During apoptosis, the pro-apoptotic Bcl-2 family proteins BAK and BAX form large oligomeric pores in the mitochondrial outer membrane. Apoptotic factors, including cytochrome c, are released through these pores from the mitochondrial intermembrane space into the cytoplasm where they initiate the cascade of events leading to cell death. To better understand this pivotal step toward apoptosis, a method was developed to induce membrane permeabilization by BAK in the membrane without using the full-length protein. Using a soluble form of BAK with a hexahistidine tag at the C terminus and a liposomal system containing the Ni(2+)-nitrilotriacetic acid lipid analog that can bind hexahistidine-tagged proteins, BAK oligomers were formed in the presence of the activator protein p7/p15Bid. In this system, we determined the conformational changes in BAK upon membrane insertion by applying the site-directed spin labeling method of EPR to 13 different amino acid locations. Upon membrane insertion, the BH3 domains were reorganized, and the alpha5-alpha6 helical hairpin structure was partially exposed to the membrane environment. The monomer-monomer interface in the oligomeric structure was also mapped by measuring the distance-dependent spin-spin interactions for each residue location. Spin labels attached in the BH3 domain were juxtaposed within 5-10 A distance in the oligomeric form in the membrane. These results are consistent with the current hypothesis that BAK or BAX forms homodimers, and these homodimers assemble into a higher order oligomeric pore. Detailed analyses of the data provide new insights into the structure of the BAX or BAK homodimer.

Therapy and diagnosis are two major categories in the clinical treatment of disease. Recently, the word "theranosis" has been created, combining the words to describe the implementation of these two distinct pursuits simultaneously. For successful theranosis, the efficient delivery of imaging agents and drugs is critical to provide sufficient imaging signal or drug concentration in the targeted disease site. To achieve this purpose, biomedical researchers have developed various nanoparticles composed of organic or inorganic materials. However, the targeted delivery of these nanoparticles in animal models and patients remains a difficult hurdle for many researchers, even if they show useful properties in cell culture condition. In this Account, we review our strategies for developing theranostic nanoparticles to accomplish in vivo targeted delivery of imaging agents and drugs. By applying these rational strategies, we achieved fine multimodal imaging and successful therapy. Our first strategy involves physicochemical optimization of nanoparticles for long circulation and an enhanced permeation and retention (EPR) effect. We accomplished this result by testing various materials in mouse models and optimizing the physical properties of the materials with imaging techniques. Through these experiments, we developed a glycol chitosan nanoparticle (CNP), which is suitable for angiogenic diseases, such as cancers, even without an additional targeting moiety. The in vivo mechanism of this particle was examined through rationally designed experiments. In addition, we evaluated and compared the biodistribution and target-site accumulation of bare and drug-loaded nanoparticles. We then focus on the targeting moieties that bind to cell surface receptors. Small peptides were selected as targeting moieties because of their stability, low cost, size, and activity per unit mass. Through phage display screening, the interleukin-4 receptor binding peptide was discovered, and we combined it with our nanoparticles. This product accumulated efficiently in atherosclerotic regions or tumors during both imaging and therapy. We also developed hyaluronic acid nanoparticles that can bind efficiently to the CD44 antigen receptors abundant in many tumor cells. Their delivery mechanism is based on both physicochemical optimization for the EPR effect and receptor-mediated endocytosis by their hyaluronic acid backbone. Finally, we introduce the stimuli-responsive system related to the chemical and biological changes in the target disease site. Considering the relatively low pH in tumors and ischemic sites, we applied pH-sensitive micelle to optical imaging, magnetic resonance imaging, anticancer drug delivery, and photodynamic therapy. In addition, we successfully evaluated the in vivo imaging of enzyme activity at the target site with an enzyme-specific peptide sequence and CNPs. On the basis of these strategies, we were able to develop self-assembled nanoparticles for in vivo targeted delivery, and successful results were obtained with them in animal models for both imaging and therapy. We anticipate that these rational strategies, as well as our nanoparticles, will be applied in both the diagnosis and therapy of many human diseases. These theranostic nanoparticles are expected to greatly contribute to optimized therapy for individual patients as personalized medicine, in the near future.

Plants synthesize the osmoprotectant glycine betaine via the route choline ->> betaine aldehyde ->> glycine betaine. In spinach, the first step is catalyzed by choline monooxygenase (CMO), a ferredoxin-dependent stromal enzyme that has been hypothesized to be an oligomer of identical subunits and to be an Fe-S protein. Analysis by HPLC and matrix-assisted laser desorption ionization MS confirmed that native CMO contains only one type of subunit (Mr 42,864). Determination of acid-labile sulfur and nonheme iron demonstrated that there is one [2Fe-2S] cluster per subunit, and EPR spectral data indicated that this cluster is of the Rieske type--i.e., coordinated by two Cys and two His ligands. A full-length CMO cDNA (1,622 bp) was cloned from spinach using a probe generated by PCR amplification for which the primers were based on internal peptide sequences. The ORF encoded a 440-amino acid polypeptide that included a 60-residue transit peptide. The deduced amino acid sequence included two Cys-His pairs spaced 16 residues apart, a motif characteristic of Rieske-type Fe-S proteins. Larger regions that included this motif also showed some sequence similarity (approximately 40%) to Rieske-type proteins, particularly bacterial oxygenases. Otherwise there was very little similarity between CMO and proteins from plants or other organisms. RNA and immunoblot analyses showed that the expression of CMO in leaves increased several-fold during salinization. We conclude that CMO is a stress-inducible representative of a new class of plant oxygenases.

Photosystem II (PSII), a multisubunit pigment-protein supercomplex found in cyanobacteria, algae, and plants, catalyzes a unique reaction in nature: the light-driven oxidation of water. Remarkable recent advances in the structural analysis of PSII now give a detailed picture of the static supercomplex on the molecular level. These data provide a solid foundation for future functional studies, in particular the mechanism of water oxidation and oxygen release. The catalytic core of the PSII is a tetramanganese-calcium cluster (Mn₄O₅Ca), commonly referred to as the oxygen-evolving complex (OEC). The function of the OEC rests on its ability to cycle through five metastable states (Si, i = 0-4), transiently storing four oxidizing equivalents, and in so doing, facilitates the four electron water splitting reaction. While the latest crystallographic model of PSII gives an atomic picture of the OEC, the exact connectivity within the inorganic core and the S-state(s) that the X-ray model represents remain uncertain. In this Account, we describe our joint experimental and theoretical efforts to eliminate these ambiguities by combining the X-ray data with spectroscopic constraints and introducing computational modeling. We are developing quantum chemical methods to predict electron paramagnetic resonance (EPR) parameters for transition metal clusters, especially focusing on spin-projection approaches combined with density functional theory (DFT) calculations. We aim to resolve the geometric and electronic structures of all S-states, correlating their structural features with spectroscopic observations to elucidate reactivity. The sequence of manganese oxidations and concomitant charge compensation events via proton transfer allow us to rationalize the multielectron S-state cycle. EPR spectroscopy combined with theoretical calculations provides a unique window into the tetramangenese complex, in particular its protonation states and metal ligand sphere evolution, far beyond the scope of static techniques such as X-ray crystallography. This approach has led, for example, to a detailed understanding of the EPR signals in the S₂-state of the OEC in terms of two interconvertible, isoenergetic structures. These two structures differ in their valence distribution and spin multiplicity, which has important consequences for substrate binding and may explain its low barrier exchange with solvent water. New experimental techniques and innovative sample preparations are beginning to unravel the complex sequence of substrate uptake/inclusion, which is coupled to proton release. The introduction of specific site perturbations, such as replacing Ca²⁺ with Sr²⁺, provides discrete information about the ligand environment of the individual Mn ions. In this way, we have identified a potential open coordination site for one Mn center, which may serve as a substrate binding site in the higher S-states, such as S₃ and S₄. In addition, we can now monitor the binding of the substrate water in the lower S-states (S₁ and S₂) using new EPR-detected NMR spectroscopies. These studies provided the first evidence that one of the substrates is subsumed into the complex itself and forms an oxo-bridge between two Mn ions. This result places important new restrictions on the mechanism of O-O bond formation. These new insights from nature's water splitting catalyst provide important criteria for the rational design of bioinspired synthetic catalysts.

SoxR protein, a transcriptional activator of the soxRS (superoxide response) regulon of Escherichia coli, contains autooxidizable [2Fe-2S] centers that are presumed to serve as redox sensors. In vitro transcription experiments previously demonstrated that only the oxidized form is active. Reduced SoxR was detected in overproducing strains by EPR spectroscopy of suspensions of intact cells. Oxidized Fe-S centers were determined by lysing the cells and treating them with the reducing agent sodium dithionite prior to EPR measurements. In uninduced cells, 90% of the SoxR was in the reduced form. Treatment with the redox cycling agents phenazine methosulfate or plumbagin was accompanied by reversible oxidation of the Fe-S centers. Mutant SoxR derivatives that were constitutively activated existed constitutively in an oxidized state. The results indicate the presence of a cellular pathway for countering the autooxidation of SoxR and confirm the hypothesis that induction of the regulon is mediated by a shift in the redox equilibrium of SoxR rather than by assembly of its Fe-S clusters.

Recent in vivo studies indicate that ring monooxygenation is a widespread mechanism by which bacteria metabolize aromatic hydrocarbons and obtain carbon and energy. In this study, toluene 2-monooxygenase from Burkholderia (formerly Pseudomonas) cepacia G4 was purified to homogeneity and found to be a three-component enzyme system. The reconstituted enzyme system oxidized toluene to o-cresol and o-cresol to 3-methylcatechol, an important intermediate for growth of the bacterium on toluene. Steady-state kinetic parameters measured for the water-soluble substrate o-cresol were a Km of 0.8 microM and a Vmax of 131 nmol min-1 (mg of hydroxylase protein)-1. The three protein components were (1) a 40 kDa polypeptide containing one FAD and a [2Fe2S] cluster, (2) a 10.4 kDa polypeptide that contained no identifiable metals or organic cofactors, and (3) a 211 kDa alpha 2 beta 2 gamma 2 component containing five to six iron atoms. The 40 kDa flavo-iron-sulfur protein oxidized NADH and transferred electrons to cytochrome c, dyes, and the alpha 2 beta 2 gamma 2 component. It is analogous to other NADH oxidoreductase components found in a wide range of bacterial mono- and dioxygenases. The 10.4 kDa component, added to the other two components and NADH, increased toluene oxidation rates 10-fold. The alpha 2 beta 2 gamma 2 component was indicated to contain the site for toluene binding and hydroxylation by the following observations: (1) tight binding to a toluene affinity column; (2) oxidation of toluene after reduction of the protein with dithionite and adding O2; (3) H2O2-dependent toluene oxidation and catalase activity; and (4) spectroscopic studies of the iron atoms in the component. The alpha 2 beta 2 gamma 2 component had no significant absorbance in the visible region. EPR spectroscopy yielded a signal at g = 16 upon addition of>> 2 equiv of electrons per 2 Fe atoms. Taken with the quantitation of five to six iron atoms, the data suggest that the alpha 2 beta 2 gamma 2 component contains two binuclear iron centers. In total, the structural, spectroscopic, and catalytic features of toluene 2-monooxygenase are reminiscent of soluble methane monooxygenase obtained from methanotrophic bacteria. The two enzyme systems also differ in many subtle ways; for example, they oxidize toluene with completely different regiospecificity.

We have used electron paramagnetic resonance (EPR) to investigate the orientation, rotational motion, and actin-binding properties of rabbit psoas muscle cross-bridges in the presence of the nonhydrolyzable nucleotide analogue, 5'-adenylylimido-diphosphate (AMPPNP). This analogue is known to decrease muscle tension without affecting its stiffness, suggesting an attached cross-bridge state different from rigor. We spin-labeled the SH1 groups on myosin heads and performed conventional EPR to obtain high-resolution information about the orientational distribution, and saturation transfer EPR to measure microsecond rotational motion. At 4 degrees C and 100 mM ionic strength, we find that AMPPNP increases both the orientational disorder and the microsecond rotational motion of myosin heads. However, computer analysis of digitized spectra shows that no new population of probes is observed that does not match either rigor or relaxation in both orientation and motion. At 4 degrees C, under nearly saturating conditions of 16 mM AMPPNP (Kd = 3.0 mM, determined from competition between AMPPNP and an ADP spin label), 47.5 +/- 2.5% of myosin heads are dynamically disoriented (as in relaxation) without a significant decrease in rigor stiffness, whereas the remainder are rigidly oriented as in rigor. The oriented heads correspond to actin-attached heads in a ternary complex, and the disoriented heads correspond to detached heads, as indicated by EPR experiments with spin-labeled subfragment 1 (S1) that provide independent measurements of orientation and binding. We take these findings as evidence for a single-headed cross-bridge that is as stiff as the double-headed rigor cross-bridge. The data are consistent with a model in which, in the presence of saturating AMPPNP, one head of each cross-bridge binds actin about 10 times more weakly, whereas the remaining head binds at least 10 times more strongly, than extrinsic S1. Thus, although there is no evidence for heads being attached at nonrigor angles, the attached cross-bridge differs from that of rigor. The heterogeneous behavior of heads is probably due to steric effects of the filament lattice.

Cu(2+) ions are found concentrated within senile plaques of Alzheimer's disease patients directly bound to amyloid-beta peptide (Abeta) and are linked to the neurotoxicity and self-association of Abeta. The affinity of Cu(2+) for monomeric Abeta is highly disputed, and there have been no reports of affinity of Cu(2+) for fibrillar Abeta. We therefore measured the affinity of Cu(2+) for both monomeric and fibrillar Abeta(1-42) using two independent methods: fluorescence quenching and circular dichroism. The binding curves were almost identical for both fibrillar and monomeric forms. Competition studies with free glycine, l-histidine, and nitrilotriacetic acid (NTA) indicate an apparent (conditional) dissociation constant of 10(-11) M, at pH 7.4. Previous studies of Cu-Abeta have typically found the affinity 2 or more orders of magnitude weaker, largely because the affinity of competing ligands or buffers has been underestimated. Abeta fibers are able to bind a full stoichiometric complement of Cu(2+) ions with little change in their secondary structure and have coordination geometry identical to that of monomeric Abeta. Electron paramagnetic resonance studies (EPR) with Abeta His/Ala analogues suggest a dynamic view of the tetragonal Cu(2+) complex, with axial as well as equatorial coordination of imidazole nitrogens creating an ensemble of coordination geometries in exchange between each other. Furthermore, the N-terminal amino group is essential for the formation of high-pH complex II. The Abeta(1-28) fragment binds an additional Cu(2+) ion compared to full-length Abeta, with appreciable affinity. This second binding site is revealed in Abeta(1-42) upon addition of methanol, indicating hydrophobic interactions block the formation of this weaker carboxylate-rich complex. A Cu(2+) affinity for Abeta of 10(11) M(-1) supports a modified amyloid cascade hypothesis in which Cu(2+) is central to Abeta neurotoxicity.

Electron paramagnetic resonance spin trapping has become an indispensable tool for the specific detection of reactive oxygen free radicals in biological systems. In this review we describe some of the advantages as well as some experimental considerations of this technique and how it can be applied to biological systems to measure oxidative stress.

Expression of the tmoA-F gene cluster from Pseudomonas mendocina KRI in Escherichia coli BL21(DE3) produces a catalytically active form of the toluene-4-monooxygenase (T4MO) complex. Here we report the purification and characterization of four soluble proteins required for the in vitro reconstitution of T4MO catalytic activity. These proteins are a diiron hydroxylase (T4MOH), a Riesketype ferredoxin (T4MOC), an effector protein (T4MOD), and an NADH oxidoreductase (T4MOF). The T4MOH component is composed of the tmoA, tmoB, and tmoE gene products [quaternary structure (alpha beta epsilon)2, Mr approximately 220 kDa]. The T4MOA polypeptide contains two copies of the amino acid sequence motif (D/E)X(28-37)DEXRH; the same motif provides all of the protein-derived ligands to the diiron centers of ribonucleotide reductase, the soluble methane monooxygenase, and the stearoyl-ACP delta 9 desaturase. Mössbauer, optical, and EPR measurements show that the T4MOH contains diiron centers and suggest that the diiron center contains hydroxo bridge(s) in the diferric state, as observed for methane monooxygenase. Mössbauer and EPR measurements also show that the T4MOC contains a Rieske-type iron-sulfur center. This assignment is in accord with the presence of the amino acid sequence motif CPHX(15-17)CX2H, which has also been found in the bacterial, chloroplastic, and mitochondrial Rieske proteins as well as the bacterial NADH-dependent cis-dihydrodiol-forming aromatic dioxygenases. While single-turnover catalytic studies confirm the function of the T4MOH as the hydroxylase, the NADH-dependent multiple-turnover hydroxylation activity is increased by more than 100-fold in the presence of the T4MOC, which mediates highly specific electron transfer between the T4MOF and the T4MOH. The T4MOD can be purified as an 11.6 kDa monomeric protein devoid of cofactors or redox-active metal ions; this component is also detected as a substoichiometric consitutent of the purified T4MOH. The rate of the hydroxylation reaction can be mildly stimulated by the further addition of separately purified T4MOD to the T4MOH, implying the formation of a high affinity, catalytically competent complex between these two components. These characterizations define a novel, four-component oxygenase combining elements from the soluble methane oxidation complex of the methanotrophic bacteria and the aromatic hydroxylation complexes of the soil pseudomonads.

An extensive series of ligand complexes of ferric cytochrome P-450-CAM has been examined by UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy in an attempt to identify the ligand trans to cysteinate in the six-coordinate resting state of the enzyme. Thus, the ligands used have been chosen to serve as models for coordination by potential endogenous amino acids and include alcohol, amide and carboxylate oxygen donors, amine, imidazole and indole nitrogen donors and disulfide, thioether, thiol, and thiolate sulfur donors. As this investigation has been by nature an empirical one, the conclusions are strengthened by the concurrent use of three different spectroscopic techniques. All of the complexes formed except those resulting from thiolate addition display spectroscopic properties that are broadly similar to those of low spin, six-coordinate P-450. Of the sulfur donor adducts, disulfide and thioether-bound P-450 have properties that are different enough in detail to distinguish them from native P-450. While the spectral features of the thiol-bound species and of low spin ferric P-450 are alike, the former are pH dependent due to interconversion to bound thiolate, whereas the latter display essentially no spectral changes with pH. Of the oxygen donor complexes, all but carboxylate have spectra that very closely match those of the resting enzyme. Adducts formed with most nitrogenous ligands, including several imidazole derivatives, exhibit spectra that are sufficiently different from native P-450 to exclude them as candidates for the sixth ligand. Interestingly, the spectral properties of a complex formed with an imidazole derivative having a bulky electron-withdrawing substituent in the alpha position are comparable to those native P-450 except for the line shape of the EPR spectrum. Previously published theoretical work suggests that the spectral differences seen between this imidazole derivative and the other examined are electronic and not steric in origin. As no similar electronic mechanism exists for the protein to reduce the electron-donating ability or histidine, it is felt that coordination of histidine in the sixth position of P-450 can be ruled out. In conclusion, close examination of all spectral data reveals that amino acid analog adducts of P-450-CAM with amides and, in particular, alcohols, produce spectra that almost exactly duplicate those of native P-450 and suggests that the ligand trans to cysteinate in the six-coordinate ferric enzyme has an oxygen donor atom.

Pseudomonas testosteroni protocatechuate 4,5-dioxygenase catalyzes extradiol-type oxygenolytic cleavage of the aromatic ring of its substrate. The essential active site Fe2+ binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2. Hyperfine broadening of the EPR resonances of the nitrosyl complex of the enzyme by protocatechuate (3,4-(OH)2-benzoate, PCA) enriched specifically with 17O (I = 5/2) in either the 3 or the 4 hydroxyl group shows that both groups can bind directly to the Fe2+ in the ternary complex. Analogous results are obtained for PCA binding to catechol 2,3-dioxygenase-NO complex suggesting that substrate binding by the Fe2+ may be a general property of extradiol dioxygenases. The protocatechuate 4,5-dioxygenase inhibitor, 4-17OH-benzoate binds directly to the Fe of the nitrosyl adduct of the enzyme through the OH group. Since previous studies have shown that water also is bound to the Fe in this ternary complex, but not in the ternary complex with PCA, the data strongly imply that there are 3 sites in the Fe coordination which can be occupied by exogenous ligands. 3-17OH-benzoate is an inhibitor of the enzyme but does not elicit detectable hyperfine broadening in the EPR spectrum of the nitrosyl adduct suggesting that it binds to the enzyme, but not to the Fe. The EPR spectra of ternary enzyme-NO complexes with PCA or 4-OH-benzoate labeled with 17O exclusively in the carboxylate substituent are not broadened, suggesting that this moiety does not bind to the Fe.

While oxygen free radicals are important mediators of brain injury, questions remain regarding which cell types and enzyme pathways trigger this radical generation. Microglial cells have been hypothesized to be an important source of radical generation; however, the magnitude, kinetics, and mechanism of this process are unknown. Oxygen radical generation by stimulated primary microglia was directly measured and characterized by electron paramagnetic resonance spin trapping. Microglia, when stimulated by phorbol ester or opsonified zymosan, gave rise to EPR spectra characteristic of superoxide. Experiments performed in the presence of superoxide dismutase, catalase, deferoxamine, and dimethyl sulfoxide excluded generation of hydroxyl radicals in significant amounts. Microglial superoxide generation was blocked by the NADPH oxidase inhibitor diphenylene iodonium in a manner similar to that seen in neutrophils, suggesting that a neutrophil like NADPH oxidase was the source of superoxide production. However, microglia produced 20 to 40 times less superoxide compared to a similar number of neutrophils during the first 30 min following stimulation, indicating a marked difference in the regulation of NADPH oxidase activation. Western blots of microglia lysates demonstrated that both large (gp91-phox) and small (p22-phox) NADPH oxidase subunits are expressed in both unstimulated and stimulated microglia. Indirect immunofluorescence demonstrated localization at the membrane surfaces of activated cells. Thus, microglial cells generate superoxide via a neutrophil-like NADPH oxidase but exhibit distinctly different time course and magnitude of activation than that seen in neutrophils.

The maltose transporter MalFGK(2) of Escherichia coli is a member of the ATP-binding cassette superfamily. A periplasmic maltose-binding protein (MBP) delivers maltose to MalFGK(2) and stimulates its ATPase activity. Site-directed spin labeling EPR spectroscopy was used to study the opening and closing of the nucleotide-binding interface of MalFGK(2) during the catalytic cycle. In the intact transporter, closure of the interface coincides not just with the binding of ATP, as seen with isolated nucleotide-binding domains, but requires both MBP and ATP, implying that MBP stimulates ATPase activity by promoting the closure of the nucleotide-binding interface. After ATP hydrolysis, with MgADP and MBP bound, the nucleotide-binding interface resides in a semi-open configuration distinct from the fully open configuration seen in the absence of any ligand. We propose that P(i) release coincides with the reorientation of transmembrane helices to an inward-facing conformation and the final step of maltose translocation into the cell.

Pseudomonas testosteroni protocatechuate 4,5-dioxygenase and Pseudomonas putida catechol 2,3-dioxygenase (metapyrocatechase) catalyze extradiol-type oxygenolytic cleavage of the aromatic ring of their substrates. The essential active site Fe2+ of each enzyme binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2. Hyperfine broadening of the EPR resonances of the nitrosyl complexes by 17O-enriched H2O shows that water is bound directly to the Fe2+ in the native enzymes, but is apparently displaced in substrate complexes. NO is not displaced by either substrates or inhibitors. The EPR spectra of several enzyme-inhibitor-NO complexes are different from those of enzyme-NO or enzyme-substrate-NO complexes and are found to be broadened by 17O-enriched water. The data show that at least 2 and perhaps 3 sites in the Fe ligation can be occupied by exogenous ligands. Furthermore, it is likely that substrates and inhibitors displace water by binding either at or near to the Fe in the nitrosyl complex. Nitric oxide binding is found to be substrate-dependent for each enzyme. Native catechol 2,3-dioxygenase exhibits KD values of 190 microM and 2.0 mM for NO binding in two types of independent sites. Only one type of site is observed in the catechol complex which exhibits a KD for NO of 3.4 microM. One type of NO binding site is observed for both the native and substrate complexed protocatechuate 4,5-dioxygenase with KD values of 360 and 3 microM, respectively. The presence of a specific site in the Fe coordination for NO which is modified in the substrate complex, suggests that O2 binding by the extradiol dioxygenases may also occur at the Fe.

Aldehyde oxidase (AO) is a cytosolic enzyme with an important role in drug and xenobiotic metabolism. Although AO has structural similarity to bacterial nitrite reductases, it is unknown whether AO-catalyzed nitrite reduction can be an important source of NO. The mechanism, magnitude, and quantitative importance of AO-mediated nitrite reduction in tissues have not been reported. To investigate this pathway and its quantitative importance, EPR spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. The kinetics and magnitude of AO-dependent NO formation were characterized. In the presence of typical aldehyde substrates or NADH, AO reduced nitrite to NO. Kinetics of AO-catalyzed nitrite reduction followed Michaelis-Menten kinetics under anaerobic conditions. Under physiological conditions, nitrite levels are far below its measured K(m) value in the presence of either the flavin site electron donor NADH or molybdenum site aldehyde electron donors. Under aerobic conditions with the FAD site-binding substrate, NADH, AO-mediated NO production was largely maintained, although with aldehyde substrates oxygen-dependent inhibition was seen. Oxygen tension, substrate, and pH levels were important regulators of AO-catalyzed NO generation. From kinetic data, it was determined that during ischemia hepatic, pulmonary, or myocardial AO and nitrite levels were sufficient to result in NO generation comparable to or exceeding maximal production by constitutive NO synthases. Thus, AO-catalyzed nitrite reduction can be an important source of NO generation, and its NO production will be further increased by therapeutic administration of nitrite.

Kinesin motors move on microtubules by a mechanism that involves a large, ATP-triggered conformational change in which a mechanical element called the neck linker docks onto the catalytic core, making contacts with the core throughout its length. Here, we investigate the thermodynamic properties of this conformational change using electron paramagnetic resonance (EPR) spectroscopy. We placed spin probes at several locations on the human kinesin neck linker and recorded EPR spectra in the presence of microtubules and either 5'-adenylylimidodiphosphate (AMPPNP) or ADP at temperatures of 4-30 degrees C. The free-energy change (DeltaG) associated with AMPPNP-induced docking of the neck linker onto the catalytic core is favorable but small, about 3 kJ/mol. In contrast, the favorable enthalpy change (DeltaH) and unfavorable entropy change (TDeltaS) are quite large, about 50 kJ/mol. A mutation in the neck linker, V331A/N332A, results in an unfavorable DeltaG for AMPPNP-induced zipping of the neck linker onto the core and causes motility defects. These results suggest that the kinesin neck linker folds onto the core from a more unstructured state, thereby paying a large entropic cost and gaining a large amount of enthalpy.

Shallow electron spin echo envelope modulations due to dipole-dipole couplings between electron spins provide information on the radial distribution function of the spins in disordered systems while angular correlations between spin pairs are negligible. Under these conditions and in the absence of orientational selection, the dipolar time evolution data can be quantitatively simulated for arbitrary radial distribution functions by shell factorization, i.e., by performing the orientational average separately for thin spherical shells and multiplying the signals of all the shells. For distances below 5 nm, a linear superposition of the signals of the shells is sufficient. The dipolar time evolution data can be separated into this linear contribution and a nonlinear background. The linear contribution can then be converted directly to a radial distribution function. For a series of shape-persistent and flexible biradicals with end-to-end distances between 2 and 5 nm, shell factorization and direct conversion of the data are in good agreement with each other and with force-field computations of the end-to-end distances. The neglect of orientation selection does not cause significant distortions of the determined distance distributions.

Various types of nanoparticles, such as liposomes, polymeric micelles, dendrimers, superparamagnetic iron oxide crystals, and colloidal gold, have been employed in targeted therapies for cancer. Both passive and active targeting strategies can be utilized for nano-drug delivery. Passive targeting is based on the enhanced permeability and retention (EPR) effect of the vasculature surrounding tumors. Active targeting relies on ligand-directed binding of nanoparticles to receptors expressed by tumor cells. Release of loaded drugs from nanoparticles may be controlled in response to changes in environmental condition such as temperature and pH. Biodistribution profiles and anticancer efficacy of nano-drugs in vivo would be different depending upon their size, surface charge, PEGylation and other biophysical properties. This review focuses on the recent development of nanoparticles for tumor targeted therapies, including physicochemical properties, tumor targeting, control of drug release, pharmacokinetics, anticancer efficacy and safety. Future perspectives are discussed as well.

Melittin spin-labeled specifically with a nitroxide at positions 7, 21, 23, or the amino terminus was bound to phospholipid membranes, and the exposure of the spin label to the aqueous phase was investigated by measurement of Heisenberg exchange with chromium oxalate in the solution. The exchange frequency was determined by saturation recovery electron paramagnetic resonance (EPR) using a loop-gap resonator. This method allows use of very low concentrations (less than 1 mM) of chromium oxalate compared with conventional measurements of EPR line broadening (typically 50 mM), thus avoiding problems associated with high metal ion concentration. Differences in exchange frequency between the various positions were also estimated by continuous wave power saturation methods. In either approach, the spin label at lysine 7 was found to be the most exposed to chromium oxalate whereas that at lysine 23 was found to be the least exposed. This is consistent with a model for the membrane bound peptide in which an amphiphilic helix lies with its axis parallel to the bilayer surface and the hydrophobic moment points toward the bilayer interior.

Electron transfer in complex I from Escherichia coli was investigated by an ultrafast freeze-quench approach. The reaction of complex I with NADH was stopped in the time domain from 90 mus to 8 ms and analyzed by electron paramagnetic resonance (EPR) spectroscopy at low temperatures. The data show that after binding of the first molecule of NADH, two electrons move via the FMN cofactor to the iron-sulfur (Fe/S) centers N1a and N2 with an apparent time constant of approximately 90 mus, implying that these two centers should have the highest redox potential in the enzyme. The rate of reduction of center N2 (the last center in the electron transfer sequence) is close to that predicted by electron transfer theory, which argues for the absence of coupled proton transfer or conformational changes during electron transfer from FMN to N2. After fast reduction of N1a and N2, we observe a slow, approximately 1-ms component of reduction of other Fe/S clusters. Because all elementary electron transfer rates between clusters are several orders of magnitude higher than this observed rate, we conclude that the millisecond component is limited by a single process corresponding to dissociation of the oxidized NAD(+) molecule from its binding site, where it prevents entry of the next NADH molecule. Despite the presence of approximately one ubiquinone per enzyme molecule, no transient semiquinone formation was observed, which has mechanistic implications, suggesting a high thermodynamic barrier for ubiquinone reduction to the semiquinone radical. Possible consequences of these findings for the proton translocation mechanism are discussed.

Posttranscriptional regulatory mechanisms superimpose "fine-tuning" control upon "on-off" switches characteristic of gene transcription. We have exploited computational modeling with experimental validation to resolve an anomalous relationship between mRNA expression and protein synthesis. The GAIT (gamma-interferon-activated inhibitor of translation) complex repressed VEGF-A synthesis to a low, constant rate independent of VEGF-A mRNA expression levels. Dynamic model simulations predicted an inhibitory GAIT-element-interacting factor to account for this relationship and led to the identification of a truncated form of glutamyl-prolyl tRNA synthetase (EPRS), a GAIT constituent that mediates binding to target transcripts. The truncated protein, EPRS(N1), shields GAIT-element-bearing transcripts from the inhibitory GAIT complex, thereby dictating a "translational trickle" of GAIT target proteins. EPRS(N1) mRNA is generated by polyadenylation-directed conversion of a Tyr codon in the EPRS-coding sequence to a stop codon (PAY(∗)). Genome-wide analysis revealed multiple candidate PAY(∗) targets, including the authenticated target RRM1, suggesting a general mechanism for production of C terminus-truncated regulatory proteins.

We have purified the Rieske iron-sulfur protein from Thermus thermophilus. Chemical analyses show that the protein contains iron, labile sulfide, and cysteine in equimolar concentrations, four of each for Mr approximately 20,000. The oxidized and reduced form of the protein have been characterized by optical, EPR, CD, magnetic CD and Mössbauer spectroscopies. Our data suggest the presence of a unique iron-sulfur center. Mössbauer studies of the oxidized and reduced protein demonstrate unambiguously that the protein contains clusters with [2Fe-2S] cores. The iron analyses and the Mössbauer data, taken together, suggest that the protein has two [2Fe-2S] clusters. This is supported by the observation that two electrons are required for complete reduction of the protein and that the g = 1.94-type signal of the reduced protein has a spin concentration of one spin (S = 1/2) per 2Fe. Within the excellent resolution of the Mössbauer and EPR data, the two clusters are identical. Our results thus suggest that each [2Fe-2S] cluster is coordinated by at most two cysteine residues. The Mössbauer spectra of the reduced protein were analyzed with an S = 1/2 spin Hamiltonian. The hyperfine parameters obtained are very similar to those reported for putidaredoxin. The main difference is that the Rieske protein exhibits an increased isomer shift at the Fe2+ site, suggesting that non-cysteine ligands are coordinated to the site that becomes reduced to Fe2+ upon reduction. A comparison of our data with those reported for various NADH-dependent dioxygenases suggest that these enzymes contain a Rieske-type [2Fe-2S] center.

Targeted nanoparticle-based technologies show increasing prevalence in radiotracer design. As a consequence, quantitative contribution of nonspecific accumulation in the target tissue, mainly governed by the enhanced permeability and retention (EPR) effect, becomes highly relevant for evaluating the specificity of these new agents. This study investigated the influence of different tumor phenotypes on the EPR effect, hypothesizing that a baseline level of uptake must be exceeded to visualize high and specific uptake of a targeted macromolecular radiotracer.

METHODS

These preliminary studies use (89)Zr-labeled mouse serum albumin ((89)Zr-desferrioxamine-mAlb) as a model radiotracer to assess uptake and retention in 3 xenograft models of human prostate cancer (CWR22rv1, DU-145, and PC-3). Experiments include PET and contrast-enhanced ultrasound imaging to assess morphology, vascularization, and radiotracer uptake; temporal ex vivo biodistribution studies to quantify radiotracer uptake over time; and histologic and autoradiographic studies to evaluate the intra- and intertumoral distribution of (89)Zr-desferrioxamine-mAlb.

RESULTS

Early uptake profiles show statistically significant but overall small differences in radiotracer uptake between different tumor phenotypes. By 20 h, nonspecific radiotracer uptake was found to be independent of tumor size and phenotype, reaching at least 5.0 percentage injected dose per gram in all 3 tumor models.

CONCLUSIONS

These studies suggest that minimal differences in tumor uptake exist at early time points, dependent on the tumor type. However, these differences equalize over time, reaching around 5.0 percentage injected dose per gram at 20 h after injection. These data provide strong support for the introduction of mandatory experimental controls of future macromolecular or nanoparticle-based drugs, particularly regarding the development of targeted radiotracers.