concordance between pathological interpretations made using WSI and those made using light microscopy (LM). For this study, 5,704 consecutive surgical pathological cases from a community hospital were included. The specimens were digitized at x40 magnification for biopsy and endoscopic resection specimens or at x20 magnification for other specimens and evaluated by 11 pathologists for diagnosis using WSI. Subsequently, the specimens were signed out using LM by 3 pathologists after 2 weeks. Diagnoses using WSI were then compared with the diagnoses made by using LM. Most (96.8%) of the 5,704 specimens were obtained from the gastrointestinal tract (2,441 biopsy specimens from the esophagogastroduodenum [42.7%], 1,678 endoscopic resection specimens from the colorectum [29.4%], 1,349 biopsy specimens from the colorectum [23.6%], 133 resected specimens from the gallbladder [2.3%], 56 endoscopic resection specimens from the stomach [0.9%], 30 resected specimens from the ap- pendix [0.5%], 14 skin biopsy specimens [0.2%], and 3 other specimens [0.1%]). The overall concordance between the diagnoses made using WSI and those made using LM was 95.1%. The major and minor dis- crepancy rates for WSI were 0.1% and 4.8%, respectively. None of the discordant cases had any clinical or prognostic implications. In conclusion, this study revealed that WSI can be used for primary diagnosis of gastrointestinal biopsy and endoscopic resection specimens. To the best of our knowledge, this is one of the studies that clearly proved that diagnosis using WSI is equivalent to diagnosis using LM. [Original].

The photobase generator nifedipine as a novel matrix for the detection of polyphenols in matrix-assisted laser desorption/ionization mass spectrometry.

Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is widely used for the detection and analysis of ionizable compounds. However, the method has less potential for the analysis of neutral compounds, such as polyphenols, owing to their lack of favorable proton-attachment or -removal groups. In this study, we reported for the first time that nifedipine (2,6-dimethyl-3,5-dicarbomethoxy-4-(2-nitrophenyl)-1,4-dihydropyridine), which is a strong photobase generator commonly used in polymerization, can abstract protons from neutral compounds in negative mode-MALDI experiments. When nifedipine (5 mg/ml) was used as a matrix reagent, the limit of detection (LOD) for epigallocatechin-3-O-gallate (EGCG) was determined to be 100 fmol/spot, which constitutes >50-fold improvement compared to the LOD obtained when trans-3-indoleacrylic acid, a matrix reagent previously reported for polyphenol detection, was used. Of the dihydropyridines investigated, only nifedipine facilitated the detection of EGCG, suggesting that the nitrosophenyl pyridine derivative of nifedipine formed by photoreduction under laser irradiation at 355 nm plays a crucial role in detecting polyphenols in negative mode. Reduced MS detection of 5-O-methylnaringenin indicated that nifedipine may preferably remove a proton from the 5-position OH group in the A ring of the flavonoid skeleton. The significant MS detection by nifedipine was extensively observed for polyphenols including flavones, flavonones, chalcones, stilbenoids and phenolic acids. In conclusion, nifedipine can act as a novel matrix for improving polyphenol detection by MALDI-MS in negative mode. Copyright © 2016 John Wiley & Sons, Ltd.

Identification of peptides in wheat germ hydrolysate that demonstrate calmodulin-dependent protein kinase II inhibitory activity.

Hydrolysis of wheat germ by proteases resulted in bioactive peptides that demonstrated an inhibitory effect against the vasoconstrictive Ca(2+)-calmodulin (CaM)-dependent protein kinase II (CaMK II). The hydrolysate by thermolysin (1.0wt%, 5h) showed a particularly potent CaMK II inhibition. As a result of mixed mode high-performance liquid chromatography of thermolysin hydrolysate with pH elution gradient ranging between 4.8 and 8.9, the fraction eluted at pH 8.9 was the most potent CaMK II inhibitor. From this fraction, Trp-Val and Trp-Ile were identified as CaMK II inhibitors. In Sprague-Dawley rats, an enhanced aortic CaMK II activity by 1µM phenylephrine was significantly (p<0.05) suppressed by 15-min incubation with 300µM Trp-Val or Trp-Ile. On the basis of Ca(2+)-chelating fluorescence and CaMK II activity assays, it was concluded that Trp-Val and Trp-Ile competed with Ca(2+)-CaM complex to bind to CaMK II with Ki values of 5.4 and 3.6µM, respectively.

Adenine attenuates the Ca(2+) contraction-signaling pathway via adenine receptor-mediated signaling in rat vascular smooth muscle cells.

Our previous study demonstrated that adenine (6-amino-6H-purine) relaxed contracted rat aorta rings in an endothelial-independent manner. Although adenine receptors (AdeRs) are expressed in diverse tissues, aortic AdeR expression has not been ascertained. Thus, the aims of this study were to clarify the expression of AdeR in rat vascular smooth muscle cells (VSMCs) and to investigate the adenine-induced vasorelaxation mechanism(s). VSMCs were isolated from 8-week-old male Wistar-Kyoto rats and used in this study. Phosphorylation of myosin light chain (p-MLC) was measured by western blot. AdeR mRNA was detected by RT-PCR. Intracellular Ca(2+) concentration ([Ca(2+)]i) was measured by using Fura-2/AM. Vasorelaxant adenine (10-100 µM) significantly reduced p-MLC by angiotensin II (Ang II, 10 µM) in VSMCs (P < 0.05). We confirmed the expression of aortic AdeR mRNA and the activation of PKA in VSMCs through stimulation of AdeR by adenine by ELISA. Intracellular Ca(2+) concentration ([Ca(2+)]i) measurement demonstrated that adenine inhibits Ang II- and m-3M3FBS (PLC agonist)-induced [Ca(2+)]i elevation. In AdeR-knockdown VSMCs, PKA activation and p-MLC reduction by adenine were completely abolished. These results firstly demonstrated that vasorelaxant adenine can suppress Ca(2+) contraction signaling pathways via aortic AdeR/PKA activation in VSMCs.

Ferulic acid enhances nitric oxide production through up-regulation of argininosuccinate synthase in inflammatory human endothelial cells.

AIM: In this study, we investigated the protective effect of ferulic acid (FA) on nitric oxide (NO) production in tumor necrosis factor (TNF)-α-stimulated inflammatory human umbilical vein endothelial cells (HUVECs), and elucidated the mechanism(s) involved. MAIN METHODS: The TNF-α-stimulated inflammatory HUVECs were treated with acetylcholine (ACh) and/or FA. NO productions were measured by monitoring nitrite and nitrate using a 2,3-diaminonaphthalene Kit. Expressions of mRNA and proteins were evaluated by RT-PCR and Western blotting, respectively. KEY FINDINGS: FA treatment resulted in a dose-dependent (10-200µM) restoration of ACh-mediated NO production in TNF-α-treated HUVECs, whereas treatment with the FA analogues, coumaric acid, and apocynin resulted in no significant effect. FA treatment had no effect on O2(-) production in TNF-α-stimulated HUVECs. N(G)-monomethyl-l-arginine acetate (a nitric oxide synthase (NOS) inhibitor) and α-methyl-dl-aspartic acid (an argininosuccinate synthase (ASS) inhibitor) counteracted the effects of FA on the NO production. While FA treatment did not significantly affect the protein expression of p-eNOS or eNOS, the protein expression of ASS as well as mRNA expression was restored to normal levels upon exposure to FA in TNF-α-stimulated HUVECs. In nucleus, FA attenuated the increase of nuclear factor-kappa B (NF-κB) expression by TNF-α. SIGNIFICANCE: FA treatment rescues the defect in ACh-induced NO production resulting from TNF-α-stimulation in inflammatory HUVECs. This effect was likely due, in part, to the FA-mediated up-regulation of ASS expression via the suppression of NF-κB inflammatory signaling cascade.