High Oxidative Stress Conditions Research Articles

Alterations in intracellular and extracellular activities of antioxidant enzymes during suspension culture of sweetpotato

Cultured plant cells are a good system for the study of antioxidant mechanisms and for the mass production of antioxidants, because they can be grown under conditions of high oxidative stress. Alterations in the intracellular and extracellular activities of three antioxidant enzymes, superoxide dismutase (SOD), guaiacol-type peroxidase (POD), and glutathione peroxidase (GPX), were investigated in suspension cultures of sweetpotato ( Ipomoea batatas) during cell growth. Intracellular SOD activities (units/mg protein) at 15 days after subculture (DAS) and 30 DAS were 10 and 20 times higher, respectively, compared with the SOD activity at 1 DAS, whereas intracellular specific POD and GPX activities did not significantly increase until after 15 DAS, when they rapidly increased. The extracellular activities of the three enzymes in culture medium were much higher than were the intracellular activities. The change in extracellular SOD activity was similar to that of extracellular GPX during cell growth. Those activities showed high levels until 5 DAS and then significantly decreased. Extracellular POD activity had an almost constant level regardless of the cell growth stage. In addition, intracellular SOD and POD isozymes were quite different from those isozymes in the culture medium. The changes in SOD and POD isozymes observed here suggest that different isozymes might modulate the levels of reactive oxygen intermediates during cell growth. Characterization of extracellular antioxidant enzymes discovered here would provide a new understanding for defense mechanism in plants.

PhgABC , a Three-Gene Operon Required for Growth of Streptococcus pneumoniae in Hyperosmotic Medium and In Vivo

To cause disease, bacterial pathogens need to be able to adapt to the physiological conditions found within the host, including an osmolality of approximately 290 mosmol kg(-1). While investigating Streptococcus pneumoniae genes contained within pneumococcal pathogenicity island 1, we identified a three-gene operon of unknown function termed phgABC. PhgC has a domain with similarity to diacylglycerol kinases of eukaryotes and is the first described member of a family of related proteins found in many gram-positive bacteria. phgA and phgC mutant strains were constructed by insertional duplication mutagenesis and found to have impaired growth under conditions of high osmotic and oxidative stress. The compatible solutes proline and glycine betaine improved growth of the wild-type and the phgA mutant strains in hyperosmolar medium, and when analyzed by electron microscopy, the cellular morphology of the phgA mutant strain was unaffected by osmotic stress. The phgA and phgC mutant strains were reduced in virulence in models of both systemic and pulmonary infection. As the virulence of the phgA mutant strain was not restored in gp91phox(-/-) mice and the phgA and phgC mutant strains had reduced growth in both blood and serum, the reduced virulence of these strains is unlikely to be due to increased sensitivity to the respiratory burst of phagocytes but is, instead, due to impaired growth at physiological osmolality.

Ascorbic acid might play a role in the sclerotial differentiation of Sclerotium rolfsii

Certain phytopathogenic fungi differentiate by forming sclerotia by an unclear biochemical mechanism. We have proposed that sclerotial differentiation might be regulated by fungal antioxidant defense. Part of this defense might be ascorbic acid, which in its reduced form is a well-known antioxidant. This natural antioxidant was studied in Sclerotium rolfsii in relation to oxidative-growth conditions, developmental stages and strain-differentiating ability. The transition of a sclerotial strain from the undifferentiated to the differentiated stage was accompanied by a sharp shift in the ratio of reduced/oxidized ascorbate toward the oxidized form. Ascorbate profiles and lipid peroxidation levels were different between the sclerotial strain grown under high- and low-oxidative stress conditions, as well as between a nonsclerotial S. rolfsii strain grown under high-oxidative stress conditions. In addition, the ratio of reduced/oxidized ascorbate in the nonsclerotial strain remained unchanged throughout growth. Lipid peroxidation under high-oxidative stress conditions in sclerotial S. rolfsii colonies one day before differentiation was 3.6-fold higher than in same-day colonies of this strain grown under low-oxidative stress conditions and 2.5-fold higher than in similar-day colonies of the nonsclerotial strain grown under high-oxidative stress conditions. Exogenous ascorbate caused a concentration-dependent reduction of lipid peroxidation and a proportional inhibition of the degree of sclerotial differentiation in the sclerotial strain grown under high-oxidative stress conditions by lowering its lipid peroxidation before differentiation to levels similar to the strain grown under low-oxidative stress conditions and to the nonsclerotial strain. Ascorbic acid might be produced by the sclerotial strain to reduce oxidative stress, although less efficiently than the nondifferenting strain. The data of this study support our theory that oxidative stress might be the triggering factor of sclerotial differentiation in phytopathogenic fungi.

The chemistry of melatonin's interaction with reactive species.

Melatonin has been shown to be an effective antioxidant in a number of experimental models both in vitro and in vivo. Considering the data available, it is now clear that the indoleamine is involved in antioxidative mechanisms more complex than originally envisaged. These range from the direct radical scavenging of a variety of radicals and reactive species to the control and/or modulation of a number of processes which may trigger a redox imbalance between antioxidant and prooxidant species. This review focuses on the direct radical scavenging activity of melatonin and provides a summary of the mechanisms of the reactions between the indoleamine and reactive species in pure chemical solutions. These actions likely account for at least some of the protective actions of melatonin under conditions of high oxidative stress.

DNA from bronchial secretions modulates elastase inhibition by alpha(1)-proteinase inhibitor and oxidized secretory leukoprotease inhibitor.

Previously we reported that DNA from sputum promotes the inhibition of human leukocyte elastase (HLE) by native secretory leukoprotease inhibitor (SLPI). This study shows that sputum DNA also promotes the inhibition by oxidized SLPI, a form of SLPI that may occupy a large fraction of the inhibitor in the lungs under conditions of high oxidative stress. With sputum DNA at 5 microg/ml, a concentration much lower than those in vivo, the inhibition constant (K(i) ) of oxidized SLPI against HLE is reduced from 31 nM to 23 to 920 pM, as compared with the K(i) of native SLPI, 58 pM, under the same conditions. On the other hand, sputum DNA retards inhibition of HLE by alpha(1)-proteinase inhibitor (alpha(1)-PI). The association rate of alpha(1)-PI and HLE is decreased from 1 x 10(7) M(-1) s(-1) in the absence of DNA to 2 to 6 x 10(6) M(-1) s(-1) in the presence of sputum DNA at 100 microg/ml. On the basis of results with an elastase-specific oligonucleotide aptamer, it was found that the downregulation of alpha(1)-PI activity can be attributed to an interaction between sputum DNA and multiple DNA-binding sites on HLE. DNA-binding sites on HLE also participate in the upregulation of oxidized SLPI activity. Data from this and our previous studies demonstrate that sputum DNA facilitates the association of HLE with native and oxidized SLPI, whereas it delays the association of HLE with alpha(1)-PI. We conclude that by modulating the inhibition of HLE, sputum DNA directly affects the balance between proteases and antiproteases in the lungs.

Large accumulation of mRNA and DNA point modifications in a plant senescent tissue

Although nucleic acids are the paradigm of genetic information conservation, they are inherently unstable molecules that suffer intrinsic and environmental damage. Oxidative stress has been related to senescence and aging and, recently, it has been shown that mutations accumulate at high frequency in mitochondrial DNA with age. We investigated RNA and DNA modifications in cork, a senescent plant tissue under high endogenous oxidative stress conditions. When compared to normally growing young tissue, cork revealed an unexpected high frequency of point modifications in both cDNA (Pn=1/1784) and nuclear DNA (Pn=1/1520). Cork should be viewed as a mosaic of genetically heterogeneous cells. This has biological implications: it supports somatic mutation models for aging and challenges ‘single cDNA clone’ as descriptor for the molecular genetics of senescent tissues.

Increased Expression but Reduced Activity of Antioxidant Enzymes in Alzheimer's Disease.

A growing body of data suggests that free radicals are involved in the pathogenesis of Alzheimer's disease (AD). Increased expression of antioxidant enzymes, such as superoxide dismutase (SOD), and their co-localization to senile plaques and dystrophic neurites have established a firm association between free-radical mediated injury and the disease neuropathology. While several studies have confirmed these findings, there is conflicting information regarding the activity of some of the enzymes. In the current report, we assayed the activity of superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px) from the same areas of the tissue showing increased expression of SOD1 and SOD2 (parallel sequential slices). Nine brains with neuropathologically confirmed AD and six neuropathologically normal, age-matched, controls were examined. Despite marked increased expression of SOD1 and SOD2 within senile plaques in all the cases studied, the activities of SOD, GSH-Px and catalase were significantly lower in AD than in control brains. The difference was most profound in the case of catalase followed by GSH-Px and SOD. These data are in qualitative agreement with that of several laboratories, and support a decrease rather than an increase, in antioxidant enzyme activity. The findings suggest two main possibilities. On one hand, the observed reduced activity along with antigenically increased expression may be consistent with inactivation of excess protein that has been synthesized under conditions of high oxidative stress. Increased protein oxidation coupled with enzyme inactivation is a documented, aging-associated phenomenon. Alternatively, the increased immuno-reactivity may reflect a redistribution phenomenon as the enzymes become more concentrated at the sites of increased oxidative stress, despite an over all reduction in their activity.

Influence of oxygen radical injury on DNA methylation.

One of the most prevalent products of oxygen radical injury in DNA is 8-hydroxyguanosine. Cells must be able to withstand damage by oxygen radicals and possess specific repair mechanisms that correct this oxidative lesion. However, when these defenses are oversaturated, such as under conditions of high oxidative stress, or when repair is inefficient, the miscoding potential of this lesion can result in mutations in the mammalian genome. In addition to causing genetic changes, active oxygen species can lead to epigenetic alterations in DNA methylation, without changing the DNA base sequence. Such changes in DNA methylation patterns can strongly affect the regulation of expression of many genes. Although DNA methylation patterns have been found to be altered during carcinogenesis, little is known about the mechanism(s) that produce this loss of epigenetic controls of gene expression in tumors. Replacement of guanine with the oxygen radical adduct 8-hydroxyguanine profoundly alters methylation of adjacent cytosines, suggesting a role for oxidative injury in the formation of aberrant DNA methylation patterns during carcinogenesis. In this paper, we review both the genetic and epigenetic mechanisms of oxidative DNA damage and its association with the carcinogenic process, with special emphasis on the influence of free radical injury on DNA methylation.