Neurite elongation is blocked if microtubule polymerization is inhibited in PC12 cells

We have injected process-bearing PC12 cells with colchicine-tubulin mixed with either fluorescein-dextran or a rhodamine-labelled tubulin analogue to determine the role of microtubule polymerization in neurite elongation. Colchicine-tubulin is a specific, substoichiometric poison of microtubule assembly. We have shown that colchicine-tubulin does not cause existing PC12 microtubules to disassemble, and yet can inhibit the assembly of rhodamine-tubulin injected along with it. In population studies of neurite outgrowth in injected and uninjected cells, we find that colchicine-tubulin substantially inhibits neurite extension from injected cells over a wide variety of concentrations. In acute time-course studies of injected cells, we find that colchicine-tubulin does not block neurite outgrowth until the injectate reaches the neurite tip. Thereafter, however, it blocks process elongation completely. Thus we can conclude that microtubule polymerization in the region of the growth cone is an important element in neurite elongation. While polymerization at the cell body may be important in supplying subunits to the distal neurite, it does not play a direct role in process extension.     

Superoxide, amine buffers and tetranitromethane: a novel free radical chain reaction

The amine buffer Tris slowly reduces tetranitromethane (TNM) to the nitroform anion in a non-accelerating reaction. The amine buffers HEPES and MOPS also (slowly) react with TNM but the dialkylaminoalkyl radicals formed from these two buffers undergo further reactions resulting in a rapid, accelerating, free radical chain process whereby the amine is oxidized and TNM reduced. The chemical functionality in any reaction component, not necessarily the buffer, required for this radical chain mechanism is >N-CH<. In the presence of such groups, the quantification of superoxide by TNM is impossible.     

Superoxide,amine buffers and tetranitromethane: A novel free radical chain reaction

The amine buffer Tris slowly reduces tetranitromethane (TNM) to the nitroform anion in a non-accelerating reaction. The amine buffers HEPES and MOPS also (slowly) react with TNM but the dialkylaminoalkyl radicals formed from these two buffers undergo further reactions resulting in a rapid, accelerating, free radical chain process whereby the amine is oxidized and TNM reduced. The chemical functionality in any reaction component, not necessarily the buffer, required for this radical chain mechanism is >N-CH<. In the presence of such groups, the quantification of superoxide by TNM is impossible.     

Superoxide dismutase and catalase protect cultured hepatocytes from the cytotoxicity of acetaminophen

Superoxide dismutase, catalase and mannitol prevent the killing of cultured hepatocytes by acetaminophen in the presence of an inhibitor of glutathione reductase, BCNU. Under these conditions, the cytotoxicity of acetaminophen depends upon its metabolism, since beta-naphthoflavone, an inhibitor of mixed function oxidation, prevents the cell killing. In hepatocytes made resistant to acetaminophen by pretreatment with the ferric iron chelator, deferoxamine, addition of ferric or ferrous iron restores the sensitivity to acetaminophen. In such a situation, both superoxide dismutase and catalase prevent the killing by acetaminophen in the presence of ferric iron. By contrast, catalase, but not superoxide dismutase, prevents the cell killing dependent upon addition of ferrous iron. These results document the participation of both superoxide anion and hydrogen peroxide in the killing of cultured hepatocytes by acetaminophen and suggest that hydroxyl radicals generated by an iron catalyzed Haber-Weiss reaction mediate the cell injury.     

Flavonoids protect LDL from oxidation and attenuate atherosclerosis

Consumption of some plant-derived flavonoids results in their absorption and appearance in plasma and tissues. The inverse relationship between dietary flavonoids consumption and cardiovascular diseases may be associated with the ability of flavonoids to attenuate LDL oxidation, macrophage foam cell formation and atherosclerosis. The effect of flavonoids on arterial cell-mediated oxidation of LDL is determined by their accumulation in the lipoprotein and in arterial cells, such as macrophages. Flavonoids can reduce LDL lipid peroxidation by scavenging reactive oxygen/nitrogen species, chelation of transition metal ions and sparing of LDL-associated antioxidants. They can also reduce macrophage oxidative stress by inhibition of cellular oxygenases [such as nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase] or by activating cellular antioxidants (such as the glutathione system). Thus, plant flavonoids, as potent natural antioxidants that protect against lipid peroxidation in arterial cells and lipoproteins, significantly attenuate the development of atherosclerosis.     

Superoxide radical initiates the autoxidation of dihydroxyacetone

The aerobic xanthine oxidase reaction causes the cooxidation of dihydroxyacetone in a process which is strongly inhibited by superoxide dismutase but not by catalase, HO X scavengers, or iron-inactivating chelating agents. Several molecules of the sugar can be oxidized per O2- introduced. A free radical chain mechanism, in which O2- acts both as an initiator and as a chain propagator, is proposed. Simple sugars capable of tautomerizing to enediols may now be added to the list of biologically relevant targets for O2-.     

Superoxide generated by glutathione reductase initiates a vanadate-dependent free radical chain oxidation of NADH.

Vanadate V(V) markedly stimulated the oxidation of NADPH by GSSG reductase and this oxidation was accompanied by the consumption of O2 and the accumulation of H2O2. Superoxide dismutases completely eliminated this effect of V(V), whereas catalase was without effect, as was exogenous H2O2 added to 0.1 mM. These effects could be seen equally well in phosphate- or in 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid-buffered solutions. Under anaerobic conditions there was no V(V)-stimulated oxidation of NADPH. Approximately 4% of the electrons flowing from NADPH to O2, through GSSG reductase, resulted in release of O2-. The average length of the free radical chains causing the oxidation of NADPH, initiated by O2- plus V(V), was calculated to be in the range 140-200 NADPH oxidized per O2- introduced. We conclude that GSSG reductase, and by extension other O2(-)-producing flavoprotein dehydrogenases such as lipoyl dehydrogenase and ferredoxin reductase, catalyze V(V)-stimulated oxidation of NAD(P)H because they release O2- and because O2- plus V(V) initiate a free radical chain oxidation of NAD(P)H. There is no reason to suppose that these enzymes can act as NAD(P)H:V(V) oxidoreductases.     

Proteins protect lipid membranes from oxidation by thiyl radicals

Oxidation of polyunsaturated fatty acids by thiyl radicals derived from GSH or Cys is believed to be responsible for some of the biological damage resulting from lipid oxidation under oxidative stress. However, this has not been demonstrated in complex biological systems. In this study, we measured the formation of lipid hydroperoxides in liposomes exposed to radicals generated by gamma radiation from GSH, GSSG, GSMe, Cys and Met. In the absence of proteins, the radicals oxidized the liposome lipids. In the presence of proteins, the thiyl radicals failed to react with the liposomes, even though the protein radicals efficiently oxidized the S-compounds. It appears that the thiyl and other S-radicals were effectively scavenged by the protein before initiating lipid oxidation. The results suggest that membrane lipid oxidation in vivo by thiyl radicals is unlikely to be a significant event.     

Red blood cells protect albumin from cigarette smoke-induced oxidation

Different studies reported the presence of oxidized (carbonylated) albumin in the extravascular pool, but not in the intravascular one of cigarette smokers. In this study we attempted to explain this apparent discrepancy exposing human serum albumin (HSA) to aqueous cigarette smoke extract (CSE). CSE induces HSA carbonylation and oxidation of the HSA Cys34 sulfhydryl group. An antioxidant action of glutathione, cysteine, and its synthetic derivative N-acetylcysteine was observed only at supra-physiological concentrations, suggesting that physiological (plasma) concentrations of glutathione and cysteine in the low micromolar range are ineffective in preventing cigarette smoke-induced oxidation of HSA. Differently, human erythrocytes resulted to be protective towards CSE-induced oxidation (carbonylation and thiol oxidation) of both HSA and total human plasma proteins.     

2-Imidazolethiones protect ascorbic acid from oxidation induced by copper

The oxidation of ascorbic acid to dehydroascorbic acid was accelerated by metal ions such as copper. This stimulation of ascorbate oxidation was inhibited by the addition of 2-imidazolethiones and other sulfhydryl-containing compounds, but not by 2-imidazolones or phenytoin. Although the 2-imidazolethiones interacted with copper, as shown by a decrease in the ultraviolet absorbance of the compounds, the product formed still protected ascorbate from oxidation. The 2-imidazolethiones are proposed to complex copper through their free -SH groups.     

Superoxide radical production in plasma membrane samples from regressing rat corpora lutea

Plasma membrane samples prepared from regressing rat corpora lutea were examined for production of the superoxide radical. A procedure was developed to purify membrane samples that were enriched approximately 15-fold with the plasma membrane marker enzyme, and superoxide radical levels were determined using electron spin resonance to measure Tiron semiquinone. During prostaglandin F2 alpha-induced and spontaneous regression, there was a significant increase in formation of superoxide radical that was not observed in plasma membrane samples from nonregressing corpora lutea. Plasma membrane incubation experiments indicated that the increase in production was temperature sensitive and reduced with inhibitors of phospholipase A2 and cyclooxygenase. Addition of superoxide dismutase or vitamin E abolished superoxide radical formation in vitro. Following the rise in superoxide radical levels during regression, there was also a significant decrease in the activity of the plasma membrane enzyme, Na+-K+ ATPase. These results indicate that the production of superoxide radical increases in plasma membrane samples prepared from regressing rat corpora lutea and that this increase is mainly due to the products of phospholipase A2 and cyclooxygenase activity.     

Structure of the radical from one-electron oxidation of 4-hydroxycinnamate

Radicals from one-electron oxidation of 4-hydroxycinnamate, ferulate and 3,4-dihydroxycinnamate have been formed by reaction with the oxidising triplet state of duroquinone. All three compounds react with triplet duroquinone with second order rate constants close to the diffusion-controlled limit. The identity of the resulting radicals is confirmed by observation of their characteristic visible absorption spectra. Time-resolved resonance Raman (TR³) spectra of the radical from 4-hydroxycinnamate were measured using a probe laser wavelength of 600 nm, to be in resonance with the long wavelength absorption band of the radical. The TR³ spectra contain prominent bands ascribed to the C-O and ring C-C stretching vibrations. The spectra are interpreted as indicating strong delocalisation of the radical site to the double bond in conjugation with the aromatic ring in 4-hydroxycinnamate. This contributes to the low reduction potential of the radical and the antioxidant properties of hydroxycinnamates.     

Superoxide dismutase, catalase and scavengers of hydroxyl radical protect against the toxic action of alloxan on pancreatic islet cells in vitro.

Experiments with isolated pancreatic islets or dispersed islet cells from non-inbred ob/ob mice were performed to test the hypothesis that free radicals, notably OH., mediate the diabetogenic toxicity of alloxan. Accumulation of 86Rb+ by whole islets and exclusion of Trypan Blue by dispersed cells were used as previously validated criteria of islet-cell viability. Alloxan alone drastically inhibited the Rb+ accumulation and significantly decreased the frequency of cells excluding Trypan Blue. Enzymic scavengers of O2.- and H2O2 or non-enzymic scavengers of OH. or singlet oxygen were added to the incubation medium and tested for their ability to protect against these effects of alloxan. Superoxide dismutase, catalase, dimethyl sulphoxide, benzoate, and mannitol counteracted the effects of alloxan in both cytotoxicity assays. Significant protection of the Rb+-accumulating capacity was also afforded by butanol, caffeine, theophylline, NADH, NADPH and, to a small extent, NAD+. Urea has a poor affinity for OH. and did not protect against alloxan. No effect was obtained with the singlet-oxygen scavenger, histidine. Except for the protection by NADH and NADPH, which may be due to a direct reaction with alloxan in the medium, the results strongly support the hypothesis. beta-Cells may be particularly vulnerable to alloxan because their metabolic specialization facilitates reduction of the drug and perhaps of other substrates for O2.--yielding redox cycles.     

Voltage-regulated calcium channels involved in the regulation of enkephalin synthesis are blocked by phorbol ester treatment

Treatment of bovine chromaffin cells with 40 mM KCl stimulates a 3-fold increase in total methionine enkephalin immunoreactivity (medium plus cells) and a 4-fold increase in proenkephalin mRNA (mRNAenk). These effects of KCl, which are dependent on extracellular calcium, can be blocked by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), although release of methionine enkephalin appears less affected. Using fura-2-loaded chromaffin cells and a dual-excitation wavelength spectrofluorometer, we have examined whether the actions of KCl and TPA on methionine enkephalin synthesis and release can be explained by changes in intracellular free calcium ([Ca2+]i). KCl produced a rapid 600 nM increase in [Ca2+]i from resting levels of approximately 170 nM. Subsequently, [Ca2+]i declined to a new steady-state plateau which was approximately 275 nM higher than the original resting levels. The postdepolarization plateau of [Ca2+]i was reduced by TPA, (-)-(R)-202,791 (a dihydropyridine calcium channel antagonist), and LaCl3 (a nonselective calcium channel blocker). TPA also inhibited potentiation of the KCl-stimulated plateau of [Ca2+]i due to (+)-(S)-202,791, a calcium channel agonist. In contrast, TPA had no effect on resting [Ca2+]i and only slightly inhibited the initial rapid KCl-stimulated increase in [Ca2+]i. The inhibitory effects were maintained for 24 h in the continuous presence of TPA. We conclude 1) that TPA inhibits enkephalin synthesis by inactivating dihydropyridine-sensitive voltage-dependent calcium channels, 2) that these channels alone maintain elevated [Ca2+]i following KCl depolarization, and 3) that sustained elevation in [Ca2+]i is necessary in order to increase enkephalin synthesis in KCl-treated chromaffin cells.     

Superoxide is responsible for the vanadate stimulation of NAD(P)H oxidation by biological membranes

Vandate augments the oxidation of NAD(P)H, but not of NMNH, by rat liver microsomes. Paraquat increases the vanadate effect on NADPH, but not on NADH, oxidation. Substoichiometric levels of NADPH caused the co-oxidation of NADH or NMNH and SOD inhibited in all cases. The ratio of NADH or NMNH co-oxidized per NADPH added allowed estimation of average chain length, which increased as the pH was lowered from 8.0 to 7.1. The initial rate of this co-oxidation of NMNH was a saturating function of the concentration of microsomes, reflecting a decrease in chain length with an increase in number of concomitant reaction chains, and due to increasing radical-radical termination reactions. Mitochondrial outer membranes behaved like the microsomal membranes, but mitochondrial inner membranes catalyzed a rapid oxidation of NADH which could be augmented by vanadate, whose action was enhanced by paraquat and inhibited by antimycin or rotenone. These and related observations support the view that vanadate stimulates NAD(P)H oxidation by biological membranes, not by virtue of interacting with enzymes, but rather by interacting with O-2.     

Superoxide generation from mitochondrial NADH dehydrogenase induces self-inactivation with specific protein radical formation

Mitochondrial superoxide (O(2)(.)) production is an important mediator of oxidative cellular injury. While NADH dehydrogenase (NDH) is a critical site of this O(2)(.) production; its mechanism of O(2)(.) generation is not known. Therefore, the catalytic function of NDH in the mediation of O(2)(.) generation was investigated by EPR spin-trapping. In the presence of NADH, O(2)(.) generation from NDH was observed and was inhibited by diphenyleneiodinium chloride (DPI), indicating involvement of the FMN-binding site of NDH. Addition of FMN increased O(2)(.) production. Destruction of the cysteine ligands of iron-sulfur clusters decreased O(2)(.) generation, suggesting a secondary role of this site. This inhibitory effect was reversed by addition of FMN. However, FMN addition could not reverse the inhibition of NDH by either DPI or heat denaturation, demonstrating involvement of both FMN and its FMN-binding protein moiety in the catalysis of O(2)(.) generation. O(2)(.) production by NDH also induced self-inactivation. Immunospin-trapping with anti-DMPO antibody and subsequent mass spectrometry was used to define the sites of oxidative damage of NDH. A DMPO adduct was detected on the 51-kDa subunit and was O(2)(.)-dependent. Alkylation of the cysteine residues of NDH significantly inhibited NDH-DMPO spin adduct formation, indicating involvement of protein thiyl radicals. LC/MS/MS analysis of a tryptic digest of the 51-kDa polypeptide revealed that cysteine (Cys(206)) and tyrosine (Tyr(177)) were specific sites of NDH-derived protein radical formation. Thus, two domains of the 51-kDa subunit, Gly(200)-Ala-Gly-Ala-Tyr-Ile-Cys(206)-Gly-Glu-Glu-Thr-Ala-Leu-Ile-Glu-Ser-Ile-Glu-Gly-Lys(219) and Ala(176)-Tyr(177)-Glu-Ala-Gly-Leu-Ile-Gly-Lys(184), were demonstrated to be susceptible to oxidative attack, and their oxidative modification results in decreased electron transfer activity.     

On the mechanism and rate of substrate oxidation by amine oxidase from lentil seedlings

The kinetics of reaction between lentil seedlings amine oxidase and two amine substrates, namely putrescine and dimethylaminomethylbenzylamine, have been studied by rapid mixing with diode array detection. In this way several wavelengths can be monitored at once allowing the simultaneous measurement of enzyme bleaching and formation of a yellow radical intermediate. The two substrates are oxidized at rates that differ by one order of magnitude in favor of putrescine. Of the individual five rate constants measured and/or calculated from the experimental ones, k2 alone, the monomolecular transformation of ES to EP accounts for this difference. The reoxidation step is instead not rate limiting and identical for the two substrates.     

Ethanolamine plasmalogens protect cholesterol-rich liposomal membranes from oxidation caused by free radicals

The aim of the present study is to investigate the effect of ethanolamine plasmalogens on the oxidative stability of cholesterol-rich membranes by comparing it with that of diacyl glycerophosphoethanolamine, using bovine brain ethanolamine plasmalogen (BBEP) or egg yolk phosphatidylethanolamine (EYPE)-containing large unilamellar vesicles (LUVs) and the water-soluble radical initiator AAPH. Electron microscopic observation and particle size measurement visually demonstrated that ethanolamine plasmalogens protect cholesterol-rich phospholipid bilayers from oxidative collapse. Lipid analyses suggested that the effect of ethanolamine plasmalogens in stabilizing membranes against oxidation is partly due to the antioxidative action of plasmalogens involved in scavenging radicals at vinyl ether linkage.     

Polycomb group repression is blocked by the Drosophila suppressor of Hairy-wing [su(Hw)] insulator.

The suppressor of Hairy-wing [SU(HW)] binding region disrupts communication between a large number of enhancers and promoters and protects transgenes from chromosomal position effects. These properties classify the SU(HW) binding region as an insulator. While enhancers are blocked in a general manner, protection from repressors appears to be more variable. In these studies, we address whether repression resulting from the Polycomb group genes can be blocked by the SU(HW) binding region. The effects of this binding region on repression established by an Ultrabithorax Polycomb group Response Element were examined. A transposon carrying two reporter genes, the yellow and white genes, was used so that repression and insulation could be assayed simultaneously. We demonstrate that the SU(HW) binding region is effective at preventing Polycomb group repression. These studies suggest that one role of the su(Hw) protein may be to restrict the range of action of repressors, such as the Polycomb group proteins, throughout the euchromatic regions of the genome.