Revealing salt concentration for microbial balance and metabolite enrichment in secondary fortified fermented soy sauce: A multi-omics perspective.

This study examined the impact of varying salt concentrations on microbiota, physicochemical properties, and metabolites in a secondary fortified fermentation process using multi-omics techniques. It aimed to determine the influence of salt stress on microbiota shifts and metabolic activities. The findings demonstrated that moderate salt reduction (MS) was found to enhance moromi's flavor and quality, while mitigating the negative effects of excessive low salt (LS). MS samples had 1.22, 1.13, and 2.92 times more amino acid nitrogen (AAN), non-volatiles, and volatiles, respectively, than high salt (HS) samples. In contrast, lactic acid and biogenic amines in LS samples were 1.56 g/100 g and 4115.11 mg/kg, respectively, decreasing to 0.15 g/100 g and 176.76 mg/kg in MS samples. Additionally, the contents of ethanol and small peptides increased in MS due to the growth of specific functional microorganisms such as Staphylococcus gallinarum, Weissella confusa, and Zygosaccharomyces rouxii, while food-borne pathogens were inhibited. Network analysis revealed that the core microbial interactions were enhanced in MS samples, promoting a balanced fermentation environment. Redundancy analysis (RDA) and correlation analyses underscored that the physicochemical properties significantly impacted bacterial community structure and the correlations between key microbes and flavor compounds. These findings provided a theoretical foundation for developing innovative reduced-salt fermentation techniques, contributing to the sustainable production of high-quality soy sauce.

Efficacy of freeze-chilled storage combined with tea polyphenol for controlling melanosis, quality deterioration, and spoilage bacterial growth of Pacific white shrimp (Litopenaeus vannamei).

This study aimed to investigate melanosis, quality attributes, and bacterial growth of freeze-chilled Pacific white shrimp (Litopenaeus vannamei) during 6 days of chilled storage, as well as the preservative effects of tea polyphenol on shrimp. The results showed that freeze-chilled storage retarded the growth of bacteria and the accumulation of putrescine in shrimp. The growth of spoilage bacteria Photobacterium and Shewanella were inhibited. However, freeze-chilled storage aggravated melanosis and lipid oxidation. The total volatile basic nitrogen (TVB-N) slightly accumulated in the thawed shrimp. The incorporation of tea polyphenol preserved freeze-chilled shrimp. Melanosis and lipid oxidation of shrimp were alleviated. The accumulation of biogenic amines, TVB-N, hypoxanthine riboside, and hypoxanthine were retarded. Meanwhile, the growth of spoilage bacteria Pseudoalteromonas, Photobacterium, Psychrobacter, and Carnobacterium were inhibited. Based on sensory analysis, the shelf-life of chilled, freeze-chilled, and freeze-chilled tea polyphenol shrimp were 4 days, 3 days, and 6 days, respectively.

A strategy of ultrasound-assisted processing to improve the performance of bio-based coating preservation for refrigerated carp fillets (Ctenopharyngodon idellus).

Effects of ultrasound-assisted chitooligosaccharides (COS-UA) coating on the quality attributes and microbial composition of refrigerated grass carp fillets were evaluated. The results showed that COS and COS-UA coatings retarded quality deterioration of fillets during storage. Compared to COS coatings, COS-UA treated samples had lower contents of BAs, simultaneously their levels of total volatile base nitrogen (TVB-N), K value and total viable counts (TVC) were further decreased by 13.6%, 4.2% and 7.8% on day 12, respectively. High-throughput sequencing showed that Aeromonas and Shewanella increased rapidly in control samples and became the main microbiota at day 12. By contrast, both coatings changed the microbial composition and reduced the proportion of spoilage organisms. Based on multiple evaluations, COS-UA extended shelf life of fillets by another 2 days when compared to COS. Therefore, ultrasonic treatment could be considered as an effective supplementary to improve the preservation effect of COS-based coatings for fresh preprocessed fish.

Effects of ethyl lauroyl arginate hydrochloride on microbiota, quality and biochemical changes of container-cultured largemouth bass (Micropterus salmonides) fillets during storage at 4 °C.

This study aimed to investigate preservative effects of ethyl lauroyl arginate hydrochloride (LAE) on microbiota, quality, and physiochemical changes of container-cultured largemouth bass fillets stored at 4 °C. The results showed LAE treatment was effective in reducing bacterial growth and attenuating physiochemical changes (flesh color, trichloroacetic acid (TCA)-soluble peptides, total volatile basic nitrogen (TVB-N), ammonia concentration, and biogenic amines) of bass fillets, while had relatively weak effect on the degradation of ATP-related compounds. As a result, LAE treatment retarded the deterioration of sensory attributes, and thus prolonged the shelf-life of largemouth bass fillets for 4 days. In addition, LAE treatment decreased the relative abundance of Pseudomonas in bass fillets, and thus changed the microbial composition. Moreover, correlation analysis between physiochemical changes and bacterial genera showed that Pseudomonas was well correlated with TCA-soluble peptides, TVB-N, ammonia, putrescine and histamine, while Aeromonas tended to have strong potentials in producing ammonia and cadaverine.

Effects of onion or caraway on the formation of biogenic amines during sauerkraut fermentation and refrigerated storage.

The effects of onion or caraway on changes in the content of biogenic amines were examined in sauerkraut during a fermentation process at 18 °C or 31 °C for 14 days and subsequent storage at 4 °C for 12 weeks. The amines were analysed by high-performance liquid chromatography with pre-column benzoylation. Total biogenic-amine concentration at the end of the fermentation was lower at 31 °C than at 18 °C. However, at this lower temperature, the presence of caraway or onion more significantly (than at 31 °C) reduced the total biogenic-amine content as compared to the control sample without an additive. After 12 weeks of refrigerated storage, concentrations of phenethylamine, tryptamine, and tyramine in the sauerkraut fermented with caraway (and concentrations of putrescine and tryptamine in the sauerkraut fermented with onion) at 31 °C increased as compared to the samples on the last day of fermentation, but did not pose a risk for consumer health.

Characterization of the microbial composition and quality of lightly salted grass carp (Ctenopharyngodon idellus) fillets with vacuum or modified atmosphere packaging.

The effect of air packaging (AP), vacuum packaging (VP) and modified atmosphere packaging (MAP, 75% CO2/25% N2) on the composition of the microbial community of lightly salted grass carp (Ctenopharyngodon idellus) fillets stored at 4 °C was studied. Physicochemical characteristics (total volatile basic nitrogen (TVB-N) value, pH value and biogenic amines (BA)) were also monitored. Based on sensory evaluation, the shelf life of fillets under AP, VP, and MAP was 8, 16 and 24 days, respectively. High-throughput sequencing showed that Acinetobacter and Pseudomonas dominated the microflora of fresh grass carp. At the sensory rejection time, Pseudomonas and Psychrobacter dominated the AP products. Lactococcus became the predominant genus of both VP and MAP fillets, and there was no obvious difference in the composition of the dominant microbiota between these two treatments at the end of the shelf life.

Highly selective colorimetric detection of putrescine in fish products using o-phthalaldehyde derivatization reaction.

A highly selective colorimetric detection method for putrescine on the basis of an optimized derivatization reaction was established. With the aids of o-phthalaldehyde (OPA) and thioglycolic acid (TGA), putrescine was derived to become red derivatives (PUT-RD), the form that can be detected qualitatively and quantitatively by visual inspection and UV-vis spectrophotometer at λmax of 490 nm, respectively. Key parameters affecting the experiment were investigated by one-factor-at-a-time and response surface analysis. At the optimal condition, this colorimetric method achieved good linearity at putrescine concentrations ranging from 0.8 to 200 µM with detection limit of 0.44 µM. The method also has good selectivity when common amino acids and inorganic ions, as well as ethylenediamine, 1,3-propanediamine, 1,5-pentanediamine, and 1,6-hexanediamine were used as interferences. The established colorimetric method was successfully employed for the detection of putrescine in 10 commercial fish products.

The tumor-suppressor cholesterol metabolite, dendrogenin A, is a new class of LXR modulator activating lethal autophagy in cancers.

Dendrogenin A (DDA) is a mammalian cholesterol metabolite recently identified that displays tumor suppressor properties. The discovery of DDA has revealed the existence in mammals of a new metabolic branch in the cholesterol pathway centered on 5,6α-epoxycholesterol and bridging cholesterol metabolism with histamine metabolism. Metabolic studies showed a drop in DDA levels in cancer cells and tumors compared to normal cells, suggesting a link between DDA metabolism deregulation and oncogenesis. Importantly, complementation of cancer cells with DDA induced 1) cancer cell re-differentiation, 2) blockade of 6-oxo-cholestan-3ß,5α-diol (OCDO) production, an endogenous tumor promoter and 3) lethal autophagy in tumors. Importantly, by binding the liver X receptor (LXR), DDA activates the expression of genes controlling autophagy. These genes include NR4A1, NR4A3, LC3 and TFEB. The canonical LXR ligands 22(R)hydroxycholesterol, TO901317 and GW3965 did not induce these effects indicating that DDA delineates a new class of selective LXR modulator (SLiM). The induction of lethal autophagy by DDA was associated with the accumulation in cancer cells of lysosomes and of the pro-lysosomal cholesterol precursor zymostenol due to the inhibition of the 3ß-hydroxysteroid-Δ8Δ7-isomerase enzyme (D8D7I). The anti-cancer efficacy of DDA was established on different mouse and human cancers such as breast cancers, melanoma and acute myeloid leukemia, including patient derived xenografts, and did not discriminate bulk cancer cells from cancer cell progenitors. Together these data highlight that the mammalian metabolite DDA is a promising anticancer compound with a broad range of anticancer applications. In addition, DDA and LXR are new actors in the transcriptional control of autophagy and DDA being a "first in line" driver of lethal autophagy in cancers via the LXR.

Improving the efficacy of hormone therapy in breast cancer: The role of cholesterol metabolism in SERM-mediated autophagy, cell differentiation and death.

Breast cancer (BC) is one of the most common female cancers in the world, with estrogen receptor (ER)-positive BC the most frequent subtype. Tamoxifen (Tam) is an effective drug that competitively binds to the ER and is routinely used for the treatment of ER-positive BC. However, a number of ER-positive BC do not respond to Tam treatment and acquired resistance is often observed, constituting a major challenge for extending patient life expectancy. The mechanisms responsible for these treatment failures remain unclear, indicating the requirement for other targets and better predictors for patient response to Tam. One of Tam's off-targets of interest is the microsomal antiestrogen binding site (AEBS), a multiproteic complex made up of the cholesterol-5,6-epoxide hydrolase (ChEH) enzymes that are involved in the late stages of cholesterol biosynthesis. Tam and other selective ER modulators stimulate oxidative stress and inhibit the ChEH subunits at pharmacological doses, triggering the production and accumulation of cholesterol-5,6-epoxide metabolites responsible for BC cell differentiation and death. However, inhibition of the cholesterogenic activity of the AEBS subunits also induces the accumulation of sterol precursors, which triggers a survival autophagy to impair Tam's efficacy. Altogether, these studies have highlighted the involvement of cholesterol metabolism in the pharmacology of Tam that has provided new clues on how to improve its therapeutic efficacy in both BC and other cancers as well as offering a new rationale for developing more efficient drugs for BC treatment.

Relaxation of isolated guinea-pig trachea by apigenin, a constituent of celery, via inhibition of phosphodiesterase.

Apigenin, was reported to have vasodilatory effects by inhibiting Ca2+ influx through both voltage- and receptor-operated calcium channels, but not by inhibiting cAMP- or cGMP-phosphodiesterases (PDEs) in rat thoracic aorta. However, apigenin was reported to inhibit PDE1, 2 and 3 in guinea-pig lung and heart. The aim of this study was to clarify that guinea-pig tracheal relaxation by apigenin whether via PDE inhibition. We isometrically recorded the tension of isolated guinea-pig tracheal segments on a polygraph. Antagonistic effects of apigenin against cumulative contractile agents or Ca2+ induced contractions of the trachealis in normal or isotonic high-K+, Ca2+-free Krebs solution, respectively. Effects of apigenin (15 and 30µM) on the cumulative forskolin- and nitroprusside-induced relaxations to histamine (30µM)-induced precontraction were performed. The inhibitory effects of 30-300µM apigenin and 3-isobutyl-1-methylxanthine (IBMX, positive control) on the cAMP- and cGMP-PDEs were determined. Apigenin concentration-dependently but non-competitively inhibited cumulative histamine-, carbachol- or Ca2+-induced contractions in normal or in the depolarized (K+, 60mM) trachealis, suggesting that Ca2+ influx through voltage-dependent calcium channels is inhibited. However, apigenin (15-30µM) parallel leftward shifted the concentration-response curves of forskolin and nitroprusside, and significantly increased the pD2 values of these two cyclase activators. Both apigenin and IBMX, a reference drug, concentration (10-300µM)-dependently and significantly, but non-selectively inhibited the activities of cAMP- and cGMP-PDEs in the trachealis. In conclusion, the relaxant effect of apigenin may be due to inhibition of both enzyme activities and reduction of intracellular Ca2+ by inhibiting Ca2+ influx in the trachealis.

Three-dimensional structure of Gly m 5 (ß-conglycinin) plays an important role in its stability and overall allergenicity.

To date, there is insufficient knowledge regarding the relationship of the allegenicity and the three-dimensional structure of Gly m 5 (ß-conglycinin), a major allergen in soybean. In the present study, allergen Gly m 5 was demonstrated to generate three major digestion-resistant fragments when it was subjected to in vitro digestion. The largest fragment corresponded to the main body of the monomer and two smaller fragments corresponded to the main bodies of two modules of the monomer. Two major protease cleavage sites were located in the regions near to the connection between two modules. Coincidentally, the major digestion-resistant fragments were demonstrated to contain intact IgE epitopes and be capable to induce basophil histamine release in Gly m 5 sensitised piglets, indicating that the three-dimensional structure of Gly m 5 afforded the molecule some protection from complete degradation into small peptides and amino acids, and contributed to its overall allergenicity.

Tuneable surface enhanced Raman spectroscopy hyphenated to chemically derivatized thin-layer chromatography plates for screening histamine in fish.

Reliable screening of histamine in fish was of urgent importance for food safety. This work presented a highly selective surface enhanced Raman spectroscopy (SERS) method mediated by thin-layer chromatography (TLC), which was tailored for identification and quantitation of histamine. Following separation and derivatization with fluram, plates were assayed with SERS, jointly using silver nanoparticle and NaCl. The latter dramatically suppressed the masking effect caused by excessive fluram throughout the plate, thus offering clear baseline and intensive Raman fingerprints specific to the analyte. Under optimized conditions, the usability of this method was validated by identifying the structural fingerprints of both targeted and unknown compounds in fish samples. Meanwhile, the quantitative results of this method agreed with those by an HPLC method officially suggested by EU for histamine determination. Showing remarkable cost-efficiency and user-friendliness, this facile TLC-SERS method was indeed screening-oriented and may be more attractive to controlling laboratories of limited resource.

Effect of cinnamon essential oil on bacterial diversity and shelf-life in vacuum-packaged common carp (Cyprinus carpio) during refrigerated storage.

The present study investigated the effect of cinnamon essential oil on the quality of vacuum-packaged common carp (Cyprinus carpio) fillets stored at 4±1°C in terms of sensory scores, physicochemical characteristics (total volatile basic nitrogen (TVB-N), biogenic amines, and color), and presence of spoilage microbiota. A total of 290,753 bacterial sequences and 162 different genera belonging to 14 phyla were observed by a high-throughput sequencing technique targeting the V3-V4 region of 16S rDNA, which showed a more comprehensive estimate of microbial diversity in carp samples compared with microbial enumeration. Before storage, Macrococcus and Aeromonas were the prevalent populations in the control samples, but cinnamon essential oil decreased the relative abundance of Macrococcus in the treated samples. Variability in the predominant microbiota in different samples during chilled storage was observed. Aeromonas followed by Lactococcus were the major contaminants in the spoiled control samples. Microbial enumeration also observed relatively higher counts of Aeromonas than other spoilage microorganisms. Compared with the control samples, cinnamon essential oil inhibited the growth of Aeromonas and Lactococcus were the predominant components in the treated samples on day 10; plate counts also revealed a relatively high level of lactic acid bacteria during refrigerated storage. However, there were no significant differences (P>0.05) in the composition of dominant microbiota between these two treatments at the end of the shelf-life. Furthermore, cinnamon essential oil treatment was more effective in inhibiting the increase of TVB-N and the accumulation of biogenic amines (especially for putrescine and cadaverine levels). Based primarily on sensory analysis, the use of cinnamon essential oil extended the shelf-life of vacuum-packaged common carp fillets by about 2days.

A UHPLC method for the simultaneous analysis of biogenic amines, amino acids and ammonium ions in beer.

This paper reports a novel UHPLC method for simultaneously quantifying nine biogenic amines, 21 amino acids, and ammonium ions, in beer. Precision values of standard curves slopes were lower than 3.4% and recovery was between 85% and 106%, indicating the absence of matrix effect. Linear calibration curves were obtained for analyte concentrations between two and four orders of magnitude (R(2)>0.996). Repeatability tests returned mean variations of 3.2% and 0.5% for beer and a standard solution, respectively. Sensitivity ranged between 0.03mg/L and 0.63mg/L for the biogenic amines, and 0.05mg/L and 5.19mg/L for other compounds. Original data on the habitual presence of ethanolamine in beers are presented. The method allows for more samples to be assayed per unit time, it uses less solvent than other techniques and therefore reduces costs and the associated waste. It could be a valuable tool for monitoring the safety and quality of beers.

Histamine prevents radiation-induced mesenchymal changes in breast cancer cells.

Radiotherapy is a prime option for treatment of solid tumors including breast cancer though side effects are usually present. Experimental evidence shows an increase in invasiveness of several neoplastic cell types through conventional tumor irradiation. The induction of epithelial to mesenchymal transition is proposed as an underlying cause of metastasis triggered by gamma irradiation. Experiments were conducted to investigate the role of histamine on the ionizing radiation-induced epithelial to mesenchymal transition events in breast cancer cells with different invasive phenotype. We also evaluated the potential involvement of Src phosphorylation in the migratory capability of irradiated cells upon histamine treatment. MCF-7 and MDA-MB-231 mammary tumor cells were exposed to a single dose of 2Gy of gamma radiation and five days after irradiation mesenchymal-like phenotypic changes were observed by optical microscope. The expression and subcellular localization of E-cadherin, ß-catenin, vimentin and Slug were determined by immunoblot and indirect immunofluorescence. There was a decrease in the epithelial marker E-cadherin expression and an increase in the mesenchymal marker vimentin after irradiation. E-cadherin and ß-catenin were mainly localized in cytoplasm. Slug positive nuclei, matrix metalloproteinase-2 activity and cell migration and invasion were significantly increased. In addition, a significant enhancement in Src phosphorylation/activation could be determined by immunoblot in irradiated cells. MCF-7 and MDA-MB-231 cells also received 1 or 20µM histamine during 24h previous to be irradiated. Notably, pre-treatment of breast cancer cells with 20µM histamine prevented the mesenchymal changes induced by ionizing radiation and also reduced the migratory behavior of irradiated cells decreasing phospho-Src levels. Collectively, our results suggest that histamine may block events related to epithelial to mesenchymal transition in irradiated mammary cancer cells and open a perspective for the potential use of histamine to improve radiotherapy efficacy.

Quality changes of salmon by-products during storage: Assessment and quantification by NMR.

Safe utilization of fish by-products is an important task due to increasing fish consumption. It can provide new valuable food/feed and will increase the economical profit and sustainability of the fishery industry. NMR spectroscopy is a reliable tool able to monitor qualitative and quantitative changes in by-products. In this work the trichloroacetic acid extracts of salmon backbones, heads and viscera stored at industrially relevant temperatures (4 and 10°C) were studied using NMR. Twenty-five metabolites were detected and the possibility of salmon by-products utilization as a source of anserine, phosphocreatine and taurine was discussed. Statistical data elaboration allowed determining the main processes occurring during by-products storage: formation of trimethylamine and biogenic amines, proteolysis and different types of fermentations. By-products freshness was evaluated using a multi-parameter approach: the trimethylamine and biogenic amines concentration changes were compared with Ki and H-values and safe temperatures and times for storage of salmon by-products were proposed.

Effects of different stunning methods on the flesh quality of grass carp (Ctenopharyngodon idellus) fillets stored at 4 °C.

The effects of stunning methods (percussion (T1), stunning at -22 °C in a freezer (T2), and immersion in ice slurry (T3)) on the flesh quality of grass carp fillets were evaluated in terms of K-value, total volatile base nitrogen (TVB-N), electrical conductivity (EC), total viable counts (TVC), biogenic amines, and sensory scores during storage. Moreover, fillets were analyzed periodically for pH, hardness, acid phosphatase activity, and the content of glycogen, adenosine triphosphate (ATP), and lactate. Significantly slower ATP depletion, slower reduction in pH, slower lactate formation, and higher initial glycogen level were observed in T3 compared to T1 and T2 (P<0.05). Grass carp stunned by percussion showed higher TVB-N, EC, TVC, total biogenic amines, and lower sensory scores. Significantly lower TVC (P<0.05) was exhibited in T3, which indicated that stunning in ice slurry could improve the quality and prolong the shelf life of grass carp.

Mast cells in rheumatic disease.

Rheumatoid Arthritis is a chronic autoimmune disease with a complex disease pathogenesis leading to inflammation and destruction of synovial tissue in the joint. Several molecules lead to activation of immune pathways, including autoantibodies, Toll-Like Receptor ligands and cytokines. These pathways can cooperate to create the pro-inflammatory environment that results in tissue destruction. Each of these pathways can activate mast cells, inducing the release of a variety of inflammatory mediators, and in combination can markedly enhance mast cell responses. Mast cell-derived cytokines, chemokines, and proteases have the potential to induce recruitment of other leukocytes able to evoke tissue remodeling or destruction. Likewise, mast cells can secrete a plethora of factors that can contribute to tissue remodeling and fibroblast activation. Although the functional role of mast cells in arthritis pathogenesis in mice is not yet elucidated, the increased numbers of mast cells and mast cell-specific mediators in synovial tissue of rheumatoid arthritis patients suggest that mast cell activation in rheumatoid arthritis may contribute to its pathogenesis.

Rapid and quantitative detection of 4(5)-methylimidazole in caramel colours: A novel fluorescent-based immunochromatographic assay.

A novel fluorescence-based immunochromatographic assay (ICA) for rapid detecting 4(5)-methylimidazole (4-MI) is presented in this study. In our work, the conjugates of fluorescent microspheres (FMs) and 4-MI monoclonal antibody were used as probe for ICA. Under optimal conditions, a standard curve of ICA-based detection of 4-MI was developed, linear detection ranged from 0.50 to 32.0 mg/L. The cross-reactivities were observed less than 3.93% by detecting 6 selected structural analogues of 4-MI. The recoveries of 4-MI in caramels detection were ranged from 82.85% to 102.31%, with the coefficient of variation (n = 3) below 9.06%. Quantitative comparison of the established fluorescence-based ICA with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) analysis of real caramel colour samples indicated a good correlation among the methods. Therefore, our developed fluorescence-based ICA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of 4-MI in food safety control.

Flavonoid galetin 3,6-dimethyl ether attenuates guinea pig ileum contraction through K(+) channel activation and decrease in cytosolic calcium concentration.

Flavonoid galetin 3,6-dimethyl ether (FGAL) has been isolated from the aerial parts of Piptadenia stipulaceae and has shown a spasmolytic effect in guinea pig ileum. Thus, we aimed to characterize its relaxant mechanism of action. FGAL exhibited a higher relaxant effect on ileum pre-contracted by histamine (EC50=1.9±0.4×10(-7) M) than by KCl (EC50=2.6±0.5×10(-6) M) or carbachol (EC50=1.8±0.4×10(-6) M). The flavonoid inhibited the cumulative contractions to histamine, as well as to CaCl2 in depolarizing medium nominally Ca(2+)-free. The flavonoid relaxed the ileum pre-contracted by S-(-)-Bay K8644 (EC50=9.5±1.9×10(-6) M) but less potently pre-contracted by KCl or histamine. CsCl attenuated the relaxant effect of FGAL (EC50=1.1±0.3×10(-6) M), but apamin or tetraethylammonium (1mM) had no effect (EC50=2.6±0.2×10(-7) and 1.6±0.3×10(-7) M, respectively), ruling out the involvement of small and big conductance Ca(2+)-activated K(+) channels (SKCa and BKCa, respectively). Either 4-aminopyridine or glibenclamide attenuated the relaxant effect of FGAL (EC50=1.8±0.2×10(-6) and 1.5±0.5×10(-6) M, respectively), indicating the involvement of voltage- and ATP-sensitive K(+) channels (KV and KATP, respectively). FGAL did not alter the viability of intestinal myocytes in the MTT assay and decreased (88%) Fluo-4 fluorescence, indicating a decrease in cytosolic Ca(2+) concentration. Therefore, the relaxant mechanism of FGAL involves pseudo-irreversible noncompetitive antagonism of histaminergic receptors, KV and KATP activation and blockade of CaV1, thus leading to a reduction in cytosolic Ca(2+) levels.