A disulfide-linked nitroxide side chain (R1) used in site-directed spin labeling of proteins often exhibits an EPR spectrum characteristic of a weakly ordered z-axis anisotropic motion at topographically diverse surface sites, including those on helices, loops and edge strands of beta-sheets. To elucidate the origin of this motion, the first crystal structures of R1 that display simple z-axis anisotropic motion at solvent-exposed helical sites (131 and 151) and a loop site (82) in T4 lysozyme have been determined. Structures of 131R1 and 151R1 determined at cryogenic or ambient temperature reveal an intraresidue C(alpha)--H...S(delta) interaction that immobilizes the disulfide group, consistent with a model in which the internal motions of R1 are dominated by rotations about the two terminal bonds (Columbus, Kálai, Jeko, Hideg, and Hubbell, Biochemistry 2001;40:3828-3846). Remarkably, the 131R1 side chain populates two rotamers equally, but the EPR spectrum reflects a single dominant dynamic population, showing that the two rotamers have similar internal motion determined by the common disulfide-backbone interaction. The anisotropic motion for loop residue 82R1 is also accounted for by a common disulfide-backbone interaction, showing that the interaction does not require a specific secondary structure. If the above observations prove to be general, then significant variations in order and rate for R1 at noninteracting solvent-exposed helical and loop sites can be assigned to backbone motion because the internal motion is essentially constant.

In mammals and birds, sulfite oxidase (SO) is a homodimeric molybdenum enzyme consisting of an N-terminal heme domain and a C-terminal molybdenum domain (EC ). In plants, the existence of SO has not yet been demonstrated, while sulfite reductase as part of sulfur assimilation is well characterized. Here we report the cloning of a plant sulfite oxidase gene from Arabidopsis thaliana and the biochemical characterization of the encoded protein (At-SO). At-SO is a molybdenum enzyme with molybdopterin as an organic component of the molybdenum cofactor. In contrast to homologous animal enzymes, At-SO lacks the heme domain, which is evident both from the amino acid sequence and from its enzymological and spectral properties. Thus, among eukaryotes, At-SO is the only molybdenum enzyme yet described possessing no redox-active centers other than the molybdenum. UV-visible and EPR spectra as well as apparent K(m) values are presented and compared with the hepatic enzyme. Subcellular analysis of crude cell extracts showed that SO was mostly found in the peroxisomal fraction. In molybdenum cofactor mutants, the activity of SO was strongly reduced. Using antibodies directed against At-SO, we show that a cross-reacting protein of similar size occurs in a wide range of plant species, including both herbacious and woody plants.

OBJECTIVE

This study was carried out to determine the biodistribution profiles and tumor localization potential of poly(ethylene oxide) (PEO)-modified poly(beta-amino ester) (PbAE) as a novel, pH-sensitive biodegradable polymeric nanoparticulate system for tumor-targeted drug delivery.

METHODS

The biodistribution studies of PEO-modified PbAE and PEO-modified poly(epsilon-caprolactone) (PCL), a non-pH-sensitive polymer, nanoparticle systems were carried out in normal mice using 111indium-oxine [111In] as a lipophilic radiolabel encapsulated within the polymeric matrix, and the distribution of the nanoparticles was studied in plasma and all the vital organs following intravenous administration. Solid tumors were developed on nude mice using human ovarian carcinoma xenograft (SKOV-3) and the change in concentrations of tritium [3H]-labeled paclitaxel encapsulated in polymeric nanoparticles was examined in blood, tumor mass, and liver.

RESULTS

Study in normal mice with a gamma-emitting isotope [111In] provided a thorough biodistribution analysis of the PEO-modified nanoparticulate carrier systems, whereas 3H-paclitaxel was useful to understand the change in concentration and tumor localization of anticancer compound directly in major sites of distribution. Both PEO-PbAE and PEO-PCL nanoparticles showed long systemic circulating properties by virtue of surface modification with PEO-containing triblock block copolymer (Pluronic stabilizer. Although the PCL nanoparticles showed higher uptake by the reticuloendothelial system, the PbAE nanoparticles effectively delivered the encapsulated payload into the tumor mass.

CONCLUSIONS

PEO-modified PbAE nanoparticles showed considerable passive tumor targeting potential in early stages of biodistribution via the enhanced permeation and retention (EPR) mechanism. This prompts a detailed biodistribution profiling of the nanocarrier for prolonged periods to provide conclusive evidence for superiority of the delivery system.

Beef heart aconitase, as isolated under aerobic conditions, is inactive and contains a [3Fe-4S]1+ cluster. On incubation at pH greater than 9.5 (or treatment with 4-8 M urea) the color of the protein changes from brown to purple. This purple form is stable and can be converted back in good yield to the active [4Fe-4S]2+ form by reduction in the presence of iron. Active aconitase is converted to the purple form at alkaline pH only after oxidative inactivation. The Fe/S2- ratio of purple aconitase is 0.8, indicating the presence of [3Fe-4S] clusters. The number of SH groups readily reacting with 5,5'-dithiobis(2-nitrobenzoic acid) is increased from approximately 1 in the enzyme as isolated to 7-8 in the purple form, indicating a partial unfolding of the protein. On conversion of inactive aconitase to the purple form, the EPR signal at g = 2.01 (S = 1/2) is replaced by signals at g = 4.3 and 9.6 (S = 5/2). Mössbauer spectroscopy shows that purple aconitase has high-spin ferric ions, each residing in a tetrahedral environment of sulfur atoms. The three iron sites are exchange-coupled to yield a ground state with S = 5/2. Analysis of the data within a spin coupling model shows that J13 congruent to J23 and 2 J12 less than J13, where the Jik describe the antiferromagnetic (J greater than 0) exchange interactions among the three iron pairs. Comparison of our data with those reported for synthetic Fe-S clusters (Hagen, K. S., Watson, A. D., and Holm, R. H., (1983) J. Am. Chem. Soc. 105, 3905-3913) shows that purple aconitase contains a linear [3Fe-4S]1+ cluster, a structural isomer of the S = 1/2 cluster of inactive aconitase. Our studies also show that protein-bound [2Fe-2S] clusters can be generated under conditions where partial unfolding of the protein occurs.

The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14-subunit transmembrane complex associated with a Ni-Fe hydrogenase. Ten ORFs (mbhA-I and mbhM) encode hydrophobic, membrane-spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni-Fe hydrogenase, however, the small subunit can coordinate only a single iron-sulfur cluster, corresponding to the proximal [4Fe-4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe-4S] protein. With P. furiosus soluble [4Fe-4S] ferredoxin as the electron donor the membranes produce H2, and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane-bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely high ratio of H2 evolution to H2 uptake activity compared with other hydrogenases. The activity is sensitive to inhibition by dicyclohexylcarbodiimide, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron-sulfur clusters with Em approximately -0.33 V. The genome contains a second putative operon, mbxABCDFGHH'MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni-Fe-cluster in the large hydrogenase subunit. Kinetic studies of membrane-bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activities allow the formulation of a comprehensive working hypothesis of H2 metabolism in P. furiosus in terms of three pools of reducing equivalents (ferredoxin, NADPH, H2) connected by devices for transduction, transfer, recovery and safety-valving of energy.

Macromolecular drugs (also referred to as polymeric drugs) are a diverse group of drugs including polymer-conjugated drugs, polymeric micelles, liposomal drugs and solid phase depot formulations of various agents. In this review we will consider only water-soluble macromolecular drugs. In common, such drugs have high molecular weights, more than 40 kDa, which enables them to overcome renal excretion. Consequently, this group of drugs can attain prolonged plasma or local half-lives. The prolonged circulating time of these macromolecules enables them to utilise the vascular abnormalities of solid tumour tissues, a phenomenon called the enhanced permeability and retention (EPR) effect. The EPR effect facilitates extravasation of polymeric drugs more selectively at tumour tissues, and this selective targeting to solid tumour tissues may lead to superior therapeutic benefits with fewer systemic adverse effects. This contrasts with conventional low-molecular-weight drugs, where intratumour concentration diminishes rapidly in parallel with plasma concentration. The EPR effect is also operative in inflammatory tissues, which justifies the development and use of this class of drugs in infectious and inflammatory conditions. At the present time, several polymeric drugs have been approved by regulatory agencies. These include zinostatin stimalamer (copolymer styrene maleic acid-conjugated neocarzinostatin, or SMANCS) and polyethyleneglycol-conjugated interferon-alpha-2a. This article discusses these and other polymeric drugs in the setting of targeting to solid tumours.

Ribonucleotide reductase has been demonstrated to be inhibited by NO synthase product(s). The experiments reported here show that nitric oxide generated from sodium nitroprusside, S-nitrosoglutathione and the sydnonimine SIN-1 inhibits ribonucleotide reductase activity present in cytosolic extracts of TA3 mammary tumor cells. Stable derivatives of these nitric oxide donors were either inactive or much less inhibitory. EPR experiments show that the tyrosyl radical of the small subunit of E. Coli or mammalian ribonucleotide reductase is efficiently scavenged by these NO donors.

The heme domain (iNOS(heme)) of inducible nitric oxide synthase (NOS) was expressed in Escherichia coli and purified to homogeneity. Rapid freeze-quench (RFQ) EPR was used to monitor the reaction of the reduced iNOS(heme) with oxygen in the presence and absence of substrate. In these reactions, heme oxidation occurs at a rate of approximately 15 s(-)(1) at 4 degrees C. A transient species with a g = 2.0 EPR signal is also observed under these conditions. The spectral properties of the g = 2.0 signal are those of an anisotropic organic radical with S = (1)/(2). Comparison of the EPR spectra obtained when iNOS(heme) is reconstituted with N5-(14)N- and (15)N-substituted tetrahydrobiopterin (H(4)B) shows a hyperfine interaction with the pterin N5 nitrogen and identifies the radical as the one-electron oxidized form (H(3)B.) of the bound H(4)B. Substitution of D(2)O for H(2)O reveals the presence of hyperfine-coupled exchangeable protons in the H(4)B radical. This radical forms at a rate of 15-20 s(-)(1), with a slower decay rate that varies (0.12-0.7 s(-)(1)) depending on the substrate. At 127 ms, H(3)B. accumulates to a maximum of 80% of the total iNOS(heme) concentration in the presence of arginine but only to approximately 2.8% in the presence of NHA. Double-mixing RFQ experiments, where NHA is added after the formation of H(3)B., show that NHA does not react rapidly with H(3)B. and suggest that NHA instead prevents the formation of the H(4)B radical. These data constitute the first direct evidence for an NOS-bound H(3)B. and are most consistent with a role for H(4)B in electron transfer in the NOS reaction.

Pyruvate formate-lyase (formate acetyltransferase; EC 2.3.1.54) of Escherichia coli cells is post-translationally interconverted between inactive and active forms. Conversion of the inactive to the active form is catalyzed by an Fe2+-dependent activating enzyme and requires adenosylmethionine and dihydroflavodoxin. This process is shown here to introduce a paramagnetic moiety into the structure of pyruvate formate-lyase. It displays an EPR signal at g = 2 with a doublet splitting of 1.5 mT and could comprise an organic free radical located on an amino acid residue of the polypeptide chain. Hypophosphite was discovered as a specific reagent that destroys both the enzyme radical and the enzyme activity; it becomes covalently bound to the protein. The enzymatic generation of the radical, which is linked to adenosylmethionine cleavage into 5'-deoxyadenosine and methionine, possibly occurs through an Fe-adenosyl complex. These results suggest a radical mechanism for the catalytic cycle of pyruvate formate-lyase.

We tested the hypothesis that low specific tension (force/cross-sectional area) in skeletal muscle from aged animals results from structural changes in myosin that occur with aging. Permeabilized semimembranosus fibers from young adult and aged rats were spin labeled site specifically at myosin SH1 (Cys-707). Electron paramagnetic resonance (EPR) was then used to resolve and quantify the structural states of the myosin head to determine the fraction of myosin heads in the strong-binding (force generating) structural state during maximal isometric contraction. Fibers from aged rats generated 27 +/- 0.8% less specific tension than fibers from younger rats (P < 0.001). EPR spectral analyses showed that, during contraction, 31.6 +/- 2.1% of myosin heads were in the strong-binding structural state in fibers from young adult animals but only 22.1 +/- 1.3% of myosin heads in fibers from aged animals were in that state (P = 0.004). Biochemical assays indicated that the age-related change in myosin structure could be due to protein oxidation, as indicated by a decrease in the number of free cysteine residues. We conclude that myosin structural changes can provide a molecular explanation for age-related decline in skeletal muscle force generation.

The reactions of nitric oxide (NO) with both oxidized and reduced cytochrome c oxidase are reported. NMR and mass spectroscopy were utilized to determine the products of the reactions; EPR and optical spectroscopy were employed to determine the states of the enzyme produced in each of these reactions. It was found that the enzyme catalyzes the consecutive oxidation and reduction of NO. A different cycle was observed when NO was added to the reduced enzyme, to the oxidized enzyme, or to the oxidized enzyme in the presence of azide. It was possible to observe the state of the enzyme at several points in each of these three cycles by varying the concentration of NO. The reactions of NO all involved a one- or two-electron redox step and could be accounted for by the involvement of only cytochrome a3 and Cua3. On the basis of these results, a mechanism for the reduction of dioxygen by the enzyme is proposed in which cytochrome a3 functions to anchor dioxygen and intermediates while remaining in the ferrous state, whereas Cua3 functions to accept electroins from cytochrome a/Cua and transfer them to dioxygen.

Electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and Mössbauer spectroscopies and other physical methods have provided important new insights into the radical-SAM superfamily of proteins, which use iron-sulfur clusters and S-adenosylmethionine to initiate H atom abstraction reactions. This remarkable chemistry involves the generation of the extremely reactive 5'-deoxyadenosyl radical, the same radical intermediate utilized in B12-dependent reactions. Although early speculation focused on the possibility of an organometallic intermediate in radical-SAM reactions, current evidence points to novel chemistry involving a site-differentiated [4Fe-4S] cluster. The focus of this forum article is on one member of the radical-SAM superfamily, pyruvate formate-lyase activating enzyme, and how physical methods, primarily EPR and ENDOR spectroscopies, are contributing to our understanding of its structure and mechanism. New ENDOR data supporting coordination of the methionine moiety of SAM to the unique site of the [4Fe-4S]2+/+ cluster are presented.

Thermoanaerobacter tengcongensis is a thermophilic Gram-positive bacterium able to dispose of the reducing equivalents generated during the fermentation of glucose to acetate and CO(2) by reducing H(+) to H(2). A unique combination of hydrogenases, a ferredoxin-dependent [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase, were found to be responsible for H(2) formation in this organism. Both enzymes were purified and characterized. The tightly membrane-bound [NiFe] hydrogenase belongs to a small group of complex-I-related [NiFe] hydrogenases and has highest sequence similarity to energy-converting [NiFe] hydrogenase (Ech) from Methanosarcina barkeri. A ferredoxin isolated from Ta. tengcongensis was identified as the physiological substrate of this enzyme. The heterotetrameric Fe-only hydrogenase was isolated from the soluble fraction. It contained FMN and multiple iron-sulfur clusters, and exhibited a typical H-cluster EPR signal after autooxidation. Sequence analysis predicted and kinetic studies confirmed that the enzyme is an NAD(H)-dependent Fe-only hydrogenase. When H(2) was allowed to accumulate in the culture, the fermentation was partially shifted to ethanol production. In cells grown at high hydrogen partial pressure [p(H(2))] the NADH-dependent hydrogenase activity was fourfold lower than in cells grown at low p(H(2)), whereas aldehyde dehydrogenase and alcohol dehydrogenase activities were higher in cells grown at elevated p(H(2)). These results indicate a regulation in response to the p(H(2)).

The R2 protein of class I ribonucleotide reductase generates and stores a tyrosyl radical essential for ribonucleotide reduction and, thus, DNA synthesis. X-ray structures of the protein have enabled detailed mechanistic suggestions, but no structural information has been available for the active radical-containing state of the protein. Here we report on methods to generate the functional tyrosyl radical in single crystals of R2 from Escherichia coli (Y122(*)). We further report on subsequent high-field EPR experiments on the radical-containing crystals. A full rotational pattern of the spectra was collected and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical in the crystal frame. The EPR data are discussed in comparison with a 1.42-A x-ray structure of the met (oxidized) form of the protein, also presented in this paper. Comparison of the orientation of the radical Y122(*) obtained from high-field EPR with that of the reduced tyrosine Y122-OH reveals a significant rotation of the tyrosyl side chain, away from the diiron center, in the active radical state. Implications for the radical transfer connecting the diiron site in R2 with the substrate-binding site in R1 are discussed. In addition, the present study demonstrates that structural and functional information about active radical states can be obtained by combined x-ray and high-field EPR crystallography.

Purebred dog health is thought to be compromised by an increasing occurence of inherited diseases but inadequate prevalence data on common disorders have hampered efforts to prioritise health reforms. Analysis of primary veterinary practice clinical data has been proposed for reliable estimation of disorder prevalence in dogs. Electronic patient record (EPR) data were collected on 148,741 dogs attending 93 clinics across central and south-eastern England. Analysis in detail of a random sample of EPRs relating to 3,884 dogs from 89 clinics identified the most frequently recorded disorders as otitis externa (prevalence 10.2%, 95% CI: 9.1-11.3), periodontal disease (9.3%, 95% CI: 8.3-10.3) and anal sac impaction (7.1%, 95% CI: 6.1-8.1). Using syndromic classification, the most prevalent body location affected was the head-and-neck (32.8%, 95% CI: 30.7-34.9), the most prevalent organ system affected was the integument (36.3%, 95% CI: 33.9-38.6) and the most prevalent pathophysiologic process diagnosed was inflammation (32.1%, 95% CI: 29.8-34.3). Among the twenty most-frequently recorded disorders, purebred dogs had a significantly higher prevalence compared with crossbreds for three: otitis externa (P = 0.001), obesity (P = 0.006) and skin mass lesion (P = 0.033), and popular breeds differed significantly from each other in their prevalence for five: periodontal disease (P = 0.002), overgrown nails (P = 0.004), degenerative joint disease (P = 0.005), obesity (P = 0.001) and lipoma (P = 0.003). These results fill a crucial data gap in disorder prevalence information and assist with disorder prioritisation. The results suggest that, for maximal impact, breeding reforms should target commonly-diagnosed complex disorders that are amenable to genetic improvement and should place special focus on at-risk breeds. Future studies evaluating disorder severity and duration will augment the usefulness of the disorder prevalence information reported herein.

Photodynamic therapy (PDT) employs the combination of nontoxic photosensitizers (PS) and harmless visible light to generate reactive oxygen species (ROS) and kill cells. Most clinically studied PS are based on the tetrapyrrole structure of porphyrins, chlorines, and related molecules, but new nontetrapyrrole PS are being sought. Fullerenes are soccer-ball shaped molecules composed of 60 or 70 carbon atoms and have attracted interest in connection with the search for biomedical applications of nanotechnology. Fullerenes are biologically inert unless derivatized with functional groups, whereupon they become soluble and can act as PS. We have compared the photodynamic activity of six functionalized fullerenes with 1, 2, or 3 hydrophilic or 1, 2, or 3 cationic groups. The octanol-water partition coefficients were determined and the relative contributions of Type I photochemistry (photogeneration of superoxide in the presence of NADH) and Type II photochemistry (photogeneration of singlet oxygen) were studied by measurement of oxygen consumption, 1270-nm luminescence and EPR spin trapping of the superoxide product. We studied three mouse cancer cell lines: (J774, LLC, and CT26) incubated for 24 h with fullerenes and illuminated with white light. The order of effectiveness as PS was inversely proportional to the degree of substitution of the fullerene nucleus for both the neutral and the cationic series. The monopyrrolidinium fullerene was the most active PS against all cell lines and induced apoptosis 4-6 h after illumination. It produced diffuse intracellular fluorescence when dichlorodihydrofluorescein was added as an ROS probe, suggesting a Type I mechanism for phototoxicity. We conclude that certain functionalized fullerenes have potential as novel PDT agents and phototoxicity may be mediated both by superoxide and by singlet oxygen.

Nanotechnology offers several attractive design features that have prompted its exploration for cancer diagnosis and treatment. Nanosized drugs have a large loading capacity, the ability to protect the payload from degradation, a large surface on which to conjugate targeting ligands, and controlled or sustained release. Nanosized drugs also leak preferentially into tumor tissue through permeable tumor vessels and are then retained in the tumor bed due to reduced lymphatic drainage. This process is known as the enhanced permeability and retention (EPR) effect. However, while the EPR effect is widely held to improve delivery of nanodrugs to tumors, it in fact offers less than a 2-fold increase in nanodrug delivery compared with critical normal organs, resulting in drug concentrations that are not sufficient for curing most cancers. In this Review, we first overview various barriers for nanosized drug delivery with an emphasis on the capillary wall's resistance, the main obstacle to delivering drugs. Then, we discuss current regulatory issues facing nanomedicine. Finally, we discuss how to make the delivery of nanosized drugs to tumors more effective by building on the EPR effect.

Photosystem II enriched membranes were depleted of Ca2+ and the 17- and 23-kDa polypeptides by treatment with NaCl and EGTA. The 17- and 23-kDa polypeptides were then reconstituted. This preparation was incapable of O2 evolution until Ca2+ was added. An EPR study revealed the presence of two new EPR signals. One of these is a modified S2 multiline signal with an isotropic g value of 1.96 with at least 26 hyperfine peaks (average spacing 55 G) distributed over approximately 1600 G. The other is a near-Gaussian signal with an isotropic g value of 2.004, which is attributed to a formal S3 state. Experiments involving the interconversion of these signals and the effect of Ca2+ and Sr2+ rebinding provide evidence for these assignments. From these results the following conclusions are drawn: (1) These results are consistent with our earlier demonstration that charge accumulation is blocked after formation of S3 when Ca2+ is deficient. (2) Binding of the 17- and 23-kDa polypeptides to photosystem II in the absence of Ca2+ results in the perturbation of the Mn cluster. This is taken as a further indication that the Ca2+-binding site is close to or even an integral part of the Mn cluster. (3) The S3 signal may arise from an organic free radical interacting magnetically with the Mn cluster. However, other possible origins for this signal, including the Mn cluster itself, must also be considered.

A simple method is presented for calculating the parameters of the hole model for distorted octahedral, low spin (t2g)5 complexes. In the case of negligible covalent bonding explicit formulas for the coefficients of the Kramer's doublet, (formula: see text). The two numerically largest g values define the plane and orientation of the orbital with the largest coefficient, which in turn indicates the directions of maximal unpaired spin density. The energy of eta with respect to xi (in units of lambda, the spin-orbit coupling constant) is (formula: see text). The product gzgygx, independent of axes, and positive for free electrons, is shown to be positive for tetragonal and negative for nearly octahedral complexes. It is considered positive for hemes. In this method coefficients will only be normalized when there is no covalency. For the majority of published cases they are, to about 1%. Since this discrepancy is larger than can be caused by propagated errors, covalency must be the rule. For comparative purposes A and B, uncorrected for covalency, should still be useful. Examination of published complete g tensors for five hemes shows that the largest g value is nearly normal to the heme plane. If the g values are taken positive and labelled so that gz greater than gy greater than gx, then the proper tetragonal axis is roughly normal to plane of the ring in hemes, but not in chlorins.

The measurement of pO2 in vivo using EPR has some features which have already led to very useful applications and this approach is likely to have increasingly wide and effective use. It is based on the effect of oxygen on EPR spectra which provides a sensitive and accurate means to measure pO2 quantitatively. The development of oxygen-sensitive paramagnetic materials which are very stable, combined with instrumental developments, has been crucial to the in vivo applications of this technique. The physical basis and biological applications of in vivo EPR oximetry are reviewed, with particular emphasis on the use of EPR spectroscopy at 1 GHz using particulate paramagnetic materials for the repetitive and non-invasive measurement of pO2 in tissues. In vivo EPR has already produced some very useful results which have contributed significantly to solving important biological problems. The characteristics of EPR oximetry which appear to be especially useful are often complementary to existing techniques for measuring oxygen in tissues. These characteristics include the capability of making repeated measurements from the same site, high sensitivity to low levels of oxygen, and non-invasive options. The existing techniques are especially useful for studies in small animals, where the depth of measurements is not an overriding issue. In larger animals and potentially in human subjects, non-invasive techniques seem to be immediately applicable to study phenomena very near the surface (within 10 mm) while invasive techniques have some very promising uses. The clinical uses of EPR oximetry which seem especially promising and likely to be undertaken in the near future are long-term monitoring of the status and response to treatment of peripheral vascular disease and optimizing cancer therapy by enabling it to be modified on the basis of the pO2 measured in the tumour.

Decreased toxicity via selective delivery of cancer therapeutics to tumors has become a hallmark achievement of nanotechnology. In order to be optimally efficacious, a systemically administered nanomedicine must reach cancer cells in sufficient quantities to elicit a response and assume its active form within the tumor microenvironment (e.g., be taken up by cancer cells and release a toxic component once within the cytosol or nuclei). Most nanomedicines achieve selective tumor accumulation via the enhanced permeability and retention (EPR) effect or a combination of the EPR effect and active targeting to cellular receptors. Here, we review how the fundamental physicochemical properties of a nanomedicine (its size, charge, hydrophobicity, etc.) can dramatically affect its distribution to cancerous tissue, transport across vascular walls, and retention in tumors. We also discuss how nanoparticle characteristics such as stability in the blood and tumor, cleavability of covalently bound components, cancer cell uptake, and cytotoxicity contribute to efficacy once the nanoparticle has reached the tumor's interstitial space. We elaborate on how tumor vascularization and receptor expression vary depending on cancer type, stage of disease, site of implantation, and host species, and review studies which have demonstrated that these variations affect tumor response to nanomedicines. Finally, we show how knowledge of these properties (both of the nanoparticle and the cancer/tumor under study) can be used to design meaningful in vivo tests to evaluate nanoparticle efficacy.

The interaction of A beta peptides with the lipid matrix of neuronal cell membranes plays an important role in the pathogenesis of Alzheimer's disease. By using EPR and CD spectroscopy, we found that in the presence of Cu(2+) or Zn(2+), pH, cholesterol, and the length of the peptide chain influenced the interaction of these peptides with lipid bilayers. In the presence of Zn(2+), A beta 40 and A beta 42 both inserted into the bilayer over the pH range 5.5-7.5, as did A beta 42 in the presence of Cu(2+). However, A beta 40 only penetrated the lipid bilayer in the presence of Cu(2+) at pH 5.5-6.5; at higher pH there was a change in the Cu(2+) coordination sphere that inhibited membrane insertion. In the absence of the metals, insertion of both peptides only occurred at pH < 5.5. Raising cholesterol to 0.2 mol fraction of the total lipid inhibited insertion of both peptides under all conditions investigated. Membrane insertion was accompanied by the formation of alpha-helical structures. The nature of these structures was the same irrespective of the conditions used, indicating a single low energy structure for A beta in membranes. Peptides that did not insert into the membrane formed beta-sheet structures on the surface of the lipid.

The crystal structure of an enzyme having polychlorinated-biphenyl degrading activity, the BphC enzyme from Pseudomonas sp. strain KKS102, has been solved as a free form at 1.8 A resolution. This is the first three-dimensional structure among the extradiol-type dioxygenases. Based on 34,387 reflections (10.0 to 1.8 A, completeness 87.8%), a current R-factor of 20.4% (with a free R-factor of 24.3%) was obtained with a model obeying standard geometry within 0.011 A in bond lengths and 1.91 degrees in bond angles. The BphC enzyme is a homo-octamer and each subunit is composed of two domains: Domain 1 (N-terminal part) and Domain 2 (C-terminal part). Each domain contains two repetitions of a novel folding motif (the "beta alpha beta beta beta" motif) each consisting of ca 55 amino acid residues. A single Fe ion in the active site coordinates the side-chains of three amino acid residues (His145, His209 and Glu260) and two solvent molecules. The coordination geometry is that of a square pyramid. In addition to the free form of the BphC enzyme, we have solved two three-dimensional structures of the BphC enzyme complexed with its substrates, 2,3-dihydroxybiphenyl (2,3-DHBP) or 3-methylcatechol (3-MCT). These substrates were found intact in the active site probably because of the oxidation of the Fe ion into ferric form (as judged by EPR spectra) in the present crystals. In both of the two substrate complexes, the two hydroxyl groups of the substrate, together with the three enzymatic side-chain ligands, were found to form a penta-coordinated system around the Fe ion roughly arranged in a trigonal bipyramidal configuration. The active site structures appear to be essentially consistent with the reaction mechanism proposed so far.

31P NMR revealed that the complex of p21ras with the GTP analog GppNHp.Mg2+ exists in two conformational states, states 1 and 2. In wild-type p21ras the equilibrium constant K1(12) between the two states is 1.09. The population of these states is different for various mutants but independent of temperature. The activation enthalpy delta H ++ and activation entropy delta S ++ for the conformational transitions were determined by full-exchange matrix analysis for wild-type p21ras and p21ras(S65P). For the wild-type protein one obtains delta H ++ = 89 +/- 2 kJ mol-1 and delta S ++ = 102 +/- 20 J mol-1 K-1 and for the mutant protein delta H ++ = 93 +/- 7 kJ mol-1 and delta S ++ = 138 +/- 30 J mol-1 K-1. The study of various p21ras mutants suggests that the two states correspond to different conformations of loop L2, with Tyr-32 in two different positions relative to the bound nucleotide. High-field EPR at 95 GHz suggest that the observed conformational transition does not directly influence the coordination sphere of the protein-bound metal ion. The influence of this transition on loop L4 was studied by 1H NMR with mutants E62H and E63H. There was no indication that L4 takes part in the transition described in L2, although a reversible conformational change could be induced by decreasing the pH value. The exchange between the two states is slow on the NMR time scale (< 10 s-1): at approximately pH 5 the population of the two states is equal. The interaction of p21ras-triphosphate complexes with the Ras-binding domain (RBD) of the effector protein c-Raf-1, Raf-RBD, and with the GTPase activating protein GAP was studied by 31P NMR spectroscopy. In complex with Raf-RBD the second conformation of p21ras (state 2) is stabilized. In this conformation Tyr-32 is located in close proximity to the phosphate groups of the nucleotide, and the beta-phosphate resonance is shifted upfield by 0.7 ppm. Spectra obtained in the presence of GAP suggest that in the ground state GAP does not interact directly with the nucleotide bound to p21ras and does not induce larger conformational changes in the neighborhood of the nucleotide. The experimental data are consistent with a picture where GAP accelerates the exchange process between the two states and simultaneously increases the population of state 1 at higher temperature.