The influence of reactive oxygen species on cell cycle progression in mammalian cells

Cell cycle regulation is performed by cyclins and cyclin dependent kinases (CDKs). Recently, it has become clear that reactive oxygen species (ROS) influence the presence and activity of these enzymes and thereby control cell cycle progression. In this review, we first describe the discovery of enzymes specialized in ROS production: the NADPH oxidase (NOX) complexes. This discovery led to the recognition of ROS as essential players in many cellular processes, including cell cycle progression. ROS influence cell cycle progression in a context-dependent manner via phosphorylation and ubiquitination of CDKs and cell cycle regulatory molecules. We show that ROS often regulate ubiquitination via intermediate phosphorylation and that phosphorylation is thus the major regulatory mechanism influenced by ROS. In addition, ROS have recently been shown to be able to activate growth factor receptors. We will illustrate the diverse roles of ROS as mediators in cell cycle regulation by incorporating phosphorylation, ubiquitination and receptor activation in a model of cell cycle regulation involving EGF-receptor activation. We conclude that ROS can no longer be ignored when studying cell cycle progression.     

Macroevolutionary trends of atomic composition and related functional group proportion in eukaryotic and prokaryotic proteins

To fully explore the trends of atomic composition during the macroevolution from prokaryote to eukaryote, five atoms (oxygen, sulfur, nitrogen, carbon, hydrogen) and related functional groups in prokaryotic and eukaryotic proteins were surveyed and compared. Genome-wide analysis showed that eukaryotic proteins have more oxygen, sulfur and nitrogen atoms than prokaryotes do. Clusters of Orthologous Groups (COG) analysis revealed that oxygen, sulfur, carbon and hydrogen frequencies are higher in eukaryotic proteins than in their prokaryotic orthologs. Furthermore, functional group analysis demonstrated that eukaryotic proteins tend to have higher proportions of sulfhydryl, hydroxyl and acylamino, but lower of sulfide and carboxyl. Taken together, an apparent trend of increase was observed for oxygen and sulfur atoms in the macroevolution; the variation of oxygen and sulfur compositions and their related functional groups in macroevolution made eukaryotic proteins carry more useful functional groups. These results will be helpful for better understanding the functional significances of atomic composition evolution.     

The structural biochemistry of the superoxide dismutases

The discovery of superoxide dismutases (SODs), which convert superoxide radicals to molecular oxygen and hydrogen peroxide, has been termed the most important discovery of modern biology never to win a Nobel Prize. Here, we review the reasons this discovery has been underappreciated, as well as discuss the robust results supporting its premier biological importance and utility for current research. We highlight our understanding of SOD function gained through structural biology analyses, which reveal important hydrogen-bonding schemes and metal-binding motifs. These structural features create remarkable enzymes that promote catalysis at faster than diffusion-limited rates by using electrostatic guidance. These architectures additionally alter the redox potential of the active site metal center to a range suitable for the superoxide disproportionation reaction and protect against inhibition of catalysis by molecules such as phosphate. SOD structures may also control their enzymatic activity through product inhibition; manipulation of these product inhibition levels has the potential to generate therapeutic forms of SOD. Markedly, structural destabilization of the SOD architecture can lead to disease, as mutations in Cu,ZnSOD may result in familial amyotrophic lateral sclerosis, a relatively common, rapidly progressing and fatal neurodegenerative disorder. We describe our current understanding of how these Cu,ZnSOD mutations may lead to aggregation/fibril formation, as a detailed understanding of these mechanisms provides new avenues for the development of therapeutics against this so far untreatable neurodegenerative pathology.     

The glutathione thiyl radical does not react with nitrogen monoxide

Laser flash photolysis experiments shows that the rate constant for the reaction of the glutathione thiyl radical with nitrogen monoxide to give S-nitrosoglutathione is lower than 2.8+/-0.6 x 10(7)M(-1)s(-1). The conversion of the thiyl radical to its carbon-centred form at 10(3)s(-1) exceeds the formation of S-nitrosoglutathione when physiological concentrations of nitrogen monoxide are taken into account.     

l-2-oxothiazolidine-4-carboxylate influence on age- and heat exposure-dependent peroxidation in rat's liver and kidney

To investigate the impact of acute heat exposure on maintenance of redox homeostasis and antioxidant balance related to aging, we have determined the GSH levels in the liver and kidney, and the activity of antioxidant enzymes in the same organs from Wistar rats at two different ages, 35 days and 18 months. The animals were housed individually in a special heated chamber maintaining a constant temperature of 40±0.5 °C. The results showed that the level of endogenous GSH was signi?cantly lower in aged than in young animals. In general, the activity of antioxidant enzymes in investigated tissues displayed an age-dependent decline. Indeed, we found unchanged CAT activity and decreased GPx activity with age. On the other hand acute heat exposure led to disproportion between peroxide metabolizing enzymes (CAT, GPx) and GR, thus promoting H₂O₂ accumulation and prooxidative state in the liver of young animals. The results for the impact of l-2-oxothiazolidine-4-carboxylate in combined stress model suggested that in spite of restore levels of GSH, the restoration of oxido-reductive balance might have only been partial due to irreversible alterations in antioxidant enzymes set by acute heat exposure and aging. Interestingly, young animals appeared to be more sensitive to the supplementation of the l-2-oxothiazolidine-4-carboxylate, likely because of the more extensive increase of GSH observed in young l-2-oxothiazolidine-4-carboxylate treated animals.     

Mixed-valence porphyrin π-cation radical derivatives: Electrochemical investigations

The electrochemistry of [Cu(OEP)] and [Ni(OEP)] are compared with the mixed-valence π-cations and . These electrochemical studies, carried out with cyclic voltammetry and hydrodynamic voltammetry, show that the mixed valence π-cations have distinct electrochemical properties, although the differences between the [M(OEP)]^+/0 and processes are subtle.     

Antioxidative responses in Vitis vinifera infected by grapevine fanleaf virus

The antioxidative response of grapevine leaves (Vitis vinifera cv. Trebbiano) affected by the presence of grapevine fanleaf virus was studied during the summer of 2010 at three different harvest times (July 1st and 26th, and August 30th). At the first and second harvest, infected leaves showed increases in the concentration of superoxide radical and hydrogen peroxide, the latter increasing for enhanced activity of superoxide dismutase. In contrast, at the last harvest time, increases in the ascorbate pool and ascorbate peroxidase activity maintained hydrogen peroxide to control levels. The glutathione pool was negatively affected as summer progressed, showing a decrease in its total and reduced form amounts. At the same time, increases in the ascorbate pool were observed, making antioxidant defenses of grapevine effective also at the last harvest time. Increases in phenolic acids, and in particular in p-hydroxybenzoic acid, at the first and second harvest might have enhanced the efficiency of the antioxidant system through an interrelation between a peroxidase/phenol/ascorbate system and the NADPH/glutathione/ascorbate cycle. The lack of increase in p-hydroxybenzoic acid at the third harvest could be due instead to the enhanced utilization of this acid for hydrogen peroxide detoxification. With time, grapevine plants lost their capacity to contrast the spread of grapevine fanleaf virus, but acquired a greater ability to counteract pathogen-induced oxidative stress, being endowed with more reduced antioxidant pools.     

Binding modes of V(O)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various synthetic DNAs studied by polarized spectroscopy

The interactions between water-soluble cationic oxovanadyl[meso-tetrakis(4-N-methylpyridiumyl)]porphyrin (VOTMPyP) and various synthetic polynucleotide including poly[d(A–T)₂], poly[d(G–C)₂], and poly[d(I–C)₂] were studied using absorption, circular dichroism (CD), and linear dichroism (LD) spectroscopy. When VOTMPyP formed a complex with poly[d(A–T)₂] and poly[d(I–C)₂], a positive CD signal at low [VOTMPyP]/[DNA] ratios (R ratios) and strong excitonic CD signals at above R ≥ 0.15 were induced. The appearance of the CD spectra of the VOTMPyP-poly[d(G–C)₂] complex were very different: a small negative CD at low R ratios and very small excitonic CD at high R ratios were observed. Considering the facts that the minor grooves of the former two polynucleotides resemble and the major groove of poly[d(I–C)₂] is similar with that of poly[d(G–C)₂], it is conclusive that VOTMPyP binds to the minor groove of all DNA at lower R ratios while they stack at the outside of DNA at higher R ratios. The binding geometry of VOTMPyP to all polynucleotides studied by LD seemed to be homogenous, irrespective of the R ratio. It has been found that VOTMPyP can have five- and six-fluxional coordination states. Comparing the absorption spectra of VOTMPyP complexed with poly[d(A–T)₂] and poly[d(G–C)₂], the distinctive absorptions of the five- and six-coordinated species were observed at lower R ratios which centered at 420–430 nm and 442 nm, respectively. While the six-coordinated VOTMPyP favored the poly[d(A–T)₂], the five-coordinated species favored the poly[d(G–C)₂] at the low R ratios. As the stacked species increased with an increasing R ratio, the six-coordinated species became the major bound species. These observations lead us to conclude that the guanine base′ amino group plays a crucial role not only in determining the binding mode of VOTMPyP but also in the conversion of the six-coordinated species to the five-coordinated species.     

Regulation of Ras proteins by reactive nitrogen species

Ras GTPases have been a subject of intense investigation since the early 1980s, when single point mutations in Ras were shown to cause deregulated cell growth control. Subsequently, Ras was identified as the most prevalent oncogene found in human cancer. Ras proteins regulate a host of pathways involved in cell growth, differentiation, and apoptosis by cycling between inactive GDP-bound and active GTP-bound states. Regulation of Ras activity is controlled by cellular factors that alter guanine nucleotide cycling. Oncogenic mutations prevent protein regulatory factors from down-regulating Ras activity, thereby maintaining Ras in a chronically activated state. The central dogma in the field is that protein modulatory factors are the primary regulators of Ras activity. Since the mid-1990s, however, evidence has accumulated that small molecule reactive nitrogen species (RNS) can also influence Ras guanine nucleotide cycling. Herein, we review the basic chemistry behind RNS formation and discuss the mechanism through which various RNS enhance nucleotide exchange in Ras proteins. In addition, we present studies that demonstrate the physiological relevance of RNS-mediated Ras activation within the context of immune system function, brain function, and cancer development. We also highlight future directions and experimental methods that may enhance our ability to detect RNS-mediated activation in cell cultures and in vivo. The development of such methods may ultimately pave new directions for detecting and elucidating how Ras proteins are regulated by redox species, as well as for targeting redox-activated Ras in cancer and other disease states.     

Ascorbic acid induced degradation of beta-glucan: Hydroxyl radicals as intermediates studied by spin trapping and electron spin resonance spectroscopy

The formation of hydroxyl radicals in beta-glucan solutions treated with ascorbic acid and iron(II) was demonstrated by ESR spin trapping based methods. Two different spin traps were tested, namely DMPO which is commonly used to detect hydroxyl radicals, and POBN often used to detect carbon centered radicals. The experiments performed showed that the presence of iron(II) with DMPO led to low DMPO-OH adduct stability and further to DMPO dimerization. The level of hydroxyl radicals formed during the beta-glucan radical mediated degradation was evaluated using two ESR spin trapping methods based on the use POBN together with either 2% (v/v) EtOH or DMSO. The addition of ascorbic acid together with iron(II) in beta-glucan solution led to an immediate maximal production of hydroxyl radicals while the presence of ascorbic acid alone led to a progressive production of radical. Further hydroxyl radicals were found to be formed when iron(II) was added alone in beta-glucan solutions. The viscosity loss observed in the three last mentioned beta-glucan solutions were found to relate with the formation of hydroxyl radicals. These data confirm the involvement of hydroxyl radical in the beta-glucan degradation.     

Triple mutation of Cys26, Trp35, and Cys126 in stromal ascorbate peroxidase confers H2O2 tolerance comparable to that of the cytosolic isoform

Ascorbate peroxidase (APX) isoforms localized in the stroma and thylakoid of the chloroplast play a principle role in detoxifying hydrogen peroxide (H₂O₂) generated in photosystem I; however, once the ascorbate is depleted, the enzyme is attacked by H₂O₂ and rapidly loses its activity. Here, we report that radical transfer across the porphyrin moiety and amino acid residues in the reaction intermediate and H₂O₂-mediated enzyme inactivation involve cooperative interactions of the Cys26, Trp35, and Cys126 residues of stromal APX. The wild-type enzyme had a half-time of inactivation of <10 s, while the triple mutant of the three residues retained 50% of the initial activity after H₂O₂ treatment for 3 min. The H₂O₂ tolerance of this mutant was comparable to that of the H₂O₂-tolerant APX isoform localized in the cytosol.     

Detection and characterisation of radicals in biological materials using EPR methodology

Background

Electron paramagnetic resonance (EPR) spectroscopy (also known as electron spin resonance, ESR, spectroscopy) is widely considered to be the “gold standard” for the detection and characterisation of radicals in biological systems.

Scope of review

The article reviews the major positive and negative aspects of EPR spectroscopy and discusses how this technique and associated methodologies can be used to maximise useful information, and minimise artefacts, when used in biological studies. Consideration is given to the direct detection of radicals (at both ambient and low temperature), the use of spin trapping and spin scavenging (e.g. reaction with hydroxylamines), the detection of nitric oxide and the detection and quantification of some transition metal ions (particularly iron and copper) and their environment.

Major conclusions

When used with care this technique can provide a wealth of valuable information on the presence of radicals and some transition metal ions in biological systems. It can provide definitive information on the identity of the species present and also information on their concentration, structure, mobility and interactions. It is however a technique that has major limitations and the user needs to understand the various pitfalls and shortcoming of the method to avoid making errors.

General significance

EPR remains the most definitive method of identifying radicals in complex systems and is also a valuable method of examining radical kinetics, concentrations and structure. This article is part of a Special Issue entitled Current methods to study reactive oxygen species — pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Kinetics of the plastoquinone pool oxidation following illumination Oxygen incorporation into photosynthetic electron transport chain

The oxidation of the PQ-pool after illumination with 50 or 500 micromol quantam(-2)s(-1) was measured in isolated thylakoids as the increase in DeltaA(263), i.e., as the appearance of PQ. While it was not observed under anaerobic conditions, under aerobic conditions it was biphasic. The first faster phase constituted 26% or 44% of total reappearance of PQ, after weak or strong light respectively. The dependence on oxygen presence as well as the correlation with the rate of oxygen consumption led to conclusion that this phase represents the appearance of PQ from PQ(*-) produced in the course of PQH(2) oxidation by superoxide accumulated in the light within the membrane.     

Relationships between single nucleotide polymorphisms of antioxidant enzymes and disease

The presence and progression of numerous diseases have been linked to deficiencies in antioxidant systems. The relationships between single nucleotide polymorphisms (SNPs) arising from specific antioxidant enzymes and diseases associated with elevated oxidative stress have been studied with the rationale that they may be useful in screening for diseases. The purpose of this narrative review is to analyse evidence from these studies. The antioxidant enzyme SNPs selected for analysis are based on those most frequently investigated in relation to diseases in humans: superoxide dismutase (SOD2) Ala16Val (80 studies), glutathione peroxidise (GPx1) Pro197Leu (24 studies) and catalase C-262T (22 studies). Although the majority of evidence supports associations between the SOD2 Ala16Val SNP and diseases such as breast, prostate and lung cancers, diabetes and cardiovascular disease, the presence of the SOD2 Ala16Val SNP confers only a small, clinically insignificant reduction (if any) in the risk of these diseases. Other diseases such as bladder cancer, liver disease, nervous system pathologies and asthma have not been consistently related to this SOD SNP genotype. The GPx1 Pro197Leu and catalase C-262T SNP genotypes have been associated with breast cancer, but only in a small number of studies. Thus, currently available evidence suggests antioxidant enzyme SNP genotypes are not useful for screening for diseases in humans.     

Responses of antioxidant gene, protein and enzymes to salinity stress in two genotypes of perennial ryegrass (Lolium perenne) differing in salt tolerance

Salinity could damage cellular membranes through overproduction of reactive oxygen species (ROS), while antioxidant capacities play a vital role in protecting plants from salinity caused oxidative damages. The objective of this study was to investigate the toxic effect of salt on the antioxidant enzyme activities, isoforms and gene expressions in perennial ryegrass (Lolium perenne L.). Salt-tolerant ‘Quickstart II’ and salt-sensitive ‘DP1′ were subjected to 0 and 250 mM NaCl for 12 d. Salt stress increased the content of lipid peroxidation (MDA), electrolyte leakage (EL) and hydrogen peroxide (H₂O₂), to a greater extent in salt-sensitive genotype. Salt-stressed plant leaves exhibited a greater activity of superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11) at 4 d after treatment (DAT), but a lower level of enzyme activity at 8 and 12 d, when compared to the control. Catalase (CAT, EC 1.11.1.6) activity was greater at 4 DAT and thereafter decreased in salt tolerant genotype relative to the control, whereas lower than the control during whole experiment period for salt-sensitive genotype. There were different patterns of five isoforms of SOD, POD and two isoforms of APX between two genotypes. Antioxidant gene expression was positively related to isoenzymatic and total enzymatic activities during 12-d salt-treated leaves of two genotypes, with a relatively higher level in salt-tolerant genotype. Thus, salt tolerance could be related to the constitutive/induced antioxidant gene, leading to more efficient enzyme stimulation and protection in perennial ryegrass.     

Relationship between the action of reactive oxygen and nitrogen species on bilayer membranes and antioxidants

Membrane lipid peroxidation (LPO) induced by hydroxyl (*OH) and ascorbyl (*Asc) radicals and by peroxynitrite (ONOO-) was investigated in asolectin (ASO), egg phosphatidylcholine (PC) and PC/phosphatidic acid mixtures (PC:PA) liposomes and rat liver microsomes (MC). Enthalpy variation (DeltaH) of PC:PA at different molar ratios were obtained by differential scanning calorimetry. It was also evaluated the LPO inhibition by quercetin, melatonin and Vitamin B6. The oxidant effect power follows the order *OH approximately *Asc > ONOO- on PC and MC; whilst on ASO liposomes, it follows *Asc > *OH approximately ONOO-. Increasing amounts of PA in PC liposomes resulted in lower levels of LPO. The DeltaH values indicate a more ordered membrane arrangement as a function of PA amount. The results were discussed in order to provide a complete view involving the influence of membranes, oxidants and antioxidants intrinsic behavior on the LPO dynamics.     

NO, NO, NO! Nitrosyl siblings from [IrCl5(NO)]

Pentachloronitrosyliridate(III) ([IrCl₅(NO)]⁻), the most electrophilic NO⁺ known to date, can be reduced chemically and/or electrochemically by one or two electrons to produce the NO and HNO/NO⁻ forms. The nitroxyl complex can be formed either by hydride attack to the NO⁺ in organic solvent, or by decomposition of iridium-coordinated nitrosothiols in aqueous solutions, while NO is produced electrochemically or by reduction of [IrCl₅(NO)]⁻ with H₂O₂. Both NO and HNO/NO⁻ complexes are stable under certain conditions but tend to labilize the trans chloride and even the cis ones after long periods of time. As expected, the NO⁺ is practically linear, although the IrNO moiety is affected by the counterions due to dramatic changes in the solid state arrangement. The other two nitrosyl redox states comprise bent structures.