The effect of in vivo treatment with triiodothyronine on the in vitro synthesis of protein-poly(ADP)-ribose adducts by isolated cardiocyte nuclei and the separation of poly(ADP)-ribosylated proteins by phenol extraction and electrophoresis

In vivo treatment of rats with triiodothyronine (30 micrograms/100 g of body weight for 4 consecutive days) inhibited poly(ADP)-ribose polymerase activity of cardiocyte nuclei, but low enzymatic activity of nuclei of noncardiocyte origin remained unaffected. RNA synthesis in cardiocyte nuclei isolated from triiodothyronine-treated rats was augmented. A positive correlation was observed between the degree of inhibition of poly(ADP)-ribose polymerase and cardiac ventricular enlargement in triiodothyronine-treated animals. RNA synthesis in isolated cardiocyte nuclei was inhibited by in vitro poly(ADP)-ribosylation only when cardiocyte nuclei were obtained from triiodothyronine-treated animals. In vitro poly(ADP)-ribosylated proteins were isolated from cardiocyte nuclei by solvent partitioning between phenol and aqueous phases. About 90% of the protein-poly(ADP)-ribose adducts partitioned into the aqueous fraction, and the chain length of polymers in this phase was between medium (n = 4-9) and long (n greater than 32), whereas the phenol phase contained protein-oligomer and monomer adducts. Not only the chain length of oligomers but the nature of modified proteins appeared to participate in determining the partitioning of polymer-protein adducts, and different proteins were separated from the two phases by gel electrophoresis. More than 90% of protein-polymer adducts formed by cardiocyte nuclei were not extracted by 0.25 N HCl, indicating prevalence of nonhistone proteins as polymer acceptors. Gel electrophoresis and near quantitative recovery of adducts in a gel system that protected from degradation of adducts to free polymers confirmed the predominance of nonhistone proteins as main acceptors and demonstrated an artifact of autoradiography that seemed to indicate histone H1 as a significant acceptor. Treatment with triiodothyronine diminished poly(ADP)-ribosylation of certain groups of proteins more than others, implying some degree of selectivity of action of the hormone. Catabolism of the polymer in vitro was not affected by triiodothyronine treatment.     

Tumor promoter phorbol-12-myristate-13-acetate induces poly(ADP)-ribosylation in fibroblasts.

The tumor promoter phorbol-12-myristate-13-acetate (PMA) causes an increase in pol(ADP)-ribosylation in mouse and human fibroblasts via the intermediate formation of active oxygen. In contrast to poly(ADP)-ribosylation induced by the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine, de novo RNA and protein synthesis are required and the accumulation of the polymer occurs in the absence of detectable DNA strand breakage. Our results suggest a mechanism for PMA-induced modulation of chromatin structure and gene expression.     

Salsolinol, a tetrahydroisoquinoline-derived neurotoxin, induces oxidative modification of neurofilament-L protection by histidyl dipeptides

Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) is a compound derived from dopamine metabolism and is capable of causing dopaminergic neurodegeneration. Oxidative modification of neurofilament proteins has been implicated in the pathogenesis of neurodegenerative disorders. In this study, oxidative modification of neurofilament-L (NF-L) by salsolinol and the inhibitory effects of histidyl dipeptides on NF-L modification were investigated. When NF-L was incubated with 0.5 mM salsolinol, the aggregation of protein was increased in a time-dependent manner. We also found that the generation of hydroxyl radicals (?OH) was linear with respect to the concentrations of salsolinol as a function of incubation time. NF-L exposure to salsolinol produced losses of glutamate, lysine and proline residues. These results suggest that the aggregation of NF-L by salsolinol may be due to oxidative damage resulting from free radicals. Carnosine, histidyl dipeptide, is involved in many cellular defense processes, including free radical detoxification. Carnosine, and anserine were shown to significantly prevent salsolinol- mediated NF-L aggregation. Both compounds also inhibited the generation of ?OH induced by salsolinol. The results indicated that carnosine and related compounds may prevent salsolinol-mediated NF-L modification via free radical scavenging.     

Characterization of altered regulation variants of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum

In Rhodospirillum rubrum, nitrogenase activity is subject to posttranslational regulation through the adenosine diphosphate (ADP)-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG). To study the posttranslational regulation of DRAG, its gene was mutagenized and colonies screened for altered DRAG regulation. Three different mutants were found and the DRAG variants displayed different biochemical properties including an altered affinity for divalent metal ions. Taken together, the results suggest that the site involved in regulation is physically near the metal binding site of DRAG.     

Prevention of Cancer-by Agents that Suppress Oxygen Radical Formation

The prevention of cancer by agents in our diet has led to the concept that oxygen radicals are a necessary component of a variety of human cancers including breast, colon and prostatic cancer. These cancers are putatively promoted by estradiol, bile acids and androgens. Epidemiological studies have shown that these cancers are suppressed in vegetarian populations. Vegetable components that may be responsible for this cancer prevention are Vitamin A, retinoids and protease inhibitors (PIS). These agents have been shown to suppress the formation of hydrogen peroxide in promoter-induced neutrophils. They also have been shown to block two-stage carcinogenesis and breast cancer when fed to animals. PIS also suppress experimentally-induced colon cancer and spontaneous liver cancer. Moreover, a new series of cancer-preventive agents, Sarcophytols (isolated by Fujiki and co-workers), are capable of suppressing two-stage carcinogenesis, breast and colon cancers in rodents when given in low concentrations. Sarcophytols were also active suppressors of H₂O₂ formation of 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced neutrophils. These observations point to an essential role of oxygen radicals in carcinogenesis. Suppression of the oxygen radical response of neutrophils in relation to cancer preventive agents is a facile assay of these important substances. The mechanism of action of oxygen radicals in promoting carcinogenesis is a multiple one. including: (1) activation of oncogenes, (2) modification of DNA bases, and (3) formation of single-strand breaks leading to poly(ADP)ribose polymerase activation.     

Oxidative modification of rat eye lens proteins by peroxyl radicals in vitro: protection by the chain-breaking antioxidants stobadine and Trolox

In an attempt to model the processes of free radical-mediated cataractogenesis, we investigated the oxidative modification of rat eye lens proteins by peroxyl radicals generated by thermal decomposition of 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH) under aerobic conditions. When incubated with AAPH, the soluble eye lens proteins precipitated in a time-dependent manner. The insolubilisation was accompanied by the accumulation of protein free carbonyls and the diminution of sulfhydryls, yet the processes were shifted in time. The SDS-PAGE analysis of the AAPH-treated proteins revealed the presence of high molecular weight cross-links and, to a lesser extent, fragments. The aggregation and cross-linking of proteins along with the generation of free carbonyls was significantly inhibited by the chain-breaking antioxidants stobadine and Trolox. On the other hand, the AAPH-initiated sulfhydryl consumption was much less sensitive to the antioxidants studied. The results point to a complex mechanism of peroxyl-radical-mediated modification of eye lens proteins with implications for cataract development and they indicate a potentially protective role of antioxidants.     

Radical production from free and peptide-bound methionine sulfoxide oxidation by peroxynitrite and hydrogen peroxide/iron(II)

Methionine sulfoxide is a post-translational protein modification that has been receiving increasing attention in the literature. Here we used electron paramagnetic resonance spin trapping techniques to show that free and peptide-bound methionine sulfoxide is oxidized by hydrogen peroxide/iron(II)-EDTA and peroxynitrite through the intermediacy of the hydroxyl radical to produce both *CH3 and *CH2CH2CH radicals. The results indicate that methionine sulfoxide residues are important targets of reactive oxygen- and nitrogen-derived species in proteins. Since the produced protein-derived radicals can propagate oxidative damage, the results add a new antioxidant route for the action of the enzyme peptide methionine sulfoxide reductase.     

ADP-iron as a Fenton reactant: radical reactions detected by spin trapping, hydrogen abstraction, and aromatic hydroxylation

A mixture of ADP, ferrous ions, and hydrogen peroxide (H2O2) generates hydroxyl radicals (OH) that attack the spin trap DMPO (5,5-dimethyl-pyrollidine-N-oxide) to yield the hydroxyl free radical spin-adduct, degrade deoxyribose and benzoate with the release of thiobarbituric acid-reactive material, and hydroxylate benzoate to give fluorescent products. Inhibition studies, with scavengers of the OH radical, suggest that the behavior of iron-ADP in the reaction is complicated by the formation of ternary complexes with certain scavengers and detector molecules. In addition, iron-ADP reacting with H2O2 appears to release a substantial number of OH radicals free into solution. During the generation of OH radicals the ADP molecule was, as expected, damaged by the iron bound to it. Damage to the iron ligand in this way is not normally monitored in reaction systems that use specific detector molecules for OH radical damage. Under certain reaction conditions the ligand may be the major recipient of OH radical damage thereby leading to the incorrect assumption that the iron ligand is a poor Fenton reactant.     

Nucleotide Availability in Maize (Zea mays L.) Root Tips (Estimation of Free and Protein-Bound Nucleotides Using 31P-Nuclear Magnetic Resonance and a Novel Protein-Ligand-Binding Assay)

Sequestration of nucleotides in cells through protein binding could influence the availability of nucleotides and free energy for metabolic reactions and, therefore, affect rates of physiological processes. We have estimated the proportion of nucleotides bound to proteins in maize (Zea mays L.) root tips. Binding of nucleoside mono- and diphosphates to total root-tip protein was studied in vitro using high-performance liquid chromatography and a new ligand-binding technique. We estimate that approximately 40% of the ADP, 65% of the GDP, 50% of the AMP, and virtually all the GMP in aerobic cells are bound to proteins. In hypoxic cells, free concentrations of these nucleotides increase proportionately much more than total intracellular concentrations. Little or no binding of CDP, UDP, CMP, and UMP was observed in vitro. Binding of nucleoside triphosphate (NTP) to protein was estimated from in vivo 31P-nuclear magnetic resonance relaxation measurements. In aerobic root tips most (approximately 70%) of the NTP is free, whereas under hypoxia NTP appears predominantly bound to protein. Our results indicate that binding of nucleotides to proteins in plant cells will significantly influence levels of free purine nucleotides available to drive and regulate respiration, protein synthesis, ion transport, and other physiological processes.     

Effects of pelargonidine and a benzocaine analogue p-diethylaminoethyl benzoate on mitochondrial K(ATP) channel.

ATP-dependent K+ (K(ATP)) channel was purified from the inner mitochondrial membrane and reconstituted into the bilayer lipid membrane. The K(ATP) activity was inhibited by high concentrations of ATP and ADP, but was activated by low concentrations (up to 200 microM) of ADP. p-Diethylaminoethyl benzoate (DEB) acted as a K(ATP) opener: at micromolar concentrations, it reversed the inhibitory effect of ATP and ADP, and also prevented K(ATP) rundown. Pelargonidine extracted from Pelargonium flowers reduced the spontaneous activity of K(ATP) channels and attenuated DEB-induced channel activation. The opposite action of DEB and pelargonidine on K(ATP) corresponded to the opposite redox properties of the two agents in reactions with free radicals: DEB behaved as an electron donor, while pelargonidine acted as an electron acceptor. We suggest that the thiol groups in mitochondrial K(ATP) are targets for redox-active ligands.     

Ischemia/Reperfusion-induced oxidative stress causes structural changes of brain membrane proteins and lipids

Oxidative stress is a recognized factor of ischemia reperfusion injury. It shares damage of lipids (LPO) and proteins (PPO), and consequently might cause changes in activity of transport systems. Global 15 min ischemia followed by 2, 24 and 48 hour reperfusion was induced by four-vessel occlusion in Wistar rats of both sexes. Levels of TBARS and conjugated dienes as parameters of LPO were analyzed in forebrain homogenates. Concentrations of total free sulfhydryl (SH) groups and emission spectra of tryptophan were measured to quantify PPO. Our results indicate that lipid peroxidation and protein oxidation occurs mainly during the period of reperfusion. However, significant increase in the level of conjugated dienes can be detected already after 15 min ischemia. Attack of proteins by free radicals leads to modification in structure of proteins seen as a decrease of free SH groups and tryptophan fluorescence. Ischemia/reperfusion induces formation of lipid peroxidation products as well as protein modifications.     

Free radical generation by early glycation products: a mechanism for accelerated atherogenesis in diabetes

Non-enzymatic glycation of reactive amino groups in model proteins increased the rate of free radical production at physiologic pH by nearly fifty-fold over non-glycated protein. Superoxide generation was confirmed by electron paramagnetic resonance measurements with the spin-trap phenyl-t-butyl-nitrone. Both Schiff base and Amadori glycation products were found to generate free radicals in a ratio of 1:1.5. Free radicals generated by glycated protein increased peroxidation of membranes of linoleic/arachidonic acid vesicles nearly 2-fold over control, suggesting that the increased glycation of proteins in diabetes may accelerate vascular wall lipid oxidative modification.     

Modulation of fibroblast proliferation by oxygen free radicals.

The major unexplained phenomenon in fibrotic conditions is an increase in replicating fibroblasts. In this report we present evidence that oxygen free radicals can both stimulate and inhibit proliferation of cultured human fibroblasts, and that fibroblasts themselves release superoxide (O2.-) free radicals. Fibroblasts released O2.- in concentrations which stimulated proliferation, a finding confirmed by a dose-dependent inhibition of proliferation by free radical scavengers. Oxygen free radicals released by a host of agents may thus provide a very fast, specific and sensitive trigger for fibroblast proliferation. Prolonged stimulation may result in fibrosis, and agents which inhibit free radical release may have a role in the prevention of fibrosis.     

Carnosine and related dipeptides protect human ceruloplasmin against peroxyl radical-mediated modification

Ceruloplasmin (CP) is the major plasma antioxidant and copper transport protein. In a previous study, we showed that the aggregation of human ceruloplasmin was induced by peroxyl radicals. We investigated the effects of antioxidant dipeptides carnosine, homocarnosine and anserine on peroxyl radical-mediated ceruloplasmin modification. Carnosine, homocarnosine and anserine significantly inhibited the aggregation of CP induced by peroxyl radicals. When CP was incubated with peroxyl radicals in the presence of three compounds, ferroxidase activity, as measured by the activity staining method, was protected. All three compounds also inhibited the formation of dityrosine in peroxyl radicals-treated CP. The results suggest that carnosine and related compounds act as peroxyl radical scavenger to protect the protein modification. It is proposed that carnosine and related peptides might be explored as potential therapeutic agents for pathologies that involve CP modification mediated by peroxyl radicals generated in the lipid peroxidation.     

Hypoxia-Induced Generation of Nitric Oxide Free Radicals in Cerebral Cortex of Newborn Guinea Pigs

Previous studies have shown that brain tissue hypoxia results in increased N-methyl-D-aspartate (NMDA) receptor activation and receptor-mediated increase in intracellular calcium which may activate Ca⁺⁺-dependent nitric oxide synthase (NOS). The present study tested the hypothesis that tissue hypoxia will induce generation of nitric oxide (NO) free radicals in cerebral cortex of newborn guinea pigs. Nitric oxide free radical generation was assayed by electron spin resonance (ESR) spectroscopy. Ten newborn guinea pigs were assigned to either normoxic (FiO₂ = 21%, n = 5) or hypoxic (FiO₂ = 7%, n = 5) groups. Prior to exposure, animals were injected subcutaneously with the spin trapping agents diethyldithiocarbamate (DETC, 400 mg/kg), FeSO₄.7H₂O (40 mg/kg) and sodium citrate (200mg/kg). Pretreated animals were exposed to either 21% or 7% oxygen for 60 min. Cortical tissue was obtained, homogenized and the spin adducts extracted. The difference of spectra between 2.047 and 2.027 gauss represents production of NO free radical. In hypoxic animals, there was a difference (16.75 ± 1.70 mm/g dry brain tissue) between the spectra of NO spin adducts identifying a significant increase in NO free radical production. In the normoxic animals, however, there was no difference between the two spectra. We conclude that hypoxia results in Ca²⁺- dependent NOS mediated increase in NO free radical production in the cerebral cortex of newborn guinea pigs. Since NO free radicals produce peroxynitrite in presence of superoxide radicals that are abundant in the hypoxic tissue, we speculate that hypoxia-induced generation of NO free radical will lead to nitration of a number of cerebral proteins including the NMDA receptor, a potential mechanism of hypoxia-induced modification of the NMDA receptor resulting in neuronal injury.     

Contribution of haemoglobin and membrane constituents modification to human erythrocyte damage promoted by peroxyl radicals of different charge and hydrophobicity

We have investigated the influence of the free radical initiator characteristics on red blood cell lipid peroxidation, membrane protein modification, and haemoglobin oxidation. 2,2'-Azobis(2-amidinopropane) (AAPH) and 4,4'-azobis(4-cyanovaleric acid) (ACV) were employed as free radical sources. Both azo-compounds are water-soluble, although ACV presents a lowed hydrophilicity, as evaluated from octanol/water partition constants. At physiological pH, they are a di-cation and a di-anion, respectively.

AAPH and ACV readily oxidise purified oxyhemoglobin in a very efficient free radical-mediated process, particularly for ACV-derived radicals, where nearly one heme moiety was modified per radical introduced into the system, suggesting that negatively charged radicals react preferentially at the heme group. The radicals derived from both azo-compounds lead to different oxidation products. Methemoglobin, hemichromes and choleglobin were produced in AAPH-promoted hemoglobin oxidation, while ACV-derived radicals predominantly form hemichromes, with very low production of choleglobin.

Red cell damage was evaluated at the level of hemoglobin and membrane constituents modification, and was expressed in terms of free radical doses. Before the onset of the lytic process, ACV leads to more lipid peroxidation than AAPH, and induces a moderate oxidation of intracellular Hb. This intracellular oxidation is markedly increased if ACV hydrophilicity is decreased by lowering the pH. On the other hand, AAPH-derived radicals are considerable more efficient in promoting protein band 3 modification and cell lysis, without significant intracellular hemoglobin oxidation. These results show that the lytic process is not triggered by lipid peroxidation or hemichrome formation, and suggest that membrane protein modification is the relevant factor leading to red blood cell lysis.     

Histochemical visualization of oxidant stress

Free radicals induce oxidative modification in distinct components of the living matter (lipid, proteins, and DNA). For qualitative and quantitative determination of free radical-induced modifications, different, more or less sensitive biochemical methods are available. Because of the high reactivity and short life of free radicals, ongoing oxidative damage is generally analyzed by measurement of secondary products-such as H(2)O(2), oxidized proteins, peroxidized lipids, and their breakdown products, oxidized DNA-or by fluorographic analysis in combination with fluorescent dyes such as dichlorofluorescin (DCFH). In addition, the determination of free radical-related oxidation products is usually carried out in plasma, urine, or, less frequently, in bioptic material. Consequently, biochemical data seldom reflect the effects of free radical insults in situ. The histochemical visualization of selected molecular markers of oxidative damage can often provide more valuable information concerning the in vivo distribution of oxidative processes. This review summarizes the methods currently available for histochemical detection and indirect visualization of free radical-induced alterations in tissues and isolated cells.     

Free RNA-binding cytoplasmic proteins are detected in a labile association with polyribosomes

A special fraction of RNA-binding proteins with a non-specific affinity for RNA is present in the extracts of eukaryotic cells. Earlier these proteins were considered exclusively as a pool of free informosomal proteins. It has been shown that a significant part (about 1/3) of RNA-binding proteins is found in labile association with mono- and polyribosome mass, respectively. The labile-associated proteins dissociate from the complex with mono- and polyribosomes with an increase in the ionic RNA-binding proteins bind to particles due to the non-specific affinity for the exposed part of RNA of mono- and polyribosomes. The decrease of the ionic strength leads to the stabilization of the RNA-binding proteins-polyribosomes complexes and enables purification of these complexes. A direct comparison by the O'Farrell two-dimensional analysis has shown that practically all the proteins that are labile-associated with polyribosomes are present within the preparation of free RNA-binding proteins.     

Heart phosphofructokinase. Allosteric kinetics following affinity labeling modification of the enzyme by 8-[m-(m-fluorosulfonylbenzamido) benzylthio] adenine.

An adenine analog 8-[m-(m-fluorosulfonylbenzamido)benzylthio]adenine (FSB-adenine) reacts covalently with sheep heart phosphofructokinase. Under conditions optimal for allosteric kinetics the modified enzyme is less sensitive to inhibition by ATP and insensitive to activation by AMP, cyclic AMP, and ADP. The concentration of fructose-6-P necessary for half-maximal activity is markedly decreased, while the cooperativity to the same substrate is not changed under the same conditions. The modified enzyme is more stable at pH 6.5 when compared with the native enzyme. Changes in the allosteric kinetics of the enzyme are proportional to the extent of modification reaching maximal effect when 3.2 mol of the reagent were bound/mol of tetrameric enzyme. Affinity labeling of the enzyme by the adenine derivative does not affect significantly the catalytic site. This is evidenced by the demonstration that under assay conditions optimal for Michaelian kinetics neither the Km for ATP nor for fructose-6-P is significantly changed following chemical modification. Maximal activity of the modified enzyme was 60% of the native enzyme. ADP gives the best protection, while AMP gives less protection against modification by the reagent. ATP slows the rate of the reaction and causes a slight decrease in maximum binding of the reagent to the enzyme. Modification of the enzyme caused a marked reduction of AMP and ADP binding. The evidence indicates that the modified site is a nucleotide mono- and diphosphate activation site.     

The hydroxyl free radical reactions of ascorbyl palmitate as measured in various in vitro models.

The OH(*) free radical scavenging properties of ascorbyl palmitate (AP), water-solubilized in the presence of a surfactant (Brij 35), were tested in various systems: (1) The inhibition of polymerization of bovine serum albumin by OH(*) free radicals generated by the Fenton reaction indicated AP exerts a considerable protective effect against polymerization by scavenging the OH(*) free radicals. (2) ESR spin trapping comparisons of DMPO with AP were conducted. Using the Fenton reaction as a source of OH(*) free radicals, AP was 1 order of magnitude faster in scavenging these radicals than DMPO. (3) Oxidative modification of BSA by (60)Co-gamma irradiation of 80 krad, results in a strong increase in protein carbonyl content. AP inhibits carbonyl formation very efficiently, indicating that AP may be utilized as a biological OH(*) free radical scavenger in human therapy.