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.     

Oral supplementation with a combination of l-citrulline and l-arginine rapidly increases plasma l-arginine concentration and enhances NO bioavailability

Background

Chronic supplementation with l-citrulline plus l-arginine has been shown to exhibit anti-atherosclerotic effects. However, the short-term action of this combination on the nitric oxide (NO)–cGMP pathway remains to be elucidated. The objective of the present study was to investigate the acute effects of a combination of oral l-citrulline and l-arginine on plasma l-arginine and NO levels, as well as on blood circulation.

Methods

Rats or New Zealand white rabbits were treated orally with l-citrulline, or l-arginine, or a combination of each at half dosage. Following supplementation, plasma levels of l-arginine, NO_x, cGMP and changes in blood circulation were determined sequentially.

Results

l-Citrulline plus l-arginine supplementation caused a more rapid increase in plasma l-arginine levels and marked enhancement of NO bioavailability, including plasma cGMP concentrations, than with dosage with the single amino acids. Blood flow in the central ear artery in rabbits was also significantly increased by l-citrulline plus l-arginine administration as compared with the control.

Conclusion

Our data show for the first time that a combination of oral l-citrulline and l-arginine effectively and rapidly augments NO-dependent responses at the acute stage. This approach may have clinical utility for the regulation of cardiovascular function in humans.     

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.     

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.     

Anti-inflammatory effects of IL-4 and dynamic compression in IL-1beta stimulated chondrocytes

Mechanical loading can counteract inflammatory pathways induced by IL-1beta by inhibiting *NO and PGE2, catabolic mediators known to be involved in cartilage degradation. The current study investigates the potential of dynamic compression, in combination with the anti-inflammatory cytokine, IL-4, to further abrogate the IL-1beta induced effects. The data presented demonstrate that IL-4 alone can inhibit nitrite release in the presence and absence of IL-1beta and partially reverse the IL-1beta induced PGE2 release. When provided in combination, IL-4 and dynamic compression could further abrogate the IL-1beta induced nitrite and PGE2 release. IL-1beta inhibited [3H]thymidine incorporation and this effect could be reversed by IL-4 or dynamic strain alone or both in combination. By contrast, 35SO4 incorporation was not influenced by IL-4 and/or dynamic strain in IL-1beta stimulated constructs. IL-4 and mechanical loading may therefore provide a potential protective mechanism for cartilage destruction as observed in OA.     

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.     

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.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

H, C NMR and UV spectroscopy studies of gold(III)-tetracyanide complex with l-cysteine, glutathione, captopril, l-methionine and dl-seleno-methionine in aqueous solution

Auricyanide [Au(CN)₄]⁻ interaction with biologically important thiols, thioether and selenoether were carried out and monitored using ¹H, ¹³C NMR and UV spectroscopy. These ligands include l-cysteine, glutathione, captopril, l-methionine and dl-seleno-methionine. Thiols show very strong affinity to be oxidized into the disulfide by auricyanide, which gets reduced to aurocyanide [Au(CN)₂]⁻. l-cysteine reaction mechanism with [Au(CN)₄]⁻ was found to be dependent on reactants mole ratio. While l-methionine was completely inert toward auricyanide, dl-Se-methionine showed some reactivity with [Au(CN)₄]⁻ after raising solution pH to 12 that facilitated cyanide exchange.     

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.     

A synopsis of the process of lipid peroxidation since the discovery of the essential fatty acids

Eighty years ago, Burr and Burr, introduced for the first time the concept of essential fatty acids. Now is very well known that requirements for polyunsaturated fatty acids PUFAs can not be met by de novo metabolic processes within mammalian tissues. Animals are absolutely dependent on plants for providing the two major precursors of the n-6 and n-3 fatty acids, C18:2n-6; linoleic and C18:3n-3; α-linolenic acids. In animal tissues these precursors are transformed to fatty acids containing three to six double bonds. During the last four decades the interest in polyunsaturated fatty acids has augmented manifolds, and the number of published studies is rising each year. The current impetus for this interest has been mainly the observation that PUFAs and their metabolites have several physiological roles including: energy provision, membrane structure, cell signaling and regulation of gene expression. In addition the observation that PUFAs are targets of lipid peroxidation opens a new important area of investigation. Melatonin, the main secretory product of the pineal gland, efficiently scavenges both the hydroxyl and peroxyl radicals counteracting lipid peroxidation in biological membranes. In addition the two key pineal biochemical functions, lipoxygenation and melatonin synthesis may be synergistically regulated by the status of n-3 essential fatty acids. At the retina level, free radicals may preferentially react with the membrane polyunsaturated fatty acids leading to the release of lipoperoxide radicals. These lipoperoxides can induce oxidative stress linked to membrane lysis, damage to neuronal membranes may be related to alteration of visual function.     

The influence of selected O-alkyl derivatives of cyclodextrins on the enzymatic decomposition of l-tryptophan by l-tryptophan indole-lyase

A series of O-alkyl derivatives of cyclodextrin: heksakis[2,3,6-tri-O-(2′-methoxyethyl)]-α-cyclodextrin; heksakis(2,3-di-O-methyl)-α-cyclodextrin; heptakis(2,3-di-O-methyl)-β-cyclodextrin; heksakis[2,3-di-O-methyl-6-O-(2′-methoxyethyl)]-α-cyclodextrin; heptakis[2,3-di-O-methyl-6-O-(2′-methoxyethyl)]-β-cyclodextrin; heksakis[2,3-di-O-(2′-methoxyethyl)]-α-cyclodextrin and heptakis[2,3-di-O-(2′-methoxyethyl)]-β-cyclodextrin have been synthesized. Purity and composition of the obtained substances were examined. The cyclodextrin derivatives listed above as well as (2-hydroxypropyl)-α-cyclodextrin and (2-hydroxypropyl)-β-cyclodextrin, the two commercially available ones, have been investigated as the additives in the course of enzymatic decomposition of l-tryptophan by l-tryptophan indole-lyase. It has been found that each of cyclodextrin derivatives causes the inhibition of enzymatic process, both competitive and non-competitive. The competitive inhibition is connected with the formation of inclusion complexes between cyclodextrins and l-tryptophan, related to the geometry of these complexes. The mechanism of the non-competitive inhibition is not so evident; it could be related to the formation of the cyclodextrin complexes on the surface of the enzyme, leading to the change in the flexibility of the enzyme molecule.     

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.     

Characterization of novel 2-oxoglutarate dependent dioxygenases converting l-proline to cis-4-hydroxy-l-proline

Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing l-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-l-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-l-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel l-proline cis-4-hydroxylases converting free l-proline to cis-4-hydroxy-l-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected l-proline cis-4-hydroxylase activity in both proteins. Likewise l-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).     

Study and application of flow injection spectrofluorimetry with a fluorescent probe of 2-(2-pyridil)-benzothiazoline for superoxide anion radicals

This paper presents an automatic spectrofluorimetric method (flow injection spectrofluorimetry) using a novel fluorescent probe named H. Py. Bzt (2-(2-pyridil)-benzothiazoline) for determining superoxide dismutase (SOD) activity. The fluorescent probe was synthesized in house and fully characterized by elemental analysis and by infrared and (1)H nuclear magnetic resonance spectra. It could specially identify and trap O(2)(*-) and was oxidized by O(2)(*-) to form a strong fluorescence product. Based on this reaction, the flow injection spectrofluorimetric method was proposed and successfully used to determine SOD activity. The proposed method has a better selectivity in the determination of reactive oxygen species because the probe can be oxidized only by O(2)(*-) excluding H(2)O(2). As a kind of simple, rapid, precise, sensitive and automatic technique, it was applied to measurement of SOD activity in scallion, garlic, and onion with satisfactory results.