Glucanase gene diversity in prokaryotic and eukaryotic organisms

A number of bacteria and eukaryotes produce extracellular enzymes that degrade various types of polysaccharides including the glucans starch, cellulose and hemicellulose (xylan). The similarities in the modes of expression and specificity of enzyme classes, such as amylase, cellulose and xylanase, suggest common genetic origins for particular activities. Our determination of the extent of similarity between these glucanases suggests that such data may be of very limited use in describing the early evolution of these proteins. The great diversity of these proteins does allow identification of their most highly conserved (and presumably functionally important) regions.     

Uric acid oxidation by peroxynitrite: multiple reactions, free radical formation, and amplification of lipid oxidation

Uric acid has been considered to be an efficient scavenger of peroxynitrite but the reaction between urate and peroxynitrite has been only partially characterized. Also, previous studies have indicated that urate may increase peroxynitrite-mediated oxidation of low density lipoprotein (LDL). Here, we examined the reaction between urate and peroxynitrite by combining kinetic, oxygen consumption, spin trapping, and product identification studies; in parallel, we tested the effect of urate upon peroxynitrite-mediated lipid oxidation. Our results demonstrated that urate reacts with peroxynitrite with an apparent second order rate constant of 4.8 x 10(2) M(-1). s(-1) in a complex process, which is accompanied by oxygen consumption and formation of allantoin, alloxan, and urate-derived radicals. The main radical was identified as the aminocarbonyl radical by the electrospray mass spectra of its 5, 5-dimethyl-l-pyrroline N-oxide adduct. Mechanistic studies suggested that urate reacts with peroxynitrous acid and with the radicals generated from its decomposition to form products that can further react with peroxynitrite anion. These many reactions may explain the reported efficiency of urate in inhibiting some peroxynitrite-mediated processes. Production of the aminocarbonyl radical, however, may propagate oxidative reactions. We demonstrated that this radical is likely to be the species responsible for the effects of urate in amplifying peroxynitrite-mediated oxidation of liposomes and LDL, which was monitored by the formation of lipid peroxides and thiobarbituric acid-reactive substances. The aminocarbonyl radical was not detectable during urate attack by other oxidants and consequently it is unlikely to be responsible for all previously described prooxidant effects of uric acid.     

Function of prokaryotic and eukaryotic ABC proteins in lipid transport

ATP binding cassette (ABC) proteins of both eukaryotic and prokaryotic origins are implicated in the transport of lipids. In humans, members of the ABC protein families A, B, C, D and G are mutated in a number of lipid transport and metabolism disorders, such as Tangier disease, Stargardt syndrome, progressive familial intrahepatic cholestasis, pseudoxanthoma elasticum, adrenoleukodystrophy or sitosterolemia. Studies employing transfection, overexpression, reconstitution, deletion and inhibition indicate the transbilayer transport of endogenous lipids and their analogs by some of these proteins, modulating lipid transbilayer asymmetry. Other proteins appear to be involved in the exposure of specific lipids on the exoplasmic leaflet, allowing their uptake by acceptors and further transport to specific sites. Additionally, lipid transport by ABC proteins is currently being studied in non-human eukaryotes, e.g. in sea urchin, trypanosomatides, arabidopsis and yeast, as well as in prokaryotes such as Escherichia coli and Lactococcus lactis. Here, we review current information about the (putative) role of both pro- and eukaryotic ABC proteins in the various phenomena associated with lipid transport. Besides providing a better understanding of phenomena like lipid metabolism, circulation, multidrug resistance, hormonal processes, fertilization, vision and signalling, studies on pro- and eukaryotic ABC proteins might eventually enable us to put a name on some of the proteins mediating transbilayer lipid transport in various membranes of cells and organelles. It must be emphasized, however, that there are still many uncertainties concerning the functions and mechanisms of ABC proteins interacting with lipids. In particular, further purification and reconstitution experiments with an unambiguous role of ATP hydrolysis are needed to demonstrate a clear involvement of ABC proteins in lipid transbilayer asymmetry.     

Ascorbate free radical and its role in growth control

Summary Ascorbate and its free radical potentiates proliferation of HL-60 cells in serum-limiting media. Dehydroascorbate does not affect growth. This stimulation of growth is due to a general shortening of the cell cycle. The incubation of HL-60 cells with ascorbate free radical produces a significant change of the redox potential of cells. The presence of cells in culture media avoids the total oxidation of ascorbate, and also HL-60 cells induce the short-term stabilization of ascorbate. Ascorbate free radical potentiates also the onset of DNA synthesis in CCL39 cells induced by fetal calf serum, although itself does not affect quiescense to proliferation transition. This transition induced by fetal calf serum also potentiates the capacity of CCL39 cells to stabilize ascorbate. We discuss here the role of ascorbate free radical on growth control by its reduction by the plasma membrane redox system and its meaning for cell physioslogy.     

The effect of carnosine on indicators of free radical lipid oxidation during acute stress in rats

The effect of carnosine intraperitoneal injection in rats (in doses 0.2, 2.0 or 20 mg/kg) on the vegetative parameters (arterial blood pressure, Hildebrandt index), the content of free radical oxidation (FRO) products and superoxide dismutase activity in serum and brain homogenates and brain lipid composition under normal condition and after different stress forms have been investigated. The carnosine injection in dose 20 mg/kg preserves and increase in arterial pressure and Hildebrandt index at all steps of stress development. The phase non-unidirectional changes in studied biochemical parameters have been revealed depending on the level of stress development in animals under control. The unidirectional and dose-dependent changes of phospholipid content and the level of brain lipids, decrease of FRO products in tissue and brain cholesterol, the increase of the superoxide dismutase activity of serum and brain homogenates have been found in intact and stressed animals after carnosine injection. A comparison of carnosine pharmacokinetics with concentration dependences of the antioxidative effect under in vitro and in vivo experiments comes to conclusion concerning the carnosine indirect adaptogenic action.     

Vitamin D3 and 20-hydroxyecdysone -- inhibitors of free radical lipid oxidation during D-hypervitaminosis

Under D-hypovitaminosis (control) conditions the statistically reliable increase of blood serum lipids free radical oxidation was revealed in comparison with the intact animals. Administration of vitamin D3 to the animals suffering from D-hypovitaminosis leads for statistically reliable decrease of blood serum lipids free radical oxidation, while 20-hydroxyecdysone in quantity of 0.02 mg per 1 kg of the animal body weight displays the antioxidative properties. Its antioxidative effect is characterized by a statistically reliable increase of Tind chemiluminescence kinetical parameter as compared with the control. Under D-hypovitaminosis conditions in the mitochondrial membranes the products of lipids free radical oxidation--dien conjugates are accumulated. In the case of administrating to the animals suffering from D-hypovitaminosis D3 or 20-hydroxyecdysone these oxidation products are absent. 20-Hydroxyecdysone under these conditions have been revealed as inducing accumulation in the mitochondrial and microsomal membranes of the substances with lambda 225 nm.     

Comparison of characteristics and function of translation termination signals between and within prokaryotic and eukaryotic organisms

Six diverse prokaryotic and five eukaryotic genomes were compared to deduce whether the protein synthesis termination signal has common determinants within and across both kingdoms. Four of the six prokaryotic and all of the eukaryotic genomes investigated demonstrated a similar pattern of nucleotide bias both 5′ and 3′ of the stop codon. A preferred core signal of 4 nt was evident, encompassing the stop codon and the following nucleotide. Codons decoded by hyper-modified tRNAs were over-represented in the region 5′ to the stop codon in genes from both kingdoms. The origin of the 3′ bias was more variable particularly among the prokaryotic organisms. In both kingdoms, genes with the highest expression index exhibited a strong bias but genes with the lowest expression showed none. Absence of bias in parasitic prokaryotes may reflect an absence of pressure to evolve more efficient translation. Experiments were undertaken to determine if a correlation existed between bias in signal abundance and termination efficiency. In Escherichia coli signal abundance correlated with termination efficiency for UAA and UGA stop codons, but not in mammalian cells. Termination signals that were highly inefficient could be made more efficient by increasing the concentration of the cognate decoding release factor.     

The structure of eukaryotic and prokaryotic complex I

The structures of the NADH dehydrogenases from Bos taurus and Aquifex aeolicus have been determined by 3D electron microscopy, and have been analyzed in comparison with the previously determined structure of Complex I from Yarrowia lipolytica. The results show a clearly preserved domain structure in the peripheral arm of complex I, which is similar in the bacterial and eukaryotic complex. The membrane arms of both eukaryotic complexes show a similar shape but also significant differences in distinctive domains. One of the major protuberances observed in Y. lipolytica complex I appears missing in the bovine complex, while a protuberance not found in Y. lipolytica connects in bovine complex I a domain of the peripheral arm to the membrane arm. The structural similarities of the peripheral arm agree with the common functional principle of all complex Is. The differences seen in the membrane arm may indicate differences in the regulatory mechanism of the enzyme in different species.     

The role of free sugars and amino acids in the regulation of biomass partitioning and plant growth

Theoretical plant growth models postulate an important role for growth substrates such as sugars and amino acids. To test this experimentally, spinach plants were grown under controlled conditions and with nitrogen added daily, following different exponential addition schemes. Plants were harvested during exponential growth. Free amino acid levels or free sugar levels were only weakly correlated with growth and biomass partitioning. Factor analysis showed however that the product of free sugar concentration and amino acid concentration yielded a parameter adequately reflecting the plant's nutritional state.It is concluded that growth and biomass partitioning under limiting N conditions cannot be modelled solely based on N substrate levels.     

Perferryl complex of nitric oxide synthase: role in secondary free radical formation

Neuronal nitric oxide synthase (NOS I) has been shown to generate nitric oxide (NO*) and superoxide (O(2)*-)during enzymatic cycling, the ratio of each free radical is dependent upon the concentration of L-arginine. Using spin trapping and electron paramagnetic resonance (EPR) spectroscopy, we recently reported that NOS I can oxidize ethanol (EtOH) to alpha-hydroxyethyl radical (CH(3)*CHOH). We speculated that the perferryl complex of NOS, (NOS-[Fe(5+)[double bond]O](3+)) was responsible for the generation of CH(3)*CHOH. Using potassium monopersulfate (KHSO(5)) to oxidize the heme of NOS I to NOS-[Fe(5+)[double bond]O](3+), we were able to demonstrate that this perferryl complex can oxidize L-arginine to L-citrulline and NO*. Even in the absence of L-arginine, EtOH was oxidized to CH(3)*CHOH by NOS-[Fe(5+)[double bond]O](3+). Sodium cyanide (NaCN), a heme blocker, inhibited the formation of CH(3)*CHOH by NOS.     

The enzymatic oxidation of Desferal to a nitroxide free radical

Desferrioxamine mesylate (Desferal), a transition metal ion chelator, has been used to inhibit the in vitro redox cycling of transition metal ions. ESR spectroscopy was utilized to detect and identify Desferal's one-electron oxidation product. We demonstrate that a horseradish peroxidase/H₂O₂ system, a xanthine oxidase/hypoxanthine system, and a hydroxyl radical-generating system are all capable of oxidizing Desferal to a nitroxide free radical. The same 9-line ESR spectrum (g = 2.0065, a^N = 7.85 G, a^H(2) = 6.35 G) was detected in all of the above systems. We, therefore, stress that care must be taken when using Desferal as a transition metal ion chelator to keep its concentration low enough to minimize these reactions, or to use a different metal ion chelator.     

Mitochondria and the regulation of free radical damage in the eye

Neuronal cell death can be determined by the overall level of reactive oxygen species (ROS) resulting from the combination of extrinsic sources and intrinsic production as a byproduct of oxidative phosphorylation. Key controllers of the intrinsic production of ROS are the mitochondrial uncoupling proteins (UCPs). By allowing a controlled leak of protons across the inner mitochondrial membrane activation of these proteins can decrease ROS and promote cell survival. In both primate models of Parkinson’s disease and mouse models of seizures, increased activity of UCP2 significantly increased neuronal cells survival. In the retina UCP2 is expressed in many neurons and glial cells, but was not detected in rod photoreceptors. Retinal ganglion cell survival following excitotoxic damage was much greater in animals overexpressing UCP2. Traditional Chinese medicines, such as an extract of Cistanche tubulosa, may provide benefit by altering mitochondrial metabolism.     

The role of calcium in the regulation of free radical reduction by thioredoxin reductase at the surface of the skin

Membrane associated thioredoxin reductase has been previously shown to reduce free radicals on the outer plasma membranes of human keratinocytes and melanocytes to provide a possible first line of defense against free radical damage at the surface of the skin. Preliminary experiments with cell cultures of human keratinocytes and melanocytes grown in serum-free medium showed that the enzyme activity depends on extracellular calcium concentration in the medium. Thioredoxin reductase activity at the surface of the skin, at the surface of human keratinocytes and melanocytes, and purified thioredoxin reductase from E. coli and adult human keratinocytes all exhibited calcium-dependent allosteric control. Since thioredoxin reductase contains two extremely reactive thiolate groups at the active site with pK values close to neutrality, both of these anions can form covalent complexes with N-ethylmaleimide by nucleophilic attack on the double bond. In our experiments we used spin-labeled maleimide [4-maleimido-tempo] to examine the local environment in the active site of thioredoxin reductase in the presence and absence of calcium. Both spin-labeled thioethers are distinguishable by EPR spectroscopy, with one site being significantly more immobilized than the other. Hence, it has been possible to observe direct evidence for active site closure by calcium. These results suggest that extracellular calcium may play an important role in regulation of thioredoxin reductase activity for the defense mechanism against UV-mediated free radical damage at the surface of human skin.     

A free radical scavenger role for daunomycin: inhibition of nonenzymatic oxidation of NADH

The effect of daunomycin(Dm) on the nonenzymatic oxidation of NADH in the presence of metal ions, H2O2 and various ligands was examined. The results show that daunomycin inhibits the oxidation of NADH by reacting with one or more of the reaction intermediates which include OH., HO2., O2. and NAD.. This reaction is a reductive one involving the anthraquinone moiety of daunomycin as confirmed by control experiments by comparative UV-Vis analysis of the spectral character of daunomycin after reduction by NaBH4. Daunomycin, once reduced, apparently does not transfer its electron to another species such as O2 and is thus thought to play the role of free radical scavenger.     

Sonochemical free radical formation in aqueous solutions

The phenomena of stable and transient acoustic cavitation in liquids exposed to ultrasound are briefly explained. The role of micronuclei, resonant bubble size, and rectified diffusion in the initiation of transient cavitation is reviewed. In aqueous solutions transient cavitation initially generates hydrogen atoms and hydroxyl radicals that may recombine to form hydrogen and H2O2 or may react with solutes in the gas phase, at the gas-liquid boundary, or in the bulk of the solution. The analogies and differences between sonochemistry and ionizing radiation chemistry are explored. The use of spin trapping and electron spin resonance to conclusively identify hydrogen atoms and hydroxyl radicals and to detect cavitation produced by continuous wave and by pulsed ultrasound is described in detail.     

The role of free radical generation in increasing cerebrovascular permeability

The brain endothelium constitutes a barrier to the passive movement of substances from the blood into the cerebral microenvironment, and disruption of this barrier after a stroke or trauma has potentially fatal consequences. Reactive oxygen species (ROS), which are formed during these cerebrovascular accidents, have a key role in this disruption. ROS are formed constitutively by mitochondria and also by the activation of cell receptors that transduce signals from inflammatory mediators, e.g., activated phospholipase A₂ forms arachidonic acid that interacts with cyclooxygenase and lipoxygenase to generate ROS. Endothelial NADPH oxidase, activated by cytokines, also contributes to ROS. There is a surge in ROS following reperfusion after cerebral ischemia and the interaction of the signaling pathways plays a role in this. This review critically evaluates the literature and concludes that the ischemic penumbra is a consequence of the initial edema resulting from the ROS surge after reperfusion.     

The role of free radical reactions with haemoglobin and thalassaemia

It is well known that all living systems depend on iron to transport (haemoglobin), store (myoglobin) and utilize (cytochromes, cytochrome oxide) oxygen for respiration. Iron is an essential component in the active sites of the enzyme that protects against oxidation, such as the iron superoxide dismutase, in bacteria and plants. In normal human plasma almost all iron loading of transferrin is 20-30% maximum. In this presentation we review and summarize recent developments in our understanding of iron transport and storage in living systems.     

The role of iron in oxygen radical mediated lipid peroxidation

The role of iron in the peroxidation of polyunsaturated fatty acids is reviewed, especially with respect to the involvement of oxygen radicals. The hydroxyl radical can be generated by a superoxide-driven Haber-Weiss reaction or by Fenton's reaction; and the hydroxyl radical can initiate lipid peroxidation. However, lipid peroxidation is frequently insensitive to hydroxyl radical scavengers or superoxide dismutase. We propose that the hydroxyl radical may not be involved in the peroxidation of membrane lipids, but instead lipid peroxidation requires both Fe2+ and Fe3+. The inability of superoxide dismutase to affect lipid peroxidation can be explained by the fact that the direct reduction of iron can occur, exemplified by rat liver microsomal NADPH-dependent lipid peroxidation. Catalase can be stimulatory, inhibitory or without affect because H2O2 may oxidize some Fe2+ to form the required Fe3+, or, alternatively, excess H2O2 may inhibit by excessive oxidation of the Fe2+. In an analogous manner reductants can form the initiating complex by reduction of Fe3+, but complete reduction would inhibit lipid peroxidation. All of these redox reactions would be influenced by iron chelation.