Xuewu Duan

Redox Control of the DNA Damage-inducible Protein DinG Helicase Activity via Its Iron-Sulfur Cluster

The Escherichia coli DNA damage-inducible protein DinG, a member of the superfamily 2 DNA helicases, has been implicated in the nucleotide excision repair and recombinational DNA repair pathways. Combining UV-visible absorption, EPR, and enzyme activity measurements, we demonstrate here that E. coli DinG contains a redox-active [4Fe-4S] cluster with a midpoint redox potential (Em) of –390 ± 23 mV (pH 8.0) and that reduction of the [4Fe-4S] cluster reversibly switches off the DinG helicase activity. Unlike the [4Fe-4S] cluster in E. coli dihydroxyacid dehydratase, the DinG [4Fe-4S] cluster is stable, and the enzyme remains fully active after exposure to 100-fold excess of hydrogen peroxide, indicating that DinG could be functional under oxidative stress conditions. However, the DinG [4Fe-4S] cluster can be efficiently modified by nitric oxide (NO), forming the DinG-bound dinitrosyl iron complex with the concomitant inactivation of helicase activity in vitro and in vivo. Reassembly of the [4Fe-4S] cluster in NO-modified DinG restores helicase activity, indicating that the iron-sulfur cluster in DinG is the primary target of NO cytotoxicity. The results led us to propose that the iron-sulfur cluster in DinG may act as a sensor of intracellular redox potential to modulate its helicase activity and that modification of the iron-sulfur cluster in DinG and likely in other DNA repair enzymes by NO may contribute to NO-mediated genomic instability.

Effects of Various Temperatures and pH Values on the Extraction Yield of Phenolics from Litchi Fruit Pericarp Tissue and the Antioxidant Activity of the Extracted Anthocyanins

Litchi fruit pericarp tissue is considered an important source of dietary phenolics. This study consisted of two experiments. The first was conducted to examine the effects of various extraction temperatures (30, 40, 50, 60, 70 and 80 °C) and pH values (2, 3, 4, 5 and 6) on the extraction yield of phenolics from litchi fruit pericarp. Extraction was most efficient at pH 4.0, while an extraction temperature of 60 °C was the best in terms of the combined extraction yield of phenolics and the stability of the extracted litchi anthocyanins. The second experiment was carried out to further evaluate the effects of various temperatures (25, 35, 45, 55 and 65 °C) and pH values (1, 3, 5 and 7) on the total antioxidant ability and scavenging activities of DPPH radicals, hydroxyl radical and superoxide anion of the extracted anthocyanins. The results indicated that use of 45–60 °C or pH 3–4 exhibited a relatively high antioxidant activity. The study will help improve extraction yield of phenolics from litchi fruit pericarp and promote better utilization of the extracted litchi anthocyanins as antioxidants.

Reactivity of Nitric Oxide with the [4Fe-4S] Cluster of Dihydroxyacid Dehydratase from Escherichia coli

Although the NO (nitric oxide)-mediated modification of iron-sulfur proteins has been well-documented in bacteria and mammalian cells, specific reactivity of NO with iron-sulfur proteins still remains elusive. In the present study, we report the first kinetic characterization of the reaction between NO and iron-sulfur clusters in protein using the Escherichia coli IlvD (dihydroxyacid dehydratase) [4Fe-4S] cluster as an example. Combining a sensitive NO electrode with EPR (electron paramagnetic resonance) spectroscopy and an enzyme activity assay, we demonstrate that NO is rapidly consumed by the IlvD [4Fe-4S] cluster with the concomitant formation of the IlvD-bound DNIC (dinitrosyl-iron complex) and inactivation of the enzyme activity under anaerobic conditions. The rate constant for the initial reaction between NO and the IlvD [4Fe-4S] cluster is estimated to be (7.0+/-2.0)x10(6) M(-2) x s(-1) at 25 degrees C, which is approx. 2-3 times faster than that of the NO autoxidation by O2 in aqueous solution. Addition of GSH failed to prevent the NO-mediated modification of the IlvD [4Fe-4S] cluster regardless of the presence of O2 in the medium, further suggesting that NO is more reactive with the IlvD [4Fe-4S] cluster than with GSH or O2. Purified aconitase B [4Fe-4S] cluster from E. coli has an almost identical NO reactivity as the IlvD [4Fe-4S] cluster. However, the reaction between NO and the endonuclease III [4Fe-4S] cluster is relatively slow, apparently because the [4Fe-4S] cluster in endonuclease III is less accessible to solvent than those in IlvD and aconitase B. When E. coli cells containing recombinant IlvD, aconitase B or endonuclease III are exposed to NO using the Silastic tubing NO delivery system under aerobic and anaerobic conditions, the [4Fe-4S] clusters in IlvD and aconitase B, but not in endonuclease III, are efficiently modified forming the protein-bound DNICs, confirming that NO has a higher reactivity with the [4Fe-4S] clusters in IlvD and aconitase B than with O2 or GSH. The results suggest that the iron-sulfur clusters in proteins such as IlvD and aconitase B may constitute the primary targets of the NO cytotoxicity under both aerobic and anaerobic conditions.

Identification of a novel phenolic compound in litchi (Litchi chinensis Sonn.) pericarp and bioactivity evaluation

Litchi (Litchi chinensis Sonn.) is a delicious fruit widely accepted by consumers all over the world. In this work, phytochemical investigation of litchi pericarp methanol extracts led to the isolation of a novel phenolic, 2-(2-hydroxyl-5-(methoxycarbonyl) phenoxy) benzoic acid, together with kaempferol, isolariciresinol, stigmasterol, butylated hydroxytoluene, 3,4-dihydroxyl benzoate, methyl shikimate and ethyl shikimate. Most were found in litchi pericarp for the first time. Their structures were mainly elucidated by NMR and MS evidences. Antioxidant activities of the eight compounds were determined by a DPPH radical scavenging assay and the results showed that 2-(2-hydroxy-5-(methoxycarbonyl) phenoxy)benzoic acid, kaempferol, isolariciresinol, butylated hydroxytoluene and 3,4-dihydroxy benzoate exhibited good antioxidant activities. An interesting finding was that butylated hydroxytoluene was detected as a natural antioxidant in this work, which was usually taken as a synthesized antioxidant. Furthermore, the novel compound exhibited no inhibitory effects against tyrosinase and α-glucosidase activities.

Effect of Nitric Oxide on Ethylene Synthesis and Softening of Banana Fruit Slice during Ripening

The effects of nitric oxide (NO) on ethylene synthesis and softening of ripening-initiated banana slice were investigated. Fruit firmness, color, and contents of starch and acid-soluble pectin (ASP) were measured. In addition, ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) content, expression and activities of ACC synthase (ACS) and ACC oxidase (ACO), and activities of cell-wall-modifying enzymes, polygalacturonase (PG), pectin methylesterase (PME), and endo-beta-1,4-glucanase, were analyzed. Application of NO reduced ethylene production, inhibited degreening of the peel and delayed softening of the pulp. The decrease of ethylene production was associated with the reduction in the activity of ACO and the expression of the MA-ACO1 gene. Moreover, the NO-treated fruit showed a lower expression of the MA-ACS1 gene but higher ACS activity and ACC content. In addition, NO treatment decreased the activities of PG, PME, and endo-beta-1,4-glucanase and maintained higher contents of ASP and starch, which may account for the delay of softening. We proposed that the inhibition of ACO activity and transcription of gene MA-ACO1 by NO resulted in decreased ethylene synthesis and the delay of ripening of banana slice.

Energy status of ripening and postharvest senescent fruit of litchi (Litchi chinensis Sonn.)

BackgroundRecent studies have demonstrated that cellular energy is a key factor switching on ripening and senescence of fruit. However, the factors that influence fruit energy status remain largely unknown.ResultsHPLC profiling showed that ATP abundance increased significantly in developing preharvest litchi fruit and was strongly correlated with fruit fresh weight. In contrast, ATP levels declined significantly during postharvest fruit senescence and were correlated with the decrease in the proportion of edible fruit. The five gene transcripts isolated from the litchi fruit pericarp were highly expressed in vegetative tissues and peaked at 70 days after flowering (DAF) consistent with fruit ADP concentrations, except for uncoupling mitochondrial protein 1 (UCP1), which was predominantly expressed in the root, and ATP synthase beta subunit (AtpB), which was up-regulated significantly before harvest and peaked 2 days after storage. These results indicated that the color-breaker stage at 70 DAF and 2 days after storage may be key turning points in fruit energy metabolism. Transcript abundance of alternative oxidase 1 (AOX1) increased after 2 days of storage to significantly higher levels than those of LcAtpB, and was down-regulated significantly by exogenous ATP. ATP supplementation had no significant effect on transcript abundance of ADP/ATP carrier 1 (AAC1) and slowed the changes in sucrose non-fermenting-1-related kinase 2 (SnRK2) expression, but maintained ATP and energy charge levels, which were correlated with delayed senescence.ConclusionsOur results suggest that senescence of litchi fruit is closely related with energy. A surge of LcAtpB expression marked the beginning of fruit senescence. The findings may provide a new strategy to extend fruit shelf life by regulating its energy level.

Natural Occurrence, Analysis, and Prevention of Mycotoxins in Fruits and their Processed Products

Mycotoxins are small toxic chemical products formed as the secondary metabolites by fungi that readily contaminate foods with toxins in the field or after harvest. The presence of mycotoxins, such as aflatoxins, ochratoxin A, and patulin, in fruits and their processed products is of high concern for human health due to their properties to induce severe acute and chronic toxicity at low-dose levels. Currently, a broad range of detection techniques used for practical analysis and detection of a wide spectrum of mycotoxins are available. Many analytical methods have been developed for the determination of each group of these mycotoxins in different food matrices, but new methods are still required to achieve higher sensitivity and address other challenges that are posed by these mycotoxins. Effective technologies are needed to reduce or even eliminate the presence of the mycotoxins in fruits and their processed products. Preventive measures aimed at the inhibition of mycotoxin formation in fruits and their processed products are the most effective approach. Detoxification of mycotoxins by different physical, chemical, and biological methods are less effective and sometimes restricted because of concerns of safety, possible losses in nutritional quality of the treated commodities and cost implications. This article reviewed the available information on the major mycotoxins found in foods and feeds, with an emphasis of fruits and their processed products, and the analytical methods used for their determination. Based on the current knowledge, the major strategies to prevent or even eliminate the presence of the mycotoxins in fruits and their processed products were proposed.

Effects of short-term anoxia treatment on browning of fresh-cut Chinese water chestnut in relation to antioxidant activity

The effects of short-term anoxia pre-treatment on browning of fresh-cut Chinese water chestnut (CWC), stored at 4°C, in relation to antioxidant activity were investigated. CWC slices were exposed to pure N2 for 4h and then stored at 4°C for 18d. Anoxia significantly inhibited browning of CWC slices during storage, accompanied by lower contents of malondialdehyde, H2O2, and lipoxygenase activity. Furthermore, anoxia induced the activities of superoxide dismutase and ascorbate peroxidase, which could benefit scavenging reactive oxygen species and alleviating lipid peroxidation. In addition, better maintenance of reducing power and free-radical-scavenging activities against α,α-diphenyl-β-picrylhydrazy (DPPH), superoxide anions and hydroxyl was observed in N2-treated CWC slices, with higher phenolic compounds and ascorbic acid contents. Collectively, these finds suggest that N2 pre-treatment enhanced enzymatic and non-enzymatic antioxidant activity in CWC slices, and thereby contributed to alleviating lipid peroxidation and maintenance of storage quality.

Identification of Major Phenolic Compounds of Chinese Water Chestnut and their Antioxidant Activity

Chinese water chestnut (CWC) is one of the most popular foods among Asian people due to its special taste and medical function. Experiments were conducted to test the antioxidant activity and then determine the major phenolic compound components present in CWC. CWC phenolic extract strongly inhibited linoleic acid oxidation and exhibited a dose-dependent free-radical scavenging activity against alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radicals, superoxide anions and hydroxyl radicals, which was superior to ascorbic acid and butylated hydroxytoluene (BHT), two commercial used antioxidants. Furthermore, the CWC extract was found to have a relatively higher reducing power, compared with BHT. The major phenolic compounds present in CWC tissues were extracted, purified and identified by high-performance liquid chromatograph (HPLC) as (-)-gallocatechin gallate, (-)-epicatechin gallate and (+)-catechin gallate. This study suggests that CWC tissues exhibit great potential for antioxidant activity and may be useful for their nutritional and medicinal functions.

Role of hydroxyl radical in modification of cell wall polysaccharides and aril breakdown during senescence of harvested longan fruit.

The effects of reactive oxygen species (ROS), especially hydroxyl radical (OH), on cellular wall disassembly in vitro and in vivo, and aril breakdown in longan fruit, were investigated. OH caused the disassembly of cell wall material (CWM) in vitro, demonstrated by the increases of total sugars and uronic acid released, and the downshift in molecular mass of CDTA soluble pectin (CSP) and Na2CO3 soluble pectin (CO3-SP). In addition, OH resulted in decreased CO3-SP content and increased CSP content in CWM suspension, suggesting the conversion of CO3-SP to CSP. Application of exogenous OH accelerated aril breakdown in longan fruit while the process was delayed by l-cysteine·HCl, a ROS scavenger. Furthermore, lower CWM content and decreased molecular mass of pectins were observed in OH-treated fruit. These results indicated that OH contributed to the degradation of cell wall polysaccharides, resulting in aril breakdown in longan fruit.