Electrochemical Resistive-Pulse Sensing.

Resistive-pulse sensing with biological or solid-state nanopores and nanopipettes has been widely employed in detecting single molecules and nanoparticles. The analytical signal in such experiments is the change in ionic current caused by the molecule/particle translocation through the pipet orifice. This paper describes a new version of the resistive-pulse technique based on the use of carbon nanopipettes (CNP). The measured current is produced by electrochemical oxidation/reduction of redox molecules at the carbon surface and responds to the particle translocation. In addition to counting single entities, this technique enables qualitative and quantitative analysis of the electroactive material they contain. Using liposomes as a model system, we demonstrate the capacity of CNPs for (1) conventional resistive-pulse sensing of single liposomes, (2) electrochemical resistive-pulse sensing, and (3) electrochemical identification and quantitation of redox species (e.g., ferrocyanide, dopamine, and nitrite) contained in a single liposome. The small physical size of a CNP suggests the possibility of single-entity measurements in biological systems.

Direct Transfection of Fatty Acid Conjugated siRNAs and Knockdown of the Glucose-Regulated Chaperones in Prostate Cancer Cells.

The emerging field of RNAi nanotechnology has led to rapid advances in the applications of siRNAs in chemical biology, medicinal chemistry, and biotechnology. In our RNAi approach, bioconjugation of linear, V-, and Y-shaped RNA templates were designed using a series of saturated and unsaturated fatty acids to improve cell uptake and knockdown efficacy of the oncogenic glucose regulated proteins (GRPs) in prostate (PC-3) cancer cells. An optimized HCTU-coupling procedure was developed for tagging variable saturated and unsaturated fatty acids onto the 5'-ends of linear and V-shaped RNA templates that were constructed by semiautomated solid phase RNA synthesis. Hybridization and self-assembly of complementary strands yielded linear, V-, and Y-shaped fatty acid-conjugated siRNAs which were characterized by native PAGE. CD spectroscopy confirmed their A-type helix conformations. RP IP HPLC provided trends in amphiphilic properties, whereas DLS and TEM confirmed multicomponent self-assembled structures that were prone to aggregation. Subsequently, the fatty acid conjugated siRNA bioconjugates were tested for their RNAi activity by direct transfection within PC-3 cells known to overexpress oncogenic GRP activity. The siRNA bioconjugates with sense strand modifiers provided more potent GRP knockdown relative to the antisense modified siRNAs, but to a lesser extent when compared to the unconjugated siRNA controls that were transfected with the commercial Trans-IT X2 dynamic delivery system. Flow cytometry revealed that the latter may be at least in part attributed to limited cell uptake of the fatty acid conjugated siRNAs. Nonetheless, these new constructs represent an entry point in modifying higher-order siRNA constructs that may lead to the generation of more efficient siRNA bioconjugates for screening important oncogene targets and for cancer gene therapy applications.

Nitroxide radicals as research tools: Elucidating the kinetics and mechanisms of catalase-like and "suicide inactivation" of metmyoglobin.

BACKGROUND: Metmyoglobin (MbFe(III)) reaction with H(2)O(2) has been a subject of study over many years. H(2)O(2) alone promotes heme destruction frequently denoted "suicide inactivation," yet the mechanism underlying H(2)O(2) dismutation associated with MbFe(III) inactivation remains obscure. METHODS: MbFe(III) reaction with excess H(2)O(2) in the absence and presence of the nitroxide was studied at pH 5.3-8.1 and 25°C by direct determination of reaction rate constants using rapid-mixing stopped-flow technique, by following H(2)O(2) depletion, O(2) evolution, spectral changes of the heme protein, and the fate of the nitroxide by EPR spectroscopy. RESULTS: The rates of both H(2)O(2) dismutation and heme inactivation processes depend on [MbFe(III)], [H(2)O(2)] and pH. Yet the inactivation stoichiometry is independent of these variables and each MbFe(III) molecule catalyzes the dismutation of 50±10 H(2)O(2) molecules until it is inactivated. The nitroxide catalytically enhances the catalase-like activity of MbFe(III) while protecting the heme against inactivation. The rate-determining step in the absence and presence of the nitroxide is the reduction of MbFe(IV)O by H(2)O(2) and by nitroxide, respectively. CONCLUSIONS: The nitroxide effects on H(2)O(2) dismutation catalyzed by MbFe(III) demonstrate that MbFe(IV)O reduction by H(2)O(2) is the rate-determining step of this process. The proposed mechanism, which adequately fits the pro-catalytic and protective effects of the nitroxide, implies the intermediacy of a compound I-H(2)O(2) adduct, which decomposes to a MbFe(IV)O and an inactivated heme at a ratio of 25:1. GENERAL SIGNIFICANCE: The effects of nitroxides are instrumental in elucidating the mechanism underlying the catalysis and inactivation routes of heme proteins.

RNAi Screening of the Glucose-Regulated Chaperones in Cancer with Self-Assembled siRNA Nanostructures.

The emerging field of RNA nanotechnology has been used to design well-programmed, self-assembled nanostructures for applications in chemistry, biology, and medicine. At the forefront of its utility in cancer is the unrestricted ability to self-assemble multiple siRNAs within a single nanostructure formulation for the RNAi screening of a wide range of oncogenes while potentiating the gene therapy of malignant tumors. In our RNAi nanotechnology approach, V- and Y-shape RNA templates were designed and constructed for the self-assembly of discrete, higher-ordered siRNA nanostructures targeting the oncogenic glucose regulated chaperones. The GRP78-targeting siRNAs self-assembled into genetically encoded spheres, triangles, squares, pentagons and hexagons of discrete sizes and shapes according to TEM imaging. Furthermore, gel electrophoresis, thermal denaturation, and CD spectroscopy validated the prerequisite siRNA hybrids for their RNAi application. In a 24 sample siRNA screen conducted within the AN3CA endometrial cancer cells known to overexpress oncogenic GRP78 activity, the self-assembled siRNAs targeting multiple sites of GRP78 expression demonstrated more potent and long-lasting anticancer activity relative to their linear controls. Extending the scope of our RNAi screening approach, the self-assembled siRNA hybrids (5 nM) targeting of GRP-75, 78, and 94 resulted in significant (50-95%) knockdown of the glucose regulated chaperones, which led to synergistic effects in tumor cell cycle arrest (50-80%) and death (50-60%) within endometrial (AN3CA), cervical (HeLa), and breast (MDA-MB-231) cancer cell lines. Interestingly, a nontumorigenic lung (MRC5) cell line displaying normal glucose regulated chaperone levels was found to tolerate siRNA treatment and demonstrated less toxicity (5-20%) relative to the cancer cells that were found to be addicted to glucose regulated chaperones. These remarkable self-assembled siRNA nanostructures may thus encompass a new class of potent siRNAs that may be useful in screening important oncogene targets while improving siRNA therapeutic efficacy and specificity in cancer.

Synthesis, characterization and anti-cancer activity of a peptide nucleolipid bioconjugate.

The synthesis, characterization and anti-cancer activity of a novel peptide nucleolipid bioconjugate is reported in this study. The prerequisite 5'-carboxy derived nucleolipid was synthesized following a five-step solution-phase approach and then coupled to the cytotoxic D-(KLAKLAK)2 sequence by solid-phase bioconjugation. The biophysical and structural properties of the peptide-nucleolipid bioconjugate were evaluated and compared to the peptide controls. These characterization studies revealed that the amphiphilic peptides favored helical-type secondary structures and well-defined nanoparticle formulations that were found to be contributive towards their biological activity. The peptide-nucleolipid bioconjugate displayed greater lethality in comparison to the native D-(KLAKLAK)2AK sequence when treated within the human A549 non-small cell lung carcinoma cell line. Thus, the amphiphilic peptide-nucleolipid forms a new class of anti-cancer peptides that may be developed into promising leads in the fight against cancer.

The mechanism underlying nitroxyl and nitric oxide formation from hydroxamic acids.

BACKGROUND: The pharmacological effects of hydroxamic acids (RC(O)NHOH, HX) are partially attributed to their ability to serve as HNO and/or NO donors under oxidative stress. Given the development and use of HXs as therapeutic agents, elucidation of the oxidation mechanism is needed for more educated selection of HX-based drugs. METHODS: Acetohydroxamic and glycine-hydroxamic acids were oxidized at pH 7.0 by a continuous flux of radiolytically generated (·)OH or by metmyoglobin and H(2)O(2) reactions system. Gas chromatography and spectroscopic methods were used to monitor the accumulation of N(2)O, N(2), nitrite and hydroxylamine. RESULTS: Oxidation of HXs by (·)OH under anoxia yields N(2)O, but not nitrite, N(2) or hydroxylamine. Upon the addition of H(2)O(2) to solutions containing HX and metmyoglobin, which is instantaneously and continuously converted into compound II, nitrite and, to a lesser extent, N(2)O are accumulated under both anoxia and normoxia. CONCLUSIONS: Oxidation of HXs under anoxia by a continuous flux of (·)OH, which solely oxidizes the hydroxamate moiety to RC(O)NHO(·), forms HNO. This observation implies that bimolecular decomposition of RC(O)NHO(·) competes efficiently with unimolecular decomposition processes such as internal disproportionation, hydrolysis or homolysis. Oxidation by metmyoglobin/H(2)O(2) involves relatively mild oxidants (compounds I and II). Compound I reacts with HX forming RC(O)NHO(·) and compound II, which oxidizes HX, RC(O)NHO(·), HNO and NO. The latter reaction is the main source of nitrite. GENERAL SIGNIFICANCE: HXs under oxidative stress release HNO, but can be considered as NO-donors provided that HNO oxidation is more efficient than its reaction with other biological targets.

Synthesis, DNA binding and anti-leukemic activity of an aminoacyl nucleolipid.

The synthesis and characterization of a new class of DNA binding molecule exhibiting potent and selective anti-leukemic activity is described. The synthesis of an aminoacyl nucleolipid was developed from an efficient EEDQ coupling strategy, in which a series of seven bioconjugates were synthesized in yields of 53-78%. Guanosine bioconjugate 7, was used as building block for the synthesis of a target aminoacyl nucleolipid 14. Its GRP78 DNA binding affinity was confirmed by gel shift assay, CD spectroscopy, Tm measurements and dynamic light scattering experiments. Moreover, in a single dose (10 µM) screen against a panel of 60 cancer cell lines, aminoacyl nucleolipid 14 was found to selectively trigger greater than 90% cell death in a SR human leukemia cancer cell line. The reported aminoacyl nucleolipid represents a useful model for a new class of DNA binding molecules for the development of potent and selective anti-cancer agents.

Cyclic Hydroxylamines as Monitors of Peroxynitrite and Superoxide-Revisited.

There is a considerable need for methods that allow quantitative determination in vitro and in vivo of transient oxidative species such as peroxynitrite (ONOOH/ONOO-) and superoxide (HO2•/O2•-). Cyclic hydroxylamines, which upon oxidation yield their respective stable nitroxide radicals, have been suggested as spin probes of peroxynitrite and superoxide. The present study investigated this approach by following the kinetics of peroxynitrite decay in the absence and presence of various 5-membered and 6-membered ring hydroxylamines, and comparing the yield of their respective nitroxides using electron paramagnetic spectroscopy. The results demonstrate that hydroxylamines do not react directly with peroxynitrite, but are oxidized to their respective nitroxides by the radicals formed during peroxynitrite self-decomposition, namely •OH and •NO2. The accumulated nitroxides are far below their expected yield, had the hydroxylamines fully scavenged all these radicals, due to multiple competing reactions of the oxidized forms of the hydroxylamines with •NO2 and ONOO-. Therefore, cyclic hydroxylamines cannot be used for quantitative assay of peroxynitrite in vitro. The situation is even more complex in vivo where •OH and •NO2 are formed also via other oxidizing reactions systems. The present study also compared the yield of accumulated nitroxides under constant flux of superoxide in the presence of various cyclic hydroxylamines. It is demonstrated that certain 5-membered ring hydroxylamines, which their respective nitroxides are poor SOD-mimics, might be considered as stoichiometric monitors of superoxide in vitro at highest possible concentrations and pH.

On the distinction between nitroxyl and nitric oxide using nitronyl nitroxides.

A better understanding of the origins of NO and HNO and their activities and biological functions requires accurate methods for their detection and quantification. The unique reaction of NO with nitronyl nitroxides such as 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (C-PTIO), which yields the corresponding imino nitroxides, is widely used for NO detection (mainly by electron paramagnetic resonance spectroscopy) and for modulation of NO-induced physiological functions. The present study demonstrates that HNO readily reacts with nitronyl nitroxides, leading to the formation of the respective imino nitroxides and hydroxylamines via a complex mechanism. Through the use of the HNO donor Angeli's salt (AS) with metmyoglobin as a competing agent, the rate constant for C-PTIO reduction by HNO has been determined to be (1.4 +/- 0.2) x 10(5) M(-1) s(-1) at pH 7.0. This reaction yields the corresponding nitronyl hydroxylamine C-PTIO-H and NO, which is trapped by C-PTIO to form (*)NO(2) and the corresponding imino nitroxide, C-PTI. (*)NO(2) oxidizes the nitronyl and imino nitroxides to their respective oxoammonium cations, which decay mainly via comproportionation with the nitronyl and imino hydroxylamines. When [AS] > [C-PTIO], the reduction of C-PTI by HNO proceeds, eventually converting C-PTIO to the corresponding imino hydroxylamine, C-PTI-H. Similar results were obtained for 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). It is concluded that nitronyl nitroxide is readily reduced by HNO to nitronyl hydroxylamine and is eventually converted into imino nitroxide and imino hydroxylamine. The yield of the imino hydroxylamine increases at the expense of the imino nitroxide as the ratio [AS](0)/[nitronyl nitroxide](0) is increased. Since the reaction of NO with nitronyl nitroxide yields only the corresponding imino nitroxide, nitronyl nitroxide can discriminate NO from HNO only when present at a concentration much lower than the total production of HNO.

Bifunctional Au-templated RNA nanoparticles enable direct cell uptake detection and GRP75 knockdown in prostate cancer.

Nucleic acids templated on gold (Au) surfaces have led to a wide range of functional materials ranging from microarrays, sensors and probes in addition to drug delivery and treatment. In this application, we describe a simple and novel method for templating amino-functionalized RNA onto Au surfaces and their self-assembly into small, discrete nanoparticles. In our method, sample hybridization with a complementary RNA strand with and without a fatty acid (palmitamide) appendage produced functionalized double-stranded RNA on the Au surface. The resulting Au-functionalized RNA particles were found to be stable under reducing conditions according to UV-Vis spectroscopy. Sample characterization by DLS and TEM confirmed self-assembly into primarily small (∼10-40 nm) spherical shaped nanoparticles expected to be amenable to cell biology. However, fluorescence emission (λexc: 350 nm, λem: 650 nm) revealed radiative properties which limited cell uptake detection. Introduction of FITC within the Au-functionalized RNA particles produced a bifunctional probe, in which FITC fluorescence emission (λexc: 494 nm, λem: 522 nm) facilitated cell uptake detection, in a time-dependent manner. The dual encapsulation-release profiles of the FITC-labeled Au-functionalized RNA particles were validated by time-dependent UV-Vis spectroscopy and spectrofluorimetry. These experiments respectively indicated an increase in FITC absorption (λabs: 494 nm) and fluorescence emission (λem: 522 nm) with increased sample incubation times, under physiological conditions. The release of Au-functionalized siRNA particles in prostate cancer (PC-3) cells resulted in concomitant knockdown of GRP75, which led to detectable levels of cell death in the absence of a transfection vector. Thus, the formulation of stable, small and discrete Au-functionalized RNA nanoparticles may prove to be valuable bifunctional probes in the theranostic study of cancer cells.

Size Matters: Arginine-Derived Peptides Targeting the PSMA Receptor Can Efficiently Complex but Not Transfect siRNA.

Oligoarginine sequences conjugated to a short cancer-targeting peptide (CTP) selective for the prostate-specific membrane antigen (PSMA) receptor was developed for selective small interfering RNA (siRNA) delivery to a human metastatic/castration-resistant prostate cancer (PCa) cell line, which expresses PSMA on the surface. The PSMA-Rn (n = 6 and 9) peptides were synthesized by solid-phase peptide synthesis, characterized by liquid chromatography-mass spectrometry (LC-MS) and condensed with glucose-regulated protein (GRP)-silencing siRNAs. Native gels showed formation of stable CTP:siRNA ionic complexes. Furthermore, siRNA release was effected by heparin competition, supporting the peptides' capabilities to act as condensing and releasing agents. However, dynamic light scattering (DLS) and transmission electron microscopy (TEM) studies revealed large anionic complexes that were prone to aggregation and limited cell uptake for RNAi activity. Taken together, these data support the notion that the development of efficient peptide-based siRNA delivery systems is in part contingent on the formulation of discrete nanoparticles that can effectively condense and release siRNA in cells.

Ligand recombination and a hierarchy of solvent slaved dynamics: the origin of kinetic phases in hemeproteins.

Ligand recombination studies play a central role both for characterizing different hemeproteins and their conformational states but also for probing fundamental biophysical processes. Consequently, there is great importance to providing a foundation from which one can understand the physical processes that give rise to and modulate the large range of kinetic patterns associated with ligand recombination in myoglobins and hemoglobins. In this work, an overview of cryogenic and solution phase recombination phenomena for COMb is first reviewed and then a new paradigm is presented for analyzing the temperature and viscosity dependent features of kinetic traces in terms of multiple phases that reflect which tier(s) of solvent slaved protein dynamics is (are) operative on the photoproduct population during the time course of the measurement. This approach allows for facile inclusion of both ligand diffusion among accessible cavities and conformational relaxation effects. The concepts are illustrated using kinetic traces and MEM populations derived from the CO recombination process for wild type and mutant myoglobins either in sol-gel matrices bathed in glycerol or in trehalose-derived glassy matrices.

A hierarchy of functionally important relaxations within myoglobin based on solvent effects, mutations and kinetic model.

Geminate CO rebinding in myoglobin is studied for two viscous solvents, trehalose and sol-gel (bathed in 100% glycerol) at several temperatures. Mutations in key distal hemepocket residues are used to eliminate or enhance specific relaxation modes. The time-resolved data are analyzed with a modified Agmon-Hopfield model which is capable of providing excellent fits in cases where a single relaxation mode is dominant. Using this approach, we determine the relaxation rate constants of specific functionally important modes, obtaining also their Arrhenius activation energies. We find a hierarchy of distal pocket modes controlling the rebinding kinetics. The "heme access mode" (HAM) is responsible for the major slow-down in rebinding. It is a solvent-coupled cooperative mode which restricts ligand return from the xenon cavities. Bulky side-chains, like those His64 and Trp29 (in the L29W mutant), operate like overdamped pendulums which move over and block the binding site. They may be either unslaved (His64) or moderately slaved (Trp29) to the solvent. Small side-chain relaxations, most notably of leucines, are revealed in some mutants (V68L, V68A). They are conjectured to facilitate inter-cavity ligand motion. When all relaxations are arrested (H64L in trehalose), we observe pure inhomogeneous kinetics with no temperature dependence, suggesting that proximal relaxation is not a factor on the investigated timescale.

Geminate rebinding in trehalose-glass embedded myoglobins reveals residue-specific control of intramolecular trajectories.

It is becoming increasingly apparent that hydrophobic cavities (also referred to as xenon cavities) within proteins have significant functional implications. The potential functional role of these cavities in modulating the internal dynamics of carbon monoxide in myoglobin (Mb) is explored in the present study by using glassy matrices derived from trehalose to limit protein dynamics and to eliminate ligand exchange between the solvent and the protein. By varying the temperature (-15 to 65 degrees C) and humidity for samples of carbonmonoxy myoglobin embedded in trehalose-glass, it is possible to observe a hierarchy of distinct geminate recombination phases that extend from nanosecond to almost seconds that can be directly associated with rebinding from specific hydrophobic cavities. The use of mutant forms of Mb reveals the role of key residues in modulating ligand access between these cavities and the distal hemepocket.

Proteins in motion: resonance Raman spectroscopy as a probe of functional intermediates.

Elucidating proteins function at a level that allows for intelligent design and manipulation is essential in realization of their potential role in biomedical and industrial applications. It has become increasingly apparent though, that probing structures and functionalities under equilibrium conditions is not sufficient. Rather, many aspects of protein behavior and reactivity are rooted in protein dynamics. Thus, there is a growing effort to probe intermediate structures that occur transiently during the course of a proteins function in particular linked to the binding or release of a ligand or substrate. However, studies following the sequence of conformational changes triggered by the binding of substrate/ligand and the concomitant change in functional properties are inherently difficult because often the diffusion times are of the order of conformational relaxation times. This chapter describes methodologies for generating resonance Raman spectra from transient forms of hemoglobin under conditions that allow for the systematic exploration of conformational relaxation and functionality. Special consideration is given to Raman compatible protocols based on sol-gel encapsulation that allow for the preparation, trapping and temporal tuning of nonequilibrium population generated from either the addition or the removal of ligands/substrates.

Theoretical Analysis of the Stereoselectivity in the Ozonolysis of Olefins. Evidence for a Modified Criegee Mechanism.

The Criegee mechanism of the ozonolysis of cis and trans symmetric alkenes of the type RCH=CHR is investigated by semiempirical AM1 calculations. The observed stereoselectivity trends are qualitatively explained in terms of the original Criegee mechanism. However, it is shown that in order to elucidate some subtle stereochemical effects as well as to account for the vain attempts to detect the carbonyloxide intermediate, a modification is required. This is done by adopting a proposal made by Cremer et al. (Chem. Phys. Lett. 1991, 187, 491) for the specific case of ethylene and applying it to all systems. The carbonyl oxide and aldehyde formed upon dissociation of the primary ozonide are not separated but form a strongly bound complex that subsequently transforms to the secondary ozonide.

Synergistic activity of acetohydroxamic acid on prokaryotes under oxidative stress: the role of reactive nitrogen species.

One-electron oxidation of acetohydroxamic acid (aceto-HX) initially gives rise to nitroxyl (HNO), which can be further oxidized to nitric oxide (NO) or react with potential biological targets such as thiols and metallo-proteins. The distinction between the effects of NO and HNO in vivo is masked by the reversible redox exchange between the two congeners and by the Janus-faced behavior of NO and HNO. The present study examines the ability of aceto-HX to serve as an HNO donor or an NO donor when added to Escherichia coli and Bacillus subtilis subjected to oxidative stress by comparing its effects to those of NO and commonly used NO and HNO donors. The results demonstrate that: (i) the effects of NO and HNO on the viability of prokaryotes exposed to H2O2 depend on the type of the bacterial cell; (ii) NO synergistically enhances H2O2-induced killing of E. coli, but protects B. subtilis depending on the extent of cell killing by H2O2; (iii) the HNO donor Angeli׳s salt alone has no effect on the viability of the cells; (iv) Angeli׳s salt synergistically enhances H2O2-induced killing of B. subtilis, but not of E. coli; (v) aceto-HX alone (1-4 mM) has no effect on the viability of the cells; (vi) aceto-HX enhances the killing of both cells induced by H2O2 and metmyoglobin, which may be attributed in the case of B. subtilis to the formation of HNO and to further oxidation of HNO to NO in the case of E. coli; (vii) the synergistic activity of aceto-HX on the killing of both cells induced by H2O2 alone does not involve reactive nitrogen species. The effect of aceto-HX on prokaryotes under oxidative stress is opposite to that of other hydroxamic acids on mammalian cells.

Pro-oxidative synergic bactericidal effect of NO: kinetics and inhibition by nitroxides.

NO plays diverse roles in physiological and pathological processes, occasionally resulting in opposing effects, particularly in cells subjected to oxidative stress. NO mostly protects eukaryotes against oxidative injury, but was demonstrated to kill prokaryotes synergistically with H2O2. This could be a promising therapeutic avenue. However, recent conflicting findings were reported describing dramatic protective activity of NO. The previous studies of NO effects on prokaryotes applied a transient oxidative stress while arbitrarily checking the residual bacterial viability after 30 or 60min and ignoring the process kinetics. If NO-induced synergy and the oxidative stress are time-dependent, the elucidation of the cell killing kinetics is essential, particularly for survival curves exhibiting a "shoulder" sometimes reflecting sublethal damage as in the linear-quadratic survival models. We studied the kinetics of NO synergic effects on H2O2-induced killing of microbial pathogens. A synergic pro-oxidative activity toward gram-negative and gram-positive cells is demonstrated even at sub-µM/min flux of NO. For certain strains, the synergic effect progressively increased with the duration of cell exposure, and the linear-quadratic survival model best fit the observed survival data. In contrast to the failure of SOD to affect the bactericidal process, nitroxide SOD mimics abrogated the pro-oxidative synergy of NO/H2O2. These cell-permeative antioxidants, which hardly react with diamagnetic species and react neither with NO nor with H2O2, can detoxify redox-active transition metals and catalytically remove intracellular superoxide and nitrogen-derived reactive species such as (•)NO2 or peroxynitrite. The possible mechanism underlying the bactericidal NO synergy under oxidative stress and the potential therapeutic gain are discussed.

The use of cyclic nitroxide radicals as HNO scavengers.

Reduction of cyclic stable nitroxides (RNO) by HNO to the respective hydroxylamines (RNO-H) has been demonstrated using EPR spectrometry. HNO shows low reactivity toward piperidine, pyrrolidine and nitronyl nitroxides with rate constants below 1.4 × 10(5)M(-1)s(-1) at pH 7.0, despite the high driving force for these reactions. The rate constants can be predicted assuming that the reactions take place via a concerted proton-electron transfer pathway and significantly low self-exchange rate constants for HNO/NO and RNO-H/RNO. NO does not react with piperidine and pyrrolidine nitroxides, but does add to HNO forming the highly oxidizing and moderately reducing hyponitrite radicals. In this work, the radicals are produced by pulse radiolysis and the rate constants of their reactions with 2,2,6,6,-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPOL) and 3-carbamoyl-PROXYL have been determined at pH 6.8 to be (2.4 ± 0.2)× 10(6), (9.8 ± 0.2)× 10(5), (5.9 ± 0.5)× 10(5)M(-1)s(-1), respectively. This low reactivity implies that NO competes efficiently with these nitroxides for the hyponitrite radical. The ability of TEMPOL and 2-(4-carboxyphenyl)-4,4,5,5,-tetramethyl-imidazoline-1-oxyl-3-oxide (C-PTIO) to oxidize HNO and their different reactivity toward NO are used to quantify HNO formed via acetohydroxamic acid oxidation. The extent of TEMPOL or C-PTIO reduction was similar to the yield of HNO formed upon oxidation by ()OH under anoxia, but not by the metmyoglobin and H(2)O(2) reaction system where both nitroxides catalytically facilitate H(2)O(2) depletion and nitrite accumulation. In this system the conversion of C-PTIO into 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl (C-PTI) is a minor reaction, which does not provide any mechanistic insight.

Reactive oxygen species mediate hepatotoxicity induced by the Hsp90 inhibitor geldanamycin and its analogs.

Geldanamycin (GM), a benzoquinone ansamycin antibiotic, is a natural product inhibitor of Hsp90 with potent and broad anti-cancer properties. Because of its adverse effects on liver, its less toxic derivatives 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) are currently being evaluated for the treatment of cancer. Previously, it has been demonstrated that the redox cycling of GM by NADPH-cytochrome P450 reductase leads to the formation of the GM semiquinone and superoxide radicals, the latter being identified using spin-trapping. We hypothesized that the different hepatotoxicity induced by GM, 17-AAG and 17-DMAG reflects the redox active properties of the quinone moiety and possibly the extent of superoxide formation, which may stimulate cellular oxidative injury. Our data demonstrate that superoxide can be efficiently trapped during the reduction of GM, 17-AAG and 17-DMAG by NADPH-cytochrome P450 reductase, and that superoxide formation rate followed the order 17-DMAG > 17-AAG > GM. In the absence of superoxide scavengers, the rate of NADPH oxidation followed the order 17-DMAG > GM > 17-AAG. The half-wave one-electron reduction potentials (E(1/2)) of GM, 17-AAG and 17-DMAG in DMSO have been determined to be -0.37, -0.13 and -0.015V (vs. Ag/AgCl), respectively. If the same order of E(1/2) follows in neutral aqueous media, thermodynamic considerations imply that 17-DMAG is more readily reduced by the P450 reductase as well as by superoxide. The order of the drug cytotoxicity toward rat primary hepatocytes, as determined by their effect on cell viability and on intracellular oxidant level, was opposite to the order of E(1/2) of the respective quinone/semiquinone couples. These results suggest that hepatotoxicity exhibited by the Hsp90 inhibitors belonging to benzoquinone ansamycins could be attributed to superoxide. The apparent discrepancy between the order of toxicity and the orders of superoxide formation rate, which is correlated with E(1/2), is discussed.