Mulberry Ethanol Extract and Rutin Protect Alcohol-Damaged GES-1 Cells by Inhibiting the MAPK Pathway.

Mulberry extract has been proven to have the effect of resisting alcohol damage, but its mechanism is still unclear. In this study, the composition of mulberry ethanol extract (MBE) was identified by LC-MS/MS and the main components of MBE were ascertained by measuring. Gastric mucosal epithelial (GES-1) cells were used to elucidate the mechanism of MBE and rutin (the central part of MBE) helped protect against alcohol damage. The results revealed that phenolics accounted for the majority of MBE, accounting for 308.6 mg/g gallic acid equivalents and 108 substances were identified, including 37 flavonoids and 50 non-flavonoids. The treatment of 400 µg/mL MBE and 320 µM rutin reduced early cell apoptosis and the content of intracellular reactive oxygen species, malondialdehyde and increased glutathione. The qPCR results indicated that the MBE inhibits the expression of genes in the mitogen-activated protein kinase (MAPK) pathway, including p38, JNK, ERK and caspase-3; rutin inhibits the expression of p38 and caspase-3. Overall, MBE was able to reduce the oxidative stress of GES-1 cells and regulated apoptosis-related genes of the MAPK pathway. This study provides information for developing anti-ethanol injury drugs or functional foods.

The red wine component ellagic acid induces autophagy and exhibits anti-lung cancer activity in vitro and in vivo.

Red wine consists of a large amount of compounds such as resveratrol, which exhibits chemopreventive and therapeutic effects against several types of cancers by targeting cancer driver molecules. In this study, we tested the anti-lung cancer activity of 11 red wine components and reported that a natural polyphenol compound ellagic acid (EA) inhibited lung cancer cell proliferation at an efficacy approximately equal to that of resveratrol. EA markedly increased the expression of the autophagosomal marker LC3-II as well as inactivation of the mechanistic target of rapamycin signalling pathway. EA elevated autophagy-associated cell death by down-regulating the expression of cancerous inhibitor of protein phosphatase 2A (CIP2A), and CIP2A overexpression attenuated EA-induced autophagy of lung cancer cells. Treating tumour-bearing mice with EA resulted in significant inhibition of tumour growth with suppression of CIP2A levels and increased autophagy. In addition, EA potentiated the inhibitory effects of the natural compound celastrol on lung cancer cells in vitro and in vivo by enhancing autophagy and down-regulating CIP2A. These findings indicate that EA may be a promising chemotherapeutic agent for lung cancer, and that the combination of EA and celastrol may have applicability for the treatment of this disease.

Effect of Cultivar, Temperature, and Environmental Conditions on the Dynamic Change of Melatonin in Mulberry Fruit Development and Wine Fermentation.

High levels of melatonin have been reported in various foods but not in mulberry or its wine. This study investigated the dynamic changes of melatonin levels during mulberry fruit development and ethanol fermentation of 2 different colored mulberry cultivars ("Hongguo2Ë® Morus nigra, black and "BaiyuwangË® Morus alba, white) at 2 fermentation temperatures (16 and 25 °C). Our results showed that the melatonin level increased in the beginning of mulberry development but decreased in the end. The MnTDC gene expression level correlated with melatonin production, which implied that TDC may be the rate-limiting enzyme of the melatonin biosynthetic process in mulberries. During mulberry fermentation, the melatonin concentration increased rapidly in the beginning and then decreased gradually. Low temperature delayed the melatonin production during fermentation. A relatively high level of melatonin was found in "Hongguo2Ë® compared with "BaiyuwangË® during fruit development and fermentation. The variation of melatonin correlated with the ethanol production rate, suggesting that melatonin may participate in physiological regulation of Saccharomyces cerevisiae during the fermentation stage.

Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation.

At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu2+ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu2+ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu2+ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China's stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress.

Changes of resveratrol and antioxidant enzymes during UV-induced plant defense response in peanut seedlings.

Plants have evolved mechanisms to avoid and repair UV radiation damage, and the free radicals caused by UV tend to be involved in the induction of antioxidant defense systems. In this study, changes in resveratrol and antioxidant enzymes were investigated in relation to UV damage in peanut seedlings. Accumulation of endogenous resveratrol and stilbene synthase mRNA occurred rapidly and significantly in response to UV-C irradiation. Applying resveratrol before UV-C irradiation mitigated rusty spots and wilting of peanut leaves, and inhibition of resveratrol by applying 3,4-methylenedioxycinnamic acid worsened UV-C damage, an effect that was found to be concentration dependent. Correspondingly, the effect of resveratrol on malondialdehyde was similar to changes in the apparent morphology of seedling leaves. Changes in H(2)O(2), O(2)(-), and antioxidant enzymes showed some similarities after either UV-C irradiation or resveratrol treatment. Activities of superoxide dismutases, glutathione reductase, and catalase were more than 2-fold higher during the first 1h after treatments. Ascorbate peroxidase activity increased to more than 3-fold higher 24h after irradiation, whereas it was more than 2-fold higher 8h after resveratrol treatment. Activities of dehydroascorbate reductase and monodehydroascorbate reductase increased by 40% during 8-24h after treatments. Consequently, we proposed that changes in endogenous resveratrol and in antioxidant enzymes may have been involved in oxidative stress induced by UV-C exposure in peanut seedlings.

Comparison of phenolic acids and flavan-3-ols during wine fermentation of grapes with different harvest times.

To explore the effects of harvest time on phenolic compounds during wine fermentation, grape berries (Vitis vinifera L. cv. Vidal) were harvested at 17.5, 22.8 and 37.2 masculine Brix and were used to make dry wine, semi-sweet wine and icewine with low alcohol levels, respectively. Phenolic acids and flavan-3-ols were assayed during the fermentation of wines by means of reverse phase-high performance liquid chromatography (RP-HPLC). The results showed that concentrations of most of the phenolic acids and flavan-3-ol in musts increased with harvest time delay and higher total levels of these species were detected in all wines, compared with those measured before fermentation (the total phenolic acid content in wines was 1.5-2.0 fold that of in musts). Except for p-coumaric acid and (-)-epicatechin, other phenolic acids and flavan-3-ols had similar variation patterns (wave-like rise) during fermentation in dry wine and semi-sweet wine. However, some detected compounds, including gentisic acid, p-hydroxybenzoic acid, caffeic acid, p-coumaric acid and sinapic acid showed obviously different trends from the other two wines in the icewine making process. It is thus suggested that the harvest time has a decisive effect on phenols in final wines and influences the evolution of phenolic acids and flavan-3-ols during wine fermentation.

Involvement of phospholipase D in the low temperature acclimation-induced thermotolerance in grape berry.

Both phospholipase D (PLD, EC 3.1.4.4) and salicylic acid (SA) play important roles in response to external stimulation and activating defense system in plants. However, roles of the two signals in plants during the development of thermotolerance induced by low temperature acclimation remain unclear. In the experiment presented in the paper, grape berries (Vitis vinifera L. cv. Chardonnay) were pretreated at 8 degrees C for 3h and then transferred to 45 degrees C for heat stress. Compared with the control without low temperature pretreatment, membrane permeability and malondialdehyde (MDA) contents were reduced and the expression of HSP73 increased in the low temperature-pretreated berries under heat stress. During low temperature acclimation, PLD, SA and HSP73 could be activated. Meanwhile, the expression of HSP73 and the accumulation of free SA induced by low temperature can be inhibited by PLD activity inhibitor. All these results suggest that the activation of PLD is an early response to low temperature, and it is involved in the accumulation of free SA and the development of thermotolerance induced by low temperature acclimation.

Prokaryotic expression, polyclonal antibody preparation of the stilbene synthase gene from grape berry and its different expression in fruit development and under heat acclimation.

Stilbene synthase (STS, EC 2.3.1.95) leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. STS expression and regulation are important. Temperature is one of the main external factors affecting phytoalexin accumulation in plant tissues, the effect of temperature on resveratrol synthesis and stilbene synthase expression in grape berries has not been reported before. Here we cloned the full-length sts cDNA with 1179bp from grape berry via PCR, and then introduced into an expressed plasmid pET-30a(+) vector at the EcoRI and XhoI restriction sites. With the isopropyl-beta-d-thiogalactoside (IPTG) induced, the pET-sts was highly expressed in Escherichia coli BL21 (DE3) pLysS cells. A fusion protein with the His-Tag was purified by Ni-NTA His.Bind Resin and then used as the antigen to immunize a New Zealand rabbit. Furthermore, the antiserum was precipitated by 50% saturated ammonium sulfate and DEAE-Sephadex A-50 chromatography to obtain the immunoglobulin G (IgG) fraction. These results provide a substantial basis for the further studies of the STS in grape berry as well as in other species of plants. The sts expression in fruit development and in response to heat acclimation was then assayed. The results indicated STS was regulated in fruits depending on the developmental stage and significantly accumulation of STS mRNA and synthesis of new STS protein during the early of heat acclimation, this work offers an important basis for further investigating the mechanism of post-harvest fruit adaptation to environmental stresses.

Cloning of phospholipase D from grape berry and its expression under heat acclimation.

To investigate whether phospholipase D (PLD, EC 3.1.4.4) plays a role in adaptive response of post-harvest fruit to environment, a PLD gene was firstly cloned from grape berry (Vitis Vinifera L. cv. Chardonnay) using RT-PCR and 3'- and 5'-RACE. The deduced amino acid sequence (809 residues) showed 84.7% identity with that of PLD from Ricinus communis. The secondary structures of this protein showed the characteristic C2 domain and two active sites of a phospholipid-metabolizing enzyme. The PLD activity and its expression in response to heat acclimation were then assayed. The results indicated PLD was significantly activated at enzyme activity, as well as accumulation of PLD mRNA and synthesis of new PLD protein during the early of heat acclimation, primary suggesting that the grape berry PLD may be involved in the heat response in post-harvest grape berry. This work offers an important basis for further investigating the mechanism of post-harvest fruit adaptation to environmental stresses.

Activity and subcellular localization of glucose-6-phosphate dehydrogenase in peach fruits.

The subcellular distribution and activity of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) were studied in developing peach (Prunus persica L. Batsch cv. Zaoyu) fruit. Fruit tissues were separated by differential centrifugation at 15,000g into plastidic and cytosolic fractions. There was no serious loss of enzyme activity (or activation) during the preparation of fractions. G6PDH activity was found in both the plastidic and cytosolic compartments. Moreover, DTT had no effect on the plastidic G6PDH activities, that is, the redox regulatory mechanism did not play an important role in the peach fleshy tissue. Results from the immunogold electron-microscope localization revealed that G6PDH isoenzymes were mainly present in the cytosol, the secondary wall and plastids (chloroplasts and chromoplasts), but scarcely found in the starch granules or the cell wall. In addition to a decrease in fruit firmness, the G6PDH activity in the cytotolic and plastidic fractions increased, and anthocyanin started to accumulate during fruit maturation. These results suggest that G6PDH, by providing precursors for metabolic processes, might be associated with the red coloration that occurs in peach fruit.

Novel interrelationship between salicylic acid, abscisic acid, and PIP2-specific phospholipase C in heat acclimation-induced thermotolerance in pea leaves.

Increasing evidence suggests that heat acclimation and exogenous salicylic acid (SA) and abscisic acid (ABA) may lead to the enhancement of thermotolerance in plants. In this study, the roles that free SA, conjugated SA, ABA, and phosphatidylinositol-4,5-bisphosphate (PIP(2))-specific phospholipase C (PLC) play in thermotolerance development induced by heat acclimation (38 degrees C) were investigated. To evaluate their potential functions, three inhibitors of synthesis or activity were infiltrated into pea leaves prior to heat acclimation treatment. The results showed that the burst of free SA in response to heat acclimation could be attributed to the conversion of SA 2-O-D-glucose, the main conjugated form of SA, to free SA. Inhibition of ABA biosynthesis also resulted in a defect in the free SA peak during heat acclimation. In acquired thermotolerance assessment, the greatest weakness of antioxidant enzyme activity and the most severe heat injury (malondialdehyde content and degree of wilting) were found in pea leaves pre-treated with neomycin, a well-known inhibitor of PIP(2)-PLC activity. PsPLC gene expression was activated by exogenous ABA, SA treatments, and heat acclimation after pre-treatments with a SA biosynthesis inhibitor. From these results, PIP(2)-PLC appears to play a key role in free SA- and ABA-associated reinforcement of thermotolerance resulting from heat acclimation.

Expression of the chalcone synthase gene from grape and preparation of an anti-CHS antibody.

Flavonoids are closely related to a plant's antioxidative ability. Because chalcone synthase (CHS) is the first enzyme to act as part of the flavonoid biosynthesis pathway, its expression and regulation are important. Here we present the expression of a full-length chs cDNA with 1225bp from grape seedlings as well as the preparation of an antibody against the expressed protein. A full-length chs cDNA was introduced into an expressed plasmid pET-30a(+) vector at the EcoRI and SalI restriction sites. pET-chs was found to be highly expressed in Escherichia coli BL21(DE3) pLysS cells with isopropyl-beta-d-thiogalactoside (IPTG) induction. A fusion protein with the His.tag label was purified by Ni-NTA His. Bind Resin and then used as the antigen to immunize a New Zealand rabbit. The resulting antiserum was then further precipitated by 50% saturated ammonium sulfate and DEAE-Sepharose FF column chromatography to obtain the immunoglobulin G (IgG) fraction. The resulting antibody was found capable of immuno-recognizing the CHS of the crude protein extracts from different grape tissues with a molecular mass of 43kDa.

Contributions of PIP(2)-specific-phospholipase C and free salicylic acid to heat acclimation-induced thermotolerance in pea leaves.

The relationship between the accumulation in endogenous free salicylic acid (SA) induced by heat acclimation (37 degrees C) and the activity of PIP(2)-phospholipase C (PIP(2)-PLC; EC 3.1.4.3) in the plasma membrane fraction was investigated in pea (Pisum sativum L.) leaves. We focused our attention on the hypothesis that positive SA signals induced by heat acclimation may be relayed by PIP(2)-PLC. Heat acclimation induced an abrupt elevation of free SA preceding the activation of PLC toward PIP(2). Immunoblotting indicated a molecular mass with 66.5kDa PLC plays key role in the development of thermotolerance in pea leaves. In addition, some characterizations of PLC toward PIP(2) isolated from pea leaves with two-phase purification containing calcium concentration, pH and a protein concentration were also studied. Neomycin sulfate, a well-known PIP(2)-PLC inhibitor, was employed to access the involvement of PIP(2)-PLC in the acquisition of heat acclimation induced-thermotolerance. We were able to identify a PIP(2)-PLC, which was similar to a conventional PIP(2)-PLC in higher plants, from pea leaves suggesting that PIP(2)-PLC was involved in the signal pathway that leads to the acquisition of heat acclimation induced-thermotolerance. On the basis of these results, we conclude that the involvement of free SA may function as the upstream event in the stimulation of PIP(2)-PLC in response to heat acclimation treatment.