Effects of fatty acid hydroperoxides produced by lipoxygenase in wheat cultivars during dough preparation on volatile compound formation.

The formation of volatile compounds affects the flavor of processed wheat flour products. Herein, the effects of the composition of fatty acid hydroperoxides and the differences in the antioxidant contents among wheat cultivars on the flavor of wheat flour products were clarified. For this purpose, the volatile compounds in wheat flour doughs, LOX activity, fatty acid hydroperoxide composition from fractionated LOX, and antioxidant content were analyzed. Norin61 exhibited a high LOX activity and 9-fatty acid hydroperoxide production. Unsaturated aldehydes derived from 9-fatty acid hydroperoxides contributed significantly to the volatile compound profile of Norin61. Moreover, the lowest lutein content was observed in Norin61 among the analyzed cultivars. The LOX activity and composition of the fatty acid hydroperoxides produced by LOX affected the production of volatile compounds, whereas carotenoids had a suppressive effect. This study provides useful information for product design with the desired flavor for developing various processed wheat flour products.

Mouse TMC4 is involved in the detection of chloride taste of salts.

Licking behavior with various salts in transmembrane channel-like 4 (Tmc4) knockout (KO) mice was observed. In Tmc4 KO mice, a significant decrease in sensitivity to chloride salts, such as NaCl, KCl, and NH4Cl, was observed, while no significant decrease in sensitivity to Na-gluconate was observed. This finding suggests that TMC4 may be involved in the detection of chloride taste.

Mastication stimuli regulate the heartbeat rate through rhythmic regulation by the hypothalamic-autonomic system; molecular and telemetric studies in weaning-stage rats.

Mastication stimuli have been demonstrated to affect memory function and autonomic nerve activity; however, this process has not been well studied during weaning compared to old age. Previously, we conducted molecular analyses of the thalamus and hippocampus to elucidate the mechanisms underlying this memory-enhancing effect in weaning-stage rats. In this study, we aimed to evaluate the effect of masticatory stimuli on the regulation of heartbeat rate (HR) through the hypothalamic-autonomic system. Three-week-old male rats were administered a powdered diet (P group) or chow-diet (C group) for 10 days. Thereafter, transcriptome analysis was performed. Vasopressin, cocaine-amphetamine-regulated transcript prepropeptide, corticotropin-releasing hormone, and thyrotropin-releasing hormone, which are involved in sympathetic activation of heart rate, were downregulated in the C group. Electrocardiograms were recorded continuously for 12 days under the same condition. Interestingly, rats in the C group had a significantly lower HR than those in the P group on day 11. We checked several parameters representing the autonomic regulation of HR. The C group had higher values for the high-frequency band integration of the HR power spectrum (parasympathetic marker) and root mean square successive difference of R-wave intervals (parasympathetic marker) relative to the P group. Such findings provide a molecular and physiological basis for understanding the regulation of cardiovascular function in response to masticatory stimuli in the autonomic nervous system.

Genes related to cell wall metabolisms are targeted by miRNAs in immature tomato fruits under drought stress.

The metabolism of tomato fruits changes when plants experience drought stress. In this study, we investigated changes in microRNA (miRNA) abundance and detected 32 miRNAs whose expression changes in fruit. The candidate target genes for each miRNA were predicted from the differentially expressed genes identified by transcriptome analysis at the same fruit maturation stage. The predicted targeted genes were related to cell wall metabolisms, response to pathogens, and plant hormones. Among these, we focused on cell wall metabolism-related genes and performed a dual luciferase assay to assess the targeting of their mRNAs by their predicted miRNA. As a result, sly-miR10532 and sly-miR7981e suppress the expression of mRNAs of galacturonosyltransferase-10 like encoding the main enzyme of pectin biosynthesis, while sly-miR171b-5p targets ß-1,3-glucosidase mRNAs involved in glucan degradation. These results will allow the systematic characterization of miRNA and their target genes in the tomato fruit under drought stress conditions.

Molecular logic of salt taste reception in special reference to transmembrane channel-like 4 (TMC4).

The taste is biologically of intrinsic importance. It almost momentarily perceives environmental stimuli for better survival. In the early 2000s, research into taste reception was greatly developed with discovery of the receptors. However, the mechanism of salt taste reception is not fully elucidated yet and many questions still remain. At present, next-generation sequencing and genome-editing technologies are available which would become pivotal tools to elucidate the remaining issues. Here we review current mechanisms of salt taste reception in particular and characterize the properties of transmembrane channel-like 4 as a novel salt taste-related molecule that we found using these sophisticated tools.

Mastication stimuli enhance the learning ability of weaning-stage rats, altering the hippocampal neuron transcriptome and micromorphology.

Mastication stimuli are known to relieve senile dementia in human and animal studies. However, few studies have focused on its effect on weaning-stage animals and the underlying molecular processes. In this study, 3-week-old male rats were raised on a powdered (P-group) or chow (C-group) diet for 8 days, and their behavior was examined using the Y-maze and novel object recognition tests. In the Y-maze test, the C-group rats showed a larger alternation ratio than the P-group rats. In the novel object recognition test, the C-group rats exhibited a significantly larger discrimination index for novel objects than for familiar objects, but the P-group rats did not. We then compared the hippocampal neuron morphology and transcriptome between the groups. C-group rats exhibited larger dendrite branch numbers in the apical dendrites of pyramidal cells in the cornu ammonis 1 (CA1) region and a larger spine density in the basal dendrites of CA1 neurons than the P-group rats. Using DNA microarray analysis, we identified 621 (P < C) and 96 (P > C) genes that were differentially expressed between the groups. These genes were enriched in functional terms related to dendrite growth and included the Igf2, RhoA, and Rho GEF genes, most of which were upregulated in the C-group. These results suggest that the mastication stimuli during the weaning period can enhance the learning ability of rats by increasing the dendrite branches of hippocampal CA1 neurons and by regulating genes related to dendrite growth.

Rosmarinic Acid and Sodium Citrate Have a Synergistic Bacteriostatic Effect against Vibrio Species by Inhibiting Iron Uptake.

BACKGROUND: New strategies are needed to combat multidrug-resistant bacteria. The restriction of iron uptake by bacteria is a promising way to inhibit their growth. We aimed to suppress the growth of Vibrio bacterial species by inhibiting their ferric ion-binding protein (FbpA) using food components. METHODS: Twenty spices were selected for the screening of FbpA inhibitors. The candidate was applied to antibacterial tests, and the mechanism was further studied. RESULTS: An active compound, rosmarinic acid (RA), was screened out. RA binds competitively and more tightly than Fe3+ to VmFbpA, the FbpA from V. metschnikovii, with apparent KD values of 8 µM vs. 17 µM. Moreover, RA can inhibit the growth of V. metschnikovii to one-third of the control at 1000 µM. Interestingly, sodium citrate (SC) enhances the growth inhibition effect of RA, although SC only does not inhibit the growth. The combination of RA/SC completely inhibits the growth of not only V. metschnikovii at 100/100 µM but also the vibriosis-causative pathogens V. vulnificus and V. parahaemolyticus, at 100/100 and 1000/100 µM, respectively. However, RA/SC does not affect the growth of Escherichia coli. CONCLUSIONS: RA/SC is a potential bacteriostatic agent against Vibrio species while causing little damage to indigenous gastrointestinal bacteria.

Transmembrane channel-like 4 is involved in pH and temperature-dependent modulation of salty taste.

Human susceptibility to NaCl varies depending on temperature and pH, the molecular mechanisms of which remain unclear. The voltage-dependent chloride channel, transmembrane channel-like 4 (TMC4), is activated at approximately 40 °C and is suppressed at pH 5.5. As these are similar in character to human sensory evaluations, human TMC4 may be involved in human salt taste reception.

TMC4 is a novel chloride channel involved in high-concentration salt taste sensation.

"Salty taste" sensation is evoked when sodium and chloride ions are present together in the oral cavity. The presence of an epithelial cation channel that receives Na+ has previously been reported. However, no molecular entity involving Cl- receptors has been elucidated. We report the strong expression of transmembrane channel-like 4 (TMC4) in the circumvallate and foliate papillae projected to the glossopharyngeal nerve, mediating a high-concentration of NaCl. Electrophysiological analysis using HEK293T cells revealed that TMC4 was a voltage-dependent Cl- channel and the consequent currents were completely inhibited by NPPB, an anion channel blocker. TMC4 allowed permeation of organic anions including gluconate, but their current amplitudes at positive potentials were less than that of Cl-. Tmc4-deficient mice showed significantly weaker glossopharyngeal nerve response to high-concentration of NaCl than the wild-type littermates. These results indicated that TMC4 is a novel chloride channel that responds to high-concentration of NaCl.

Ibuprofen inhibits oral NaCl response through transmembrane channel-like 4.

Nonsteroidal anti-inflammatory drugs, such as ibuprofen, are known to modify salty taste perception in humans. However, the underlying molecular mechanisms remain unknown. We investigated the inhibitory effect of ibuprofen on the NaCl stimulation of epithelium sodium channel (ENaC) and transmembrane channel-like 4 (TMC4), which are involved in salty taste detection. Although ibuprofen only minimally inhibited the response of the ENaC to NaCl, it significantly inhibited the TMC4 response to NaCl with an IC50 at 1.45 mM. These results suggest that ibuprofen interferes with detection of salty taste via inhibition of TMC4.

De novo transcriptome analysis and comparative expression profiling of genes associated with the taste-modifying protein neoculin in Curculigo latifolia and Curculigo capitulata fruits.

BACKGROUND: Curculigo latifolia is a perennial plant endogenous to Southeast Asia whose fruits contain the taste-modifying protein neoculin, which binds to sweet receptors and makes sour fruits taste sweet. Although similar to snowdrop (Galanthus nivalis) agglutinin (GNA), which contains mannose-binding sites in its sequence and 3D structure, neoculin lacks such sites and has no lectin activity. Whether the fruits of C. latifolia and other Curculigo plants contain neoculin and/or GNA family members was unclear. RESULTS: Through de novo RNA-seq assembly of the fruits of C. latifolia and the related C. capitulata and detailed analysis of the expression patterns of neoculin and neoculin-like genes in both species, we assembled 85,697 transcripts from C. latifolia and 76,775 from C. capitulata using Trinity and annotated them using public databases. We identified 70,371 unigenes in C. latifolia and 63,704 in C. capitulata. In total, 38.6% of unigenes from C. latifolia and 42.6% from C. capitulata shared high similarity between the two species. We identified ten neoculin-related transcripts in C. latifolia and 15 in C. capitulata, encoding both the basic and acidic subunits of neoculin in both plants. We aligned these 25 transcripts and generated a phylogenetic tree. Many orthologs in the two species shared high similarity, despite the low number of common genes, suggesting that these genes likely existed before the two species diverged. The relative expression levels of these genes differed considerably between the two species: the transcripts per million (TPM) values of neoculin genes were 60 times higher in C. latifolia than in C. capitulata, whereas those of GNA family members were 15,000 times lower in C. latifolia than in C. capitulata. CONCLUSIONS: The genetic diversity of neoculin-related genes strongly suggests that neoculin genes underwent duplication during evolution. The marked differences in their expression profiles between C. latifolia and C. capitulata may be due to mutations in regions involved in transcriptional regulation. Comprehensive analysis of the genes expressed in the fruits of these two Curculigo species helped elucidate the origin of neoculin at the molecular level.

Identification and functional analysis of cadmium-binding protein in the visceral mass of Crassostrea gigas.

The Pacific oyster, Crassostrea gigas, is a traditional food worldwide. The soft body of the oyster can easily accumulate heavy metals such as cadmium (Cd). To clarify the molecular mechanism of Cd accumulation in the viscera of C. gigas, we identified Cd-binding proteins. 5,10,15,20-Tetraphenyl-21H,23H-porphinetetrasulfonic acid, disulfuric acid, tetrahydrate, and Cd-binding competition experiments using immobilized metal ion affinity chromatography revealed the binding of water-soluble high molecular weight proteins to Cd, including C. gigas protein disulfide isomerase (cgPDI). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed two CGHC motifs in cgPDI. The binding between Cd and rcgPDI was confirmed through a Cd-binding experiment using the TPPS method. Isothermal titration calorimetry (ITC) revealed the binding of two Cd ions to one molecule of rcgPDI. Circular dichroism (CD) spectrum and tryptophan fluorescence analyses demonstrated that the rcgPDI bound to Cd. The binding markedly changed the two-dimensional or three-dimensional structures. The activity of rcgPDI measured by a PDI Activity Assay Kit was more affected by the addition of Cd than by human PDI. Immunological analyses indicated that C. gigas contained cgPDI at a concentration of 1.0 nmol/g (viscera wet weight). The combination of ITC and quantification results revealed that Cd-binding to cgPDI accounted for 20% of the total bound Cd in the visceral mass. The findings provide new insights into the defense mechanisms of invertebrates against Cd.

Transcriptomic and Metabolomic Analyses Provide Insights into the Upregulation of Fatty Acid and Phospholipid Metabolism in Tomato Fruit under Drought Stress.

Transcriptome and metabolome analysis in tomato (Solanum lycopersicum) fruits cultivated under drought conditions showed that drought stress promoted fatty acid synthesis and increased the content of fatty acids in fruits. The accumulation of some phospholipids composed of palmitic acid and oleic acid also was significantly increased, especially in seeds. Moreover, inositol, which is a component of cell membranes and cell walls, was increased through the activity of the myoinositol monophosphatase 1-mediated pathway. In mature fruits, the levels of metabolic regulators such as ß-alanine and 4-aminobutyric acid were elevated. These results showed that these compounds are drought-responsive and enhance drought tolerance and subsequently they could enhance the nutritional value and health benefits of tomato fruit.

Quercetin 3,5,7,3',4'-pentamethyl ether from Kaempferia parviflora directly and effectively activates human SIRT1.

Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, is a crucial regulator that produces multiple physiological benefits, such as the prevention of cancer and age-related diseases. SIRT1 is activated by sirtuin-activating compounds (STACs). Here, we report that quercetin 3,5,7,3',4'-pentamethyl ether (KPMF-8), a natural STAC from Thai black ginger Kaempferia parviflora, interacts with SIRT1 directly and stimulates SIRT1 activity by enhancing the binding affinity of SIRT1 with Ac-p53 peptide, a native substrate peptide without a fluorogenic moiety. The binding affinity between SIRT1 and Ac-p53 peptide was enhanced 8.2-fold by KPMF-8 but only 1.4-fold by resveratrol. The specific binding sites of KPMF-8 to SIRT1 were mainly localized to the helix2-turn-helix3 motif in the N-terminal domain of SIRT1. Intracellular deacetylase activity in MCF-7 cells was promoted 1.7-fold by KPMF-8 supplemented in the cell medium but only 1.2-fold by resveratrol. This work reveals that KPMF-8 activates SIRT1 more effectively than resveratrol does.

Yeast-based reporter assay system for identifying the requirements of intramembrane proteolysis by signal peptide peptidase of Arabidopsis thaliana.

Signal peptide peptidase (SPP) is an aspartic protease with two active sites, YD and GXGD, in the transmembrane domain. SPP cleaves signal peptides, and the released fragments play key roles in the immune system, embryo development and protein turnover in cells. Despite SPP having an important function, a general system to identify the requirements of intramembrane proteolysis by SPP has not been developed because proteolysis occurs in the membrane. In this study, we first established a reporter assay system in yeast to verify the cleavage activity of the Arabidopsis thaliana SPP (AtSPP). Next, we screened candidate substrates of AtSPP from A. thaliana pollen and roots. In the pollen, 13 signal peptides with 'pollen' and 'cell wall' as gene ontology terms were selected. In the roots, mutants overexpressing AtSPP were constructed, and gene expression changes were compared with the wild-type. Nine signal peptides expressed in the roots were selected. Then we used the candidate substrates in our reporter assay system to determine the requirements for proteolysis by AtSPP. Fifteen of 22 signal peptides were cleaved by AtSPP. The absence of the positively charged amino acids, His and Lys on the C terminus of the signal sequence, was observed in cleaved substrates. Moreover, mutation of a helix breaker-to-Leu substitution in the intramembrane region in substrates prevented cleavage by AtSPP. These results indicated that substrates of AtSPP required the helix breaker structure to be cleaved.

Changes in Whole-Blood microRNA Profiles during the Onset and Treatment Process of Cerebral Infarction: A Human Study.

Circulating miRNA species are promising symptom markers for various diseases, including cardiovascular disease. However, studies regarding their role in the treatment process are limited, especially concerning cerebral infarction. This study aimed to extract miRNA markers to investigate whether they reflect both onset and treatment process of cerebral infarction. A total of 22 patients (P-group) and 22 control subjects (C-group) were examined for their whole-blood miRNA profiles using DNA GeneChip™ miRNA 4.0 Array, with six patients examined after treatment (T-group). A total of 64 miRNAs were found to be differentially expressed between the C- and P-groups. Out of 64 miRNAs, the expression levels of two miRNAs correlated with hypertension. A total of 155 miRNAs were differentially expressed between the P- and T-groups. Five common miRNAs were found among the 64 and 155 miRNAs identified. Importantly, these common miRNAs were inversely regulated in each comparison (e.g., C < P > T), including miR-505-5p, which was previously reported to be upregulated in aortic stenosis patients. Our previous study using rat cerebral infarction models detected the downregulation of an apoptosis repressor, WDR26, which was repressed by one of the five miRNAs. Our results provide novel information regarding the miRNA-based diagnosis of cerebral infarction in humans. In particular, the five common miRNAs could be useful makers for the onset and the treatment process. Trial registration: This study was registered in the UMIN Clinical Trials Registry (UMIN000038321).

Functional characterisation of two ferric-ion coordination modes of TtFbpA, the periplasmic subunit of an ABC-type iron transporter from Thermus thermophilus HB8.

The ferric ion binding protein A of Thermus thermophilus HB8 (TtFbpA) is the periplasmic subunit of an ABC-type iron transporter. Two Fe3+-bound crystal structures at pH 5.5 and pH 7.5 and one apo structure have been reported for TtFbpA. In addition to three Tyr residues, TtFbpA coordinates with Fe3+ using two monodentate HCO3- and one H2O to form a six-coordinated mode at pH 5.5 or one bidentate CO32- to form a five-coordinated mode at pH 7.5. We investigated the biological significance of these Fe3+-bound forms of TtFbpA and the synergistic anions (HCO3- and CO32-). Quantum mechanical calculations in silico indicated that only these coordination modes were plausible out of six possibilities. Comparison of the crystal structures revealed a key motif, RZX1X2L(I/V), that would couple the Fe3+ coordination mode and the TtFbpA protein conformation. Both gel filtration chromatography and isothermal titration calorimetry showed that TtFbpA could bind Fe3+ at pH 7.5 but not at pH 5.5. Isothermal titration calorimetry also revealed that the binding at pH 7.5 was a three-step endothermic reaction that required NaHCO3. These results indicate that the holo structure at pH 5.5 is unstable in solution and may correspond to a transition state of TtFbpA-Fe3+ binding at pH 7.5 because HCO3- is much more abundant than CO32- at both pH values. Reorganisation of the synergistic ions and coupled protein conformational change will occur to form the stable TtFbpA-Fe3+ complex at pH 7.5, but not at pH 5.5. Identification of such a transition state will contribute to molecular design of novel FbpA inhibitors.

Oryza sativa Brittle Culm 1-like 6 modulates ß-glucan levels in the endosperm cell wall.

The endosperm cell wall affects post-harvest grain quality by affecting the mechanical fragility and water absorption of the grain. Therefore, understanding the mechanism underlying endosperm cell wall synthesis is important for determining the growth and quality of cereals. However, the molecular machinery mediating endosperm cell wall biosynthesis is not well understood. In this study, we investigated the role of Oryza sativa Brittle Culm 1-like 6 (OsBC1L6), a member of the COBRA-like protein family, in cellulose synthesis in rice. OsBC1L6 mRNA was expressed in ripening seeds during endosperm enlargement. When OsBC1L6-RFP was expressed in Arabidopsis cell cultures, this fusion protein was transported to the plasma membrane. To investigate the target molecules of OsBC1L6, we analyzed the binding interactions of OsBC1L6 with cellohexaose and the analogs using surface plasmon resonance, determining that cellohexaose bound to OsBC1L6. The ß-glucan contents were significantly reduced in OsBC1L6-RNAi calli and OsBC1L6-deficient seeds from a Tos insertion mutant, compared to their wild-type counterparts. These findings suggest that OsBC1L6 modulates ß-glucan synthesis during endosperm cell wall formation by interacting with cellulose moieties on the plasma membrane during seed ripening.

Novel indole and benzothiophene ring derivatives showing differential modulatory activity against human epithelial sodium channel subunits, ENaC ß and γ.

The epithelial sodium channel (ENaC) plays a pivotal role in sodium homeostasis, and the development of drugs that modulate ENaC activity is of great potential therapeutic relevance. We screened 6100 chemicals for their ability to activate sodium permeability of ENaC. We used a two-step strategy: a high throughput cell-based assay and an electrophysiological assay. Five compounds were identified showing common structural features including an indole or benzothiophene ring. ENaC consists of three subunits: α, ß, and γ. Changing the heteromeric combination of human and mouse ENaC αßγ subunits, we found that all five compounds activated the human ß subunit but not the mouse subunit. However, four of them exhibited lower activity when the human γ subunit was substituted by the mouse γ subunit. Our findings provide a structural basis for designing human ENaC activity modulators. Abbreviations: ENaC: Epithelial sodium channel; ΔRFU: delta relative fluorescence units; EC50: Half-maximal effective concentration; Emax: maximum effect value.

Structural and functional analyses of calcium ion response factors in the mantle of Pinctada fucata.

The pearl oyster, Pinctada fucata, is cultured for pearl production in Japan. The shell of the pearl oyster consists of calcium carbonate and a small amount of organic matrix. Despite many studies of the shell matrix proteins, the mechanism by which calcium elements are transported from the mantle to the shell remains unclear. Investigating the molecular mechanism of calcium transportation, we prepared artificial seawater with a high concentration of calcium ions (10ASW) to induce calcification in the pearl oyster. When pearl oysters were cultured in 10ASW, unusual nanoparticles were precipitated on the surface of the nacreous layer. SDS-PAGE and 2D-PAGE analyses revealed that some calcium-sensing proteins (Sarcoplasmic Ca-binding Protein (Pf-SCP) and Pf-filamin A) might be related to the synthesis of these nanoparticles. The recombinant proteins of Pf-SCP can bind to calcium ions and accumulate nanoparticles of calcium carbonate crystals. However, transcriptomic analysis of the pearl oysters grown in 10ASW showed that the matrix protein genes in the shell did not differ before and after treatment with 10ASW. These results suggest that, despite increasing calcium transportation to the shell, treatment with a high concentration of calcium ions does not induce formation of the organic framework in the shell microstructure. These findings offer meaningful insights into the transportation of calcium elements from the mantle to the shell.