Crystal structure of an RNA duplex r(G GCGC CC)2 with non-adjacent G*U base pairs.
(17/3703)
The crystal structure of a self-complementary RNA duplex r(GGGCGCUCC)2with non-adjacent G*U and U*G wobble pairs separated by four Watson-Crick base pairs has been determined to 2.5 A resolution. Crystals belong to the space group R3; a = 33.09 A,alpha = 87.30 degrees with a pseudodyad related duplex in the asymmetric unit. The structure was refined to a final Rworkof 17.5% and Rfreeof 24.0%. The duplexes stack head-to-tail forming infinite columns with virtually no twist at the junction steps. The 3'-terminal cytosine nucleosides are disordered and there are no electron densities, but the 3' penultimate phosphates are observed. As expected, the wobble pairs are displaced with guanine towards the minor groove and uracil towards the major groove. The largest twist angles (37.70 and 40.57 degrees ) are at steps G1*C17/G2*U16 and U7*G11/C8*G10, while the smallest twist angles (28.24 and 27.27 degrees ) are at G2*U16/G3*C15 and C6*G12/U7*G11 and conform to the pseudo-dyad symmetry of the duplex. The molecule has two unequal kinks (17 and 11 degrees ) at the wobble sites and a third kink at the central G5 site which may be attributed to trans alpha (O5'-P), trans gamma (C4'-C5') backbone conformations. The 2'-hydroxyl groups in the minor groove form inter-column hydrogen bonding, either directly or through water molecules. (+info)
Calcein as a fluorescent probe for ferric iron. Application to iron nutrition in plant cells.
(18/3703)
The recent use of calcein (CA) as a fluorescent probe for cellular iron has been shown to reflect the nutritional status of iron in mammalian cells (Breuer, W., Epsztejn, S., and Cabantchik, Z. I. (1995) J. Biol. Chem. 270, 24209-24215). CA was claimed to be a chemosensor for iron(II), to measure the labile iron pool and the concentration of cellular free iron(II). We first study here the thermodynamic and kinetic properties of iron binding by CA. Chelation of a first iron(III) involves one aminodiacetic arm and a phenol. The overall stability constant log beta111 of FeIIICAH is 33. 9. The free metal ion concentration is pFeIII = 20.3. A (FeIII)2 CA complex can be formed. A reversible iron(III) exchange from FeIIICAH to citrate and nitrilotriacetic acid is evidenced when these ligands are present in large excess. The kinetics of iron(III) exchange by CA is compatible with metabolic studies. The low reduction potential of FeIIICAH shows that the ferric form is highly stabilized. CA fluorescence is quenched by 85% after FeIII chelation but by only 20% using FeII. Real time iron nutrition by Arabidopsis thaliana cells has been measured by fluorimetry, and the iron buffer FeIIICAH + CA was used as source of iron. As a siderophore, FeIIICAH promotes cell growth and regreening of iron-deficient cells more rapidly than FeIIIEDTA. We conclude that CA is a good chemosensor for iron(III) in cells and biological fluids, but not for Fe(II). We discuss the interest of quantifying iron buffers in biochemical studies of iron, in vitro as well as in cells. (+info)
The effects of level of expression of a jellyfish Shaker potassium channel: a positive potassium feedback mechanism.
(19/3703)
1. When jellyfish Shaker potassium channels (jShak2) are heterologously expressed in Xenopus oocytes at different levels they demonstrate density-dependent changes in electrical and kinetic properties of macroscopic currents. 2. The activation and inactivation properties of jShak2 channels depend on the extracellular potassium concentration. In this study we present experimental data which show that expression-dependent changes in kinetic and electrical properties of jShak2 macroscopic currents can be explained by the positive feedback effect of dynamic accumulation of K+ in the perimembranal space. (+info)
Contributions of K+:Cl- cotransport and Na+/K+-ATPase to basolateral ion transport in malpighian tubules of Drosophila melanogaster.
(20/3703)
Mechanisms of Na+ and K+ transport across the basolateral membrane of isolated Malpighian tubules of Drosophila melanogaster were studied by examining the effects of ion substitution and putative inhibitors of specific ion transporters on fluid secretion rates, basolateral membrane potential and secreted fluid cation composition. Inhibition of fluid secretion by [(dihydroindenyl)oxy]alkanoic acid (DIOA) and bumetanide (10(-)4 mol l-1) suggested that a K+:Cl- cotransporter is the main route for K+ entry into the principal cells of the tubules. Differences in the effects of bumetanide on fluxes of K+ and Na+ are inconsistent with effects upon a basolateral Na+:K+:2Cl- cotransporter. Large differences in electrical potential across apical (>100 mV, lumen positive) and basolateral (<60 mV, cell negative) cell membranes suggest that a favourable electrochemical gradient for Cl- entry into the cell may be used to drive K+ into the cell against its electrochemical gradient, via a DIOA-sensitive K+:Cl- cotransporter. A Na+/K+-ATPase was also present in the basolateral membrane of the Malpighian tubules. Addition of 10(-)5 to 10(-)3 mol l-1 ouabain to unstimulated tubules depolarized the basolateral potential, increased the Na+ concentration of the secreted fluid by 50-73 % and increased the fluid secretion rate by 10-19 %, consistent with an increased availability of intracellular Na+. We suggest that an apical vacuolar-type H+-ATPase and a basolateral Na+/K+-ATPase are both stimulated by cyclic AMP. In cyclic-AMP-stimulated tubules, K+ entry is stimulated by the increase in the apical membrane potential, which drives K+:Cl- cotransport at a faster rate, and by the stimulation of the Na+/K+-ATPase. Fluid secretion by cyclic-AMP-stimulated tubules was reduced by 26 % in the presence of ouabain, suggesting that the Na+/K+-ATPase plays a minor role in K+ entry into the tubule cells. Malpighian tubules secreted a Na+-rich (150 mmol l-1) fluid at high rates when bathed in K+-free amino-acid-replete saline (AARS). Secretion in K+-free AARS was inhibited by amiloride and bafilomycin A1, but not by bumetanide or hydrochlorothiazide, which inhibit Na+:Cl- cotransport. There was no evidence for a Na+ conductance in the basolateral membrane of unstimulated or cyclic-AMP-stimulated tubules. Possible mechanisms of Na+ entry into the tubule cells include cotransport with organic solutes such as amino acids and glucose. (+info)
Non-invasive determination of bacterial single cell properties by electrorotation.
(21/3703)
So far, electrorotation and its application to the determination of single cell properties have been limited to eukaryotes. Here an experimental system is described that allows the recording of electrorotation spectra of single bacterial cells. The small physical dimensions of the developed measuring chamber combined with a single frame video analysis made it possible to monitor the rotation of objects as small as bacteria by microscopical observation despite Brownian rotation and cellular movement. Thus physical properties of distinct organelles of E. coli could be simultaneously determined in vivo at frequencies between 1 kHz and 1 GHz. Experimental data were evaluated following a three-shell model of the cell. Electrical conductivities of cytoplasm and outer membrane were determined to 4.4 mS/cm and 25 microS/cm, respectively, that of the periplasmic space was found to increase with the square root of the medium ionic strength. Specific capacitances of inner and outer membrane amounted to 1.4 microF/cm2 and 0.26 microF/cm2, respectively, the thickness of the periplasm to about 50 nm. Heat treatment of the cells lead to a reduction of cytoplasmic conductivity to 0.9 mS/cm, probably caused by an efflux of ions through the permeabilized inner membrane. (+info)
Nature of the chromophore binding site of bacteriorhodopsin: the potential role of Arg82 as a principal counterion.
(22/3703)
The nature of the chromophore binding site of light-adapted bacteriorhodopsin is analyzed by using modified neglect of differential overlap with partial single and double configuration interaction (MNDO-PSDCI) molecular orbital theory to interpret previously reported linear and nonlinear optical spectroscopic measurements. We conclude that in the absence of divalent metal cations in close interaction with Asp85 and Asp212, a positively charged amino acid must be present in the same vicinity. We find that models in which Arg82 is pointed upward into the chromophore binding site and directly stabilizes Asp85 and Asp212 are successful in rationalizing the observed one-photon and two-photon properties. We conclude further that a water molecule is strongly hydrogen bonded to the chromophore imine proton. The chromophore "1Bu*+" and "1Ag*-" states, despite extensive mixing, exhibit significantly different configurational character. The lowest-lying "1Bu*+" state is dominated by single excitations, whereas the second-excited "1Ag*-" state is dominated by double excitations. We can rule out the possibility of a negatively charged binding site, because such a site would produce a lowest-lying "1Ag*-" state, which is contrary to experimental observation. The possibility that Arg82 migrates toward the extracellular surface during the photocycle is examined. (+info)
Visualizing ion relaxation in the transport of short DNA fragments.
(23/3703)
Ion relaxation plays an important role in a wide range of phenomena involving the transport of charged biomolecules. Ion relaxation is responsible for reducing sedimentation and diffusion constants, reducing electrophoretic mobilities, increasing intrinsic viscosities, and, for biomolecules that lack a permanent electric dipole moment, provides a mechanism for orienting them in an external electric field. Recently, a numerical boundary element method was developed to solve the coupled Navier-Stokes, Poisson, and ion transport equations for a polyion modeled as a rigid body of arbitrary size, shape, and charge distribution. This method has subsequently been used to compute the electrophoretic mobilities and intrinsic viscosities of a number of model proteins and DNA fragments. The primary purpose of the present work is to examine the effect of ion relaxation on the ion density and fluid velocity fields around short DNA fragments (20 and 40 bp). Contour density as well as vector field diagrams of the various scalar and vector fields are presented and discussed at monovalent salt concentrations of 0.03 and 0.11 M. In addition, the net charge current fluxes in the vicinity of the DNA fragments at low and high salt concentrations are briefly examined and discussed. (+info)
A three-state model for connexin37 gating kinetics.
(24/3703)
The gating behavior of human connexin 37 (hCx37) is unaffected by the nature of the bathing monovalent (for Na, K, Rb). It is modified by [Mg] in the millimolar range. For fitting the kinetics, we propose a simple extension to three states of the canonical 2-state model of the hemichannel. The extra closed state allows for some immobilization of a hemichannel at high transjunctional voltages. The model is reasonably efficient at fitting data at various voltage protocols. Interpreting the fits of the data at different [Mg] is consistent with a binding site for Mg. (+info)