1. The major EPR signals from native and cytochrome c-reduced beef heart cytochrome c oxidase (EC 1.9.3.1) are characterized with respect to resonance parameters, number of components and total integrated intensity. A mistake in all earlier integrations and simulations of very anisotropic EPR signals is pointed out. 2. The so-called Cu2+ signal is found to contain at least three components, one "inactive" form and two nearly similar active forms. One of the latter forms, corresponding to about 20% of the total EPR detectable Cu, has not been observed earlier and can only be resolved in 35 GHz spectra. It is not reduced by cytochrome c and is thought to reflect some kind of inhomogeneity in the enzyme preparation. The 35 GHz spectrum of the cytochrome c reducible component shows a rhombic splitting and can be well simulated with g-values 2.18, 2.03 and 1.99. The origin of such a unique type of Cu2+ spectrum is discussed. 3. The low-spin heme signal in the oxidized enzyme (g = 3.03, 2.21, 1.45) is found to correspond closely to one heme and shows no signs of interaction with other paramagnetic centres. 4. The high-spin heme signals appearing in partly reduced oxidase are found to consist of at least three species, one axial and two rhombic types. An integration procedure is described that allows the determination of the total integral intensity of high-spin heme EPR signals only by considering the g = 6 part of the signals. In a titration with ascorbate and cytochrome c the maximum intensity of the g = 6 species corresponds to 23% of the enzyme concentration.

Lipid rafts in the plasma membrane, domains rich in cholesterol and sphingolipids, have been implicated in a number of important membrane functions. Detergent insolubility has been used to define membrane "rafts" biochemically. However, such an approach does not directly contribute to the understanding of the size and the lifetime of rafts, dynamics of the raft-constituent molecules, and the function of rafts in the membrane in situ. To address these issues, we have developed pulse EPR spin labeling and single molecule tracking optical techniques for studies of rafts in both artificial and cell membranes. In this review, we summarize our results and perspectives obtained by using these methods. We emphasize the importance of clearly distinguishing small/unstable rafts (lifetime shorter than a millisecond) in unstimulated cells and stabilized rafts induced by liganded and oligomerized (GPI-anchored) receptor molecules (core receptor rafts, lifetime over a few minutes). We propose that these stabilized rafts further induce temporal, greater rafts (signaling rafts, lifetime on the order of a second) for signaling by coalescing other small/unstable rafts, including those in the inner leaflet of the membrane, each containing perhaps one molecule of the downstream effector molecules. At variance with the general view, we emphasize the importance of cholesterol segregation from the liquid-crystalline unsaturated bulk-phase membrane for formation of the rafts, rather than the affinity of cholesterol and saturated alkyl chains. In the binary mixture of cholesterol and an unsaturated phospholipid, cholesterol is segregated out from the bulk unsaturated liquid-crystalline phase, forming cholesterol-enriched domains or clustered cholesterol domains, probably due to the lateral nonconformability between the rigid planar transfused ring structure of cholesterol and the rigid bend of the unsaturated alkyl chain at C9-C10. However, such cholesterol-rich domains are small, perhaps consisting of only several cholesterol molecules, and are short-lived, on the order of 1-100 ns. We speculate that these cholesterol-enriched domains may be stabilized by the presence of saturated alkyl chains of sphingomyelin or glycosphingolipids, and also by clustered raft proteins. In the influenza viral membrane, one of the simplest forms of a biological membrane, the lifetime of a protein and cholesterol-rich domain was evaluated to be on the order of 100 micro, again showing the short lifetime of rafts in an unstimulated state. Finally, we propose a thermal Lego model for rafts as the basic building blocks for signaling pathways in the plasma membrane.

Heme oxygenase (HO) catalyzes the O(2)- and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. In the present study, we have generated a detailed reaction cycle for the first monooxygenation step of HO catalysis, conversion of the heme to alpha-meso-hydroxyheme. We employed EPR (using both (16)O(2) and (17)O(2)) and (1)H, (14)N ENDOR spectroscopies to characterize the intermediates generated by 77 K radiolytic cryoreduction and subsequent annealing of wild-type oxy-HO and D140A, F mutants. One-electron cryoreduction of oxy-HO yields a hydroperoxoferri-HO with g-tensor, g = [2.37, 2.187, 1.924]. Annealing of this species to 200 K is accompanied by spectroscopic changes that include the appearance of a new (1)H ENDOR signal, reflecting rearrangements in the active site. Kinetic measurements at 214 K reveal that the annealed hydroperoxoferri-HO species, denoted R, generates the ferri-alpha-meso-hydroxyheme product in a first-order reaction. Disruption of the H-bonding network within the distal pocket of HO by the alanine and phenylalanine mutations of residue D140 prevents product formation. The hydroperoxoferri-HO (D140A) instead undergoes heterolytic cleavage of the O-O bond, ultimately yielding an EPR-silent compound II-like species that does not form product. These results, which agree with earlier suggestions, establish that hydroperoxoferri-HO is indeed the reactive species, directly forming the alpha-meso-hydroxyheme product by attack of the distal OH of the hydroperoxo moiety at the heme alpha-carbon.

The gene for a minor, extracellular protease has been identified in Bacillus subtilis. The gene (epr) encoded a primary product of 645 amino acids that was partially homologous to both subtilisin (Apr) and the major internal serine protease (ISP-1) of B. subtilis. Deletion analysis indicated that the C-terminal 240 amino acids of Epr were not necessary for activity. This C-terminal region exhibited several unusual features, including a high abundance of lysine residues and the presence of a partially homologous sequence of 44 amino acids that was directly repeated five times. The epr gene mapped near sacA and was not required for growth or sporulation.

The X-ray crystallographic structure of the native R2F subunit of the ribonucleotide reductase (RNR) of Corynebacterium ammoniagenes ATCC 6872 is reported, with a resolution of 1.36 A. The metal site contains an oxo/hydroxo-bridged manganese dimer, located near a tyrosine residue (Y115). The coordination of the manganese dimer and its distance to a nearby tyrosine residue resemble the di-iron metalloradical cofactor of class I RNR from Escherichia coli . Multifrequency EPR measurements of the highly active C. ammoniagenes R2F subunit show that the metal site contains a ferromagnetically exchange-coupled Mn(III)Mn(III) dimer weakly coupled to a tyrosyl radical. A mechanism for the metalloradical cofactor (Mn(III)Mn(III)Y(*)) generation is proposed. H(2)O(2) (HO(2)(-)) instead of O(2) is hypothesized as physiological oxidant for the Mn dimer which in turn oxidizes the tyrosine Y115. Changes in the ligand sphere of both manganese ions during metalloradical generation direct the complex formation of this cofactor, disfavoring alternate reaction pathways such as H(2)O(2) dismutation, as observed for manganese catalase, a structural analogue of the R2F metal site. The presented results demonstrate the importance of manganese for radical formation in this RNR and confirm the assignment of this enzyme to class Ib.

Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa alpha(2) homodimer containing, on average, 0.7 iron atoms/subunit. Mössbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K(3)(Fe(CN)(6)), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A(280)/A(656) ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.

Intersubunit intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation, which serves as the input state for reduction of FMN by electrons from NADPH and flavin adenine dinucleotide (FAD) in the reductase domain. To favor the formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct of rat neuronal NOS (nNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of IET between the FMN and heme domains in the nNOS oxyFMN construct in the presence and absence of added calmodulin (CaM) were directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single-domain heme oxygenase constructs. The IET rate constant in the presence of CaM (262 s(-)(1)) was increased approximately 10-fold compared to that in the absence of CaM (22 s(-)(1)). The effect of CaM on interdomain interactions was further evidenced by electron paramagnetic resonance (EPR) spectra. This work provides the first direct evidence of the CaM control of electron transfer (ET) between FMN and heme domains through facilitation of the FMN/heme interactions in the output state. Therefore, CaM controls IET between heme and FMN domains by a conformational gated mechanism. This is essential in coupling ET in the reductase domain in NOS with NO synthesis in the oxygenase domain.

Nitroglycerin (glyceryl trinitrate, GTN) relaxes blood vessels primarily via activation of the soluble guanylyl cyclase (sGC)/cGMP/cGMP-dependent protein kinase (cGK-I) pathway. Although the precise mechanism of sGC activation by GTN in the vascular wall is unknown, the mediatory role of nitric oxide (NO) has been postulated. We tested the GTN/NO hypothesis in different types of isolated rat and rabbit blood vessels using two novel approaches: (1) EPR spin trapping using colloid Fe(DETC)2 and (2) analysis of cGK-I-dependent phosphorylation of the vasodilator-stimulated phosphoprotein at Ser239 (P-VASP). For comparison, another organic nitrate, isosorbide dinitrate (ISDN), and endothelium-dependent vasodilator, calcium ionophore A23187, were tested. We found a marked discrepancy between GTN's strong vasoactivity (vasodilation and augmentation of P-VASP) and its poor NO donor properties. In aortas precontracted with phenylephrine, GTN, ISDN, and A23187 induced nearly full relaxations (>80%) and doubling of vascular P-VASP content at concentrations of 100 nmol/L, 100 micromol/L, and 1 micromol/L, respectively. GTN applied in vasorelaxant concentrations (10 to 1000 nmol/L) did not significantly increase the basal vascular NO production, in contrast to ISDN and A23187. The absence of GTN-derived NO was confirmed in rabbit vena cava and renal artery. A significant increase in vascular NO formation was observed only at suprapharmacological GTN concentrations (>10 micromol/L). The concentration dependency of NO formation from GTN was comparable to that of ISDN, although the latter exhibits 100-folds lower vasorelaxant potency. We conclude that GTN activates the sGC/cGMP/cGK-I pathway and induces vasorelaxation without intermediacy of the free radical NO. The full text of this article is available online at http://www.circresaha.org.

CobA from Salmonella enterica is a member of an enzymatic system responsible for the de novo biosynthesis of adenosylcobalamin (AdoCbl), catalyzing the formation of the essential Co-C bond by transferring the adenosyl group from a molecule of ATP to a transient Co(1+)corrinoid species generated in the enzyme active site. A particularly fascinating aspect of this reaction is that the flavodoxin in vivo reducing agent that serves as the electron donor to CobA possesses a reduction potential that is considerably more positive than that of the Co(2+/1+) couple of the corrinoid substrate. To explore how CobA may overcome this challenge, we have employed electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to probe the interaction between Co(3+)- and Co(2+)corrinoids and the enzyme active site. Our data reveal that while Co(3+)corrinoids interact only weakly with CobA, Co(2+)corrinoids undergo partial conversion to a new paramagnetic species that can be obtained in nearly quantitative yield when CobA is preincubated with the co-substrate ATP. This "activated" species is characterized by a distinct set of ligand field transitions in the near-IR spectral region and EPR parameters that are unprecedented for Co(2+)corrinoids. Analysis of these data on the basis of qualitative spectral correlations and density functional theory computations reveals that this unique Co(2+)corrinoid species possesses an essentially square-planar Co(2+) center that lacks any significant axial bonding interactions. Possible implications of these findings for the mechanism of Co(2+) ->> Co(1+) reduction employed by CobA and Co-C bond-forming enzymes in general are explored.

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains approximately 2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 A. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.

Doxorubicin (DOX) is an effective chemotherapeutic against a wide range of solid tumors. However, its clinical use is limited by severe side effects such as cardiotoxicity as well as inherent and acquired drug resistance of tumors. DOX encapsulation within self-assembled polymeric micelles has the potential to decrease the systemic distribution of free drug and enhance the drug accumulation in the tumor via the enhanced permeability and retention (EPR). In this study, DOX was encapsulated in micelles composed of poly (ethylene oxide)-poly [(R)-3-hydroxybutyrate]-poly (ethylene oxide) (PEO-PHB-PEO) triblock copolymers. Micelle size, DOX loading and DOX release were characterized. To evaluate DOX activity, micelles were tested in both monolayer cell cultures and three-dimensional (3-D) multicellular spheroids (MCS) that mimic solid tumors. Antitumor activity in vivo was further studied with tumor-bearing mice. The micelles improved the efficiency of Dox penetration in 3-D MCS compared with free DOX. Efficient cell killing by Dox-micelles in both monolayer cells and 3-D MCS was also demonstrated. Finally, DOX-loaded micelles mediate efficient tumor delivery from tail vein injections to tumor-bearing mice with much less toxicity compared with free DOX.

ESCRT-I is required for the sorting of integral membrane proteins to the lysosome, or vacuole in yeast, for cytokinesis in animal cells, and for the budding of HIV-1 from human macrophages and T lymphocytes. ESCRT-I is a heterotetramer of Vps23, Vps28, Vps37, and Mvb12. The crystal structures of the core complex and the ubiquitin E2 variant and Vps28 C-terminal domains have been determined, but internal flexibility has prevented crystallization of intact ESCRT-I. Here we have characterized the structure of ESCRT-I in solution by simultaneous structural refinement against small-angle X-ray scattering and double electron-electron resonance spectroscopy of spin-labeled complexes. An ensemble of at least six structures, comprising an equally populated mixture of closed and open conformations, was necessary to fit all of the data. This structural ensemble was cross-validated against single-molecule FRET spectroscopy, which suggested the presence of a continuum of open states. ESCRT-I in solution thus appears to consist of an approximately 50% population of one or a few related closed conformations, with the other 50% populating a continuum of open conformations. These conformations provide reference points for the structural pathway by which ESCRT-I induces membrane buds.

P-glycoprotein (P-gp) is involved in the ATP-dependant cellular efflux of a large number of drugs including ivermectin, a macrocyclic lactone (ML) endectocide, widely used in livestock and human antiparasitic therapy. The interactions of P-gp with ivermectin and other MLs were studied. In a first approach, the ability of ivermectin (IVM), eprinomectin (EPR), abamectin (ABA), doramectin (DOR), selamectin (SEL), or moxidectin (MOX) to inhibit the rhodamine123 efflux was measured in recombinant cells overexpressing P-gp. Then, the influence of these compounds on the P-gp ATPase activity was tested on membrane vesicles prepared from fibroblasts overexpressing P-gp. All the MLs tested increased the intracellular rhodamine123. However, the potency of MOX to inhibit P-gp function was 10 times lower than the other MLs. They all inhibited the basal and decreased the verapamil-stimulated P-gp ATPase activity. But SEL and MOX were less potent than the other MLs when competing with verapamil. According to the structural specificity of SEL and MOX, we conclude that the integrity of the sugar moiety is determinant to achieve the optimal interaction of macrocyclic lactones with P-gp. The structure-affinity relationship for interaction with P-gp is important information for improving ML bioavailability and reversal of multidrug resistance (MDR).

The mechanism of the enhanced vascular permeability and retention (EPR) effect seen in solid tumors was investigated with sarcoma 180 (S-180) in mice by using the bradykinin receptor antagonist D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin] (HOE 140), the nitric oxide (NO) scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), and the cyclooxygenase (prostaglandin synthase) inhibitor indomethacin. In the S-180 solid tumor model, administration of HOE 140 (0.65 or 1.3 microg/kg/8 h, s.c.), PTIO (167 mg/kg/2 h, four times/8 h, i.p.), or indomethacin (5 or 10 mg/kg/day, three times, i.p.) significantly suppressed the EPR effect in the tumor, and the combined administration of these agents achieved a stronger inhibition of the EPR effect than did each compound alone. Indomethacin (10 mg/kg/day, three times) plus PTIO (167 mg/kg/2 h, four times) given i.p. had the greatest inhibition (70%) on the EPR effect. When HOE 140 was administered s.c. at a dose of 13 microg/kg/12 h for 2 weeks after tumor inoculation, growth of the solid tumor was also suppressed by 32%, by tumor weight. In the ascitic form of S-180, i.p. administration of HOE 140 at 13 microg/kg/12 h initiated immediately after tumor inoculation significantly suppressed formation of S-180 tumor ascites; the life span of ascitic S-180 tumor-bearing mice was prolonged at the same dose of HOE 140. The expression of inducible NO synthase mRNA and of cyclooxygenase 2 mRNA in S-180 tumor tissue was highly elevated, as evidenced by Northern blotting and reverse transcription-PCR and by Southern blot analyses. These results indicate that bradykinin, NO, and prostaglandins play an important role in enhanced vascular permeability in tumor tissue and sustain tumor growth. More importantly, bradykinin antagonists such as HOE 140 may be beneficial for the inhibition of tumor growth.

Membrane fluidity of non-cultured lung cancer tissue was studied by electron paramagnetic resonance (EPR). EPR spectra of a lipophilic spin probe in a tissue of resected tumor samples from 51 patients were compared with computer simulated spectra, which were superimpositions of spectra characterizing membrane domains with different fluidity. The membranes of tumor tissues were more fluid, than those of normal lungs; the most fluid domains were enlarged and their order parameter decreased in comparison to normal tissue. An empirical fluidity parameter (H13) was defined as the criterion to correlate EPR and clinical data. The histology of tumor, the quantitative presence of different tumor and non-tumor cells and the pathohisthological stage of the disease had no significant influence on fluidity.

The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme's three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61.

Divergent literature data are found concerning the effect of lactate on free radical production during exercise. To clarify this point, we tested the pro- or antioxidant effect of lactate ion in vitro at different concentrations using three methods: 1) electron paramagnetic resonance (EPR) was used to study the scavenging ability of lactate toward the superoxide aion (O(2)(-).) and hydroxyl radical (.OH); 2) linoleic acid micelles were employed to investigate the lipid radical scavenging capacity of lactate; and 3) primary rat hepatocyte culture was used to study the inhibition of membrane lipid peroxidation by lactate. EPR experiments exhibited scavenging activities of lactate toward both O(2)(-). and.OH; lactate was also able to inhibit lipid peroxidation of hepatocyte culture. Both effects of lactate were concentration dependent. However, no inhibition of lipid peroxidation by lactate was observed in the micelle model. These results suggested that lactate ion may prevent lipid peroxidation by scavenging free radicals such as O(2)(-). and.OH but not lipid radicals. Thus lactate ion might be considered as a potential antioxidant agent.

The supramolecular organization of photosystem II (PSII) was characterized in distinct domains of the thylakoid membrane, the grana core, the grana margins, the stroma lamellae, and the so-called Y100 fraction. PSII supercomplexes, PSII core dimers, PSII core monomers, PSII core monomers lacking the CP43 subunit, and PSII reaction centers were resolved and quantified by blue native PAGE, SDS-PAGE for the second dimension, and immunoanalysis of the D1 protein. Dimeric PSII (PSII supercomplexes and PSII core dimers) dominate in the core part of the thylakoid granum, whereas the monomeric PSII prevails in the stroma lamellae. Considerable amounts of PSII monomers lacking the CP43 protein and PSII reaction centers (D1-D2-cytochrome b559 complex) were found in the stroma lamellae. Our quantitative picture of the supramolecular composition of PSII, which is totally different between different domains of the thylakoid membrane, is discussed with respect to the function of PSII in each fraction. Steady state electron transfer, flash-induced fluorescence decay, and EPR analysis revealed that nearly all of the dimeric forms represent oxygen-evolving PSII centers. PSII core monomers were heterogeneous, and a large fraction did not evolve oxygen. PSII monomers without the CP43 protein and PSII reaction centers showed no oxygen-evolving activity.

One major challenge in nanomedicine is the selective delivery of nanoparticles to diseased tissues. Nanoparticle delivery systems require targeting for specific delivery to pathogenic sites when enhanced permeability and retention (EPR) is not suitable or inefficient. Nanoparticle functionalization is a widely-used technique for targeting ligand conjugation; these ligands possess inherent abilities to direct nanoparticle selective binding. This review illustrates methods of ligand-nanoparticle functionalization, provides a cross-section of various ligand classes, including small molecules, peptides, antibodies, engineered proteins, or nucleic acid aptamers, and discusses some unconventional approaches currently under investigation.

Complex III Qo site semiquinone has been assigned pivotal roles in productive energy-conversion and destructive superoxide generation. After a 30-year search, a genetic heme bH knockout arrests this transient semiquinone EPR radical, revealing the natural engineering balance pitting energy-conserving, short-circuit minimizing, split electron transfer and catalytic speed against damaging oxygen reduction.

BACKGROUND

There is a growing body of evidence that a pathological diagnosis is necessary for small (<3 cm in diameter), asymptomatic, hypoechoic, subepithelial tumors (SETs) originating from the muscularis propria on EUS. However, the diagnostic efficacy of current tissue sampling techniques appears to be limited.

OBJECTIVE

To evaluate the diagnostic yield and safety of endoscopic partial resection using the unroofing technique (EPR-UT) in a subset of patients.

METHODS

A prospective case series.

METHODS

A single tertiary-care referral center.

METHODS

Between August 2007 and March 2009, 16 patients with hypoechoic SETs of <3 cm in diameter, originating from the muscularis propria on EUS (14 gastric and 2 esophageal lesions), underwent EPR-UT.

METHODS

The overlying mucosa was removed by using the unroofing technique using a conventional snare with electrical current to expose the tumor sufficiently. Next, the exposed tumor was partially resected by snaring.

METHODS

The diagnostic yield and safety of this method.

RESULTS

EPR-UT provided specimens that were sufficient for a diagnosis and the assessment of risk for malignancy in 15 out of 16 cases (diagnostic yield 93.7% [95% CI, 80.4%-100.0%]). The pathological diagnoses were leiomyoma (7 of 15, 46.6%), GI stromal tumor (6 of 15, 40.0%), aberrant pancreas (1 of 15, 6.6%), and well-differentiated neuroendocrine carcinoma (1 of 15, 6.6%). Six cases with GI stromal tumor were classified as very low risk for malignant potential (mitotic index <5/50 high-power fields). Procedural blood oozing was relatively common (9 of 16, 56.0% [95% CI, 33.0%-77.0%]); however, this minor complication was easily controlled by argon plasma coagulation. There were no procedure-related major complications (0 of 16, 0% [95% CI, 0.0%-23.0%]).

CONCLUSIONS

Single-center, noncomparative study with small sample size.

CONCLUSIONS

EPR-UT appears to be simple, safe, and effective for determining the definite pathological diagnosis and assessing malignant potential of small, hypoechoic SETs originating from the muscularis propria on EUS.

Molecular oxygen in electronic singlet state is a very powerful oxidant. Its damaging action in a variety of biological processes has been well recognized. Here we report the singlet oxygen quenching action of proline. Singlet oxygen (1O2) was produced photochemically by irradiating a solution of sensitiser and detected by following the formation of stable nitroxide radical yielded in the reaction of 1O2 with the sterically hindered amine (2,2,6,6-tetramethylpiperidine, TEMP). Illumination of a sensitiser, toluidine blue led to a time dependent increase in singlet oxygen production as detected by the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) by EPR spectrometry. Interestingly, the production of TEMPO was completely abolished by the presence of proline at concentration as low as 20mM. These results show that proline is a very effective singlet oxygen quencher. Other singlet oxygen generating photosensitizer like hematopophyrin and fluorescein also produced identical results with proline. Since proline is one of the important solutes which accumulate in many organisms when they are exposed to environmental stresses, it is likely that proline accumulation is related to the protection of these organisms against singlet oxygen production during stress conditions. A possible mechanism of singlet oxygen quenching by proline is discussed.

A free radical-coupled copper complex has been identified as the catalytic structure in the active site of glyoxal oxidase from Phanerochaete chrysosporium based on a combination of spectroscopic and biochemical studies. The native (inactive) enzyme is activated by oxidants leading to the elimination of the cupric EPR signal consistent with formation of an antiferromagnetically coupled radical-copper complex. Oxidation also leads to the appearance of a substoichiometric free radical EPR signal with an average g value (gav = 2.0055) characteristic of phenoxyl tau-radicals arising from a minority apoenzyme fraction. Optical absorption, CD, and spectroelectrochemical measurements on the active enzyme reveal complex spectra extending into the near IR and define the redox potential for radical formation (E 1/2 = 0.64 V versus NHE, pH 7.0). Resonance Raman spectra have identified the signature of a modified (cysteinyl-tyrosine) phenoxyl in the vibrational spectra of the active complex. This radical-copper motif has previously been found only in galactose oxidase, with which glyoxal oxidase shares many properties despite lacking obvious sequence identity, and catalyzing a distinct reaction. The enzymes thus represent members of a growing class of free radical metalloenzymes based on the radical-copper catalytic motif and appear to represent functional variants that have evolved to distinct catalytic roles.

While sterically stabilized liposomes (SSL) can passively accumulate into tumor tissue due to the effect of enhanced permeability and retention (EPR), the intracellular uptake of the entrapped anticancer drugs by the tumor cells should be a determinant step for their antitumor activities. Therefore, strategies that can enhance the intracellular uptake of SSL into tumor cells could lead to an improved therapeutic efficacy for the drugs. To check this possibility, RGD-mimetic-modified SSL (RGDm-SSL) were constructed aimed to achieve tumor accumulation as well as enhanced intracellular delivery, and were loaded with doxorubicin (DOX), an anticancer drug. Flow cytometry and confocal microscopy reveal that RGDm-SSL facilitated the DOX uptake into the melanoma cells via integrin-mediated endocytosis. DOX-loaded RGDm-SSL (RGDm-SSL-DOX) displayed higher cytotoxicity on melanoma cells than DOX-loaded SSL (SSL-DOX). Tissue distribution and therapeutic experiments were examined in C57BL/6 mice carrying melanoma B16 tumors. RGDm-SSL-DOX displayed similar DOX accumulation in tumor tissue to that of SSL-DOX but showed significantly lower DOX level in blood and remarkably higher DOX level in spleen than SSL-DOX. Administration of RGDm-SSL-DOX at a dose of 5 mg DOX/kg resulted in effective retardation of tumor growth and prolonged survival times compared with SSL-DOX. These results suggest that RGDm-modified SSL may be a promising intracellular targeting carrier for efficient delivery of chemotherapeutic agents into tumor cells.