Negative regulation of chitosan-induced stomatal closure by glutathione in Arabidopsis thaliana.

Chitosan (CHT) is a deacylated derivative of chitin and improves growth and yield performance, activates defensive genes, and also induces stomatal closure in plants. Glutathione (GSH) has significant functions in the growth, development, defense systems, signaling, and gene expression. Glutathione negatively regulates abscisic acid (ABA)-, methyl jasmonate (MeJA)-, and salicylic acid (SA)-induced stomatal closure. However, the negative regulation by GSH of CHT-induced stomatal closure is still unknown. Regulation of CHT-induced stomatal closure by GSH in guard cells was investigated using two GSH-deficient mutants, cad2-1 and ch1-1, and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). The cad2-1 and ch1-1 mutations and CDNB treatment enhanced CHT-induced stomatal closure. Treatment with glutathione monoethyl ester (GSHmee) restored the GSH level in the guard cells of cad2-1 and ch1-1 and complemented the stomatal phenotype of the mutants. These results indicate that GSH negatively regulates CHT-induced stomatal closure in A. thaliana.

The effect of exogenous dihydroxyacetone and methylglyoxal on growth, anthocyanin accumulation, and the glyoxalase system in Arabidopsis.

Dihydroxyacetone (DHA) occurs in wide-ranging organisms, including plants, and can undergo spontaneous conversion to methylglyoxal (MG). While the toxicity of MG to plants is well-known, the toxicity of DHA to plants remains to be elucidated. We investigated the effects of DHA and MG on Arabidopsis. Exogenous DHA at up to 10 mm did not affect the radicle emergence, the expansion of green cotyledons, the seedling growth, or the activity of glyoxalase II, while DHA at 10 mm inhibited the root elongation and increased the activity of glyoxalase I. Exogenous MG at 1.0 mm inhibited these physiological responses and increased both activities. Dihydroxyacetone at 10 mm increased the MG content in the roots. These results indicate that DHA is not so toxic as MG in Arabidopsis seeds and seedlings and suggest that the toxic effect of DHA at high concentrations is attributed to MG accumulation by the conversion to MG.

Cycloartenyl Ferulate Is the Predominant Compound in Brown Rice Conferring Cytoprotective Potential against Oxidative Stress-Induced Cytotoxicity.

Since brown rice extract is a rich source of biologically active compounds, the present study is aimed to quantify the major compounds in brown rice and to compare their cytoprotective potential against oxidative stress. The content of the main hydrophobic compounds in brown rice followed the order of cycloartenyl ferulate (CAF) (89.00 ± 8.07 nmol/g) >> α-tocopherol (αT) (19.73 ± 2.28 nmol/g) > γ-tocotrienol (γT3) (18.24 ± 1.41 nmol/g) > α-tocotrienol (αT3) (16.02 ± 1.29 nmol/g) > γ-tocopherol (γT) (3.81 ± 0.40 nmol/g). However, the percent contribution of CAF to the radical scavenging activity of one gram of whole brown rice was similar to those of αT, αT3, and γT3 because of its weaker antioxidant activity. The CAF pretreatment displayed a significant cytoprotective effect on the hydrogen peroxide-induced cytotoxicity from 10 µM, which is lower than the minimal concentrations of αT and γT required for a significant protection. CAF also enhanced the nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation coincided with the enhancement of the heme oxygenase-1 (HO-1) mRNA level. An HO-1 inhibitor, tin protoporphyrin IX (SnPP), significantly impaired the cytoprotection of CAF. The cytoprotective potential of CAF is attributable to its cycloartenyl moiety besides the ferulyl moiety. These results suggested that CAF is the predominant cytoprotector in brown rice against hydrogen peroxide-induced cytotoxicity.

Construction of benzyl isothiocyanate-loaded fish skin gelatin-luteolin compound emulsion delivery system, and its digestion and absorption characteristics.

BACKGROUND: Fish skin gelatin (FSG) and luteolin (LUT) were used as composite emulsifiers, and benzyl isothiocyanate (BITC) was used as a model of nutrient delivery to construct a stable emulsion. The storage stability of the FSG-LUT emulsion and its effect on BITC release were investigated both in vitro and ex vivo. RESULTS: LUT can quench FSG fluorophores statically and form a stable complex through hydrogen bonding and hydrophobic interactions. The FSG-LUT emulsion storage stability and embedding rate were higher than those of the FSG emulsion. The FSG-LUT emulsion microstructure was resistant to oral and gastric digestion, and the BITC retention rate and bioaccessibility were much higher than those of the FSG emulsion. Lastly, the ex vivo everted gut sac of rat intestine study demonstrated that BITC showed the highest absorption in the ileum, and the FSG-LUT emulsion absorbed BITC and sustained a controlled release in a specific position. CONCLUSION: LUT could form stable complexes with FSG, which improved the stability and bioavailability of BITC in the FSG-LUT emulsion delivery system, and promoted further intestinal BITC absorption. © 2022 Society of Chemical Industry.

Extracellular malate induces stomatal closure via direct activation of guard-cell anion channel SLAC1 and stimulation of Ca2+ signalling.

Plants secrete malate from guard cells to apoplast under stress conditions and exogenous malate induces stomatal closure. Malate is considered an extracellular chemical signal of stomatal closure. However, the molecular mechanism of malate-induced stomatal closure is not fully elucidated. We investigated responses of stomatal aperture, ion channels, and cytosolic Ca2+ to malate. A treatment with malate induced stomatal closure in Arabidopsis thaliana wild-type plants, but not in the mutants deficient in the slow (S-type) anion channel gene SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1). The treatment with malate increased S-type anion currents in guard-cell protoplasts of wild-type plants but not in the slac1 mutant. In addition, extracellular rather than intracellular application of malate increased the S-type currents of constitutively active mutants of SLAC1, which have kinase-independent activities, in a heterologous expression system using Xenopus oocytes. The treatment with malate transiently increased cytosolic Ca2+ concentration in the wild-type Arabidopsis guard cells and the malate-induced stomatal closure was inhibited by the Ca2+ channel blocker and the Ca2+ chelator. These results indicate that extracellular malate directly activates SLAC1 and simultaneously stimulates Ca2+ signalling in guard cells, resulting in steady and solid activation of SLAC1 for stomatal closure.

Benzyl isothiocyanate and its metabolites inhibit cell proliferation through protein modification in mouse preosteoclast RAW264.7 cells.

Benzyl isothiocyanate (BITC), derived from cruciferous vegetables, is an organosulfur compound exerting antiproliferative effects in several human cancer cells. In this study, we assessed BITC as a potential osteoclastogenesis inhibitor and investigated its underlying mechanism. BITC at 5 µM significantly decreased the viability of the osteoclast-like differentiating RAW264.7 cells, coinciding with the downregulation of the primary biomarkers for osteoclast differentiation, such as the tartrate-resistant acid phosphatase activity and nuclear factor of activated T-cells gene expression. Not only BITC but also its metabolites, inhibited cell proliferation in the normal RAW264.7 cells, suggesting that BITC shows an anti-osteoclastogenesis effect in vivo after its ingestion and metabolism, possibly through an antiproliferative action. Both BITC and its metabolites also enhanced the DNA fragmentation and the caspase-3 activity, whereas their higher concentrations tended to suppress these effects. BITC was intracellularly accumulated when the cells were treated with its metabolites via their degradation into the free form. A quantitative experiment using the proteolysis/high performance liquid chromatography technique showed that the amount of BITC-lysine thiourea in the cells was also increased in a time-dependent manner, suggesting that lysine modification of the cellular proteins actually took place in the cells treated by BITC. Among the cellular proteins, the cleaved caspase-3 was identified as a potential target for lysine modification by BITC. Taken together, BITC dissociated from its metabolites as well as its free form might modulate osteoclastogenesis, possibly through inhibition of cell proliferation by protein modification.

Negative regulation of salicylic acid-induced stomatal closure by glutathione in Arabidopsis thaliana.

Salicylic acid (SA) is a ubiquitous phenolic phytohormone that induces stomatal closure. Glutathione (GSH) negatively regulates stomatal closure induced by other plant hormones such as abscisic acid (ABA) and methyl jasmonate (MeJA). However, the involvement of GSH in SA-induced stomatal closure is still unknown. We investigated the regulation of SA signaling by GSH in guard cells using an Arabidopsis thaliana mutant, cad2-1, which is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase. Application of SA decreased stomatal apertures with decreasing intracellular GSH level in guard cells. Decreasing GSH by the cad2-1 mutation and by a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene, enhanced the SA-induced stomatal closure. Treatment with glutathione monoethyl ester restored the GSH level in the cad2-1 guard cells and complemented the stomatal phenotype of the mutant. These results indicate that GSH negatively modulates SA-induced stomatal closure in A. thaliana.

Malate induces stomatal closure via a receptor-like kinase GHR1- and reactive oxygen species-dependent pathway in Arabidopsis thaliana.

A primary metabolite malate is secreted from guard cells in response to the phytohormone abscisic acid (ABA) and elevated CO2. The secreted malate subsequently facilitates stomatal closure in plants. Here, we investigated the molecular mechanism of malate-induced stomatal closure using inhibitors and ABA signaling component mutants of Arabidopsis thaliana. Malate-induced stomatal closure was impaired by a protein kinase inhibitor, K252a, and also by the disruption of a receptor-like kinase GHR1, which mediates activation of calcium ion (Ca2+) channel by reactive oxygen species (ROS) in guard cells. Malate induced ROS production in guard cells while the malate-induced stomatal closure was impaired by a peroxidase inhibitor, salicylhydroxamic acid, but not by the disruption of Nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases, RBOHD and RBOHF. The malate-induced stomatal closure was impaired by Ca2+ channel blockers, verapamil, and niflumic acid. These results demonstrate that the malate signaling is mediated by GHR1 and ROS in Arabidopsis guard cells.

Antioxidative Effect of Chlorella Pyrenoidosa Protein Hydrolysates and Their Application in Krill Oil-in-Water Emulsions.

Chlorella pyrenoidosa is an excellent source of protein, and in this research, we assessed the antioxidant and emulsifying effects of Chlorella protein hydrolysate (CPH) using neutral proteases and alkaline proteases, as well as the properties of CPH-derived krill oil-in-water (O/W) emulsions. The CPHs exhibited the ability to scavenge several kinds of free radicals, including 1,1-diphenyl-2-picrylhydrazyl (DPPH), O2-, hydroxyl, and ABTS. Additionally, the CPHs (5 mg/mL) scavenged approximately 100% of the DPPH and ABTS. The CPHs showed similar emulsifying activities to Tween 20 and excellent foaming activities (max FS 74%), which helped to stabilize the krill oil-in-water emulsion. Less than 10 mg/mL CPHs was able to form fresh krill oil-in-water emulsions; moreover, the CPHs (5 mg/mL) in a krill O/W emulsion were homogenous, opaque, and stable for at least 30 days. Based on their inhibitory effects on the peroxide value (POV) and thiobarbituric acid reactive substances (TRABS), the CPHs were found to be able to inhibit lipid oxidation in both emulsifying systems and krill O/W emulsions. Thus, the CPHs could improve superoxide dismutase (SOD) activities by 5- or 10-fold and decrease the high reactive oxygen species (ROS) level caused by the addition of H2O2 in vitro. In conclusion, health-promoting CPHs could be applied in krill oil-in-water emulsions as both emulsifiers and antioxidants, which could help to improve the oxidative and physical stability of emulsions.

A Major Intestinal Catabolite of Quercetin Glycosides, 3-Hydroxyphenylacetic Acid, Protects the Hepatocytes from the Acetaldehyde-Induced Cytotoxicity through the Enhancement of the Total Aldehyde Dehydrogenase Activity.

Aldehyde dehydrogenases (ALDHs) are the major enzyme superfamily for the aldehyde metabolism. Since the ALDH polymorphism leads to the accumulation of acetaldehyde, we considered that the enhancement of the liver ALDH activity by certain food ingredients could help prevent alcohol-induced chronic diseases. Here, we evaluated the modulating effects of 3-hydroxyphenylacetic acid (OPAC), the major metabolite of quercetin glycosides, on the ALDH activity and acetaldehyde-induced cytotoxicity in the cultured cell models. OPAC significantly enhanced the total ALDH activity not only in mouse hepatoma Hepa1c1c7 cells, but also in human hepatoma HepG2 cells. OPAC significantly increased not only the nuclear level of aryl hydrocarbon receptor (AhR), but also the AhR-dependent reporter gene expression, though not the nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent one. The pretreatment of OPAC at the concentration required for the ALDH upregulation completely inhibited the acetaldehyde-induced cytotoxicity. Silencing AhR impaired the resistant effect of OPAC against acetaldehyde. These results strongly suggested that OPAC protects the cells from the acetaldehyde-induced cytotoxicity, mainly through the AhR-dependent and Nrf2-independent enhancement of the total ALDH activity. Our findings suggest that OPAC has a protective potential in hepatocyte models and could offer a new preventive possibility of quercetin glycosides for targeting alcohol-induced chronic diseases.

Immune State Conversion of the Mesenteric Lymph Node in a Mouse Breast Cancer Model.

Secondary lymphoid tissues, such as the spleen and lymph nodes (LNs), contribute to breast cancer development and metastasis in both anti- and pro-tumoral directions. Although secondary lymphoid tissues have been extensively studied, very little is known about the immune conversion in mesenteric LNs (mLNs) during breast cancer development. Here, we demonstrate inflammatory immune conversion of mLNs in a metastatic 4T1 breast cancer model. Splenic T cells were significantly decreased and continuously suppressed IFN-γ production during tumor development, while myeloid-derived suppressor cells (MDSCs) were dramatically enriched. However, T cell numbers in the mLN did not decrease, and the MDSCs only moderately increased. T cells in the mLN exhibited conversion from a pro-inflammatory state with high IFN-γ expression to an anti-inflammatory state with high expression of IL-4 and IL-10 in early- to late-stages of breast cancer development. Interestingly, increased migration of CD103+CD11b+ dendritic cells (DCs) into the mLN, along with increased (1→3)-ß-D-glucan levels in serum, was observed even in late-stage breast cancer. This suggests that CD103+CD11b+ DCs could prime cancer-reactive T cells. Together, the data indicate that the mLN is an important lymphoid tissue contributing to breast cancer development.

Influence of fish skin gelatin-sodium alginate complex stabilized emulsion on benzyl isothiocyanate stability and digestibility in vitro and in vivo.

BACKGROUND: An emulsion delivery system for benzyl isothiocyanate (BITC) was prepared using fish skin gelatin (FSG) and sodium alginate (Alg). The effects of the FSG-Alg complex on the emulsion stability and BITC release pattern from the emulsion were investigated in vitro and in vivo. RESULTS: The storage stability and embedding rate of the 10 g kg-1 FSG and 2.5 g kg-1 Alg (FSG-Alg) emulsion were the highest among all samples. The FSG-Alg complex provided BITC a better protection during in vitro digestion. The microstructure of the FSG-Alg emulsions was more stable during in vitro digestion, and the bioaccessibility and retention rate of BITC were much higher compared to those of the FSG emulsion. The results of the ex vivo everted gut sac of rat intestine study showed that the FSG-Alg emulsion significantly increased the BITC absorption rate in the duodenum. CONCLUSION: The FSG-Alg emulsion delivery system is a highly stable system for the delivery of BITC that improves the bioaccessibility of BITC and promotes its absorption in the duodenum. © 2022 Society of Chemical Industry.

Gastrointestinal digestion and absorption characterization in vitro of zinc-chelating hydrolysate from scallop adductor (Patinopecten yessoensis).

BACKGROUD: Zinc (Zn) is an essential catalytic element in the human health system but its absorption in the intestinal system can be strongly affected by gastrointestinal (GI) digestion. In this study, the food-derived potential Zn carrier, scallop adductor hydrolysates (SAHs), was produced and characterized. RESULTS: During temporary storage at 4 °C, SAH decreased in Zn-chelating capacity in the aqueous phase, whereas the SAH-Zn complex exhibited high stability. Moreover, the secondary structure of SAH had no significant alteration. Zn morphologically altered the surface structures of SAH, which was involving in carboxyl group of SAH. Results of in vitro GI digestion suggested that the SAH-Zn maintained good stability in GI system and only proportion of high molecular weight cleaved. In addition, SAH could successfully carry and transport Zn while the fluorescence staining revealed free Zn accumulation inside the tissue. Finally, three representative absorbed peptides (around 600 Da) were identified and synthesized. Three synthetic peptides exhibit higher Zn-chelating capacity than SAH and could also successfully transported through the intestine. CONCLUSION: This study provided a theoretical basis for the investigation of digestion and absorption of marine animal-derived peptides as Zn carriers. © 2021 Society of Chemical Industry.

Characterization of a synthetic zinc-chelating peptide from sea cucumber (Stichopus japonicus) and its gastrointestinal digestion and absorption in vitro.

BACKGROUND: Zinc absorption in intestinal system could be strongly affected by the gastrointestinal digestion and absorption of zinc-chelating peptides serving as zinc carriers. In this study, a novel zinc-chelating sea cucumber synthetic peptide (SCSP) was synthesized to estimate its gastrointestinal digestion and promotive effect of zinc absorption in vitro. RESULTS: Analysis of isothermal titration calorimetry suggested that the binding of SCSP and zinc (N ≈ 1) was exothermic, with relatively weak binding affinity (K = 1.0 × 10-3  mol L-1 ). The formation of SCSP-Zn complexes brought morphological changes to the peptides confirmed by scanning electron microscopy (SEM), which also indicated 6.88% of the existence of zinc element. In addition, the SCSP-Zn complexes remained stable under simulated human gastrointestinal digestion. In an in vitro study, the SCSP-Zn complex could successfully transport through the intestinal membrane in the model of everted rat gut sacs (nearly 7.5 µM cm-2 ) as well as Caco-2 cells where the zinc transport reached 0.0014 mg mL-1 carried by SCSP. Fluorescence staining experiments revealed free zinc accumulation inside the tissues and cells treated with the SCSP-Zn complex. CONCLUSIONS: The chelation SCSP-Zn had the promotion ability of zinc absorption in vitro and ex vivo experiments, which suggested a theoretical basis for the design and production of effective zinc chelating peptides as zinc carriers to improve zinc bioavailability. © 2022 Society of Chemical Industry.

A multidrug resistance-associated protein inhibitor is a potential enhancer of the benzyl isothiocyanate-induced apoptosis induction in human colorectal cancer cells.

The increasing drug efflux through the ATP-binding cassette (ABC) transporters is the most plausible mechanism that mediates resistance to the anticancer phytochemicals, such as benzyl isothiocyanate (BITC), as well as chemotherapy drugs. To identify a potential component to overcome this resistance by combinatory utilization, we focused on multidrug resistance-associated proteins (MRPs) pumping various drug metabolites with glutathione as well as the organic anions. The pharmacological treatment of an MRP inhibitor, MK571, significantly potentiated the BITC-induced antiproliferation, coincided with the enhanced accumulation of BITC and glutathione in human colorectal cancer HCT-116 cells. MK571 also enhanced the apoptosis induction as well as activation of the mitogen-activated protein kinases and caspase-3, whereas it did not affect their basal levels. These results suggested that, since MRPs might play a pivotal role in the BITC efflux, MK571 potentiates the BITC-induced antiproliferation in human colorectal cancer cells through inhibition of the glutathione-dependent BITC efflux.

Modulation of frequency and height of cytosolic calcium spikes by plasma membrane anion channels in guard cells.

Cytosolic calcium ([Ca2+]cyt) elevation activates plasma membrane anion channels in guard cells, which is required for stomatal closure. However, involvement of the anion channels in the [Ca2+]cyt elevation remains unclear. We investigated the involvement using Arabidopsis thaliana anion channel mutants, slac1-4 slah3-3 and slac1-4 almt12-1. Extracellular calcium induced stomatal closure in the wild-type plants but not in the anion channel mutant plants whereas extracellular calcium induced [Ca2+]cyt elevation both in the wild-type guard cells and in the mutant guard cells. The peak height and the number of the [Ca2+]cyt spike were lower and larger in the slac1-4 slah3-3 than in the wild type and the height and the number in the slac1-4 almt12-1 were much lower and much larger than in the wild type. These results suggest that the anion channels are involved in the regulation of [Ca2+]cyt elevation in guard cells.

White rice ethanol extract is qualitatively, but not quantitatively, equivalent to that of brown rice as an antioxidant source.

The purpose of this study is to compare the potentials to exhibit biologically active antioxidant actions between white rice (WR) and brown rice (BR) in in vitro assays and a cellular model. The Trolox equivalent (TE) per 1 mg ethanol extract of WR for the 1,1-diphenyl-2-picrylhydrazyl assay was slightly higher than that of BR, whereas the TE per 1 g whole WR was much lower than that for BR. This tendency was very comparable to those for the oxygen radical absorbance capacity and total polyphenol content. Both of the ethanol extracts also similarly suppressed the hydrogen peroxide-induced cytotoxicity and enhanced the gene expression of drug-metabolizing enzymes. Based on the α-tocopherol quantity, its contribution to the cytoprotective effect of the rice extracts is very limited. Taken together, the ethanol extract of WR might be a qualitatively, but not quantitatively, equivalent source of antioxidative phytochemicals to that of BR.

Neither glutamate nor alanine but arginine sensitizes BY-2 cells to arsenate.

Arsenic is toxic for plants. Our previous results showed that the application of proline enhanced the sensitivity of tobacco BY-2 cells to arsenate. In order to clarify the enhancement mechanism, we investigated the effects of other amino acids on the arsenate-stressed BY-2 cells. Glutamate at up to 10 m m did not affect the cell growth in the absence or presence of arsenate. Arginine at up to 10 m m did not affect the growth in the absence of arsenate but arginine at 10 m m enhanced the inhibition of the cell growth by arsenate. Alanine at up to 10 m m did not affect the cell growth under nonstressed condition but alanine at 10 m m significantly improved the cell growth under arsenate stress. These results suggest that alanine mitigates arsenate stress in BY-2 cells and that arginine like proline enhances the sensitivity of BY-2 cells to arsenate.

Cadmium uptake via apoplastic bypass flow in Oryza sativa.

It is known that rice roots take up cadmium (Cd) via the symplastic route mediated by membrane-bound mineral transporters. Here we provide evidence that apoplastic bypass flow is another Cd uptake route in rice. High concentrations of Cd rendered apoplastic bypass flow rate increased in rice seedlings. These concentrations of Cd compromised membrane integrity in the root meristem and transition zone. Polyethleneglycol and proline inhibited the Cd-induced apoplastic bypass flow and Cd transfer to the shoots. Loss-of-function mutant of the Cd uptake transporter, nramp5, showed Cd transport to the shoot comparable to the wild type. At a low Cd concentration, increased apoplastic bypass flow rate by NaCl stress resulted in an elevation of Cd transport to shoots both in the wildtype and nramp5. These observations indicate that apoplastic bypass flow in roots carries Cd transport leading to xylem loading of Cd in addition to the symplastic pathway mediated by mineral transporters under stressed conditions.

Luteolin suppresses 5-hydroxytryptamine elevation in stimulated RBL-2H3 cells and experimental colitis mice.

Increased 5-hydroxytryptamine may be associated with the development and progression of inflammatory bowel disease. In this study, we examined the suppressive effect of flavonoids on the increased intra- and extracellular 5-hydroxytryptamine levels in rat mast RBL-2H3 cells, known to produce 5-hydroxytryptamine by the phorbol 12-myristate 13-acetate stimulation. Among the flavonoids examined, luteolin and quercetin significantly reduced the cellular 5-hydroxytryptamine concentration. Gene and protein expression analyses revealed that luteolin significantly suppressed cellular tryptophan hydroxylase 1 expression induced by phorbol 12-myristate 13-acetate stimulation. Mitogen-activated protein kinase/extracellular signal-regulated kinase signaling was also suppressed by luteolin, suggesting that this pathway is one of targets of 5-hydroxytryptamine modulation by luteolin. An in vivo experimental colitis model was prepared by administering 2.5% dextran sodium sulfate in drinking water to C57BL/6 mice for seven days. The ingestion of 0.1% dietary luteolin suppressed the increasing 5-hydroxytryptamine in the colorectal mucosa. In conclusion, luteolin possesses a suppressive effect on extensive 5-hydroxytryptamine formation in both experimental RBL-2H3 cells and colitis models.