bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative determination of oLAb titers in various animal species
Authors: Tatzber F. , Wonisch W. , Schmidl E. , Esterbauer H.
Published: December 2008

It is generally accepted, that lipid peroxidation plays a pathogenic role in atherosclerosis. Furthermore, recent studies indicate that antibodies directed against oxidative modifications of Low Density Lipoprotein (oLAb) contribute to atherosclerotic processes and may have some function in other disorders. These antibodies have been determined predominantly in humans, because assays for oLAb measurement use species specific anti IgG conjugates. From such assay designs it is not possible to get directly comparable data from various animal species. Main advantages of comparable data between animal species are that results of animal experiments can be interpreted using human calibrators and that results of immunisations and production of monoclonal antibodies are directly comparable not only within, but also between animal species. The aim of this study was to find a modification for ELISAs for oLAb determination, which allows to measure sera of various animal species simultaneously.

Microtitration plates were coated with oxidised LDL and blocked with bovine serum albumine. Human and animal sera were then pipetted into the plate in logarithmic serial dilutions and incubated for 2 h at 37°C. After washing, a protein A horse‐radish peroxidase conjugate (Biomakor, Israel) was added to each well in a dilution of 1:20,000. The incubation conditions had to be optimized to achieve reliable results. After another washing step, the assay was developed with TMB. Absorptions were read at 450 nm in a microplate photometer.

Following the manufacturers incubation instructions, which recommended a duration of 1 h at room temperature, the system did not work optimally. No binding of protein A to IgG molecules bound to oxidised LDL could be observed, if the system was incubated at 37°C. In our hands, best results were achieved for several animal species, if the conjugate was incubated for two hours at 2–4°C in a refrigerator. Under these conditions, assay sensitivity was the same as in the standard method, which uses anti‐species IgG conjugates.

The protein A modification of oLAb allows direct reading of animal oLAb titres from human calibrators. With this method, results of animal experiments can be interpreted on the basis of the situation in humans. Preliminary results obtained show that immunisation experiments with oxidised LDL give serum titres in animals, which are in the same order of magnitude as human sera with high oLAb concentrations. The results of this study, in accordance with findings of other authors, give further indications that atherosclerotic processes are influenced by the specific immune system.
Biogenic 4‐hydroxy‐2‐nonenal activates transcription factor AP‐1 but not NF‐κB in cells of the macrophage lineage
Authors: Camandola Simonetta , Scavazza Antonella , Leonarduzzi Gabriella , Biasi Fiorella , Chiarpotto Elena , Azzi Angelo , Poli Giuseppe
Published: December 2008

A large spectrum of pro‐oxidant agents, including molecules with lipoperoxidative effect, can modulate gene expression through modification of the DNA binding activity of the transcription factors activator protein 1 (AP‐1) and nuclear factor kappa B (NF‐κB). In this study the effect on these redox‐sensitive factors by 4‐hydroxy‐2‐nonenal (HNE), a major aldehydic product of lipid peroxidation, was examined in two cell lines of the macrophage type. Incubation in the presence of μM concentrations of the aldehyde led to a rapid increase of AP‐1 binding with a transient maximum 30 min from HNE addition to the culture medium in both cell lines. On the contrary, HNE did not stimulate nuclear translocation of NF‐κB. The diverging effect of HNE on the two transcription factors is likely related to the demonstrated differential activation pathway of AP‐1 and NF‐κB in macrophages. The HNE‐induced activation of AP‐1 suggests the aldehyde's involvement in the regulatory mechanisms of cell proliferation and differentiation.
Polyphenols produced during red wine ageing
Authors: Brouillard R. , George F. , Fougerousse A.
Published: December 2008

Over the past few years, it has been accepted that a moderate red wine consumption is a factor beneficial to human health. Indeed, people of France and Italy, the two major wine‐producing European countries, eat a lot of fatty foods but suffer less from fatal heart strokes than people in North‐America or in the northern regions of Europe, where wine is not consumed on a regular basis. For a time, ethanol was thought to be the ‘good’ chemical species hiding behind what is known as the ‘French paradox’. Researchers now have turned their investigations towards a family of natural substances called ‘polyphenols’, which are only found in plants and are abundant in grapes. It is well known that these molecules behave as radical scavengers and antioxidants, and it has been demonstrated that they can protect cholesterol in the LDL species from oxidation, a process thought to be at the origin of many fatal heart attacks. However, taken one by one, it remains difficult to demonstrate which are the best polyphenols as far as their antioxidant activities are concerned. The main obstacle in that kind of research is not the design of the chemical and biological tests themselves, but surprisingly enough, the limited access to chemically pure and structurally elucidated polyphenolic compounds. In this article, particular attention will be paid to polyphenols of red wine made from Vitis vinifera cultivars. With respect to the ‘French paradox’, we address the following question: are wine polyphenolic compounds identical to those found in grapes (skin, pulp and seed), or are there biochemical modifications specifically taking place on the native flavonoids when a wine ages? Indeed, structural changes occur during wine conservation, and one of the most studied of those changes concerns red wine colour evolution, called ‘wine ageing’. As a wine ages, it has been demonstrated that the initially present grape pigments slowly turn into new more stable red pigments. That phenomenon goes on for weeks, months and years. Since grape and wine polyphenols are chemically distinct, their antioxidant activities cannot be the same. So, eating grapes might well lead to beneficial effects on human health, due to the variety and sometimes large amounts of their polyphenolic content. However, epidemiological surveys have focused on wines, not on grapes…
α‐tocopherol protects against expression of adhesion molecules on neutrophils and endothelial cells
Authors: Yoshikawa Toshikazu , Yoshida Norimasa , Manabe Hiroki , Terasawa Yoshimitsu , Takemura Toshiki , Kondo Motoharu
Published: December 2008

Leukocyte–endothelial cell interactions, which are mediated by various adhesion molecules, are a crucial event in inflammatory reactions including atherosclerosis. α‐tocopherol (α‐Toc) has been used for therapy of vascular diseases because of its antioxidant activity. However, the effect of α‐Toc on inflammatory reactions has not been investigated very well. In the present study, we examined the effect of α‐Toc on expression of adhesion molecules on human neutrophils and human umbilical vein endothelial cells (HUVEC). Expression of CD11a, CD11b and CD18 on neutrophils was assessed by immunofluorescence flow cytometry 30 min after the stimulation of neutrophils with 10−7 M platelet‐activating factor (PAF). Surface expression of intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) on HUVEC was evaluated by enzyme immunoassay 8 h after the incubation of HUVEC with IL‐1β (20 U/ml). PAF induced upregulation of CD11b and CD18 on neutrophils and IL‐1β increased surface expression of ICAM‐1 and VCAM‐1 on HUVEC. Coincubation of neutrophils with α‐Toc and pretreatment of HUVEC with α‐Toc significantly reduced PAF‐induced CD11b/CD18 expression and IL‐1β‐induced upregulation of ICAM‐1 and VCAM‐1, respectively. These findings indicate that α‐Toc may work as an anti‐inflammatory agent through inhibiting neutrophil–endothelial cell adhesive reactions.
Inhibition of NF‐κB transcriptional activity by α‐tocopheryl succinate
Authors: Nakamura Tetsuya , Goto Masaki , Matsumoto Akira , Tanaka Isao
Published: December 2008

The role of vitamin E in cell regulation in addition to its function as an antioxidant has attracted attention. The effects of α‐tocopherol (T) and α‐tocopheryl succinate (TS) on transcriptional activation of the tumor necrosis factor alpha (TNF‐α ) gene and nuclear factor kappa B (NF‐κ B) activation were examined. Two stable transformants were used: TR‐1 cells derived from THP‐1 cells transfected with a vector contains the human TNF‐α promoter (1.4‐kb) joined to the human placental alkaline phosphatase (PLAP) coding sequence, and B164 cells derived from the same cell line but carrying the vector containing the human β‐actin promoter (4.3‐kb) as a control.

The transfectants were cultured in the presence of TS, followed by stimulation with lipopolysaccharide (LPS). After stimulation, PLAP activity secreted into the culture medium was measured. TS reduced TNF‐α transcriptional activity in a concentration‐dependent manner, while no effect was observed on that of the β‐actin promoter.

Gel shift assay revealed that THP‐1 cells pretreated with TS and then with LPS showed inhibition of NF‐κ B activity by 43% at 50 μM versus the TS‐untreated group. Since TS did not affect activator protein‐1 (AP‐1) activity under the same conditions, the inhibitory effect of TS on NF‐κ B activation might be specific. However, T had no effect on the results of the gel shift assay.

Vitamin E transportation was analyzed by simultaneous determination of vitamin E and its derivatives using HPLC. The vitamin E recovered from culture pellets showed almost the same amounts of T and TS transferred and was recovered in unchanged form. These observations indicated that TS inhibited NF‐κ B activation and/or translocation to the nuclei in its unchanged form under the culture conditions used here.

These results suggested that vitamin E is involved in signal transduction via an effect distinct from its antioxidant function. To explain the lack of activity with T, it remains to be clarified whether physiological incorporation of T occurred.
Evidence for A1and A2Badenosine receptors in baby hamster kidney (BHK) cells
Authors: Mittal Rashmi A. , Tan Chee‐Hong , Khoo Hoon‐Eng
Published: December 2008

Adenosine is known to produce biphasic effects in the renal tissues via adenosine receptors. However, the presence of more than one subtype of adenosine receptor on a type of kidney cell or tissue has not been conclusively demonstrated. To address this issue, we investigated the presence of A1and A2adenosine receptors in baby hamster kidney (BHK) cells by use of radioligand binding and the reverse transcription‐polymerase chain reaction. Ligand binding studies with (3H)‐DPCPX revealed a single class of binding site with a KDof 9.2 ± 2.0 nM, a Bmaxof 1.7 ± 0.2 pmol/mg protein and a pharmacological profile characteristic of A1adenosine receptor on the BHK cell membrane. As the presence of A2adenosine receptors could not be conclusively determined by ligand binding studies, the more sensitive method of RT‐PCR was employed. The presence of A1and A2Badenosine receptors was detected by RT‐PCR with specific primers and the subsequent sequencing of the resultant amplification product. The sequences obtained were 75–90% homologous to the respective adenosine receptor mRNA of rat, mouse and human.
Singlet oxygen scavenging by α‐tocopherol and β‐carotene: Kinetic studies in phospholipid membranes and ethanol solution
Authors: Fukuzawa Kenji , Inokami Yasuki , Tokumura Akira , Terao Junji , Suzuki Asahi
Published: December 2008

The rate constants (ks of 1O2 scavenging for α‐tocopherol (α‐Toc) and β‐carotene (β‐Car) were measured in liposome membranes, and compared with those in EtOH solution. 1O2 was site‐specifically generated by photoirradiation using two photosensitizers, water‐soluble Rose bengal (RB) and lipid‐soluble 12‐(1‐pyrene)‐dodecanoic acid (PDA). The ks value for β‐Car in EtOH solution was 1.3 × 1010 M−1 s−1, which was 36 times that for α‐Toc (3.6 × 108 M−1 s−1), but there was no difference between their ks values in liposomes (1.8 × 107 M−1 s−1 for β‐Car and 1.2 × 107 M−1 s−1 for α‐Toc). In the liposomes, the ks value for α‐Toc was affected by the membrane site where 1O2 was generated, which depended on the localization of the photosensitizer, being high at the membrane surface in the RB‐system and low in the inner region of the membrane in the PDA‐system. In contrast, the ks value for β‐Car was not affected by the 1O2‐generating site. These differences were supposed to be caused by differences in the relative concentrations of 1O2 and active sites of α‐Toc and β‐Car in the membranes. α‐Toc and β‐Car inhibited 1O2‐dependent peroxidation of egg yolk phosphatidylcholine (egg PC). The concentrations of α‐Toc required for 50% inhibition of lipid peroxidation (IC50) were higher than those of β‐Car, being more than 6 times higher in EtOH solution and less than 2 times higher in liposomes. The ratio of the antioxidant activity of β‐Car to that of α‐Toc was more in EtOH solution than in liposomes, and was well correlated with the ratio of their 1O2 scavenging rate constants.
Effect of supplementation with vitamin E on LDL oxidizability and prevention of atherosclerosis
Authors: Suzukawa Michio , Ayaori Makoto , Shige Hideki , Hisada Tetsuya , Ishikawa Toshitsugu , Nakamura Haruo
Published: December 2008

Supplementation of LDL with vitamin E is thought to protect LDL from oxidative modification and prevent the development of atherosclerosis. Large epidemiological studies have revealed that vitamin E levels in plasma are inversely correlated to the incidence of coronary heart disease. Double‐blind placebo‐controlled trials have reported that supplementation with vitamin E decreases the incidence of coronary events in coronary heart disease (CHD) patients. However, it is not clear how high a dose of vitamin E is needed to prevent formation of atherosclerosis. In animal studies, a diet containing 0.125% vitamin E increased its levels in plasma two‐fold and prevented formation of early atherosclerotic lesions in the thoracic aorta of hypercholesterolemic rabbits. Dose‐response studies in humans have reported that 400 IU/day vitamin E increased its levels in plasma two‐fold and prolonged the lag time before LDL oxidation. It has been reported that oxidizability of LDL was correlated to the atherosclerotic score of coronary angiography in CHD patients. About 400 IU/day vitamin E, which increases its levels two‐fold and prolongs sufficiently the lag time before LDL oxidation, might be beneficial in decreasing the individual risk of CHD.
Aging and oxidative stress in neurodegeneration
Authors: Urano Shiro , Sato Yuko , Otonari Tomoyuki , Makabe Shinobu , Suzuki Shozo , Ogata Masahiro , Endo Toyoshige
Published: December 2008

The effect of oxidative stress on the function of brain synapse, the difference in susceptibility of synapse to hyperoxia with age, and the changes in vitamin E status by stress and aging were investigated. Synaptic membrane permeability to sucrose was increased with age. When rats were subjected to hyperoxia, the membrane permeability on each age increased significantly. The susceptibility of synapse of 25 month old rats exposed to stress was about 2.5 times higher than unexposed old rats. The synaptic plasma membrane fluidity decreased significantly either in response to hyperoxia or during aging. The thiobarbituric acid reactive substances (TBARS) in the synaptic plasma membranes increased with age, and those in the membranes of oxygen‐exposed rats were higher than in the unexposed rats. The cholesterol/phospholipids (C/P) ratio of the membranes increased significantly with age, and the values in the membranes of oxygen‐exposed rats increased more significantly than in unexposed rats of each age. In a measurement of fatty acid content in the membranes, the content of docosahexaenoic acid (DHA, C22:6) decreased significantly during aging and by hyperoxia. These results suggest that free radicals derived from oxygen may attack nerve terminals and peroxidize the membrane, resulting in the deterioration of function of brain synapse, and that susceptibility of synapse to oxidative stress was significantly increased with age. Vitamin E content in the synaptic plasma membranes decreased with age. When rats were subjected to oxidative stress, the content was lower in each age than in normal rat membranes. An intraperitoneal administration of vitamin E prior to stress reduced these abnormalities. It is obvious that vitamin E contributes to the protection against nerve terminal dysfunction caused by oxidative stress.
Identification by differential display PCR of the selenoprotein thioredoxin reductase as a 1α,25(OH)_2‐vitamin D_3‐responsive gene in human osteoblasts — regulation by selenite
Authors: Schütze N. , Bachthaler M. , Lechner A. , Köhrle J. , Jakob F.
Published: December 2008

1α,25(OH)2 vitamin D3 (1,25(OH)2D3) is a potent hormone, stimulating bone cell growth and differentiation. In order to detect novel targets for 1,25(OH)2D3 action, we applied differential display PCR (ddPCR) to human fetal osteoblasts (FOB cells). By ddPCR analysis, we identified the selenoprotein thioredoxin reductase (TRR) as a 1,25(OH)2D3‐responsive gene. In FOB cells, the response of TRR mRNA steady state levels to 1,25(OH)2D3 was fast and transient. Maximal stimulation was observed after one hour of 1,25(OH)2D3 treatment, thereafter TRR steady state mRNA levels declined to control levels. This transient response of TRR mRNA was not reflected at the TRR enzyme activity level upon treatment with 1,25(OH)2D3 for up to 48 h. Sodium selenite added to differentiated FOB cells increased TRR enzyme activity 2.6‐fold, whereas no selenite effect on TRR mRNA steady state levels was measurable. Our data might provide a link between the induction of a differentiation program by 1,25(OH)2D3 and the expression of the selenium responsive TRR system in human osteoblasts.
Ribonucleotide reductase in Bacillus subtilis — evidence for a Mn‐dependent enzyme
Authors: Mohamed S. F. , Gvozdiak O. R. , Stallmann D. , Griepenburg U. , Follmann H. , Auling G.
Published: December 2008

The reduction of 2′‐ribonucleotides to 2′‐deoxyribonucleotides, a unique step in DNA formation, is catalyzed by ribonucleotide reductase (RRase), an allosterically regulated, cell cycle‐dependent enzyme. This work reports a reversible impairment of DNA formation and ribonucleotide reduction upon manganese depletion in Bacillus subtilis demonstrated through in vivo labeling with nucleic acid precursors and enzyme assays with ether‐permeabilized cells. No deoxyadenosylcobalamin‐dependent reduction of ribonucleotides was detected in the cytosol, and the properties of a partially purified enzyme fraction, i.e., sensitivity towards EDTA and hydroxyurea (HU), indicated a metal‐dependent type of RRase. The enzyme was enriched by gel filtration on Superose ™ 12 from glycerol‐ or fumarate‐grown cells and submitted to Q‐band electron paramagnetic resonance (EPR) spectroscopy for further characterization of the metal center. A distinct Mn(II) signal was obtained in both preparations characteristic of a protein‐bound manganese in a mononuclear metal center with axial symmetry. The intensity of this Mn signal was not affected by addition of the radical scavenger HU (10 mM) but reduced in the presence of 2.5 mM EDTA. On the basis of these results, we suggest that Bacillus subtilis has a Mn‐dependent ribonucleotide reductase.
Oxidative stress, antioxidant defences and aging
Authors: Lenaz Giorgio , Cavazzoni Marika , Genova Maria Luisa , D'Aurelio Marilena , Pich Milena Merlo , Pallotti Francesco , Formiggini Gabriella , Marchetti Mario , Castelli Giovanna Parenti , Bovina Carla
Published: December 2008

Apoptosis and aging share common mechanisms in oxidative stress and mitochondrial involvement. Treatment of cultured neuroblastoma cells with a radical initiator induced apoptosis; raise in hydrogen peroxide and release of cytochrome c from mitochondria preceded collapse of mitochondrial potential and cell death. In rat hepatocytes treated with adriamycin incubation with exogenous Coenzyme Q10 counteracted the drug‐induced increase of hydrogen peroxide and the fall of the mitochondrial potential, thus demonstrating the quinone antioxidant effect.

Complex I activity and its rotenone sensitivity decreased in brain cortex non‐synaptic mitochondria from old rats; a 5 kb mitochondrial DNA deletion was found only in the old rats. A similar behavior was found in human platelets from old individuals. The postulated energy decline was confirmed by the inhibitor sensitivities of platelet aggregation and lactate production. The lack of the 5 kb deletion in platelets throws doubts on mitochondrial DNA lesions as the only causes of mitochondrial dysfunction in aging.
Some new aspects of creatine kinase (CK): compartmentation, structure, function and regulation for cellular and mitochondrial bioenergetics and physiology
Authors: Wallimann Theo , Dolder Max , Schlattner Uwe , Eder Michael , Hornemann Thorsten , O'Gorman Eddie , Rück Alex , Brdiczka Dieter
Published: December 2008

Creatine kinase (CK) isoenzymes, specifically located at places of energy demand and energy production, are linked by a phosphocreatine/creatine (PCr/Cr) circuit, found in cells with intermittently high energy demands. Cytosolic CKs, in close conjunction with Ca2+‐pumps, play a crucial role for the energetics of Ca2+‐homeostasis. Mitochondrial Mi‐CK, a cuboidal‐shaped octamer with a central channel, binds and crosslinks mitochondrial membranes and forms a functionally coupled microcompartment with porin and adenine nucleotide translocase for vectorial export of PCr into the cytosol. The CK system is regulated by AMP‐activated protein kinase via PCr/Cr and ATP/AMP ratios. Mi‐CK stabilizes and cross‐links cristae‐ or inner/outer membranes to form parallel membrane stacks and, if overexpressed due to creatine depletion or cellular energy stress, forms those crystalline intramitochondrial inclusions seen in some mitochondrial cytopathy patients. Mi‐CK is a prime target for free radical damage by peroxynitrite. Mi‐CK octamers, together with CK substrates have a marked stabilizing and protective effect against mitochondrial permeability transition pore opening, thus providing a rationale for creatine supplementation of patients with neuromuscular and neurodegenerative diseases.
Imaging the mitochondrial permeability transition pore in intact cells
Authors: Petronilli Valeria , Miotto Giovanni , Canton Marcella , Colonna Raffaele , Bernardi Paolo , Lisa Fabio Di
Published: December 2008

The involvement of mitochondrial permeability transition pore (MTP) in cellular processes is generally investigated by indirect means, such as changes in mitochondrial membrane potential or pharmacological inhibition. However, such effects could not be related univocally to MTP. In addition, source of errors could be represented by the increased retention of membrane potential probes induced by cyclosporin A (CsA) and the interactions between fluorescent probes.

We developed a direct technique for monitoring MTP. Cells were co‐loaded with calcein‐AM and CoCl2, resulting in the quenching of the cytosolic signal without affecting the mitochondrial fluorescence. MTP inducers caused a rapid decrease in mitochondrial calcein fluorescence which, however, was not completely prevented by CsA. Besides the large and rapid efflux of calcein induced by MTP agonists, we also observed a constant and spontaneous decrease of mitochondrial calcein which was completely prevented by CsA. Thus, MTP likely fluctuates between open and closed states in intact cells.
The mitochondrial permeability transition
Authors: Bernardi Paolo , Colonna Raffaele , Costantini Paola , Eriksson Ove , Fontaine Eric , Ichas François , Massari Stefano , Nicolli Annamaria , Petronilli Valeria , Scorrano Luca
Published: December 2008

This review summarizes recent work on the regulation of the permeability transition pore, a cyclosporin A‐sensitive mitochondrial channel that may play a role in intracellular calcium homeostasis and in a variety of forms of cell death. The basic bioenergetics aspects of pore modulation are discussed, with some emphasis on the links between oxidative stress and pore dysregulation as a potential cause of mitochondrial dysfunction that may be relevant to cell injury.
Confocal microscopy of the mitochondrial permeability transition in necrotic cell killing, apoptosis and autophagy
Authors: Lemasters John J. , Qian Ting , Elmore Steven P. , Trost Lawrence C. , Nishimura Yoshiya , Herman Brian , Bradham Cynthia A. , Brenner David A. , Nieminen Anna‐Liisa
Published: December 2008

Onset of the cyclosporin‐A‐sensitive mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by laser scanning confocal microscopy. The MPT is a causative event in many types of necrotic and apoptotic cell death, including oxidative stress, ischemia/reperfusion injury, Ca2+ionophore toxicity and tumor necrosis factor alpha (TNFα) induced apoptosis, and may contribute to Reye's‐related drug toxicity. Pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and increased mitochondrial Ca2+and pH can each promote onset of the MPT in situ. The MPT can also be directly visualized during TNFα‐induced apoptosis to hepatocytes. Mitochondria spontaneously depolarize in situ after nutrient deprivation before entering an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. We propose a model in which onset of the MPT to increasing numbers of mitochondria leads progressively to autophagy, apoptosis and necrotic cell death.
Neuronal excitotoxicity: the role of mitochondria
Authors: Nicholls David G. , Budd Samantha L.
Published: December 2008

Chronic activation of NMDA receptors by glutamate is toxic to cultured neurons. The extensive Ca2+entry accompanying receptor activation is largely accumulated by the intracellular mitochondria, with resultant effects on mitochondrial membrane potential, ATP synthesis, glycolysis, reactive oxygen species generation and ultimately failure of cytoplasmic Ca2+homeostasis and cell death. Each of these parameters is inter‐related and in this review we describe attempts to separate out each factor to establish the sequence of events following NMDA receptor activation. The conclusion is that mitochondrial Ca2+accumulation is a key event in glutamate excitotoxicity, and that cells maintained by glycolysis in the absence of a mitochondrial membrane potential are highly resistant to glutamate excitotoxicity.
Selenophosphate as a substrate for mammalian selenocysteine synthase, its stability and toxicity
Authors: Mizutani Takaharu , Kanaya Kazuo , Tanabe Kazutaka
Published: December 2008

The mechanism of selenocysteine synthesis on tRNASec in mammals was previously studied by means of HSe− as a Se donor to synthesize selenocysteine. It has been recently established that HSe− in E. coli is activated by ATP to become selenophosphate (SeP). In this study, we provide evidence that [75Se]selenocysteine is produced by bovine selenocysteine synthase from Ser‐tRNASec and [75Se]SeP, synthesized from elemental 75Se and Tris(trimethylsilyl)phosphite. We also studied the stability of SeP by NMR measurement. SeP was stable during storage under nitrogen at ‐80°C for 3 months in 0.2 M Hepes buffer at pH 6.8. However, SeP decomposed at 0°C in air (half life 32 hrs) or at 22°C under nitrogen (half life 30 hrs) at pH 6.8. The half lives of SeP at ‐19°C in air and at 0°C under nitrogen at pH 6.8 were 740 and 840 hrs, respectively. At pH 4 under nitrogen at 22°C, the half life was 240 hrs. The half life was only 9.2 hrs at pH 9 under nitrogen at 0°C. Thus, SeP was proved to be stable at low temperature, under acidic and anaerobic conditions, but labile under neutral and alkaline conditions.The LD50 of SeP administered i.p. to mice was 37.5 mg/kg body weight.
ATP‐sensitive association of mortalin with the IL‐1 receptor type I
Authors: Sacht Gudrun , Brigelius‐Flohé Regina , Kiess Michael , Sztajer Helena , Flohé Leopold
Published: December 2008

Interleukin‐1 (IL‐1) is a major proinflammatory cytokine mediating local and systemic responses of the immune system. Two types of IL‐1 receptors are known, but only the IL‐1 receptor type I initiates biological responses. Here we show that two proteins with nucleic acid binding potential and mortalin, a member of the HSP70‐family, are associated with the IL‐1 receptor type I irrespective of IL‐1 binding. The association of mortalin with the IL‐1 receptor type I is specifically reversed by ATP concentrations in the physiological range. Other nucleotides are not or much less effective. The in vitro dissociation of mortalin effects neither the receptor association nor the activity of IRAK, which initiates the IL‐1‐dependent phosphorylation cascade. The roles of the receptor‐associated proteins are therefore discussed in the context of receptor internalisation.
Structure and reaction mechanisms of multifunctional mitochondrial cytochrome bc1 complex
Authors: Yu Chang‐An , Zhang Li , Deng Kai‐Ping , Tian Hna , Xia Di , Kim Hoeon , Deisenhofer Johann , Yu Linda
Published: December 2008

The cytochrome bc1 complex from bovine heart mitochondria is a multi‐functional enzyme complex. In addition to electron and proton transfer activity, the complex also processes an activatable peptidase activity and a superoxide generating activity. The crystal structure of the complex exists as a closely interacting functional dimer. There are 13 transmembrane helices in each monomer, eight of which belong to cytochrome b, and five of which belong to cytochrome c1, Rieske iron‐sulfur protein (ISP), subunits 7, 10 and 11, one each. The distances of 21 Å between bL heme and bH heme and of 27 Å between bL heme and the iron‐sulfur cluster (FeS), accommodate well the observed fast electron transfers between the involved redox centers. However, the distance of 31 Å between heme c1 and FeS, makes it difficult to explain the high electron transfer rate between them. 3D structural analyses of the bc1 complexes co‐crystallized with the Qo site inhibitors suggest that the extramembrane domain of the ISP may undergo substantial movement during the catalytic cycle of the complex. This suggestion is further supported by the decreased in the cytochrome bc1 complex activity and the increased in activation energy for mutants with increased rigidity in the neck region of ISP.
Characterization ofSaccharomyces cerevisiaeubiquinone‐deficient mutants
Authors: Schultz Jeffery R. , Clarke Catherine F.
Published: December 2008

Ubiquinol (QH2) is a lipid‐soluble molecule that participates in cellular redox reactions. Previous studies have shown that yeast mutants lacking QH2are hypersensitive to treatment with polyunsaturated fatty acids (PUFAs) indicating that QH2can function as an antioxidant in vivo. In this study the effect of 1 mM linolenic acid on levels of Q6and Q6H2is assessed in both wild‐type and respiration‐deficient (atp2Δ) strains. The response of Q‐deficient mutants to other forms of oxidative stress is further characterized to define those conditions where QH2acts as an antioxidant. Endogenous antioxidant defense systems were also assessed in wild‐type, Q‐deficient, andatp2Δ strains. Superoxide dismutase (SOD) activity decreased and catalase activity increased in both Q‐deficient andatp2Δ mutants compared to wild‐type cells, suggesting that such changes result from the loss of respiration rather than the lack of Q.
Dynamics of antioxidant action of ubiquinol: A reappraisal
Authors: Shi Honglian , Noguchi Noriko , Niki Etsuo
Published: December 2008

The dynamics of action of ubiquinol as an antioxidant against lipid peroxidation was reinvestigated and compared with that ofα‐tocopherol. It was found that ubiquinol was 2.5 and 1.9 times more reactive thanα‐tocopherol toward phenoxyl and peroxyl radicals, respectively, at 25°C in ethanol and that it was capable of donating two hydrogen atoms toward oxygen radicals but that the apparent stoichiometric number decreased in the inhibition of lipid peroxidation, to even smaller than 1, due to its autoxidation. The autoxidation of ubiquinol proceeded even in the micelles and liposomal membranes in aqueous dispersions as well as in organic homogeneous solution. The apparent antioxidant activity of ubiquinol was smaller than that ofα‐tocopherol against lipid peroxidation in organic solution as judged from either rate of oxidation or duration of inhibition period. They exerted similar antioxidant potency against lipid peroxidation in the membranes and micelles in aqueous dispersions. The combination of ubiquinol andα‐tocopherol was suggested to be effective.
Protective role of ubiquinone in vitamin E and selenium‐deficient plasma membranes
Authors: Navarro Francisco , Arroyo Antonio , Martín Sergio F. , Bello Rosario I. , De Cabo Rafael , Burgess John R. , Navas Plácido , Villalba José M.
Published: December 2008

We have studied the effects of dietary depletion of vitamin E and selenium on endogenous ubiquinone‐dependent antioxidant system. Deficiency induced an increase in both coenzyme Q9 and Q10 in liver tissue, reaching a maximum between 4 and 7 weeks of deficient diet consumption. Cytochrome b5 reductase polypeptide was also enriched in membranes after 5 weeks of deficient diet consumption. Substantial DT‐diaphorase activity was found in deficient, but not in control plasma membranes. Deficient membranes were very sensitive to lipid peroxidation, although a great protection was observed after incubation with NAD(P)H. Our results show that liver cells can boost endogenous ubiquinone‐dependent protective mechanisms in response to deficiency in vitamin E and selenium.
Role of plasma membrane coenzyme Q on the regulation of apoptosis
Authors: López‐Lluch G. , Barroso M. P. , Martín S. F. , Fernández‐Ayala D. J. M. , Gómez‐Díaz C. , Villalba J. M. , Navas P.
Published: December 2008

Serum withdrawal is a model to study the mechanisms involved in the induction of apoptosis caused by mild oxidative stress. Apoptosis induced by growth factors removal was prevented by the external addition of antioxidants such as ascorbate,α‐tocopherol and coenzyme Q (CoQ). CoQ is a lipophilic antioxidant which prevents oxidative stress and participates in the regeneration ofα‐tocopherol and ascorbate in the plasma membrane. We have found an inverse relationship between CoQ content in plasma membrane and lipid peroxidation rates in leukaemic cells. CoQ10 addition to serum‐free culture media prevented both lipid peroxidation and cell death. Also, CoQ10 addition decreased ceramide release after serum withdrawal by inhibition of magnesium‐dependent plasma membrane neutral‐sphingomyelinase. Moreover, CoQ10 addition partially blocked activation of CPP32/caspase‐3. These results suggest CoQ of the plasma membrane as a regulator of initiation phase of oxidative stress‐mediated serum withdrawal‐induced apoptosis.
Improved brain and muscle mitochondrial respiration with CoQ. An in vivo study by 31P‐MR spectroscopy in patients with mitochondrial cytopathies
Authors: Barbiroli Bruno , Iotti Stefano , Lodi Raffaele
Published: December 2008

We used in vivo phosphorus magnetic resonance spectroscopy (31P‐MRS) to study the effect of CoQ10 on the efficiency of brain and skeletal muscle mitochondrial respiration in ten patients with mitochondrial cytopathies. Before CoQ, brain [PCr] was remarkably lower in patients than in controls, while [Pi] and [ADP] were higher. Brain cytosolic free [Mg2+] and Δ G of ATP hydrolysis were also abnormal in all patients. MRS also revealed abnormal mitochondrial function in the skeletal muscles of all patients, as shown by a decreased rate of PCr recovery from exercise. After six‐months of treatment with CoQ (150 mg/day), all brain MRS‐measurable variables as well as the rate of muscle mitochondrial respiration were remarkably improved in all patients. These in vivo findings show that treatment with CoQ in patients with mitochondrial cytopathies improves mitochondrial respiration in both brain and skeletal muscles, and are consistent with Lenaz's view that increased CoQ concentration in the mitochondrial membrane increases the efficiency of oxidative phosphorylation independently of enzyme deficit.
CoQ10: Could it have a role in cancer management?
Authors: Hodges S. , Hertz N. , Lockwood K. , Lister R.
Published: December 2008

Coenzyme Q10 or ubiquinone has been shown to have both anti‐cancer and immune system enhancing properties when tested in animals. Preliminary results reported here suggest that it might inhibit tumour‐associated cytokines. Clinical studies conducted with combination therapies of CoQ10 and other antioxidants are ongoing, but the results are difficult evaluate owing to the lack of proper control groups and of initial randomisation. Also on the basis of some anti‐cancer effects of antioxidants reported in literature, further animal studies and a proper clinical trial of coenzyme Q10 in cancer patients are needed.
Lycopene and β‐carotene decompose more rapidly than lutein and zeaxanthin upon exposure to various pro‐oxidants in vitro
Authors: Siems Werner G. , Sommerburg Olaf , Van Kuijk Frederik J.G.M.
Published: December 2008

Major carotenoids of human plasma and tissues were exposed to radical‐initiated autoxidation conditions. The consumption of lutein and zeaxanthin, the only carotenoids in the retina, and lycopene and β‐carotene, the most effective quenchers of singlet oxygen in plasma, were compared.

Under all conditions of free radical‐initiated autoxidation of carotenoids which were investigated, the breakdown of lycopene and β‐carotene was much faster than that of lutein and zeaxanthin. Under the influence of UV light in presence of Rose Bengal, by far the highest breakdown rate was found for β‐carotene, followed by lycopene. Bleaching of carotenoid mixtures mediated by NaOCl, addition of azo‐bis‐isobutyronitril (AIBN), and the photoirradiation of carotenoid mixtures by natural sunlight lead to the following sequence of breakdown rates: lycopene > β‐carotene > zeaxanthin > lutein.

The slow degradation of the xanthophylls zeaxanthin and lutein may be suggested to explain the majority of zeaxanthin and lutein in the retina of man and other species. In correspondence to that, the rapid degradation of β‐carotene and lycopene under the influence of natural sunlight and UV light is postulated to be the reason for the almost lack of those two carotenoids in the human retina. Nevertheless, a final proof of that theory is lacking.
(+)‐Catechin as antioxidant: mechanisms preventing human plasma oxidation and activity in red wines
Authors: Lotito Silvina B. , Fraga César G.
Published: December 2008

We evaluated the antioxidant effect of (+)‐catechin (CTCH), in the presence of physiological antioxidant levels of ascorbic acid (AA), α‐tocopherol (AT) and beta‐carotene (BC), in human plasma oxidised with AAPH. Following a five‐hour incubation, the formation of lipid oxidation products (TBARS) was almost doubled, and the concentrations of lipid soluble antioxidants were 10 to 30% from the initial levels. In these conditions, AA was consumed within the first hour of incubation. The addition of CTCH prevented AT and BC depletion and TBARS formation, but had no effect on AA consumption. When the kinetics of oxidation were analysed CTCH oxidation preceded lipid soluble antioxidant depletion, but no consumption of CTCH was associated to AA oxidation. Considering that CTCH could contribute to the antioxidant activity of red wine, we first characterised both the antioxidant capacity and CTCH content of several wines. The wines with highest content of CTCH and antioxidant activity were also the most effective in preventing AAPH‐mediated oxidation of plasma vitamin E. Results support the idea that CTCH could have a role as a physiological antioxidant in human plasma, and that CTCH of wine could contribute to the antioxidant status of human plasma.
The role of oxidative stress and NF‐κB activation in late diabetic complications
Authors: Mohamed Abdel Khalek , Bierhaus Angelika , Schiekofer Stephan , Tritschler Hans , Ziegler Reinhard , Nawroth Peter P.
Published: December 2008

A common endpoint of hyperglycemia dependent cellular changes is the generation of reactive oxygen intermediates (ROIs) and the presence of elevated oxidative stress. Therefore, oxidative stress is supposed to play an important role in the development of late diabetic complications. Formation of advanced glycation end products (AGE's) due to elevated nonenzymatic glycation of proteins, lipids and nucleic acids is accompanied by oxidative, radical‐generating reactions and thus represents a major source for oxygen free radicals under hyperglycemic conditions. Once formed, AGE's can influence cellular function by binding to several binding sites including the receptor for AGE's, RAGE. Binding of AGE's (and other ligands) to RAGE results in generation of intracellular oxidative stress and subsequent activation of the redox‐sensitive transcription factor NF‐κB in vitro and in vivo. Consistently, activation of NF‐κB in diabetic patients correlates with the quality of glycemic control and can be reduced by treatment with the antioxidant α‐lipoic acid. The development of techiques allowing for a tissue culture independent measurement of NF‐κB activation in patients with diabetes mellitus gives insights into the molecular mechanisms linking diabetes mellitus and hyperglycemia with formation of advanced glycated endproducts and generation of oxidative stress finally resulting in oxidative stress mediated cellular activation.
Effect of a diet supplemented with thioproline on murine macrophage function in a model of premature ageing
Authors: Correa R. , Blanco B. , Del Río M. , Víctor V. , Guayerbas N. , Medina S. , De La Fuente M.
Published: December 2008

A previous study has shown that diet supplementation with thioproline (thiazolidine‐4‐carboxylic acid, TCA), an intracellular sulfhydril antioxidant and free radical scavenger, stimulates lymphocyte functions in old mice. In the present work, the effect of thioproline ingestion on the phagocytic functions of peritoneal macrophages, namely adherence, chemotaxis, phagocytosis and superoxide anion production was studied in adult female OF1‐Swiss mice, that were fed thioproline (0.1% w/w) for five weeks, starting this ingestion at the age of 22 ± 2 weeks. Mice were divided into a fast and a slow group based on their exploratory activity, which was assessed by their performance in a T‐shaped maze. Slow mice showed a worse phagocytic activity with respect to fast animals. After thioproline treatment, a stimulation of all the functions studied as well as a neutralization of the superoxide radical were observed. The effect of this antioxidant was stronger in the slow than in the fast group. Thus, a diet supplemented with thioproline may enhance the immune functions, especially when they are depressed.
Thiols in formaldehyde dissimilation and detoxification
Authors: Duine Johannis A.
Published: December 2008

Glutathione is not a universal coenzyme for formaldehyde oxidation. MySH (mycothiol, 1‐O‐(2′‐[N‐acetyl‐L‐cysteinyl]amido‐2′‐deoxy‐/propto‐D‐glucopyranosyl)‐D‐myo‐inositol) is GSH's counterpart as coenzyme in formaldehyde dehydrogenase from certain Gram‐positive bacteria. However, formaldehyde dissimilation and detoxification not only proceed via thiol‐dependent but also via thiol‐independent dehydrogenases. The distinct structures and enzymatic properties of MySH‐dependent and GSH‐dependent formaldehyde dehydrogenases could provide clues for development of selective drugs against pathogenic Mycobacteria. It is to be expected that other new types of thiol‐dependent formaldehyde dehydrogenases will be discovered in the future. Indications exist that the product of thiol‐dependent formaldehyde oxidation, the thiol formate ester, is not only hydrolytically converted into thiol and formate but can also be oxidatively converted in some cases by a molybdoprotein aldehyde dehydrogenase into the corresponding carbonate ester, decomposing spontaneously into CO2 and the thiol.
AhpC, oxidative stress and drug resistance in Mycobacterium tuberculosis
Authors: Sherman David R. , Mdluli Khisimuzi , Hickey Mark J. , Barry, III Clifton E. , Kendall Stover C.
Published: December 2008

The Mycobacterium tuberculosis AhpC is similar to a family of bacterial and eukaryotic antioxidant proteins with alkylhydroperoxidase (Ahp) and thioredoxin‐dependent peroxidase (TPx) activities. AhpC expression is associated with resistance to the front‐line antitubercular drug isoniazid in the naturally resistant organisms E. coli and M. smegmatis. We identified several isoniazid‐resistant M. tuberculosis isolates with ahpC promoter mutations resulting in AhpC overexpression. These strains were more resistant to cumene hydroperoxide than were wild‐type strains. However, these strains were unchanged in their sensitivity to isoniazid, refuting a role for AhpC in detoxification of this drug. All the isoniazid‐resistant, AhpC‐overexpressing strains were also deficient in activity of the mycobacterial catalase‐peroxidase KatG. KatG, the only known catalase in M. tuberculosis, is required for activation of isoniazid. We propose that compensatory ahpC promoter mutations are selected from KatG‐deficient, isoniazid‐resistant M. tuberculosis during infections, to mitigate the added burden imposed by organic peroxides on these strains.
Superoxide production by dinitrophenyl‐ derivatized thioredoxin reductase ‐ a model for the mechanism and correlation to immunostimulation by dinitrohalobenzenes
Authors: Arnér Elias S.J.
Published: December 2008

Mammalian thioredoxin reductase catalyzes NADPH dependent reduction of a wide variety of substrates and plays a central role in redox regulation and antioxidant defence. Recently the enzyme was discovered to be a selenoprotein with a catalytically active penultimate selenocysteine residue. Dinitrohalobenzenes irreversibly inhibit the enzyme with a concomitant induction of an NADPH oxidase activity, producing superoxide. A model explaining the reactivity of dinitrohalobenzenes with thioredoxin reductase is presented, involving dinitrophenyl‐derivatization of both the selenocysteine residue and its neighboring cysteine residue, reduction by NADPH of the enzyme‐bound flavin in dinitrophenyl‐alkylated enzyme (dnp‐TrxR), followed by two consecutive one‐electron transfers from the flavin to nitro groups of the dnp‐moieties in dnp‐TrxR, forming nitro anion radicals. The nitro radicals react with oxygen to form superoxide, again generating dnp‐TrxR with an oxidized flavin, which may then follow another cycle of NADPH‐dependent superoxide production. Dinitrohalobenzene compounds are well known for their immunostimulatory properties. Here it is proposed that the inflammatory components of this immunostimulation can be mediated by interaction with the thioredoxin system, via effects on cell function by superoxide production, oxidative stress and increased extracellular levels of thioredoxin.
Vitamin B6 enzymes participating in selenium amino acid metabolism
Authors: Soda Kenji , Oikawa Tadao , Esaki Nobuyoshi
Published: December 2008

Various vitamin B6 enzymes play important roles in mammalian and microbial metabolism of selenium amino acids. Selenocysteine is synthesized from selenohomocysteine by catalysis of cystathionine β‐synthase and cystathionine γ‐lyase, which both require pyridoxal phosphate. Selenocysteine β‐lyase, a new B6‐enzyme, exclusively catalyzes β‐elimination of selenocysteine, and occurs in mammalian systems and bacteria. Methionine γ‐lyase, cysteine desulfurase, cysteine sulfinate desulfinase, and D‐selenocystine α, β‐lyase, which are B6‐enzymes, act on cysteine, cysteine sulfinate, D‐cystine, and their derivatives, and their selenium counterparts indiscriminately. Their reaction mechanisms are comparatively described.