Control of membrane curvature is required in many important cellular processes, including endocytosis and vesicular trafficking. Endophilin is a bin/amphiphysin/rvs (BAR) domain protein that induces vesicle formation by promotion of membrane curvature through membrane binding as a dimer. Using site-directed spin labeling and EPR spectroscopy, we show that the overall BAR domain structure of the rat endophilin A1 dimer determined crystallographically is maintained under predominantly vesiculating conditions. Spin-labeled side chains on the concave surface of the BAR domain do not penetrate into the acyl chain interior, indicating that the BAR domain interacts only peripherally with the surface of a curved bilayer. Using a combination of EPR data and computational refinement, we determined the structure of residues 63-86, a region that is disordered in the crystal structure of rat endophilin A1. Upon membrane binding, residues 63-75 in each subunit of the endophilin dimer form a slightly tilted, amphipathic alpha-helix that directly interacts with the membrane. In their predominant conformation, these helices are located orthogonal to the long axis of the BAR domain. In this conformation, the amphipathic helices are positioned to act as molecular wedges that induce membrane curvature along the concave surface of the BAR domain.

We used fluorescent probes and EPR to study the mechanism(s) underlying reactive oxygen species (ROS) production by endothelial cell mitochondria and the action of mitoquinol, a mitochondria-targeted antioxidant. ROS measured by fluorescence resulted from complex I superoxide released to the matrix and converted to H(2)O(2). In contrast, EPR largely detected superoxide generated at complex III and effluxed outward. ROS fluorescence by mitochondria fueled by the complex II substrate, succinate, was substantial but markedly inhibited by rotenone. Superoxide, detected by EPR, in succinate-fueled mitochondria was not inhibited by rotenone and likely derived from semiquinone formation at complex III. Mitoquinol decreased H(2)O(2) fluorescence by succinate-fueled mitochondria but had little effect on the EPR signal for superoxide. This was not associated with a detectable decrease in membrane potential. Mitoquinol markedly enhanced ROS fluorescence in mitochondria fueled by the complex I substrates, glutamate and malate. Inhibitor studies suggested that this occurred in complex I, at one or more Q binding pockets. The above effects of mitoquinol were determined in mitochondria isolated and subsequently exposed to the targeted antioxidant. However, similar effects were observed in mitochondria after antecedent exposure to mitoquinol/mitoquinone in culture, suggesting that the agent is retained after isolation of the organelles. In conclusion, ROS production in bovine aortic endothelial cell mitochondria results largely from reverse transport to complex I and through the Q cycle in complex III. Mitoquinol blocks ROS from reverse electron transport but increases superoxide production derived from forward transport. These effects likely occur at one or more Q binding sites in complex I.

Neurotransmitter release at the synapse requires membrane fusion. The SNARE complex, composed of the plasma membrane t-SNAREs syntaxin 1A and SNAP-25 and the vesicle v-SNARE synaptobrevin, mediates the fusion of 2 membranes. Synaptic vesicles contain unusually high cholesterol, but the exact role of cholesterol in fusion is not known. In this study, cholesterol was found to stimulate SNARE-mediated lipid mixing of proteoliposomes by a factor of 5 at a physiological concentration. Surprisingly, however, the stimulatory effect was more pronounced when cholesterol was on the v-SNARE side than when it was on the t-SNARE side. Site-directed spin labeling and both continuous wave (CW) and pulsed EPR revealed that cholesterol induces a conformational change of the v-SNARE transmembrane domain (TMD) from an open scissors-like dimer to a parallel dimer. When the TMD was forced to form a parallel dimer by the disulfide bond, the rate was stimulated 2.3-fold even without cholesterol, supporting the relevance of the open-to-closed conformational change to the fusion activity. The open scissors-like conformation may be unfavorable for fusion and cholesterol may relieve this inhibitory factor.

An exaggerated exercise pressor reflex (EPR) contributes to exercise intolerance and excessive sympatho-excitation in the chronic heart failure (CHF) state. However, the components of this reflex that are responsible for the exaggerated EPR in CHF remain unknown. To determine whether muscle afferent function is altered in CHF, we recorded the discharge of group III and IV afferents in response to static contraction, passive stretch and hindlimb intra-arterial injection of capsaicin in sham and CHF rats. We also investigated the roles of purinergic 2X receptor (P2X) and the transient receptor potential vanilloid 1 (VR1) in mediating the altered sensitivity of muscle afferents. Compared with sham rats, CHF rats exhibited greater responses of group III afferents to contraction and stretch whereas the responses of group IV afferents to contraction and capsaicin were blunted. Hindlimb intra-arterial infusion of pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (PPADS), a P2X antagonist, attenuated the responses of group III afferents to contraction and stretch in CHF rats to a greater extent than in sham rats. Western blot data showed that P2X3 receptors were significantly upregulated in doral root ganglion (DRG) of CHF rats whereas VR1 receptors were significantly downregulated. Immunohistochemical evidence showed that immunostaining of the P2X3 receptors was more intense in both IB4-positive (C-fibre) and NF200-positive (A-fibre) neurons in DRG of CHF rats whereas the immunostaining of the VR1 receptors was decreased in IB4-positive neurons. These data suggest that group III afferents are sensitized whereas group IV afferents are desensitized in CHF, which is related to the dysfunction of P2X and VR1 receptors.

Both enhanced vascular permeability and angiogenesis of tumor sustain rapid growth of tumor involving many vascular mediators and high vascular density. On the contrary, however, they can be utilized for macromolecular drug delivery to tumor. Impaired reticuloendothelial/lymphatic clearance of macromolecules from the tumor, or lack of such clearance, is another unique characteristic of tumor tissue, which results intratumor retention of macromolecular drugs thus delivered (Figure 1). Consequently, enhanced permeability and retention (EPR) effect is the basis for the selective targeting of macromolecular drugs to tumor, and the EPR concept is now utilized for selective delivery of many macromolecular anticancer agents in aqueous formation for i.v. or i.a. as well as oily formation for i.a. dosing, which is not possible for low-molecular-weight drugs because of rapid washout by capillary vascular blood flow. This EPR concept has been validated in clinical settings with hepatoma and other solid tumors. In our laboratories, several promising macromolecular anticancer drugs after SMANCS, such as PEG-XO, PEG-DAO, PEG-ZnPP, were developed, warranting further investigation for clinical application. More efficient drug delivery to tumor, especially of macromolecular drugs, may be possible by enhancing the EPR effect with the use of various vascular permeability mediators or potentiators. Suppression of the EPR effect by the use of appropriate inhibitors or antidotes, such as the bradykinin antagonist HOE 140 and protease inhibitors or NOS inhibitors, may also be possible. Thus, one may be able to suppress or retard tumor growth and tumor metastasis. Also, by suppressing vascular permeability with antidotes such as the bradykinin antagonist HOE 140, pleural fluid in lung cancer and ascitic fluid in abdominal carcinomatosis may be controlled and the clinical course of cancer patients may be improved. In summary, tumor vasculature can be an excellent target for delivery of macromolecular anticancer drugs; the most beneficial class of drugs in view of tumor-selective targeting based on the EPR effect in solid tumor as well as compliance of patients and ultimate therapeutic efficacy.

Cancer imaging requires selective high accumulation of contrast agents in the tumor region and correspondingly low uptake in healthy tissues. Here, by making use of a novel synthetic polymer to solubilize single-walled carbon nanotubes (SWNTs), we prepared a well-functionalized SWNT formulation with long blood circulation (half-life of ∼30 h) in vivo to achieve ultrahigh accumulation of ∼30% injected dose (ID)/g in 4T1 murine breast tumors in Balb/c mice. Functionalization dependent blood circulation and tumor uptake were investigated through comparisons with phospholipid-PEG solubilized SWNTs. For the first time, we performed video-rate imaging of tumors based on the intrinsic fluorescence of SWNTs in the second near-infrared (NIR-II, 1.1-1.4 μm) window. We carried out dynamic contrast imaging through principal component analysis (PCA) to immediately pinpoint the tumor within ∼20 s after injection. Imaging over time revealed increasing tumor contrast up to 72 h after injection, allowing for its unambiguous identification. The 3D reconstruction of the SWNTs distribution based on their stable photoluminescence inside the tumor revealed a high degree of colocalization of SWNTs and blood vessels, suggesting enhanced permeability and retention (EPR) effect as the main cause of high passive tumor uptake of the nanotubes.

Several monofloral Cuban honeys were analyzed to determine their total phenolic, flavonoid, ascorbic acid, amino acid, protein and carotenoid contents as well as their radical-scavenging activity and antimicrobial capacities. The total phenolic, flavonoid and carotenoid contents varied considerably, and the highest values were obtained for Linen vine (Govania polygama (Jack) Urb) honey, which is classified as an amber honey. The highest amino acid content was found in Morning glory (Ipomoea triloba L.) while Liven vine had the highest protein content. Similarly Linen vine honey had the highest antioxidant activity while the lowest was found in Christmas vine (Turbina corymbosa (L.) Raf). Ascorbic acid was absent. Hydroxyl radical formation was studied by EPR and spin trapping, and it was found in all honeys tested. The antimicrobial activity was screened using two Gram-positive and Gram-negative bacteria. S. aureus was the most sensitive microorganism while Pseudomonas aeruginosa presented higher minimum active dilution values. Bacillus subtilis and Escherichia coli were both moderately sensitive to honey antimicrobial activity. A correlation between radical-scavenging activity and total phenolic content was found. Correlation existed also between color vs phenolics content, vs flavonoid content or between phenolic vs flavonoid.

It has been hypothesized that free radical metabolism and oxygenation in living organs and tissues such as the heart may vary over the spatially defined tissue structure. In an effort to study these spatially defined differences, we have developed electron paramagnetic resonance imaging instrumentation enabling the performance of three-dimensional spectral-spatial images of free radicals infused into the heart and large vessels. Using this instrumentation, high-quality three-dimensional spectral-spatial images of isolated perfused rat hearts and rabbit aortas are obtained. In the isolated aorta, it is shown that spatially and spectrally accurate images of the vessel lumen and wall could be obtained in this living vascular tissue. In the isolated rat heart, imaging experiments were performed to determine the kinetics of radical clearance at different spatial locations within the heart during myocardial ischemia. The kinetic data show the existence of regional and transmural differences in myocardial free radical clearance. It is further demonstrated that EPR imaging can be used to noninvasively measure spatially localized oxygen concentrations in the heart. Thus, the technique of spectral-spatial EPR imaging is shown to be a powerful tool in providing spatial information regarding the free radical distribution, metabolism, and tissue oxygenation in living biological organs and tissues.

A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative Fe(B) site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by approximately 110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a approximately 100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the Fe(B) site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues.

Nanomaterials provide large surface areas, relativeto their volumes, on which to load functions. One challenge, however, has been to achieve precise control in loading multiple functionalities. Traditional bioconjugation techniques, which randomly target the surface functional groups of nanomaterials, have been found increasingly inadequate for such control, which is a drawback that may substantially slow down or prohibit the translational efforts. In the current study, we evaluated ferritin nanocages as candidate nanoplatforms for multifunctional loading. Ferritin nanocages can be either genetically or chemically modified to impart functionalities to their surfaces, and metal cations can be encapsulated in their interiors by association with metal binding sites. Moreover, different types of ferritin nanocages can be disassembled under acidic condition and reassembled at pH of 7.4, providing a facile way to achieve function hybridization. We were able to use combinations of these unique properties to produce a number of multifunctional ferritin nanostructures with precise control of their composition. We then studied these nanoparticles, both in vitro and in vivo, to evaluate their potential suitability as multimodality imaging probes. A good tumor targeting profile was observed, which was attributable to both the enhanced permeability and retention (EPR) effect and biovector mediated targeting. This, in combination with the generalizability of the function loading techniques, promises ferritin particles as a powerful nanoplatfom in the era of nanomedicine.

Among serum proteins albumin and transferrin have attracted the most interest as drug carriers in the past two decades. Prior to that, their potential use was overshadowed by the advent of monoclonal antibodies that was initiated by Milstein and Koehler in 1975. Meanwhile intensive pursuit of exploiting transferrin, but above all albumin as an exogenous or endogenous carrier protein for treating various diseases, primarily cancer, rheumatoid arthritis, diabetes and hepatitis has resulted in several marketed products and numerous clinical trials. While the use of transferrin has clinically been primarily restricted to immunotoxins, albumin-based drug delivery systems ranging from albumin drug nanoparticles, albumin fusion protein, prodrugs and peptide derivatives that bind covalently to albumin as well as physically binding antibody fragments and therapeutically active peptides are in advanced clinical trials or approved products. For treating diabetes, Levemir and Victoza that are myristic acid derivatives of human insulin or glucagon-like peptide 1 (GLP-1) act as long-acting peptides by binding to the fatty acid binding sites on circulating albumin to control glucose levels. Levemir from Novo Nordisk has already developed into a blockbuster since its market approval in 2004. Abraxane, an albumin paclitaxel nanoparticle as a water-soluble galenic formulation avoiding the use of cremophor/ethanol, transports paclitaxel through passive targeting as an albumin paclitaxel complex to the tumor site and is superior to conventional Taxol against metastatic breast cancer. INNO-206, an albumin-binding doxorubicin prodrug that also accumulates in solid tumors due to the enhanced permeability and retention (EPR) effect but releases the parent drug through acid cleavage, either intra- or extracellularly, is entering phase II studies against sarcoma. An expanding field is the use of albumin-binding antibody moieties which do not contain the fragment crystallizable (Fc) portion of, conventional immunoglobulin G (IgG) but are comprised of monovalent or bivalent light and/or heavy chains and incorporate an additional albumin-binding peptide or antibody domain. The most advanced antibody of this kind is ATN-103 (Ozoralizumab), a trivalent albumin-binding nanobody that neutralizes the pro-inflammatory tumor necrosis factor alpha (TNF-α) as a causative agent for exacerbating rheumatoid arthritis. ATN-103 is currently in multi-center phase II trials against this debilitating disease. In summary, because albumin as the most abundant circulating protein cannot only be used to improve the pharmacokinetic profile of therapeutically relevant peptides and the targeting moiety of antibodies but also for peptide-based targeting as well as low-molecular weight drugs to inflamed or malignant tissue, it is anticipated that R&D efforts of academia and the pharmaceutical industry in this field of drug delivery will prosper.

Rev-erbβ is a heme-binding nuclear hormone receptor that represses a broad spectrum of target genes involved in regulating metabolism, the circadian cycle, and proinflammatory responses. Here, we demonstrate that a thiol-disulfide redox switch controls the interaction between heme and the ligand-binding domain of Rev-erbβ. The reduced dithiol state of Rev-erbβ binds heme 5-fold more tightly than the oxidized disulfide state. By means of site-directed mutagenesis and by UV-visible and EPR spectroscopy, we also show that the ferric heme of reduced (dithiol) Rev-erbβ can undergo a redox-triggered switch from imidazole/thiol ligation (via His-568 and Cys-384, based on a prior crystal structure) to His/neutral residue ligation upon oxidation to the disulfide form. On the other hand, we find that change in the redox state of iron has no effect on heme binding to the ligand-binding domain of the protein. The low dissociation constant for the complex between Fe(3+)- or Fe(2+)-heme and the reduced dithiol state of the protein (K(d) = ∼ 20 nM) is in the range of the intracellular free heme concentration. We also determined that the Fe(2+)-heme bound to the ligand-binding domain of Rev-erbβ has high affinity for CO (K(d) = 60 nM), which replaces one of the internal ligands when bound. We suggest that this thiol-disulfide redox switch is one mechanism by which oxidative stress is linked to circadian and/or metabolic imbalance. Heme dissociation from Rev-erbβ has been shown to derepress the expression of target genes in response to changes in intracellular redox conditions. We propose that oxidative stress leads to oxidation of cysteine(s), thus releasing heme from Rev-erbβ and altering its transcriptional activity.

Melanins are complex, incompletely understood polymeric pigments that historically have been difficult to investigate with common chemical, histochemical, and physicochemical techniques. Because these pigments uniquely contain a stable population of organic free radicals, electron paramagnetic resonance (EPR) spectroscopy is a particularly effective method for studying them, and a set of qualitative EPR criteria has been established for their identification. However, a number of practical problems have arisen in applying these criteria to identify and characterize unknown pigments in relatively scarce pathological specimens, indicating that a standardized approach is needed. As reported here, a standardized EPR test for melanin based on the EPR criteria has been developed, guided by the requirements that it be sensitive, accurate, simple, and easy to interpret. It has been evaluated using the well-characterized synthetic melanin prepared by alkaline autooxidation of 5,6-dihydroxyphenylalanine (Dopa) and initially applied to the identification and characterization of an unknown pigment purified from an unusual malignant lung tumor.

The past decade has brought major advances in our knowledge of the structures and mechanisms of MAO A and MAO B, which are pharmacological targets for specific inhibitors. In both enzymes, crystallographic and biochemical data show their respective C-terminal transmembrane helices anchor the enzymes to the outer mitochondrial membrane. Pulsed EPR data show both enzymes are dimeric in their membrane-bound forms with agreement between distances measured in their crystalline forms. Distances measured between active site-directed spin-labels in membrane preparations show excellent agreement with those estimated from crystallographic data. Our knowledge of requirements for development of specific reversible MAO B inhibitors is in a fairly mature status. Less is known regarding the structural requirements for highly specific reversible MAO A inhibitors. In spite of their 70% level of sequence identity and similarities of C(alpha) folds, the two enzymes exhibit significant functional and structural differences that can be exploited in the ultimate goal of the development of highly specific inhibitors. This review summarizes the current structural and mechanistic information available that can be utilized in the development of future highly specific neuroprotectants and cardioprotectants.

Partially folded proteins, characterized as exhibiting secondary structure elements with loose or absent tertiary contacts, represent important intermediates in both physiological protein folding and pathological protein misfolding. To aid in the characterization of the structural state(s) of such proteins, a novel structure calculation scheme is presented that combines structural restraints derived from pulsed EPR and NMR spectroscopy. The methodology is established for the protein alpha-synuclein (alphaS), which exhibits characteristics of a partially folded protein when bound to a micelle of the detergent sodium lauroyl sarcosinate (SLAS). By combining 18 EPR-derived interelectron spin label distance distributions with NMR-based secondary structure definitions and bond vector restraints, interelectron distances were correlated and a set of theoretical ensemble basis populations was calculated. A minimal set of basis structures, representing the partially folded state of SLAS-bound alphaS, was subsequently derived by back-calculating correlated distance distributions. A surprising variety of well-defined protein-micelle interactions was thus revealed in which the micelle is engulfed by two differently arranged antiparallel alphaS helices. The methodology further provided the population ratios between dominant ensemble structural states, whereas limitation in obtainable structural resolution arose from spin label flexibility and residual uncertainties in secondary structure definitions. To advance the understanding of protein-micelle interactions, the present study concludes by showing that, in marked contrast to secondary structure stability, helix dynamics of SLAS-bound alphaS correlate with the degree of protein-induced departures from free micelle dimensions.

Early-phase reactions (EPRs) and late-phase reactions (LPRs) are characteristic features of bronchial asthma, although the pathogenetic mechanisms responsible for each of the responses are not fully defined. A murine model of EPRs and LPRs was developed to investigate the role of IL-5 and eosinophils in development of both responses. After initial intraperitoneal sensitization and airway challenge to ovalbumin (OVA), mice were provoked by additional exposure to OVA. An EPR, characterized by a transient increase in airway responsiveness, was observed 5-30 minutes after antigen provocation. This response was followed by an LPR that reached its maximum at 6 hours after challenge and was characterized by increased airway responsiveness and significant lung eosinophilia. The EPR was blocked by cromoglycate and albuterol, whereas the LPR was abolished by cromoglycate and hydrocortisone. Before provocation with allergen, administration of anti-IL-5 antibody prevented the influx of eosinophils into the lung tissue and abolished the LPR but not EPR. These results suggest that IL-5 and eosinophils are essential for development of the LPR, but not EPR, in this model.

KcsA is a prokaryotic potassium channel. The present study employs cysteine scanning mutagenesis and site-directed spin labeling to investigate the structure of the second transmembrane segment (residues 82-120) in functional tetrameric channels reconstituted in lipid bilayers. Spin-spin interactions are observed between nitroxide side chains at symmetry-related sites close to the 4-fold axis of symmetry. To aid in quantitative analysis of these interactions, a new diamagnetic analogue of the nitroxide side chain is used to prepare magnetically dilute samples with constant structure. Using constraints imposed by the spin-spin interactions, a packing model for this segment is deduced that is in excellent agreement with the recently reported crystal structure [Doyle, D., et al. (1998) Science 280, 69-77]. The relatively immobilized state of the nitroxide side chains suggests that the channel is rigid on the electron paramagnetic resonance time scale. Moreover, the poor sulfhydryl reactivity of the cysteine at many locations indicates that the channel is not subject to the low-frequency fluctuations that permit reaction of buried cysteines. At sites expected to be located in the pore, the accessibility of the side chains to collision with O(2) or nickel(II) ethylenediaminediacetate is low. This inaccessibility, together with the generally low mobility of the side chains throughout the sequence, makes it difficult to detect the presence of the pore based on these measurements. However, the presence of a solvated pore can be directly demonstrated using a polarity parameter deduced from the EPR spectra recorded at low temperature. These measurements also reveal the presence of a polarity gradient in the phospholipid bilayer.

Peroxynitrite mediates the oxidation of the thiol group of both cysteine and glutathione. This process is associated with oxygen consumption. At acidic pH and a cysteine/peroxynitrite molar ratio of < or = 1.2, there was a single fast phase of oxygen consumption, which increased with increasing concentrations of both cysteine and oxygen. At higher molar ratios the profile of oxygen consumption became biphasic, with a fast phase (phase I) that decreased with increasing cysteine concentration, followed by a slow phase (phase II) whose rate of oxygen consumption increased with increasing cysteine concentration. Oxygen consumption in phase I was inhibited by desferrioxamine and 5,5-dimethyl-1-pyrroline N-oxide, but not by mannitol; superoxide dismutase also inhibited oxygen consumption in phase I, while catalase added during phase II decreased the rate of oxygen consumption. For both cysteine and glutathione, oxygen consumption in phase I was maximal at neutral to acidic pH: in contrast, total thiol oxidation was maximal at alkaline pH. EPR spin-trapping studies using N-tert-butyl-alpha-phenylnitrone indicated that the yield of thiyl radical adducts had a pH profile comparable with that found for oxygen consumption. The apparent second-order rate constants for the reactions of peroxynitrite with cysteine and glutathione were 1290 +/- 30 M-1.S-1 and 281 +/- 6 M-1.S-1 respectively at pH 5.75 and 37 degrees C. These results are consistent with two different pathways participating in the reaction of peroxynitrite with low-molecular-mass thiols: (a) the reaction of the peroxynitrite anion with the protonated thiol group, in a second-order process likely to involve a two-electron oxidation, and (b) the reaction of peroxynitrous acid, or a secondary species derived from it, with the thiolate in a one-electron transfer process that yields thiyl radicals capable of initiating an oxygen-dependent radical chain reaction.

The oxidation of carotenoid upon illumination at low temperature has been studied in Mn-depleted photosystem II (PSII) using EPR and electronic absorption spectroscopy. Illumination of PSII at 20 K results in carotenoid cation radical (Car+*) formation in essentially all of the centers. When a sample which was preilluminated at 20 K was warmed in darkness to 120 K, Car+* was replaced by a chlorophyll cation radical. This suggests that carotenoid functions as an electron carrier between P680, the photooxidizable chlorophyll in PSII, and ChlZ, the monomeric chlorophyll which acts as a secondary electron donor under some conditions. By correlating with the absorption spectra at different temperatures, specific EPR signals from Car+* and ChlZ+* are distinguished in terms of their g-values and widths. When cytochrome b559 (Cyt b559) is prereduced, illumination at 20 K results in the oxidation of Cyt b559 without the prior formation of a stable Car+*. Although these results can be reconciled with a linear pathway, they are more straightforwardly explained in terms of a branched electron-transfer pathway, where Car is a direct electron donor to P680(+), while Cyt b559 and ChlZ are both capable of donating electrons to Car+*, and where the ChlZ donates electrons when Cyt b559 is oxidized prior to illumination. These results have significant repercussions on the current thinking concerning the protective role of the Cyt b559/ChlZ electron-transfer pathways and on structural models of PSII.

Although the erythropoietin receptor (EpR) lacks a tyrosine kinase consensus sequence within its proline-rich intracellular domain, addition of its ligand to Ep-responsive cells stimulates the rapid and transient tyrosine phosphorylation of a number of cellular proteins. The characterization of these phosphorylatable substrates, which include 5 major phosphoproteins with molecular masses of approximately 145, 130, 97, 72, and 56 Kd is an essential step in understanding the signal transduction pathways used by Ep. Recently, we and others have shown that the major 72-Kd tyrosine phosphorylated protein is the EpR itself. We now report, using both murine DA-3 and human MO7E cell lines engineered to express high levels of biologically responsive EpRs (and designated DA-ER and MO7-ER, respectively), that the major 56-Kd tyrosine phosphorylated protein is the recently identified SH2-containing protein, p52shc. Interestingly, in Ep-stimulated cells, anti-Shc antibodies coprecipitate the major 145-Kd tyrosine phosphorylated protein in both DA-ER and MO7-ER cells. Tyrosine phosphorylation of both proteins is detectable within 30 seconds of incubation with Ep at 37 degrees C, reaches a maximum between 2 and 5 minutes, and declines by 30 minutes. In addition, tyrosine phosphorylated Shc appears capable of associating with the activated EpR, but this could only be shown in MO7-ER cells. Lastly, as has been shown previously with the tyrosine kinase containing receptors for epidermal growth factor, platelet derived growth factor, and insulin, activation of the EpR leads to the association of p52shc with the 25-Kd polypeptide, Grb2. Taken together, our data suggest that the previously reported increases in rasGTP observed with Ep result, in part, from the tyrosine phosphorylation of Shc and its association with Grb2 and/or a tyrosine phosphorylated 145-Kd protein.

Gangliosides from beef brain have been spin-labeled using two different attaching groups and employed to investigate the physical nature of ganglioside behaviour in membranes. Results obtained using EPR spectroscopy indicate that, in phosphatidylcholine bilayers at physiological pH, ganglioside oligosaccharide chains are quite mobile and show a measurable tendency towards cooperative interaction amongst themselves. We suggest that the source of this interaction is the formation of H-bonds between sugar residues in adjacent ganglioside molecules. We present evidence that physiological (extracellular fluid) levels of Ca2+ and Mg2+ lead to cross-linking and condensing of ganglioside headgroups by complexing sialic acid carboxyl residues. Ganglioside headgroup interactions are not very sensitive to changes in the buffer ionic strength, suggesting that ionic interactions are of minor importance. We have found no measurable tendency for headgroup carbohydrate to penetrate hydrophobic regions of lipid bilayers. EPR spectroscopy was also used to follow the interaction of spin-labeled gangliosides with the glycoprotein, glycophorin, and with intact BHK cells. We suggest that these carbohydrate-based interactions should contribute significantly to the properties of the eucaryotic cell glycocalyx. We predict that laterally mobile carbohydrate-bearing components of cell surface will show a tendency to cluster about complex glycoprotein arrays, especially if the species involved bear accessible carboxylic acid functions.

The class I E. coli ribonucleotide reductase, composed of homodimers of R1 and R2, catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. The reduction process involves the tyrosyl radical on R2 that generates a transient thiyl radical on R1 over a proposed distance of 35 A. A mechanism-based inhibitor, 2'-azido-2'-deoxyuridine-5'-diphosphate, that reduces the tyrosyl radical on R2 and forms a nitrogen-centered radical on R1 has provided a method to measure the diagonal distance between the two subunits. PELDOR and DQC paramagnetic resonance methods give rise to a distance of 48 A, similar to that calculated from a docking model of the R1 and R2 structures.

Previously, we developed a multifunctional envelope-type nano device (MEND) for efficient delivery of nucleic acids. For tumor delivery of a MEND, PEGylation is a useful method, which confers a longer systemic circulation and tumor accumulation via the enhanced permeability and retention (EPR) effect. However, PEGylation inhibits cellular uptake and subsequent endosomal escape. To overcome this, we developed a PEG-peptide-DOPE (PPD) that is cleaved in a matrix metalloproteinase (MMP)-rich environment. In this study, we report on the systemic delivery of siRNA to tumors by employing a MEND that is modified with PPD (PPD-MEND). An in vitro study revealed that PPD modification accelerated both cellular uptake and endosomal escape, compared to a conventional PEG modified MEND. To balance both systemic stability and efficient activity, PPD-MEND was further co-modified with PEG-DSPE. As a result, the systemic administration of the optimized PPD-MEND resulted in an approximately 70% silencing activity in tumors, compared to non-treatment. Finally, a safety evaluation showed that the PPD-MEND showed no hepatotoxicity and innate immune stimulation. Furthermore, in a DNA microarray analysis in liver and spleen tissue, less gene alternation was found for the PPD-MEND compared to that for the PEG-unmodified MEND due to less accumulation in liver and spleen.

An in vivo tumor targeting test of glycol-chitosan nanoaggregates was carried out with FITC-conjugated glycol-chitosan nanoaggregates (FTC-GC) and the doxorubicin conjugated glycol-chitosan (GC-DOX). To investigate its biodistribution in tumor-bearing rats, glycol-chitosan was labeled with fluorescein isothiocyanate (FITC), which formed nanoaggregates with a diameter of about 250 nm in aqueous media. GC-DOX nanoaggregates containing acid-sensitive spacers were prepared. The GC-DOX formed micelle-like nanoaggregates spontaneously in aqueous media. GC-DOX nanoaggregates had a narrow and unimodal size distribution, and its hydrodynamic diameter measured by dynamic light scattering ranged from 250 to 300 nm. A loading content of doxorubicin into GC-DOX nanoaggregates as high as 38%, with 97% loading efficiency, could be obtained using a physical entrapment method. A tumor-bearing animal model was developed by inoculating tumor cells into the back of a rat. The FTC-GC nanoaggregates were injected into the tail vein of tumor-bearing rats and their tissue distribution was examined. The FTC-GC nanoaggregates were distributed mainly in kidney, tumor and the liver and were scarcely observed in other tissues. They were maintained at a high level for 8 days and their distribution in tumor tissues increased gradually. This suggests that chitosan nanoaggregates accumulate passively in the tumor tissue due to the enhanced permeability and retention (EPR) effect. Doxorubicin loaded GC-DOX nanoaggregates (DOX/GC-DOX) were injected into the tail vein of tumor-bearing rats and their anti-tumor effect was examined. Tumor growth was suppressed over 10 days.