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We previously characterized a H + transport pathway in medullary thick ascending limb nephron segments that when activated stimulated the production of superoxide by nicotinamide adenine dinucleotide phosphate oxidase. Importantly, the activity of this pathway was greater in Dahl salt-sensitive rats than salt-resistant (SS.13 BN ) rats, and superoxide production was enhanced in low Na + media. The goal of this study was to determine the molecular identity of this pathway and its relationship to Na + . We hypothesized that the voltage-gated proton channel, HV1, was the source of superoxide-stimulating H + currents. To test this hypothesis, we developed HV1 −/− null mutant rats on the Dahl salt-sensitive rat genetic background using zinc-finger nuclease gene targeting. HV1 could be detected in medullary thick limb from wild-type rats. Intracellular acidification using an NH 4 Cl prepulse in 0 sodium/BaCl 2 containing media resulted in superoxide production in thick limb from wild-type but not HV1 −/− rats ( P I 0.005 versus 0.002 U/s, P =0.046, respectively). Superoxide production was enhanced by low intracellular sodium (−/− rats compared with wild-type Dahl salt-sensitive rats. We conclude that HV1 is expressed in medullary thick ascending limb and promotes superoxide production in this segment when intracellular Na + is low. HV1 contributes to the development of hypertension and renal disease in Dahl salt-sensitive rats.

An intelligent constant false alarm rate (CFAR) processor to perform adaptive threshold target detection is presented. It employs a composite approach based on the well-known cell averaging CFAR (CA-CFAR), smallest of CFAR (SO-CFAR), and greatest of CFAR (GO-CFAR) processors. Data in the reference window is used to compute a second-order statistic called the variability index (VI) and the ratio of the means of the leading and lagging windows. Based on these statistics, the VI-CFAR dynamically tailors the background estimation algorithm. The VI-CFAR processor provides low loss CFAR performance in a homogeneous environment and also performs robustly in nonhomogeneous environments including multiple targets and extended clutter edges.

Intercropping is an agricultural system widely practiced in the tropics and becoming more widespread in temperate regions. The vast majority of varieties currently grown in intercrop have been developed in and for monoculture, although some breeding programs specifically focused on intercrop systems have been initiated. It is not clear if selection in monoculture is adequate to identify varieties adapted to intercrop, nor is it clear if varieties selected for intercrop performance with one companion will be adapted to another crop combination. The aims of this study were to determine the extent to which selection of corn in monoculture would identify types adapted to growth in corn—bean or corn—clover intercrop, and to determine how selection in one of the intercrops (corn—bean or corn—clover) would compare with selection in the other. The corn used consisted of two groups that had been selected in monoculture, and two that had been selected in corn—bean intercrop. All groups were evaluated in monoculture, corn-bean intercrop, and corn—clover intercrop. Analysis of variance showed that the ranking of the four selection groups was not significantly different when the two intercrops were compared, but was significantly different (P < 0.05 for comparison of clover intercrop and monoculture, and P = 0.07 for comparison of bean intercrop and monoculture) when either of the two intercrops was compared with monoculture. Correlation analysis of corn traits and corn yield in the three cropping systems revealed more similarities between the intercrops than between either intercrop and monoculture. Plant height and leaf area index were more strongly negatively correlated with days to flower in both intercrops than in monoculture. Correlation analysis also revealed some differences between the intercrops, particularly with bean and clover yields. Clover yields were not adversely affected by early growth and maturity of corn, but bean yields were. We conclude that selection of corn in monoculture is not ideal when lines adapted to cornclover intercrop or corn—bean intercrop are desired. Furthermore, selection in either intercrop will identify corn types adapted to the other, as early vigor and maturity allow best corn performance in both crop combinations. Thus, individualized breeding programs may not be required to adapt a crop to growth in association with relatively different companions.

X-ray-induced damage in Estane® 5703, a segmented poly(ester urethane) copolymer, has been investigated by thermally stimulated luminescence, radioluminescence, optical absorption and electron spin resonance techniques over a wide temperature range. Some glow curve features correlate with molecular motion of the polymer, as determined by viscoelastic measurements, indicating charge detrapping via thermal destruction of cavity traps. Spectral emission consists of four Lorentzian bands with maxima at 2.38, 2.55, 2.74 and 2.93 eV, which are attributed to triplet-to-singlet (π, π∗) electronic transitions of the phenyl group in the polymer hard segment. Absorption peaks at 3.97, 4.29 and 4.46 eV are also assigned to (π, π∗) transitions of the phenyl group. Several radicals with overlapping resonances are induced at low temperature, which, with increasing temperature, evolve into the relatively stable peroxy radical at room temperature.

Summary Thirty-six thousand four hundred barrels of CO2 gelled foam were successfully placed via an injector well into the Rangely Weber Sand Unit in Colorado. The treatment objectives were (1) to improve volumetric conformance in this CO2 flood by reducing excessive CO2 breakthrough through fractures, and (2) to increase oil recovery from the associated producers. Local reservoir characteristics indicate the need for a large-volume treatment to achieve these goals. The required treatment volume is beyond the economic limits of standard gel systems. Foam gel technology is one way to economically achieve in-depth conformance improvement at Rangely by replacing 60 to 80% of the liquid phase by the less expensive CO2 phase. A gelled foam system was specifically designed for application to Rangely conformance problems. The field-tested surfactant/gel system was designed according to the following criteria: (1) to produce strong and robust gelled foams under the harsh pH conditions of a CO2 flood, (2) to provide enough gelation delay to achieve the injection of a large foam volume with manageable injectivity reduction, and (3) to considerably reduce the unit cost of the treatment fluid relative to standard non-foamed gel systems. This paper describes the methodology used to design and test the optimum gelled foam system for Rangely. Laboratory results are presented to support the chemicals system selection, including gelation kinetics experiments, surfactant selection, and corefloods with supercritical CO2 at field conditions. The candidate well selection process is described, including injection profile surveys, offset well response, and bypassed reserves calculation. Data taken during the injection phase of the program, including injectivity history and on-site quality control monitoring of the chemical system behavior, are given. Finally a preliminary assessment of the impact of the treatment on CO2 cycling rates and incremental oil production is presented.

We used reverse transcription–coupled PCR to produce a high-resolution temporal map of fluctuations in mRNA expression of 112 genes during rat central nervous system development, focusing on the cervical spinal cord. The data provide a temporal gene expression “fingerprint” of spinal cord development based on major families of inter- and intracellular signaling genes. By using distance matrices for the pair-wise comparison of these 112 temporal gene expression patterns as the basis for a cluster analysis, we found five basic “waves” of expression that characterize distinct phases of development. The results suggest functional relationships among the genes fluctuating in parallel. We found that genes belonging to distinct functional classes and gene families clearly map to particular expression profiles. The concepts and data analysis discussed herein may be useful in objectively identifying coherent patterns and sequences of events in the complex genetic signaling network of development. Functional genomics approaches such as this may have applications in the elucidation of complex developmental and degenerative disorders.

Significance: NOX2 is important for host defense, and yet is implicated in a large number of diseases in which inflammation plays a role in pathogenesis. These include acute and chronic lung inflammatory diseases, stroke, traumatic brain injury, and neurodegenerative diseases, including Alzheimer's and Parkinson's Diseases. Recent Advances: Recent drug development programs have targeted several NOX isoforms that are implicated in a variety of diseases. The focus has been primarily on NOX4 and NOX1 rather than on NOX2, due, in part, to concerns about possible immunosuppressive side effects. Nevertheless, NOX2 clearly contributes to the pathogenesis of many inflammatory diseases, and its inhibition is predicted to provide a novel therapeutic approach. Critical Issues: Possible side effects that might arise from targeting NOX2 are discussed, including the possibility that such inhibition will contribute to increased infections and/or autoimmune disorders. The state of the field with regard to existing NOX2 inhibitors and targeted development of novel inhibitors is also summarized. Future Directions: NOX2 inhibitors show particular promise for the treatment of inflammatory diseases, both acute and chronic. Theoretical side effects include pro-inflammatory and autoimmune complications and should be considered in any therapeutic program, but in our opinion, available data do not indicate that they are sufficiently likely to eliminate NOX2 as a drug target, particularly when weighed against the seriousness of many NOX2-related indications. Model studies demonstrating efficacy with minimal side effects are needed to encourage future development of NOX2 inhibitors as therapeutic agents. Antioxid. Redox Signal. 23, 375–405.

Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca2+-dependent (cNOS) or Ca2+-independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [3H]NG-nitro-L-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin's ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors.

The response of axillary buds to floral stimulus activity in stem pieces was examined in two near-isogenic cultivars of tobacco that differ in the recessive maryland mammoth (mm) allele, which confers short-day behavior. All axillary buds from day-neutral plants assayed on six-internode stem pieces made few nodes (less than 20) before flowering, while axillary buds from plants homozygous for mm assayed on six-internode stem pieces either did not flower in noninductive conditions or made many nodes before flowering in inductive conditions. About 80% of day-neutral axillary buds grafted onto dayneutral stem pieces did not respond to floral stimulus in stem pieces, indicating that the floral stimulus in stem pieces is ephemeral. In other graft combinations, the proportion of axillary buds that did respond to floral stimulus in stem pieces was substantially reduced from the 20% of day-neutral buds on day-neutral stem pieces that responded. These results indicate that the mm allele probably reduces both the amount of floral stimulus activity in stem pieces and the competence of axillary buds to respond.

Summary Systematic time-lapse pulsed-neutron capture (PNC) logging was conducted during a 2-year steam-foam mechanistic field trial in the steamflooded Monarch reservoir, which is composed of interbedded sandstones, conglomerates, siltstones, and diatomaceous mudstones. Observation wells were drilled with one continuously cored through the reservoir interval and two emptied to facilitate better temperature logging. Uncalibrated PNC log responses in the air-filled wells were normalized to water-filled conditions and were used to generate a series of steam/gas saturation profiles that indicate clearly the dynamics of the fluid and foam in the reservoir during the experiment. Correlations between neutron capture cross sections (sigma), mineralogy, and rock chemistry from core samples were examined for additional use of the PNC logs for steamflood reservoir characterization. Sigma was found to increase with increasing clay/mica content and diatomite content. This finding may result in improved stratigraphic correlations of the more laterally continuous diatomaceous mudstones based on PNC logs. The PNC log data from the field trial, when used in conjunction with core, temperature, and pressure data, were critical in developing a better understanding of foam generation and propagation in the reservoir. Furthermore, they minimized the time and cost required to complete the field trial successfully. They also led to opportunities for vertical expansion of steamflooding in the area.

Precise spatiotemporal regulation of O2(-)-generating NADPH oxidases (Nox) is a vital requirement. In the case of Nox1-3, which depend on the small GTPase Rac, acceleration of GTP hydrolysis by GTPase activating protein (GAP) could represent a feasible temporal control mechanism. Our goal was to investigate the molecular interactions between RacGAPs and phagocytic Nox2 in neutrophilic granulocytes. In structural studies we revealed that simultaneous interaction of Rac with its effector protein p67(phox) and regulatory protein RacGAP was sterically possible. The effect of RacGAPs was experimentally investigated in a cell-free O2(-)-generating system consisting of isolated membranes and recombinant p47(phox) and p67(phox) proteins. Addition of soluble RacGAPs decreased O2(-) production and there was no difference in the effect of four RacGAPs previously identified in neutrophils. Depletion of membrane-associated RacGAPs had a selective effect: a decrease in ARHGAP1 or ARHGAP25 level increased O2(-) production but a depletion of ARHGAP35 had no effect. Only membrane-localized RacGAPs seem to be able to interact with Rac when it is assembled in the Nox2 complex. Thus, in neutrophils multiple RacGAPs are involved in the control of O2(-) production by Nox2, allowing selective regulation via different signaling pathways.

The human voltage-gated proton channel, hH V 1, appears to exist mainly as a dimer. Teleologically, this is puzzling because each protomer retains the main properties that characterize this protein: proton conduction that is regulated by conformational (channel opening and closing) changes that occur in response to both voltage and pH. The H V 1 dimer is mainly linked by C-terminal coiled-coil interactions. Several types of mutations produce monomeric constructs that open approximately five times faster than the wild-type dimeric channel but with weaker voltage dependence. Intriguingly, the quintessential function of the H V 1 dimer, opening to allow H + conduction, occurs cooperatively. Both protomers undergo a conformational change, but both must undergo this transition before either can conduct. The teleological purpose of dimerization may be to steepen the voltage dependence of channel opening, at least in phagocytes. In other cells, the purpose is not understood. Finally, several single-celled species have H V that are likely monomeric.

The topological similarity of voltage-gated proton channels (H(V)1s) to the voltage-sensing domain (VSD) of other voltage-gated ion channels raises the central question of whether H(V)1s have a similar structure. We present the construction and validation of a homology model of the human H(V)1 (hH(V)1). Multiple structural alignment was used to construct structural models of the open (proton-conducting) state of hH(V)1 by exploiting the homology of hH(V)1 with VSDs of K(+) and Na(+) channels of known three-dimensional structure. The comparative assessment of structural stability of the homology models and their VSD templates was performed using massively repeated molecular dynamics simulations in which the proteins were allowed to relax from their initial conformation in an explicit membrane mimetic. The analysis of structural deviations from the initial conformation based on up to 125 repeats of 100-ns simulations for each system reveals structural features consistently retained in the homology models and leads to a consensus structural model for hH(V)1 in which well-defined external and internal salt-bridge networks stabilize the open state. The structural and electrostatic properties of this open-state model are compatible with proton translocation and offer an explanation for the reversal of charge selectivity in neutral mutants of Asp(112). Furthermore, these structural properties are consistent with experimental accessibility data, providing a valuable basis for further structural and functional studies of hH(V)1. Each Arg residue in the S4 helix of hH(V)1 was replaced by His to test accessibility using Zn(2+) as a probe. The two outermost Arg residues in S4 were accessible to external solution, whereas the innermost one was accessible only to the internal solution. Both modeling and experimental data indicate that in the open state, Arg(211), the third Arg residue in the S4 helix in hH(V)1, remains accessible to the internal solution and is located near the charge transfer center, Phe(150).

In 1972, J. Woodland Hastings and colleagues predicted the existence of a proton selective channel (HV1) that opens in response to depolarizing voltage across the vacuole membrane of bioluminescent dinoflagellates and conducts protons into specialized luminescence compartments (scintillons), thereby causing a pH drop that triggers light emission. HV1 channels were subsequently identified and demonstrated to have important functions in a multitude of eukaryotic cells. Here we report a predicted protein from Lingulodinium polyedrum that displays hallmark properties of bona fide HV1, including time-dependent opening with depolarization, perfect proton selectivity, and characteristic ΔpH dependent gating. Western blotting and fluorescence confocal microscopy of isolated L. polyedrum scintillons immunostained with antibody to LpHV1 confirm LpHV1's predicted organellar location. Proteomics analysis demonstrates that isolated scintillon preparations contain peptides that map to LpHV1. Finally, Zn2+ inhibits both LpHV1 proton current and the acid-induced flash in isolated scintillons. These results implicate LpHV1 as the voltage gated proton channel that triggers bioluminescence in L. polyedrum, confirming Hastings' hypothesis. The same channel likely mediates the action potential that communicates the signal along the tonoplast to the scintillon.

The voltage gated proton channel, HV1, has four transmembrane segments and bears a striking resemblance to the voltage-sensing domain (VSD) of other voltage gated ion channels. One difference is that the human HV1, hHV1, has only three Arg residues in its S4 helix, Arg205 (R1), Arg208 (R2), and Arg211 (R3). Cys scanning indicates that S4 of hHV1 moves outward during channel opening (Gonzalez et al, 2010. Nat. Struct. Mol. Biol. 17:51-56). To explore the extent of this movement, we replaced each Arg individually with His, and probed accessibility by applying Zn2+ externally or internally. All mutations were carried out in a background lacking the two His residues (H140 & H193) that are responsible for inhibition of WT channels by Zn2+ (Ramsey et al., 2006. Nature 440:1213-1216). Both R1 and R2 were accessible to Zn2+ from the external solution. R3 was not accessible from the external solution, but was accessible from the internal solution, and surprisingly, remained accessible in the open state. In contrast with analogous studies of the Shaker K channel VSD (Starace & Bezanilla, 2004. Nature 427:548-553), none of the Arg→His mutants was a hyperpolarization-activated proton channel. Molecular dynamics simulations produced a similar picture of the open hHV1 channel. At the selectivity filter, Asp112 forms a salt bridge with Arg208. R1 and R3 participate in external and internal charge clusters, respectively, that stabilize the open state. Existing evidence thus suggests that the S4 helix of hHV1 moves outward during channel opening, qualitatively like S4 in K+ channels, but the extent of this movement is substantially less. This difference may reflect the fact that the conduction pathway in other channels resides in a physically separate domain (S5-S6), whereas in hHV1 it is within the VSD itself.

The voltage gated proton channel, HV1, is the most selective ion channel known, and plays an important role in innate immunity, human sperm maturation, tumorigenesis, and dinoflagellate bioluminescence, to name a few examples. When the gene was identified in 2006, HV1 was found to be homologous to the voltage sensor domains of classic voltage gated cation channels; these homologous domains do not conduct ions. Phylogenetic analysis of VSDs showed that HV1, voltage sensing phosphatases, and c15orf27 are three twigs that occupy a branch distinct from other voltage gated ion channels, and that this branch is part of the larger group comprising voltage gated sodium and calcium channels, which split from potassium channels at least a billion years ago. We used a combination of sequence and phylogenetic analysis, structural modeling, and electrophysiological experimentation to identify the proton selectivity filter of HV1, an important milestone in elucidating the functional components of the protein. We proposed a “signature sequence” that defines all presently known HV1, and used it to identify the first dinoflagellate HV1, despite only 15% identity with hHV1. We combined molecular dynamics and sampling methods with other approaches, to help us choose and experimentally validate a refined model that we are using to help us discover the determinants of proton specific conduction.

Sulfate and sulfate salts are not very soluble in borosilicate waste glass. When sulfate is present in excess it can form water soluble secondary phases and/or a molten salt layer (gall) on the melt pool surface which is purported to cause steam explosions in slurry fed melters. Therefore, sulfate can impact glass durability while formation of a molten salt layer on the melt pool can impact processing. Sulfate solubility has been shown to be compositionally dependent in various studies, (e.g. , B2O3, Li2O, CaO, MgO, Na2O, and Fe2O3 were shown to increase sulfate solubility while Al2O3 and SiO2 decreased sulfate solubility). This compositional dependency is shown to be related to the calculated melt viscosity at various temperatures and hence the melt polymerization.

Voltage gated proton channels (HV1) are the most selective channels known, with no detectable permeability to any ion besides H+. We recently identified the selectivity filter of the human voltage gated proton channel (hHV1). Mutation of an aspartate residue, Asp112, in the middle of the S1 transmembrane domain resulted in loss of proton specificity. Surprisingly, mutant channels were anion selective. Cation substitution did not affect Vrev at all. Replacing CH3SO3- by Cl- shifted Vrev negatively, showing that that Cl- is more permeable than CH3SO3-. Dilution of the bath solution by 90% with isotonic sucrose does not change pH (or pOH), but shifted the measured reversal potential positively, in both Cl- and CH3SO3- bath solutions, indicating anion selectivity. However, although 10-fold reduction in ionic strength produced large Vrev (reversal potential) shifts of up to +40 mV, it did not shift Vrev by +57 mV, as expected for pure anion selectivity. The sub-Nernstian shift means that the mutant channels must be permeable to either H+ or OH-, the only ions whose concentration does not change. Because EH and EOH are identical, it is difficult to distinguish these species. We will present and discuss several types of evidence, most of which suggests that OH- and not H+ is permeant in mutant hHV1 channels.

Voltage gated proton channels (HV1) are the most selective channels known, with no detectable permeability to any ion besides H+. Two classes of proton selectivity mechanisms have been proposed: (a) frozen water wires, and (b) a titratable residue in the permeation pathway. We have identified the selectivity filters of proton channels in two evolutionarily distant species, Karlodinium veneficum (a dinoflagellate) and Homo sapiens. In both, selectivity is imparted by the presence of an aspartate residue in the middle of the S1 transmembrane domain: Asp112 in human hHV1and Asp51 in Karlodinium kHV1. Neutralizing mutations of this key Asp residue result in loss of proton specificity. Among mutants tested, only Glu replacement of the critical Asp retained selectivity. To our complete surprise, mutant channels were anion selective in both species. Astonishingly, His at this key location also compromised proton specific conduction - His mutants in both species were permeable to Cl-. Given the strong similarity of hHV1 and the VSD (voltage-sensing domain) of voltage gated K+ and Na+ channels, we expected that a His residue located at the constriction of the channel would promote proton conduction. This expectation was based largely on the elegant studies of Starace and Bezanilla (2001, J. Gen. Physiol. 117:469-490; 2004, Nature. 427:548-553) who showed that Arg→His replacement in S4 of the Shaker K+ channel VSD results in proton conduction. Evidently, proton specific conduction in voltage gated proton channels requires an acidic group at the selectivity filter. That the same phenomenology (Asp or Glu are required, other mutants including His are anion permeable) occurred in such disparate species as dinoflagellate and human (only 15% identity in a global, pair-wise alignment) suggests that these features reflect universal properties of the proton selectivity mechanism.

Fogel and Hastings (1972, P.N.A.S. USA 69:690-693) first hypothesized the existence of voltage gated proton channels in bioluminescent dinoflagellates, where they were postulated to trigger the flash. The flash originates in discrete organelles termed scintillons (Nicolas et al., 1987 J. Cell Biol. 105: 723-735.) that contain luciferase and its substrate, luciferin. Luciferase is activated, and luciferin is released from a binding protein, at low pH. Proton channels in the membranes of the scintillon were postulated to conduct H+ from the acidic central flotation vacuole into the scintillon, activating luciferase and releasing luciferin, causing a bioluminescent flash. We have recently cloned a voltage gated proton channel (kHV1) from a non-bioluminescent dinoflagellate, Karlodinium veneficum (Smith et al., 2011 P.N.A.S.in-press). In heterologous expression systems, this channel exhibits a negative Vthreshold so that inward current is activated, consistent with the proposed role of allowing proton flux from the vacuole into the scintillon. The H+ current of heterologously expressed kHV1 is inhibited by Zn2+, but at much higher concentrations than in mammalian channels. We have developed a method to isolate functional scintillons from the bioluminescent species Noctiluca miliaris through gentle homogenization and sucrose density gradient centrifugation. The density of the scintillon fraction was found to be 1.213 g/cm3. Reducing the pH of the scintillon suspension to below pH 6 elicits a luminescent flash. Addition of divalent metals to the suspension diminishes the intensity of the flash. Zn2+, Ni2+, Cu2+, Co2+, and Cd2+ at millimolar concentrations abolished the luminescence from isolated scintillons. The sensitivity of scintillon luminescence to divalent metals is consistent with the idea that proton selective channels in the scintillon membrane mediate the activation of the flash.

An introduction to the physical background of the various aspects and commonly encountered terminology encountered in NMR experiments is presented. Illustrations using selected examples are given of contributions NMR techniques already make to the field of non-destructive testing and evaluation.

A decade before the first voltage-clamp study of voltage gated proton channels, Fogel & Hastings (1972, Proc. Natl. Acad. Sci. USA 69:690-693) proposed the existence of voltage gated proton channels in dinoflagellates, where they are thought to mediate proton flux from vacuole into the scintillon, triggering a bioluminescent flash in response to mechanical stimulation. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology of key regions of known proton channel genes. K. veneficum is a predatory, non-bioluminescent dinoflagellate notorious for producing a variety of toxins (Sheng et al, 2010, Proc. Natl. Acad. Sci. USA 107:2082-2087); its blooms coincide with fish kills in the coastal waters of four continents. This species was recently proposed as a source of biodiesel production (Fuentes-Grunewald et al, 2009, J. Ind. Microbiol. Biotechnol. 36:1215-1224). We have expressed the Karlodinium gene heterologously in both HEK-293 and COS-7 mammalian cell lines. Patch clamp studies confirm that this gene codes for a genuine voltage gated proton channel that is activated by depolarization and is extremely selective for protons. Similar to other known voltage gated proton channels, the gH-V relationship shifts positively when pHo is decreased or pHi is increased. The time constant of activation (τact) exhibits non-monotonic voltage dependence, increasing with depolarization just above Vthreshold and then decreasing with larger depolarization. In most respects, the properties of Karlodinium proton channels qualitatively resemble those of other proton channels, but a number of distinctive properties are currently under investigation.

Three polyetherpolyurethanes of known chemistry and mechanical properties were characterized by solid state nuclear magnetic resonance and small angle X-ray scattering in order to examine their morphologies. Polyetherpolyurethanes consist of hard and soft segments which can phase separate if the percentage of hard segment is large enough. It is postulated that the degree of phase separation which dictates morphology is of primary importance to the wear behaviour of these materials. In an attempt to establish this relationship, the effect of a three hour anneal at 155 ± 5 °C on the morphologies and abrasion resistance of injection moulded samples is being investigated.

Magnetic-relaxation experiments were performed on Bi[sub 2]Sr[sub 2]CaCu[sub 2]O[sub [ital x]] single crystals with the direction of the field parallel to the [ital ab] plane. Based on the relaxation data, we have obtained relationships between the activation energy [ital U] and the current density [ital j] by an approach we developed previously. We found that the activation energy has a logarithmic dependence on [ital j] in a wide regime of driving force. It has been reported that CuO[sub 2] planes in high-[ital T][sub [ital c]] superconductors can act as strong intrinsic pinning centers and that the relation [ital U][similar to][ital U][sub 0]ln([ital j][sub [ital c]]/[ital j]) may describe such a pinning mechanism. Our experimental results have shown good agreement with such a physical model of intrinsic flux pinning.

Magnetic relaxation studies have been performed on a Bi2Sr2CaCu2Op single crystal for field orientations parallel and perpendicular to the c-axis. The current density dependence of the effective flux-creep activation energy is derived from the flux-creep data through an expansion of the effective activation energy U about a magnetization current Jo. Although the current densities are much lower in the H∥ab orientation, the overall U(J) behaviors are similarly logarithmic for both field orientations. We discuss the nature of the effective activation energy and the so-called “apparent activation energy”, Uo, contrasting them to the actual activation energy, Up, and our data is used to illustrate problems pith the definition of Uo. A value ≈ 764 is found for the ratio of the critical vortex current densities in the tpo field orientations, {Jc}ab{Jc}c.

The voltage-gated proton channel exists as a dimer, although each protomer has a separate conduction pathway, and when forced to exist as a monomer, most major functions are retained. However, the proton channel protomers appear to interact during gating. Proton channel dimerization is thought to result mainly from coiled-coil interaction of the intracellular C-termini. Several types of evidence are discussed that suggest that the dimer conformation may not be static, but is dynamic and can sample different orientations. Zn(2+) appears to link the protomers in an orientation from which the channel(s) cannot open. A tandem WT-WT dimer exhibits signs of cooperative gating, indicating that despite the abnormal linkage, the correct orientation for opening can occur. We propose that C-terminal interaction functions mainly to tether the protomers together. Comparison of the properties of monomeric and dimeric proton channels speaks against the hypothesis that enhanced gating reflects monomer-dimer interconversion.

By targeting redox-sensitive amino acids in signaling proteins, the NADPH oxidase (Nox) family of enzymes link reactive oxygen species to physiological processes. We previously analyzed the sequences of 107 Nox enzymes and identified conserved regions that are predicted to have important functions in Nox structure or activation. One such region is the cytosolic B-loop, which in Nox1-4 contains a conserved polybasic region. Previous studies of Nox2 showed that certain basic residues in the B-loop are important for activity and translocation of p47(phox)/p67(phox), suggesting this region participates in subunit assembly. However, conservation of this region in Nox4, which does not require p47(phox)/p67(phox), suggested an additional role for the B-loop in Nox function. Here, we show by mutation of Nox4 B-loop residues that this region is important for Nox4 activity. Fluorescence polarization detected binding between Nox4 B-loop peptide and dehydrogenase domain (K(d) = 58 +/- 12 nm). This interaction was weakened with Nox4 R96E B-loop corresponding to a mutation that also markedly decreases the activity of holo-Nox4. Truncations of the dehydrogenase domain localize the B-loop-binding site to the N-terminal half of the NADPH-binding subdomain. Similarly, the Nox2 B-loop bound to the Nox2 dehydrogenase domain, and both the Nox2 and Nox4 interactions were dependent on the polybasic region of the B-loop. These data indicate that the B-loop is critical for Nox4 function; we propose that the B-loop, by binding to the dehydrogenase domain, provides the interface between the transmembrane and dehydrogenase domains of Nox enzymes.

11B, 27Al and 29Si magic angle spinning NMR results are reported for the boroaluminosilicate mineral grandidierite (Mg, Fe)Al3SiBO9. Three distinct aluminium sites are identified, two AlO6 and one AlO5. Despite overlap of the centrebands from these sites the use of three magnetic fields (9.4, 11.7 and 14.1 T) allows unambiguous values for the isotropic chemical shift (δiso), quadrupolar coupling constant (Cq) and quadrupolar asymmetry parameter (η) to be deduced for each site. The NMR spectrum from the AlO5 site is simulated with parameters Cq = 8.7 ± 0.1 MHz, η = 0.95 ± 0.05 and δiso = 41 ± 1 ppm which are compared to values from other well-defined AlO5 units.

A detailed 91Zr NMR investigation is presented of the five component (cubic, monoclinic, tetragonal, orthorhombic and delta) phase mixture in the transformation toughened engineering ceramic, magnesia-partially-stabilised zirconia (MgPSZ). The phases are distinguished on the basis of the quadrupole interaction displayed in the powder pattern of the 91Zr(1/2, -1/2) transition. This paper reports: (i) the first characterisation of the magnesia-fully-stabilised cubic phase at the eutectic composition (13.5 mol% MgO); (ii) the observation of a poorly ordered tetragonal phase on fast cooling ZrO2 (9.3 mol% MgO) from the cubic phase field at 1720 degrees C, and the subsequent growth and ordering of the tetragonal phase precipitates due to further annealing; (iii) the observation of the (partial) transformation of the cubic phase to the ordered delta-phase (Mg2Zr5O12) on annealing MgPSZ at 1100 degrees C for 8 h; and (iv) the observation of the transformation of the tetragonal phase into the orthorhombic phase after cooling in liquid nitrogen, and the reverse transformation after heating to 600 degrees C.

Floral initiation can be analyzed from a developmental perspective by focusing upon how developmental fates are imprinted, remembered, and expressed. This is not an altogether new perspective, since people studying flowering have been concerned for a long time with the commitment of meristems to form flowers and the morphological, cellular, and molecular changes associated with this commitment. What is novel is the emphasis on developmental states as opposed to physiological processes. This developmental focus indicates that there appear to be at least three major developmental states that are acquired and expressed in the process of a meristem initiating floral morphogenesis. The meristem cells must first become competent to respond to a developmental signal that evokes them into a florally determined state. The leaves are the usual source of this signal and a specific leaf may or may not have the capacity to be inductively active. When a leaf does develop the capacity for inductive activity, this capacity is usually correlated with the ontogeny of the leaf. Inductive activity, however, may be continually expressed as in some day-neutral plants or may be latent as in plants where the photoperiod is the external cue for activity. Competent shoot apical meristems respond to inductive leaf signal by being evoked into a florally determined state. Under permissive conditions this florally determined state is expressed as the initiation of floral morphogenesis. Many mechanisms have evolved to regulate entry into and expression of these developmental states. As we learn more about the developmental states associated with flowering and how they are acquired and expressed, we will understand better how the various patterns of flowering are related to one another as well as which developmental processes are common to all angiosperms.

The characterisation by 91Zr NMR at room temperature is reported for Zr metal (hexagonal close-packed), ZrH2 (tetragonal), ZrC (cubic), ZrCo (cubic), ZrO2 (monoclinic), ZrO2 (3 mol% Y2O3, tetragonal) and ZrO2 (9.3 mol% MgO). The density of states deduced from the Knight shift for zirconium metal agrees with band structure calculations. It is shown for the first time that the quadrupolar lineshape distinguishes between the various zirconia-based phases.

The electrical properties as well as the static and the dynamic magnetic behavior of Y2Cl3 and YCl3 were investigated with the help of powder DC susceptibility and magic angle spinning NMR measurements. Y2Cl3 is a semiconductor with a band gap of 0.85 eV, and is diamagnetic above 100 K. The 89Y resonance in Y2Cl3 exhibits considerable paramagnetic chemical shifts, +762 ppm and +506 ppm, which are distinctly different for the two Y lattice sites, and contrast the shift of −230 ppm for anhydrous YCl3. These features are related to metal-metal bonding in Y2Cl3.

Volumetric analysis of structural MR images of the brain may provide quantitative evidence of neurodegeneration and help identify patients at risk for rapid clinical deterioration. This note describes tests of a fully automated MR imaging postprocessing system for volumetric analysis of structures (such as the hippocampus) known to be affected in early Alzheimer disease (AD). The system yielded results that correlated highly with independent computer-aided manual segmentation and were sensitive to the anatomic atrophy characteristic of mild AD.

The human voltage-gated proton channel (hHv1) is a transmembrane protein responsible for selective proton permeation across cell membranes in nasal mucosa, sperm, and white blood cells. Its pathological states include male infertility, allergies, and diseases such as cystic fibrosis, asthma, and lupus. Its involvement in ischemic stroke and invasiveness of breast cancer cells has substantiated hHv1 as a therapeutic target for drug designs for which require the understanding of hHv1 structure and proton conduction mechanism. We recently constructed and validated a homology model (Kulleperuma et al., 2013) of hHv1 characterized by the presence of a salt bridge between anionic D112 and cationic R208 side chains in the narrow region of the hydrated pore. Thanks to the pairing of these and other charged residues in ionic networks, the distribution of charged and polar residues in the wild-type channel is a priori compatible with permeation of either cations or anions. However, a static-field electrostatic barrier opposing cation movement arises in neutral mutants of residue D112, consistent with the observation that these mutants are selective to anions (Musset et al., 2011). Our recent experiments show that proton selectivity is restored in double mutant D112V/V116D, while D112S and D112V/D116S are anion-selective and D112V does not conduct ions. Atomistic molecular dynamics simulations of these mutants in lipid bilayers show that their structures differ in the organization of ionic side chains in the external vestibule and suggest that, consistent with the above analysis, the distribution of charged groups plays a role in modulating the selectivity of the pore for either anions or protons. To elucidate the molecular mechanism of permeation and selectivity, we are conducting free energy simulations for the translocation of protons and other ions in both wild-type and mutant forms of hHv1.

INSULT, a novel method for the creation of insertions, deletions, and point mutations without subcloning, requires only one new primer per mutant, and produces circular plasmids, obviating the need for special "ultracompetent" cells. The method includes cycles of linear amplification with a thermophilic polymerase, and nick repair after each cycle with a thermophilic ligase. After production of multiple single-stranded copies of circular mutation-bearing plasmid DNA, addition of a "generic" primer followed by one or more polymerase reaction cycles generates double-stranded circular DNA bearing the desired mutation.

Control of nitric oxide (NO) synthesis in the constitutive nitric-oxide synthases (NOS) by calcium/calmodulin is exerted through the regulation of electron transfer from NADPH through the reductase domains. This process has been shown previously to involve the calmodulin binding site, the autoinhibitory insertion in the FMN binding domain, and the C-terminal tail. Smaller sequence elements also appear to correlate with control. Although some of these elements appear well positioned to function in control, they are poorly conserved; their role in control is neither well established nor defined by available information. In this study mutations have been induced in the small insertion of the hinge subdomain, which has been shown recently to form a beta hairpin in structural studies of the neuronal NOS reductase domains adjacent to the calmodulin site and the autoinhibitory element. Modification of the small insertion in neuronal NOS tends to increase cytochrome c reduction but not NO synthetic activity; some modifications or deletions in the corresponding region in endothelial NOS modestly increase activity under some conditions. Unexpectedly, some minor changes in the sequence introduce a loss in the content of heme relative to flavin cofactors. Taken together, these results suggest that the small insertion protects the calmodulin binding site and that it may be a modulator of NOS activity.

The interactions of neuronal nitric-oxide synthase (nNOS) with calmodulin (CaM) and mutant forms of CaM, including CaM-troponin C chimeras, have been previously reported, but there has been no comparable investigation of CaM interactions with the other constitutively expressed NOS (cNOS), endothelial NOS (eNOS), or the inducible isoform (iNOS). The present study was designed to evaluate the role of the four CaM EF hands in the activation of eNOS and iNOS. To assess the role of CaM regions on aspects of enzymatic function, three distinct activities associated with NOS were measured: NADPH oxidation, cytochrome c reduction, and nitric oxide (*NO) generation as assessed by the oxyhemoglobin capture assay. CaM activates the cNOS enzymes by a mechanism other than stimulating electron transfer into the oxygenase domain. Interactions with the reductase moiety are dominant in cNOS activation, and EF hand 1 is critical for activation of both nNOS and eNOS. Although the activation patterns for nNOS and eNOS are clearly related, effects of the chimeras on all the reactions are not equivalent. We propose that cytochrome c reduction is a measure of the release of the FMN domain from the reductase complex. In contrast, cytochrome c reduction by iNOS is readily activated by each of the chimeras examined here and may be constitutive. Each of the chimeras were co-expressed with the human iNOS enzyme in Escherichia coli and subsequently purified. Domains 2 and 3 of CaM contain important elements required for the Ca2+/CaM independence of *NO production by the iNOS enzyme. The disparity between cytochrome c reduction and *NO production at low calcium can be attributed to poor association of heme and FMN domains when the bound CaM constructs are depleted of Ca2+. In general cNOSs are much more difficult to activate than iNOS, which can be attributed to their extra sequence elements, which are adjacent to the CaM-binding site and associated with CaM control.

This report focuses on the first surrogate demonstration of a workable flowsheet for SB3 material. Two 15L glass Sludge Receipt and Adjustment Tank/Slurry Mix Evaporator (SRAT/SME) vessels at the Aiken County Technology Laboratory (ACTL) were used for the feed preparation portion of the test while the feed was vitrified in the ACTL Slurry-fed Melt Rate Furnace (SMRF). The flowsheet tested should not be considered final as further optimization is probable.

The Experimental Test Accelerator (ETA) II is a high-average-power linear induction accelerator built to support free-electron-laser research. Magnetic pulse compression and high-power thyratron modulators are used to generate the 125 kV accelerator drive pulses. The authors describe the design and testing of the 5 kHz, 3 MW thyratron modulators for the ETA II. The power conditioning system and the pulse timing requirements are described, and the operation of the ETA II system is discussed. >

In 1972, J. Woodland Hastings and colleagues predicted the existence of a proton selective channel that opens in response to depolarizing voltage (HV1) across the vacuole membrane of bioluminescent dinoflagellates and conducts protons into specialized luminescence compartments (scintillons), thus causing the pH drop that triggers the light flash. RNA-Seq data from several luminescent dinoflagellate species provided candidate HV1 genes. When expressed in mammalian cells, the predicted HV1 from Lingulodinium polyedrum displays the hallmark properties of bona fide proton channels, including time-dependent opening with depolarization, perfect proton selectivity, and characteristic ΔpH dependent gating. RT-PCR and Western blotting confirm expression of HV1 in L. polyedrum. Fluorescence confocal microscopy of L. polyedrum cells stained with antibodies to luminescence proteins luciferase and luciferin binding protein and to HV1 reveal structures consistent with HV1's proposed function in bioluminescence.[Supported by NSF: MCB-1242985, NIH: GM102336, ⋯]