Functional dynamics of ion channels: modulation of proton movement by conformational switches

Detailed comparative studies of proton relay in native and chemically modified gramicidin channels provide a unique opportunity to uncover the structural basis of biological proton transport. The function of ion channels hinges on their ability to provide surrogate solvation in narrow pore filters so as to overcome the dielectric barrier presented by biological membranes. In the potassium channel KcsA and in the cation channel gramicidin, permeant selectivity and mobility are determined by the proteinaceous matrix via hydrogen bonding, charge-dipole, and dipole-dipole interactions. In particular, main-chain carbonyl groups in these pore interiors play an essential role in the solvation of alkali ions and of protons. In this study, molecular dynamics simulations reveal how the translocation of H(+) is controlled by nanosecond conformational transitions exchanging distorted states of the peptidic backbone in the single-file region of a dioxolane-linked analogue of the gramicidin dimer. These results underline the functional role of channel dynamics and provide a mechanism for the modulation of proton currents by fluctuating dipoles.     

Efficient and simple synthesis of 3-aryl-1H-pyrazoles

Efficient preparation of 3-aryl-1H-pyrazoles by reaction of 1-protected-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazoles with (het)aryl halides is described. The choice of THP protecting group is discussed.     

Fractionation of basic nuclear proteins of human sperm by zinc chelate affinity chromatography

Immobilized metal affinity chromatography was investigated for the fractionation of basic nuclear proteins of human sperm. Human sperm nuclei essentially contain two classes of protamines: a protamine of type P1 (HPl), rich in cysteine but with only one histidine, and three protamines of type P2 (HP2, HP3, HP4), rich in cysteine and histidine (nine in protamine HP2), potential ligands for transition metal ions. The critical conditions for metal affinity chromatography were defined: choice of metal, protein material and buffer, type of elution and sample loading. Chromatography of nuclear proteins, without histones and with cysteine residues alkylated by iodoacetamide, was optimum on zinc Chelating Sepharose in a Tris-acetate buffer and elution with an increasing concentration gradient of imidazole. Under these conditions, the two classes of protamines were completely separated. The intermediate basic proteins were further purified by reversed-phase high-performance liquid chromatography. Heterogeneity of binding to zinc of protamine HP1 was demonstrated. The proposed method is simple and reproducible and the recovery of proteins is high. It may be applied to study the expression and function of P1 and P2 protamines, e.g., in the case of infertile men.     

Polymer-supported approach for solution-phase synthesis of cysteine trap protease inhibitors: procedure for straightforward optimization of the P1-P1' pocket

Peptide-based reversible and irreversible cysteine proteases inhibitors are well reported in the literature. Many of these compounds have an electrophilic carbonyl group as a cysteine trap in the place of a scissile amide moiety of the natural substrate. As a common mechanism strategy, we have designed a probe library of a cysteine trap for rapid optimization of P1-P1' pockets of different cysteine proteases. The synthesis of this library using a straightforward methodology based on polymer-supported reagents and scavengers to avoid tedious purification steps has been achieved. For the selective monobromination of diazo ketones, preparation of a new supported reagent, piperidinoaminomethylpolystyrene hydrobromide, is also described.     

In vivo application of intestinal pH measurement using 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorescence imaging

Measurement of gastrointestinal intramucosal pH (pHim) has been recognized as an important factor in the detection of hypoxia-induced dysfunctions. However, current pH measurement techniques are limited in terms of time and spatial resolutions. A major advance in accurate pH measurement was the development of the ratiometric fluorescent indicator dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). This study aimed to set up and validate a fluorescence imaging technique to measure in vivo the intramucosal pH (pHim) of the intestine. The intestine was inserted into an optical chamber placed under a microscope. Animals were injected intravenously with the pH-sensitive fluorescent dye BCECF. Fluorescence was visualized by illuminating the intestine alternately at 490 and 470 nm. The emitted fluorescence was directed to an intensified camera. The ratio of emitted fluorescence at excitation wavelengths of 490 and 470 nm was measured, corrected and converted to pHim by constructing a calibration curve. The pHim controls were performed with a pH microelectrode and were correlated with venous blood gas sampling. Results show that pHim is determined with an accuracy of +/- 0.07 pH units and a response time of 1 min. In conclusion pHim mapping of rat intestine can be obtained by fluorescence imaging using BCECF. This technology could be easily adapted for endoscopic pH measurements.     

15N,(1)H Heteronuclear correlation NMR of gramicidin A in DMPC-d(67)

We have studied gramicidin A, an environmentally sensitive polymorphic pentadecapeptide, fully 15N-labelled and dispersed in a highly deuterated phospholipid bilayer system. By submitting the sample to fast magic angle spinning, we were able to reduce the polypeptide amide hydrogen linewidths to 160 Hz, and hence to partially resolve them. By correlating these resonances with the 40 Hz wide dipolar coupled 15N in a 2D-CROPSY (cross-polarization spectroscopy) experiment, it was possible to observe the 20 partially overlapping 1H-15N signal pairs from the polypeptide backbone and sidechains. Both chemical shift distributions closely match those of the same peptide in SDS micelles, but only poorly match those of conformationally different gramicidin A in trifluoroethanol, dimethylsulfoxide, or methanol/chloroform mixture. Our results are indicative of the N-to-N right-handed beta6.3-helix conformation of gramicidin A and offer sufficient resolution to encourage development of experiments to measure orientational or distance restraints using through-space dipolar couplings.     

Effect of brain lesions on [3H]ohmefentanyl binding site densities in the rat striatum and substantia nigra.

We have recently demonstrated that [3H]ohmefentanyl, a non-peptidergic opioid ligand which was suggested to cross the blood brain barrier in contrast to other peptidergic opioid ligands, bound not only to mu opioid receptor sites but also to sigma sites. In order to examine whether [3H]ohmefentanyl can be used as a marker for mu sites, we investigated the effects of brain lesions on [3H]ohmefentanyl binding site densities, as compared with [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]DAGO), a selective mu ligand. These binding site densities were measured by quantitative autoradiography in the rat striatum and substantia nigra, two brain structures known to contain a high density of mu receptors, following lesions of the nigro-striatal dopaminergic pathway and striatal intrinsic neurons. Following unilateral nigral lesion with 6-hydroxydopamine, [3H]ohmefentanyl binding site densities were decreased in the patches (-35%) and matrix (-20%) of the ipsilateral striatum and in the lesioned substantia nigra pars compacta (-49%). Unilateral striatal lesion with quinolinic acid induced 72%, 61% and 50% decreases in [3H]ohmefentanyl binding in the patches and matrix of the lesioned striatum and in the ipsilateral substantia nigra pars reticulata, respectively. Similar results were obtained in the binding of [3H]DAGO. Indeed, a significant linear correlation was observed between [3H]ohmefentanyl and [3H]DAGO binding site densities. Therefore, mu opioid receptors may be mainly located on intrinsic neurons in the striatum, dopaminergic cell bodies in the substantia nigra pars compacta and nerve terminals of striatal efferents in the substantia nigra pars reticulata.(ABSTRACT TRUNCATED AT 250 WORDS)     

Weitere Vereinfachung der Mikrobestimmung von Jod in organischen Verbindungen

Zusammenfassung Die Mikromethode zur Jodbestimmung in organischen Substanzen durch Verbrennen nachSchöniger und nachfolgende Titration mit 0,005-n Quecksilberperchlorat wird dahingehend vereinfacht, daß die Jodabsorption nach der Verbrennung anstatt mit Natronlauge direkt mit Quecksilberperchlorat erfolgt.

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Summary The micro method for determining iodine in organic materials through combustion afterSchöniger and subsequent titration with 0.005N mercuric perchlorate is modified to the extent that after the combustion the iodine is absorbed directly in mercuric perchlorate rather than in sodium hydroxide.

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Résumé On a simplifié la microméthode pour le dosage de l'iode dans les substances organiques avec combustion suivantSchöniger et titrage consécutif par le perchlorate de mercure 0,005N. L'absorption de l'iode après la combustion s'effectue directement par le perchlorate de mercure à la place de la lessive de soude.

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Riassunto La microdeterminazione dello iodio nelle sostanze organiche per combustione secondoSchöniger e successiva titolazione con perclorato di mercurio 0,005-n, è stata semplificata riprendendo direttamente il prodotto di combustione in una soluzione di perclorato di mercurio invece di una soluzione di idrato sodico.

     

Etude des métastannates de cadmium par spectrographie d'absorption infrarouge entre 5 et 40μ. Solutions solides entre l'oxyde d'étain et Poxyde de cadmium

Résumé Par chauffage à 1000° de mélanges d'oxyde stannique et d'oxyde de cadmium en proportions diverses, comme par déshydratation de précipités mixtes des hydroxydes correspondants, il se forme une série de solutions solides de l'oxyde de cadmium dans l'oxyde d'étain. Elles sont solubles dans l'acide chlorhydrique et présentent un spectre d'absorption infrarouge caractéristique que l'on a comparé avec celui des 2 métastannates de cadmium CdSnO₃, l'un de type ilménite, l'autre de type perovskite.

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Summary By heating various mixture of stannic oxide and cadmium oxide to 1000° C, a series of solid solutions of cadmium oxide in stannic oxide results as is the case when mixed precipitates of the corresponding hydroxides are dehydrated. They are soluble in hydrochloric acid and yield a characteristic infrared absorption spectrum that was compared with that of the two cadmium métastannates CdSnO₃, one of the ilmenite type, the other of the perovskite type.

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Zusammenfassung Erhitzt man verschieden zusammengesetzte Gemische von Zinnoxid und Cadmiumoxid auf 1000° C oder werden Mischfällungen der entsprechenden Hydroxide dehydratisiert, so entsteht eine Reihe fester Lösungen von Cadmiumoxid in Zinnoxid. Sie sind in Salzsäure löslich und zeigen ein charakteristisches Absorptionsspektrum im IR. Dieses wurde mit dem Spektrum zweier Cadmium-Metastannate (CdSnO₃) vom Typus des Ilmenits bzw. Perovskits verglichen.