Oxygen is required for the l-cysteine-mediated decomposition of protein-bound dinitrosyl-iron complexes

Increasing evidence suggests that iron-sulfur proteins are the primary targets of nitric oxide (NO). Exposure of Escherichia coli cells to NO readily converts iron-sulfur proteins to protein-bound dinitrosyl-iron complexes (DNICs). Although the protein-bound DNICs are stable in vitro under aerobic or anaerobic conditions, they are efficiently repaired in aerobically growing E. coli cells even without new protein synthesis. The cellular repair mechanism for the NO-modified iron-sulfur proteins remains largely elusive. Here we report that, unlike aerobically growing E. coli cells, starved E. coli cells fail to reactivate the NO-modified iron-sulfur proteins. Significantly, the addition of l-cysteine, but not other related biological thiols, results in decomposition of the protein-bound DNICs in starved E. coli cells and in cell extracts under aerobic conditions. However, under anaerobic conditions, l-cysteine has little or no effect on the protein-bound DNICs in starved E. coli cells or in vitro, suggesting that oxygen is required for the l-cysteine-mediated decomposition of the protein-bound DNICs. Additional studies reveal that l-cysteine is able to release the DNIC from the protein and bind to it, and the l-cysteine-bound DNICs are rapidly disrupted by oxygen, resulting in the eventual decomposition of the protein-bound DNICs under aerobic conditions. © 2010 Elsevier Inc

Iron-sulfur proteins are the major source of protein-bound dinitrosyl iron complexes formed in Escherichia coli cells under nitric oxide stress

Protein-bound dinitrosyl iron complexes (DNICs) have been observed in prokaryotic and eukaryotic cells under nitric oxide (NO) stress. The identity of proteins that bind DNICs, however, still remains elusive. Here we demonstrate that iron-sulfur proteins are the major source of protein-bound DNICs formed in Escherichia coli cells under NO stress. Expression of recombinant iron-sulfur proteins, but not proteins without iron-sulfur clusters, almost doubles the amount of protein-bound DNICs formed in E. coli cells after NO exposure. Purification of recombinant proteins from the NO-exposed E. coli cells further confirms that iron-sulfur proteins, but not proteins without iron-sulfur clusters, are modified, forming protein-bound DNICs. Deletion of the iron-sulfur cluster assembly proteins IscA and SufA to block the [4Fe-4S] cluster biogenesis in E. coli cells largely eliminates the NO-mediated formation of protein-bound DNICs, suggesting that iron-sulfur clusters are mainly responsible for the NO-mediated formation of protein-bound DNICs in cells. Furthermore, depletion of the chelatable iron pool in wild-type E. coli cells effectively removes iron-sulfur clusters from proteins and concomitantly diminishes the NO-mediated formation of protein-bound DNICs, indicating that iron-sulfur clusters in proteins constitute at least part of the chelatable iron pool in cells. © 2011 Elsevier Inc. All rights reserved

Anoxia Treatment for Delaying Skin Browning, Inhibiting Disease Development and Maintaining the Quality of Litchi Fruit

Litchi fruit has a very short shelf life after harvest, so marketers and consumers alike desire longer periods of storage, transportation and distribution. To extend shelf life, anoxia treatments were used for the fruit. Litchi fruit were exposed to pure N2 for 0, 3, 6, 12 or 24 h. They were then kept individually in closed but vented containers for 6 days in the dark at 20 °C and 95–100 % relative humidity. Exposure of litchi fruit to N2 for 3 or 6 h markedly delayed skin browning, reduced rot development and maintained higher concentrations of total soluble solids, titratable acidity and ascorbic acid after 6 days of storage. Anoxia treatment for 24 h reduced browning index, but it accelerated disease development, compared to the control. Thus, a pre-storage pure N2 treatment for 3 or 6 h can be an effective means of reducing rotting while maintaining the physical quality of the fruit

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 betweenNOand the IlvD [4Fe-4S] cluster is estimated to be (7.0±2.0)×106 M-2 · s-1 at 25°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 NOmediated modification of the IlvD [4Fe-4S] cluster regardless of the presence of O2 in the medium, further suggesting that NO is more reactivewith 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 proteinbound 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. © The Authors Journal compilation © 2009 Biochemical Society

Enhanced DPPH radical scavenging activity and DNA protection effect of litchi pericarp extract by Aspergillus awamori bioconversion

BACKGROUND: Litchi (Litchi chinensis Sonn.) pericarp is a major byproduct which contains a significant amount of polyphenol. This study was designed to biotransformation litchi pericarp extract (LPE) by Aspergillus awamori to produce more bioactive compounds with stronger antioxidant activities. RESULTS: The study exhibited that the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities significantly (p < 0.05) increased from 15.53% to 18.23% in the water-extracted fraction and from 25.41% to 36.82% in the ethyl acetate-extracted fraction. Application of DNA cleavage assay further demonstrated the enhanced protection effect of the fermented phenolics on DNA damage. It is also noted that the water-extracted fraction of the fermented LPE possessed a much stronger capacity than the ethyl acetate-extracted fraction to prevent from damage of supercoiled DNA. Interestingly, it was found that some new compounds such as catechin and quercetin appeared after of A. awamori fermentation of LPE, which could account for the enhanced antioxidant activity. CONCLUSION: The DPPH radical scavenging activity and DNA protection effect of LPE were increased by Aspergillus awamori bioconversion while some compounds responsible for the enhanced antioxidant activity were identified. This study provided an effective way of utilizing fruit pericarp as a readily accessible source of the natural antioxidants in food industry and, thus, extended the application area such as fruit by-products

Effects of Pure Oxygen on the Rate of Skin Browning and Energy Status in Longan Fruit

Postharvest pericarp browning is one of the main problems resulting in reduced shelf life of longan fruit. Experiments were conducted to examine the changes in concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP), energy charge levels and activities of polyphenol oxidase (PPO) and peroxidase (POD) in relation to pericarp browning of longan fruit. Fruit kept for 6 days in pure oxygen atmosphere at 28 C showed lower browning indices and higher ATP concentrations but lower AMP concentrations and higher respiratory rates, compared to those kept in air. While energy charge decreased during storage, the decrease was delayed markedly by exposure to pure oxygen. There was a lower energy charge in the browned fruit, which was associated with rapid increase in malondialdehyde concentration. Enhanced respiration of longan fruit exposed to pure oxygen can result in the production of ATP. However, fruit exposed to pure oxygen exhibited higher activities of PPO and POD, which was not associated with reduced skin browning inhibition. These results supported the hypothesis that skin browning of postharvest longan fruit may be a consequence of membrane injury caused by the lack of maintenance energy

Effects of Pure Oxygen on the Rate of Skin Browning and Energy Status in Longan Fruit

Postharvest pericarp browning is one of the main problems resulting in reduced shelf life of longan fruit. Experiments were conducted to examine the changes in concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP), energy charge levels and activities of polyphenol oxidase (PPO) and peroxidase (POD) in relation to pericarp browning of longan fruit. Fruit kept for 6 days in pure oxygen atmosphere at 28 C showed lower browning indices and higher ATP concentrations but lower AMP concentrations and higher respiratory rates, compared to those kept in air. While energy charge decreased during storage, the decrease was delayed markedly by exposure to pure oxygen. There was a lower energy charge in the browned fruit, which was associated with rapid increase in malondialdehyde concentration. Enhanced respiration of longan fruit exposed to pure oxygen can result in the production of ATP. However, fruit exposed to pure oxygen exhibited higher activities of PPO and POD, which was not associated with reduced skin browning inhibition. These results supported the hypothesis that skin browning of postharvest longan fruit may be a consequence of membrane injury caused by the lack of maintenance energy

An exotic fruit with high nutritional value: Kadsura coccinea fruit

This research was to determine nutritional composition, essential and toxic elemental content, and major phenolic acid with antioxidant activity in Kadsura coccinea fruit. The results indicated that Kadsura coccinea fruit exhibited the high contents of total protein, total fat, ash and essential elements such as calcium (Ca), ferrum (Fe) and phosphorus (P). The levels of four common toxic elements, i.e. cadmium (Cd), mercury (Hg), arsenic (As) and lead (Pb), were lower than legal limits. By high-performance liquid chromatography (HPLC) analysis, gallic acid was identified as major phenolic acid in peel and pulp tissues. Its contents were no significant difference in both tissues. In comparison with two commercial antioxidants, the major phenolic acid extracted from Kadsura coccinea exhibited stronger 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity and reducing power. Kadsura coccinea fruit is a good source of nutrition and natural antioxidant. It is worthwhile to popularize this exotic fruit around the world

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