Temporal Control of Ion Channel Activation

Ion channels are a large class of integral membrane proteins that allow for the diffusion of ions across a cellular membrane and are found in all forms of life. Pentameric ligand-gated ion channels (pLGICs) comprise a large family of proteins that include the nicotinic acetylcholine receptor (nAChR) and the γ-aminobutyric acid (GABA) receptor. These ion channels are responsible for the fast synaptic transmission that occurs in humans and as a result are of fundamental biological importance. pLGICs bind ligands (neurotransmitters), and upon ligand-binding undergo activation. The activation event causes an ion channel to enter a new physical state that is able to conduct ions. Ion channels allow for the flux of ions across the membrane through a pore that is formed upon ion channel activation. For pLGICs to function properly both ligand-binding and ion channel activation must occur. The ligand-binding event has been studied extensively over the past few decades, and a detailed mechanism of binding has emerged. During activation the ion channel must undergo structural rearrangements that allow the protein to enter a conformation in which ions can flow through. Despite this great and ubiquitous importance, a fundamental understanding of the ion channel activation mechanism and kinetics, as well as concomitant structural arrangements, remains elusive.

This dissertation describes efforts that have been made to temporally control the activation of ligand-gated ion channels. Temporal control of ion channel activation provides a means by which to activate ion channels when desired. The majority of this work examines the use of light to activate ion channels. Several photocages were examined in this thesis; photocages are molecules that release a ligand under irradiation, and, for the work described here, the released ligand then activates the ion channel. First, a new water-soluble photoacid was developed for the activation of proton-sensitive ion channels. Activation of acid-sensing ion channels, ASIC2a and GLIC, was observed only upon irradiation. Next, a variety of Ru²⁺ photocages were also developed for the release of amine ligands. The Ru²⁺ systems interacted in a deleterious manner with a representative subset of biologically essential ion channels. The rapid mixing of ion channels with agonist was also examined. A detection system was built to monitor ion channels activation in the rapid mixing experiments. I have shown that liposomes, and functionally-reconstituted ELIC, are not destroyed during the mixing process. The work presented here provides the means to deliver agonist to ligand-gated ion channels in a controlled fashion.

Evidences Of Endocytosis Via Caveolae Following Blood-brain Barrier Breakdown By Phoneutria Nigriventer Spider Venom.

Spider venoms contain neurotoxic peptides aimed at paralyzing prey or for defense against predators; that is why they represent valuable tools for studies in neuroscience field. The present study aimed at identifying the process of internalization that occurs during the increased trafficking of vesicles caused by Phoneutria nigriventer spider venom (PNV)-induced blood-brain barrier (BBB) breakdown. Herein, we found that caveolin-1α is up-regulated in the cerebellar capillaries and Purkinje neurons of PNV-administered P14 (neonate) and 8- to 10-week-old (adult) rats. The white matter and granular layers were regions where caveolin-1α showed major upregulation. The variable age played a role in this effect. Caveolin-1 is the central protein that controls caveolae formation. Caveolar-specialized cholesterol- and sphingolipid-rich membrane sub-domains are involved in endocytosis, transcytosis, mechano-sensing, synapse formation and stabilization, signal transduction, intercellular communication, apoptosis, and various signaling events, including those related to calcium handling. PNV is extremely rich in neurotoxic peptides that affect glutamate handling and interferes with ion channels physiology. We suggest that the PNV-induced BBB opening is associated with a high expression of caveolae frame-forming caveolin-1α, and therefore in the process of internalization and enhanced transcytosis. Caveolin-1α up-regulation in Purkinje neurons could be related to a way of neurons to preserve, restore, and enhance function following PNV-induced excitotoxicity. The findings disclose interesting perspectives for further molecular studies of the interaction between PNV and caveolar specialized membrane domains. It proves PNV to be excellent tool for studies of transcytosis, the most common form of BBB-enhanced permeability.

Effect of different cleansers on the weight and ion release of removable partial denture: an in vitro study

OBJECTIVE: Removable partial dentures (RPD) require different hygiene care, and association of brushing and chemical cleansing is the most recommended to control biofilm formation. However, the effect of cleansers has not been evaluated in RPD metallic components. The aim of this study was to evaluate in vitro the effect of different denture cleansers on the weight and ion release of RPD. MATERIAL AND METHODS: Five specimens (12x3 mm metallic disc positioned in a 38x18x4 mm mould filled with resin), 7 cleanser agents [Periogard (PE), Cepacol (CE), Corega Tabs (CT), Medical Interporous (MI), Polident (PO), 0.05% sodium hypochlorite (NaOCl), and distilled water (DW) (control)] and 2 cobalt-chromium alloys [DeguDent (DD), and VeraPDI (VPDI)] were used for each experimental situation. One hundred and eighty immersions were performed and the weight was analyzed with a high precision analytic balance. Data were recorded before and after the immersions. The ion release was analyzed using mass spectrometry with inductively coupled plasma. Data were analyzed by two-way ANOVA and Tukey HSD post hoc test at 5% significance level. RESULTS: Statistical analysis showed that CT and MI had higher values of weight loss with higher change in VPDI alloy compared to DD. The solutions that caused more ion release were NaOCl and MI. CONCLUSIONS: It may be concluded that 0.05% NaOCl and Medical Interporous tablets are not suitable as auxiliary chemical solutions for RPD care.

Adsorption of amyloglucosidase from Aspergillus niger NRRL 3122 using ion exchange resin

Amyloglucosidase enzyme was produced by Aspergillus niger NRRL 3122 from solid-state fermentation, using deffated rice bran as substrate. The effects of process parameters (pH, temperature) in the equilibrium partition coefficient for the system amyloglucosidase - resin DEAE-cellulose were investigated, aiming at obtaining the optimum conditions for a subsequent purification process. The highest partition coefficients were obtained using 0.025M Tris-HCl buffer, pH 8.0 and 25ºC. The conditions that supplied the highest partition coefficient were specified, the isotherm that better described the amyloglucosidase process of adsorption obtained. It was observed that the adsorption could be well described by Langmuir equation and the values of Qm and Kd estimated at 133.0 U mL-1 and 15.4 U mL-1, respectively. From the adjustment of the kinetic curves using the fourth-order Runge-Kutta algorithm, the adsorption (k1) and desorption (k2) constants were obtained through optimization by the least square procedure, and the values calculated were 2.4x10-3 mL U-1 min-1 for k1 and 0.037 min-1 for k2 .

Characterization studies of 1-(4-cyano-2-oxo-1,2-dihydro-1-pyridyl)-3-(4-cyano-1,2-dihydro-1-pyridyl)propane formed from the reaction of hydroxide Ion with 1,3-Bis-(4-cyano pyridinium)propane

The aqueous alkaline reaction of 1,3-bis(4-cyanopyridinium)propane dibromide, a reactant constituted of two pyridinium rings linked by a three-methylene bridge, generates a novel compound,1 -(4-cyano-2-oxo-1,2-dihydro-1-pyridyl)-3-(4-cyano-1,2-dihydro-1-pyridyl)propane. The reaction pathway is attributed to the proximity of the OH- ion inserted between two pyridinium moieties, which occurs only in bis(pyridinium) derivatives connected by short methylene spacers, where charge-conformational effects are important.

The inducing NO-vasodilation by chemical reduction of coordinated nitrite ion in cis-[Ru(NO(2))L(bpy)(2)](+) complex

The synthesis of [Ru(NO(2)) L(bpy)(2)](+) (bpy = 2,2'-bipyridine and L = pyridine (py) and pyrazine (pz)) can be accomplished by addition of [Ru(NO) L(bpy) 2](PF(6))(3) to aqueous solutions of physiological pH. The electrochemical processes of [Ru(NO2) L(bpy) 2]+ in aqueous solution were studied by cyclic voltammetry and differential pulse voltammetry. The anodic scan shows a peak around 1.00 V vs. Ag/AgCl attributed to the oxidation process centered on the metal ion. However, in the cathodic scan a second peak around-0.60 V vs. Ag/AgCl was observed and attributed to the reduction process centered on the nitrite ligand. The controlled reduction potential electrolysis at-0.80 V vs. Ag/AgCl shows NO release characteristics as judged by NO measurement with a NO-sensor. This assumption was confirmed by ESI/MS(+) and spectroelectrochemical experiment where cis-[Ru(bpy)(2)L(H(2)O)](2+) was obtained as a product of the reduction of cis-[Ru(II)(NO(2)) L(bpy)(2)](+). The vasorelaxation observed in denuded aortic rings pre-contracted with 0.1 mu mol L(-1) phenylephrine responded with relaxation in the presence of cis-[RuII(NO2) L(bpy) 2]+. The potential of rat aorta cells to metabolize cis-[RuII(NO(2)) L(bpy)(2)](+) was also followed by confocal analysis. The obtained results suggest that NO release happens by reduction of cis-[RuII(NO(2)) L(bpy)(2)](+) inside the cell. The maximum vasorelaxation was achieved with 1 x 10(-5) mol L(-1) of cis-[RuII(NO(2)) L(bpy)(2)](+) complex.

Indications of Conical Emission of Charged Hadrons at the BNL Relativistic Heavy Ion Collider

Three-particle azimuthal correlation measurements with a high transverse momentum trigger particle are reported for pp, d + Au, and Au + Au collisions at root(S)NN = 200 GeV by the STAR experiment. Dijet structures are observed in pp, d + Au and peripheral Au + Au collisions. An additional structure is observed in central Au + Au data, signaling conical emission of correlated charged hadrons. The conical emission angle is found to be theta = 1.37 +/- 0.02(stat)(-0.07)(+0.06)(syst), independent of p perpendicular to.

System-Size Independence of Directed Flow Measured at the BNL Relativistic Heavy-Ion Collider

We measure directed flow (v(1)) for charged particles in Au + Au and Cu + Cu collisions at root s(NN) = 200 and 62.4 GeV, as a function of pseudorapidity (eta), transverse momentum (p(t)), and collision centrality, based on data from the STAR experiment. We find that the directed flow depends on the incident energy but, contrary to all available model implementations, not on the size of the colliding system at a given centrality. We extend the validity of the limiting fragmentation concept to v(1) in different collision systems, and investigate possible explanations for the observed sign change in v(1)(p(t)).

Spin alignment measurements of the K*(0)(892) and phi(1020) vector mesons in heavy ion collisions at root S(NN)=200 GeV

We present the first spin alignment measurements for the K*(0)(892) and phi(1020) vector mesons produced at midrapidity with transverse momenta up to 5 GeV/c at root s(NN) = 200 GeV at RHIC. The diagonal spin-density matrix elements with respect to the reaction plane in Au+Au collisions are rho(00) = 0.32 +/- 0.04 (stat) +/- 0.09 (syst) for the K*(0) (0.8 < p(T) < 5.0 GeV/c) and rho(00) = 0.34 +/- 0.02 (stat) +/- 0.03 (syst) for the phi (0.4 < p(T) < 5.0 GeV/c) and are constant with transverse momentum and collision centrality. The data are consistent with the unpolarized expectation of 1/3 and thus no evidence is found for the transfer of the orbital angular momentum of the colliding system to the vector-meson spins. Spin alignments for K(*0) and phi in Au+Au collisions were also measured with respect to the particle's production plane. The phi result, rho(00) = 0.41 +/- 0.02 (stat) +/- 0.04 (syst), is consistent with that in p+p collisions, rho(00) = 0.39 +/- 0.03 (stat) +/- 0.06 (syst), also measured in this work. The measurements thus constrain the possible size of polarization phenomena in the production dynamics of vector mesons.

rho(0) photoproduction in ultraperipheral relativistic heavy ion collisions at root s(NN)=200 GeV

Photoproduction reactions occur when the electromagnetic field of a relativistic heavy ion interacts with another heavy ion. The STAR Collaboration presents a measurement of rho(0) and direct pi(+)pi(-) photoproduction in ultraperipheral relativistic heavy ion collisions at root s(NN) = 200 GeV. We observe both exclusive photoproduction and photoproduction accompanied by mutual Coulomb excitation. We find a coherent cross section of sigma(AuAu -> Au*Au*rho(0)) = 530 +/- 19(stat.) +/- 57(syst.) mb, in accord with theoretical calculations based on a Glauber approach, but considerably below the predictions of a color dipole model. The rho 0 transverse momentum spectrum (p(T)(2)) is fit by a double exponential curve including both coherent and incoherent coupling to the target nucleus; we find sigma(inc)/sigma(coh) = 0.29 +/- 0.03 (stat.) +/- 0.08 (syst.). The ratio of direct pi(+)pi(-) to rho(0) production is comparable to that observed in gamma(p) collisions at HERA and appears to be independent of photon energy. Finally, the measured rho(0) spin helicity matrix elements agree within errors with the expected s-channel helicity conservation.

A high voltage pulse power supply for metal plasma immersion ion implantation and deposition

We describe the design and implementation of a high voltage pulse power supply (pulser) that supports the operation of a repetitively pulsed filtered vacuum arc plasma deposition facility in plasma immersion ion implantation and deposition (Mepiiid) mode. Negative pulses (micropulses) of up to 20 kV in magnitude and 20 A peak current are provided in gated pulse packets (macropulses) over a broad range of possible pulse width and duty cycle. Application of the system consisting of filtered vacuum arc and high voltage pulser is demonstrated by forming diamond-like carbon (DLC) thin films with and without substrate bias provided by the pulser. Significantly enhanced film/substrate adhesion is observed when the pulser is used to induce interface mixing between the DLC film and the underlying Si substrate. (C) 2010 American Institute of Physics. [doi:10.1063/1.3518969]

Surface plasmon resonance of gold nanoparticles formed by cathodic arc plasma ion implantation into polymer

Shallow subsurface layers of gold nanoclusters were formed in polymethylmethacrylate (PMMA) polymer by very low energy (49 eV) gold ion implantation. The ion implantation process was modeled by computer simulation and accurately predicted the layer depth and width. Transmission electron microscopy (TEM) was used to image the buried layer and individual nanoclusters; the layer width was similar to 6-8 nm and the cluster diameter was similar to 5-6 nm. Surface plasmon resonance (SPR) absorption effects were observed by UV-visible spectroscopy. The TEM and SPR results were related to prior measurements of electrical conductivity of Au-doped PMMA, and excellent consistency was found with a model of electrical conductivity in which either at low implantation dose the individual nanoclusters are separated and do not physically touch each other, or at higher implantation dose the nanoclusters touch each other to form a random resistor network (percolation model). (C) 2009 American Vacuum Society. [DOI: 10.1116/1.3231449]

Structural properties of buried conducting layers formed by very low energy ion implantation of gold into polymer

We have investigated the fundamental structural properties of conducting thin films formed by implanting gold ions into polymethylmethacrylate (PMMA) polymer at 49 eV using a repetitively pulsed cathodic arc plasma gun. Transmission electron microscopy images of these composites show that the implanted ions form gold clusters of diameter similar to 2-12 nm distributed throughout a shallow, buried layer of average thickness 7 nm, and small angle x-ray scattering (SAXS) reveals the structural properties of the PMMA-gold buried layer. The SAXS data have been interpreted using a theoretical model that accounts for peculiarities of disordered systems.

Kinetic ion effect on geodesic acoustic Alfven modes in tokamaks

Using a quasitoroidal set of coordinates with coaxial circular magnetic surfaces, the Vlasov equation is solved for collisionless plasmas, and the dielectric tensor is found for large aspect ratio tokamaks in a low frequency band. Taking into account q-profile and charge separation parallel electric field, it is found that the Alfven wave continuum is deformed by ion geodesic effects producing continuum minimum at the rational magnetic surfaces. Low frequency geodesic ion induced Alfven waves are found below the continuum minimum where collisionless damping has a gap for Maxwell distribution. In kinetic approach, the ion thermal motion defines the geodesic effect but the mode frequency is strongly corrected due to parallel motion of electrons.

Conducting polymer formed by low energy gold ion implantation

A buried conducting layer of metal/polymer nanocomposite was formed by very low energy gold ion implantation into polymethylmethacrylate. The conducting layer is similar to 3 nm deep and of width similar to 1 nm. In situ resistivity measurements were performed as the implantation proceeded, and the conductivity thus obtained as a function of buried gold concentration. The measured conductivity obeys the behavior well established for composites in the percolation regime. The critical concentration, below which the polymer remains an insulator, is attained at a dose similar to 1.0 x 10(16) atoms/cm(2) of implanted gold ions. (C) 2008 American Institute of Physics.