Flavonoids can replace alpha-tocopherol as an antioxidant

Endogenous antioxidants such as the lipid-soluble vitamin E protect the cell membranes from oxidative damage. Glutathione seems to be able to regenerate alpha-tocopherol via a so-called free radical reductase. The transient protection by reduced glutathione (GSH) against lipid peroxidation in control liver microsomes is not observed in microsomes deficient in alpha-tocopherol. Introduction of antioxidant flavonoids, such as 7-monohydroxyethylrutoside, fisetin or naringenin, into the deficient microsomes restored the GSH-dependent protection, suggesting that flavonoids can take over the role of alpha-tocopherol as a chain-breaking antioxidant in liver microsomal membranes.     

The extraordinary antioxidant activity of vitamin E phosphate

The antioxidant activities of RRR-vitamin E (VE), all-rac-vitamin E (all-rac-VE), trolox, RRR-vitamin E acetate (VEA), all-rac-vitamin E phosphate (VEP) and RRR-vitamin E succinate (VES) were compared. In this study, the rank order in the inhibition of lipid peroxidation (LPO) of VE and its derivatives was trolox>VE approximately all-rac-VE>VEA>VES. VE and trolox inhibited LPO in non-heated and heated rat liver microsomes. It has generally been accepted that this is due to scavenging of free radicals by these antioxidants, and during this protection the antioxidants are oxidized. VEA and VES have to be converted into VE by esterases to obtain antioxidant activity against LPO. VEP, however, had a potent antioxidant effect of its own without conversion to VE. In contrast to VE, VEP is not consumed during this protection. Of the compounds tested, VEP is the most potent in induction of hemolysis of erythrocytes. EPR experiments using the spin label 16-doxylstearic acid showed that VEP reduces membrane fluidity, in contrast to VE. This indicates that VEP acts as a detergent and forms a barrier that might inhibit the transfer of radicals from one polyunsaturated fatty acid to another. This new mechanism may form the basis for a new class of antioxidants.     

Aquaporin functionality in roots of Zea mays in relation to the interactive effects of boron and salinity

Zea mays L. cv. amylacea, a plant tolerant of B and salinity, was used to determine the involvement of aquaporin functionality in the interactive effects of B and salinity. Also, growth, chlorophyll concentration, and water relations were studied. While growth and chlorophyll concentration did not show noticeable changes under saline conditions, the decrease in leaf water potential and osmotic potential, together with the marked decrease of stomatal conductance and root hydraulic conductance, showed that the plants were adjusted osmotically. However, no effect of B was observed. The very weak response of the Lpc of salt-stressed roots to Hg suggested that water channels were greatly reduced in number or, if present, were non-functional. The evidence that substantial B movement can occur through diffusion and channel-mediated transport is compelling, and could account for B uptake under conditions of adequate or greater B supply. Therefore, the reduction in the functionality of aquaporins for NaCl-treated plants could be related to the reduction of B concentrations in roots and leaves in B + NaCl-treated plants, in comparison with plants treated only with B.     

Targeting nucleotide-requiring enzymes: implications for diazoxide-induced cardioprotection

Modulation of mitochondrial respiratory chain, dehydrogenase, and nucleotide-metabolizing enzyme activities is fundamental to cellular protection. Here, we demonstrate that the potassium channel opener diazoxide, within its cardioprotective concentration range, modulated the activity of flavin adenine dinucleotide-dependent succinate dehydrogenase with an IC50 of 32 microM and reduced the rate of succinate-supported generation of reactive oxygen species (ROS) in heart mitochondria. 5-Hydroxydecanoic fatty acid circumvented diazoxide-inhibited succinate dehydrogenase-driven electron flow, indicating a metabolism-dependent supply of redox equivalents to the respiratory chain. In perfused rat hearts, diazoxide diminished the generation of malondialdehyde, a marker of oxidative stress, which, however, increased on diazoxide washout. This effect of diazoxide mimicked ischemic preconditioning and was associated with reduced oxidative damage on ischemia-reperfusion. Diazoxide reduced cellular and mitochondrial ATPase activities, along with nucleotide degradation, contributing to preservation of myocardial ATP levels during ischemia. Thus, by targeting nucleotide-requiring enzymes, particularly mitochondrial succinate dehydrogenase and cellular ATPases, diazoxide reduces ROS generation and nucleotide degradation, resulting in preservation of myocardial energetics under stress.     

New method to study oxidative damage and antioxidants in the human small bowel: effects of iron application

Iron may induce oxidative damage to the intestinal mucosa by its catalyzing role in the formation of highly reactive hydroxyl radicals. This study aimed to determine iron-induced oxidative damage provoked by a single clinical dosage of ferrous sulfate and to elucidate the antioxidant defense mechanisms in the human small intestine in vivo. A double-lumen perfusion tube was positioned orogastrically into a 40-cm segment of the proximal small intestine in six healthy volunteers (25 +/- 5 yr). The segment was perfused with saline and subsequently with saline containing 80 mg iron as ferrous sulfate at a rate of 10 ml/min. Intestinal fluid samples were collected at 15-min intervals. Thiobarbituric acid reactive substances concentrations as an indicator of lipid peroxidation increased significantly from 0.07 microM (range, 0-0.33 microM) during saline perfusion to 3.35 microM (range, 1.19-7.27 microM) during iron perfusion (P < 0.05). Nonprotein antioxidant capacity increased significantly from 474 microM (range, 162-748 microM) to 1,314 microM (range, 674-1,542 microM) (P < 0.05). These data show that a single dosage of ferrous sulfate induces oxidative damage and the subsequent release of an antioxidant in the small intestine in vivo in healthy volunteers.     

Data-driven docking for the study of biomolecular complexes

With the amount of genetic information available, a lot of attention has focused on systems biology, in particular biomolecular interactions. Considering the huge number of such interactions, and their often weak and transient nature, conventional experimental methods such as X-ray crystallography and NMR spectroscopy are not sufficient to gain structural insight into these. A wealth of biochemical and/or biophysical data can, however, readily be obtained for biomolecular complexes. Combining these data with docking (the process of modeling the 3D structure of a complex from its known constituents) should provide valuable structural information and complement the classical structural methods. In this review we discuss and illustrate the various sources of data that can be used to map interactions and their combination with docking methods to generate structural models of the complexes. Finally a perspective on the future of this kind of approach is given.     

No role of DT-diaphorase (NQO1) in the protection against oxidized quercetin

Quercetin is one of the most studied alimentary antioxidants. During its antioxidant activity, quercetin becomes oxidized into its ortho-quinone/quinone methide, denoted as QQ. QQ is toxic since it is highly reactive towards thiols. DT-diaphorase (NQO1) might protect against QQ toxicity by reducing QQ to quercetin. However, conflicting data have been reported. The aim of the present study is to elucidate the role of DT-diaphorase in the protection against QQ-mediated thiol reactivity. It was found that QQ is indeed a substrate for DT-diaphorase. However, QQ reacted much faster with glutathione or protein thiols than with DT-diaphorase in experiments with isolated compounds as well as with human liver cytosol or blood plasma. This indicates that DT-diaphorase has no role in the protection against QQ.     

Exercise training and oxidative stress in the elderly as measured by antipyrine hydroxylation products.

Effects of 12 wk exercise training on oxidative stress were examined in elderly humans. We measured oxidative stress during a 45 min cycling test by using antipyrine hydroxylation products. Antipyrine breakdown is independent of blood flow to the liver, which is important during exercise. Furthermore, antipyrine reacts quickly with hydroxyl radicals to form para- and ortho-hydroxyantipyrine. Ortho-hydroxyantipyrine is not formed in man through the mono-oxygenase pathway of cytochrome P450. Twenty subjects (9 women; 60 +/- 3 y) participated in the training program. Thirteen subjects (5 women; 64 +/- 7 y) served as inactive controls. Subjects trained, twice a week for 1 h, at a fitness center. After 12 wk, maximal oxygen uptake (p < .005) and workload capacity (p < .001) were only significantly elevated in the training group. After 12 wk, both groups observed no change in the ratios of antipyrine hydroxylates, para- and ortho-hydroxyantipyrine, to native antipyrine. Furthermore, no differences were observed within or between groups in the exercise-induced increase in the plasma level of thiobarbituric acid reactive species. In conclusion, 12-wk training had no effect on exercise-induced oxidative stress in elderly humans as measured by free radical reaction products of antipyrine. Despite the fact that training in elderly humans improves functional capacity, it appears not to compromise antioxidant defense mechanisms.     

p53 overexpression in formalin-fixed, paraffin-embedded tissue detected by immunohistochemistry.

Mutation and overexpression of the p53 gene have been noted in a wide range of human cancers and are thought to play a role in malignant transformation. Previously, immunohistochemical detection of p53 has been possible only in fresh-frozen tissues. We examined p53 expression in paraffin-embedded tissues from 50 epithelial ovarian cancers and 25 primary breast cancers with a modified immunohistochemical (IHC) technique developed in this laboratory, using monoclonal antibody (MAb) PAb1801. The 75 cases were selected from a group of patients in whom the expression levels had already been assessed in a fresh-tissue IHC assay. An identical staining reactivity was observed in both formalin-fixed, paraffin-embedded tissue and fresh-frozen tissue in 48 of 50 (96%) epithelial ovarian cancers and in 23 of 25 (92%) primary breast cancers. Immunodetection of p53 in paraffin-embedded tissue blocks will be a useful alternative to the standard fresh-tissue assay and can accurately reflect the level of p53 expression in human tumors.     

Treatment of murine peritoneal macrophages with bacterial lipopolysaccharide alters expression of c-fos and c-myc oncogenes

Expression of the c-fos, c-myc, and c-fms proto-oncogenes has been studied in thioglycollate-elicited murine peritoneal macrophages after exposure to lipopolysaccharide (LPS). After incubation with LPS (20 ng/ml), a transient and rapid induction of the expression of c-fos and c-myc oncogenes could be observed, whereas the RNA levels for c-fms were not affected. Treatment with lipid A, the active moiety of the LPS molecule, increased the c-fos and c-myc expression to a comparable degree. Similar induction of c-fos and c-myc was observed after treatment with phorbol myristate acetate, suggesting that this effect of LPS on murine macrophages might be mediated through stimulation of protein kinase C. Under similar experimental conditions, LPS treatment of macrophages did not trigger DNA synthesis. Treatment with LPS blocked DNA synthesis in macrophages treated with L cell-conditioned medium containing colony-stimulating factor. Thus changes in c-fos and c-myc expression may be elements in the complex series of biochemical events that contribute to macrophage activation and are not necessarily related to induction or priming for cellular proliferation.