Monoclonal antibodies to a nitroxide lipid hapten

The isolation and characterization of a hybridoma cell line producing a monoclonal IgG₁ antibody against a spin-label nitroxide group is described. The antibody recognizes a synthetic hapten containing linked dinitrophenyl and 2,2,6,6-tetramethylpiperidinyl 1-oxy groups, having an affinity of 3.6±1.0·10⁶ M^?1 for the soluble hapten at 25°C. The antibody binds to phospholipid vesicles containing 2 mol% of spin label-derivitized lipid (lipid hapten) with an affinity of 1.5±0.2·10⁸ M^?1. This monoclonal IgG₁ mediates the binding of hapten-bearing lipid vesicles to mouse macrophage RAW264 cells bearing Fc receptors. The cellular responses to this binding are similar to those observed previously using polyclonal rabbit anti-hapten IgG. As with the heterogeneous antibodies, the monoclonal IgG₁ is more efficient in mediating cellular uptake when the vesicles are in the ‘fluid’ physical state (dimyristoylphosphatidylcholine at 37°C) compared to ‘solid’ (dipalmitoylphosphatidylcholine at 37°C). Despite the enhanced binding of ‘fluid’ phospholipid vesicles to cells, only the ‘solid’ vesicles triggered a significant respiratory burst in RAW264 macrophages.     

Antioxidant effects of water- and lipid-soluble nitroxide radicals in liposomes

Liposomes are today useful tools in different fields of science and technology. A lack of stability due to lipid peroxidation is the main problem in the extension of the use of these formulations. Recent investigative works have reported the protective effects of stable nitroxide radicals against oxidative processes in different media and under different stress conditions. Our group has focused its attention on the natural aging of liposomes and the protection provided by the water- and lipid-soluble nitroxide radicals 2,2,6,6-tetramethylpiperdine-1-oxyl (TEMPO) and doxylstearic acids (5-DSA, 12-DSA, and 16-DSA), respectively. Unilamellar liposomes were incubated under air atmosphere at 37°C, both in the absence and in the presence of these radicals. Conjugated dienes, lipid hydroperoxides, TBARS, membrane fluidity, and nitroxide ESR signal intensity were followed as a function of time. Our results demonstrated that doxylstearic acids were more efficient than TEMPO in retarding lipid peroxidation at all the concentrations tested. The inhibition percentages, depending on the total nitroxide concentration, were not proportional to the lipid–water partition coefficient. Furthermore, time-course ESR signals showed a slower decrease for doxylstearic acids than for TEMPO. No significant differences were found among 5-DSA, 12-DSA, and 16-DSA. We concluded that the nitroxide radical efficiency as antioxidant directly depends on both nitroxide concentration and lipophilicity.     

Intensity enhancement of intraligand singlet-triplet π-π transition for the coordinated β-diketonate in Ni(II) complexes with a nitroxide radical

Room temperature and low temperature magnetic circular dichroism (MCD) in the intraligand spin-forbidden singlet-triplet π-π^∗ transition for the coordinated β-diketonate ligands were observed for the β-diketonato Ni(II) complexes with a chelated imino or nitronyl nitroxide radical, but not for the β-diketonato Ni(II) complexes without the radical ligands. This is elucidated by the borrowing mechanism from the singlet-singlet π-π^∗ transition through the hypothetical interligand β-diketonate-to-radical charge transfer (LLCT) in contrast to the case of Cr(III) complexes.     

In vitro photostability and photoprotection studies of a novel 'multi-active' UV-absorber

This paper reports on the synthesis and properties of a new UV-absorber (OC-NO) based on the most popular UV filter worldwide, ethylhexyl methoxycinnamate (OMC) in which the methoxy group has been replaced with a pyrrolidine nitroxide bearing antioxidant activity. This sunscreen active has therefore both UV-absorbing and antioxidant properties which could ideally address both the UV-B and UV-A skin photo-damage. For broad-spectrum coverage, the combinations of OC-NO with two commonly used UV-A absorbers (BMDBM and DHHB) were also studied. The results obtained reveal that OC-NO: (a) is as photostable as OMC after UV-A exposure; (b) acts as free radical scavenger as demonstrated by EPR and chemical studies; (c) reduces UV-A and UV-A+BMDBM induced lipid peroxidation in liposomes and cells, measured as reduced TBARS levels and increased C11-BODIPY red fluorescence, respectively; (d) has comparable antioxidant activity to that of vitamin E and BHT commonly used in skin care formulations; (e) is non-cytotoxic to human skin fibroblasts as assessed with the MTT assay when exposed to increasing doses of UV-A; and (f) OC-NO+DHHB is a promising, photostable broad spectrum UV-filter combination that concomitantly reduces UV-induced free radical damage. These results suggest that nitroxide/antioxidant-based UV-absorbers may pave the way for the utilization of 'multi-active' ingredients in sunscreens thereby reducing the number of ingredients in these formulations.     

Synthesis, crystal structure, magnetism and electrochemical properties of two copper(II) furoyltrifluoroacetonate complexes with nitroxide radical

Two new magnetic copper compounds were obtained using the 4,4,4-trifluoro-1-furoylbutane-1,3-dione (Ftfac) ligand and two nitroxide radicals: 3-pyridyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (NITmPy) and 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-N-oxy (Tempol). The complexes with formula [Cu(Ftfac)₂(NITmPy)₂] (1) and [Cu(Ftfac)₂(Tempol)] (2) were structurally characterized by single-crystal X-ray diffraction. In compound 1, the copper ion has a distorted octahedral environment, bound to two NITmpPy ligands through the nitrogen atom of the pyridine ring. In compound 2, the copper ion has a distorted pyramidal environment in which the apical position is occupied by the oxygen atom of the Tempol hydroxyl group. The temperature dependence of the magnetic susceptibility of the two compounds was investigated. It was found that compound 1 presents ferromagnetic interaction (J = 9.1 cm⁻¹) among copper(II) ions and NITmPy radicals. As a result of the interconnection between molecular moieties through H-bonds, compound 2 presents an unusual magnetic behavior with alternating ferro- and antiferromagnetic interactions.     

Cellular accumulation and antioxidant activity of acetoxymethoxycarbonyl pyrrolidine nitroxides

Abstract

Nitroxides are widely used in biology as antioxidants, spin labels, functional spin probes for pH, oxygen and thiol levels, and tissue redox status imaging using electron paramagnetic resonance (EPR); however, biological applications of nitroxides is hindered by fast bioreduction to EPR-silent hydroxylamines and rapid clearance. In this work, we have studied pyrrolidine nitroxides with acetoxymethoxycarbonyl groups which can undergo hydrolysis by cellular esterases to hydrophilic carboxylate derivatives resistant to bioreduction. Nitroxides containing acetoxymethoxycarbonyl groups were rapidly absorbed by cells from the media, 3,4-bis-(acetoxymethoxycarbonyl)-proxyl (DCP-AM2) and 3-(2-(bis(2-(acetoxymethoxy)-2-oxoethyl)amino)acetamido)-proxyl (DCAP-AM2) showing the strongest EPR signal of the cellular fraction. Remarkably, the EPR parameters of 3,4-dicarboxy-proxyl (DCP) and its mono- and di-acetoxymethyl esters are different, and consequent intracellular hydrolysis of acetoxymethoxycarbonyl groups in DCP-AM2 can be followed by EPR. To elucidate intracellular location of the resultant DCP, the mitochondrial fraction has been isolated. EPR measurements showed that mitochondria were the main place where DCP was finally accumulated. TEMPO derivatives showed expectedly much faster decay of EPR signal in the cellular fraction, compared to pyrrolidine nitroxides. It was found that supplementation of endothelial cells with 50?nM of DCP-AM2 completely normalised the mitochondrial superoxide level. Moreover, administration of DCP-AM2 to mice (1.4?mg/kg/day) resulted in substantial nitroxide accumulation in the tissues and significantly reduced hypertension. We found that hydroxylamine derivatives of dicarboxyproxyl nitroxide DCP-AM-H can be used for the detection of superoxide in vivo in angiotensin II model of hypertension. Infusion of DCP-AM-H in mice leads to accumulation of persistent EPR signal of nitroxide in the blood and vascular tissue in angiotensin II-infused wild-type but not in SOD2 overexpressing mice. Our data demonstrate that acetoxymethoxycarbonyl group containing nitroxides accumulate in mitochondria and demonstrate site-specific antioxidant activity.     

Crystallographic structure of a helical lipopeptaibol antibiotic analogue

An X-ray diffraction analysis of the [Fmoc⁰,TOAC^4,8, Leu-OMe¹¹] analogue of thelipopeptaibol antibiotic trichogin A IV shows that the undecapeptide isfolded in a right-handed, mixed /3₁₀-helix. The helicalmolecules are connected in a head-to-tail arrangement along the b-axisthrough C=O···H-N intermolecular H-bonding. Thispacking mode generates a hydrophobic cavity where the FmocN-protecting groups are accommodated. The distances andangles between the nitroxide groups of the two TOAC residues, separated byone turn of the -helix, have been determined.     

Total syntheses in solution of TOAC-labelled alamethicin F50/5 analogues

Total syntheses in solution of a set of four selected analogues of the 19-mer component F50/5 of alamethicin, the most extensively studied among the channel-former peptaibol antibiotics, are planned and reported. All analogues bear three Glu(OMe) residues, replacing the Gln residues at positions 7, 18, and 19 of the naturally occurring compound. Three analogues are mono-labelled with the free-radical-containing amino acid residue TOAC at the strategic positions 1, 8, or 16. The fourth analogue is bis-labelled with the same EPR-active residue at both positions 1 and 16. In the native sequence, all of the positions where TOAC replacements have been introduced are characterized by residues of Aib, the prototype of the class of helicogenic C(alpha)-tetrasubstituted alpha-amino acids. All of the TOAC analogues synthesized exhibit significant membrane-modifying properties.     

The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress

The serine protease HtrA (DegP), which is indispensable for cell survival at elevated temperatures, is a peripheral membrane protein, localized on the periplasmic side of the inner membrane in Escherichia coli, and the biochemical and genetic evidence indicates that the physiological role of HtrA is to degrade denatured proteins formed in the cellular envelope during heat shock. The aim of this study was to find out if the HtrA protease contributes to protection of the cell against oxidative stress. We compared the influence of various oxidizing agents on htrA mutant cells with their effects on wild-type bacteria, and found that the htrA mutation did not increase sensitivity to hydrogen peroxide or paraquat but made the cell extremely sensitive to ferrous [Fe(II)] ions, which are known to enhance oxidation of proteins. Treatment with ferrous ions caused a larger increase in the level of protein carbonyl groups in the membrane fraction of the cell than in the periplasm and cytoplasm. Iron-induced oxidation of membrane proteins was enhanced in the htrA mutant relative to wild-type cells. Inhibition of the growth of the htrA mutant by iron could be alleviated more efficiently by a nitroxide antioxidant that localizes in the membranes (A-TEMPO) than by a derivative (4OH-TEMPO) that acts mainly in the soluble fraction of the cell. Inhibition of the growth of the htrA mutant was more pronounced following treatment with cumene hydroperoxide, which partitions into membranes, than with t-butyl hydroperoxide, which forms radical mainly in the cytosol. Both ferrous ions and cumene hydroperoxide, but not hydrogen peroxide, paraquat or t-butyl hydroperoxide, induced synthesis of HtrA. Our results show that HtrA plays a role in defense against oxidative shock and support the hypothesis that HtrA participates in the degradation of oxidatively damaged proteins localized in the cell envelope, especially those associated with the membranes. Received: 9 March 1999 / Accepted: 31 May 1999     

Identification of a single genome by electron paramagnetic resonance (EPR) with nitroxide-labeled oligonucleotide probes

Nitroxide-labeled nucleic acids are used as a molecular size sensor to identify as few as one genome under polymerase chain reaction (PCR) conditions by electron paramagnetic resonance (EPR) spectroscopy. DNA identification is based on differences in the EPR spectra of mono-nitroxide-labeled nucleic acids. The experimental data imply that rapid DNA identification can be achieved in many systems by EPR at the molecular level.