Structural characterisation of tyrosine-nitrated peptides by ultraviolet and infrared matrix-assisted laser desorption/ionisation Fourier transform ion cyclotron resonance mass spectrometry

Nitration of tyrosine residues in proteins may occur in cells upon oxidative stress and inflammation processes mediated through generation of reactive nitroxyl from peroxynitrite. Tyrosine nitration from oxidative pathways may generate cytotoxic species that cause protein dysfunction and pathogenesis. A number of protein nitrations in vivo have been reported and some specific Tyrosine nitration sites have been recently identified using mass spectrometric methods. High-resolution Fourier transform ion cyclotron resonance mass spectrometry (MALDI) FT-ICR-MS) is shown here to be a highly efficient method in the determination of protein nitrations. Following the identification of nitration of the catalytic site Tyr-430 residue of bovine prostacyclin synthase, we synthesised several model peptides containing both unmodified tyrosine and 3-nitro-tyrosine residues, using solid-phase peptide synthesis (SPPS). The structures of the nitrotyrosine peptides were characterised both by ESI- and by matrix-assisted laser desorption/ionisation (MALDI)-FT-ICR-MS, using a standard ultraviolet (UV) nitrogen nitrogen laser and a 2.97 microm Nd-YAG infrared laser. Using UV-MALDI-MS, 3-nitrotyrosyl-peptides were found to undergo extensive photochemical fragmentation at the nitrophenyl group, which may hamper or prevent the unequivocal identification of Tyr-nitrations in cellular proteins. In contrast, infrared-MALDI-FT-ICR-MS did not produce fragmentation of molecular ions of Tyr-nitrated peptides.     

Enantiopure 4- and 5-aminopiperidin-2-ones: regiocontrolled synthesis and conformational characterization as bioactive beta-turn mimetics

Starting from aspartic acid, we synthesized lactam-bridged beta- and gamma-amino acid equivalents. Using the 1,4-bis-electrophile 1b as a central intermediate, the 4- and 5-aminopiperidin-2-ones 4 and 8, respectively, were approached by regioselective functionalization and subsequent lactamization. Diastereoselective C-alkylation was performed after N-protection of the lactam functionality when exclusive trans configuration resulting in the formation of 5a-f was observed in the 4-amino series. On the other hand, cis selectivity was typical for the alkylations of the 5-amino lactams 5a,b. To investigate the ability of the lactam building blocks to induce reverse-turn structures by intramolecular hydrogen bonding, the model peptidomimetics 12 and 14 representing Homo-Freidinger lactams of type II and III were prepared from 4a and 8a, respectively. Conformational analyses in dilute solution (1 mM) by IR and NMR spectroscopy at room temperature clearly indicated that the 4-aminopiperidin-2-one derivative 12 predominantly adopts a reverse-turn structure stabilized by a CO-HN hydrogen bond in an 11-membered ring. VT NMR experiments showed a substantial temperature dependency of the terminal NH when Deltadelta(NH)/DeltaT = -6.5 indicated that the amount of intramolecular hydrogen bonding is higher at low temperature. An application in the field of medicinal chemistry was demonstrated. Thus, starting from the Homo-Freidinger lactam 11c and the enantiomer ent-11c, we synthesized the peptidomimetics 15c and 16c and investigated them as lactam-bridged analogues of the dopamine receptor modulating peptide Pro-Leu-Gly-NH(2) (PLG). Both test compounds turned out to enhance significantly the agonist binding of dopamine D2 receptors, when the isomer 15c revealed a potency comparable to the genuine ligand PLG.     

Schwermetallchelate von α-Cyano-β-amino-dithioacrylsäureestern

Heavy Metal Chelates of α-Cyano-β-amino-dithioacrylates The inner metal chelates of Pd^II, Ni^II, Co^III, and Ag^I with α-cyano-β-amino-dithioacrylates have been prepared. The coordination of the dithioester group and the amine nitrogen (S/N-coordination) has been proved by the chemical shift of the S2p and N1s electron binding energies in the ESCA spectra.     

Cellulose synthesis in maize: isolation and expression analysis of the cellulose synthase (CesA) gene family

Stalk lodging in maize results in significant yield losses. We have determined that cellulose per unit length of the stalk is the primary determinant of internodal strength. An increase in cellulose concentration in the wall might allow simultaneous improvements in stalk strength and harvest index. Cellulose formation in plants can be perturbed by mutations in the genes involved in cellulose synthesis, post-synthetic cellulose alteration or deposition, N-glycosylation, and some other genes with as yet unknown functions. We have isolated 12 members of the cellulose synthase (CesA) gene family from maize. The genes involved in primary wall formation appear to have duplicated relatively independently in dicots and monocots. The deduced amino acid sequences of three of the maize genes, ZmCesA10–12, cluster with the Arabidopsis CesA sequences that have been shown to be involved in secondary wall formation. Based on their expression patterns across multiple tissues, these three genes appear to be coordinately expressed. The remaining genes show overlapping expression to varying degrees with ZmCesA1, 7, and 8 forming one group, ZmCesA3 and 5 a second group, and ZmCesA2 and 6 exhibiting independent expression of any other gene. This suggests that the varying levels of coexpression may just be incidental except in the case of ZmCesA10–12, which may interact with each other to form a functional enzyme complex. Isolation of the expressed CesA genes from maize and their association with primary or secondary wall formation has made it possible to test their respective roles in cellulose synthesis through mutational genetics or transgenic approaches. This information would be useful in improving stalk strength.     

Separation of enzymes from Candida boidinii crude extract by continuous flow zone electrophoresis.

Separation of the enzymes formate dehydrogenase, formaldehyde dehydrogenase and methanol oxidase from Candida boidinii crude extract has been explored using continuous flow zone electrophoresis in the VaP-22 and the scaled-up VaP-220 electrophoresis apparatus. Yields up to 95% and purification factors between 3 and 7 were obtained, together with separation of cell debris from the enzymes. Multiple injections of sample were used to obtain a protein throughput of 46.2 mg/h in the VaP-22. A tenfold higher throughput was achieved using the VaP-220. Correlation of the electrophoretic mobility in continuous flow zone electrophoresis with the elution behavior in ion-exchange chromatography confirmed the primary role of net surface charge in the separation of biological molecules. Proteins and enzymes with differences greater than 0.05 M elution molarities in ion-exchange chromatography can be separated. This corresponds to a preparative scale (mg/h or g/h) separation of proteins and enzymes whose difference in apparent electrophoretic mobility is greater than 0.70 x 10(-5) cm2/(V.s).     

Analysis of RNA sequence structure maps by exhaustive enumeration I. Neutral networks

Summary Global relations between RNA sequences and secondary structures are understood as mappings from sequence space into shape space. These mappings are investigated by exhaustive folding of allGC andAU sequences with chain lengths up to 30. The computed structural data are evaluated through exhaustive enumeration and used as an exact reference for testing analytical results derived from mathematical models and sampling based on statistical methods. Several new concepts of RNA sequence to secondary structure mappings are investigated, among them that ofneutral networks (being sets of sequences folding into the same structure). Exhaustive enumeration allows to test several previously suggested relations: the number of (minimum free energy) secondary structures as a function of the chain length as well as the frequency distribution of structures at constant chain length (commonly resulting in generalized forms ofZipf's law).

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Analyse der Beziehungen zwischen RNA-Sequenzen und Sekundärstrukturen durch vollständige Faltung, 1. Mitt. Faltung, Neutrale Netzwerke

Zusammenfassung Die globalen Benziehungen zwischen RNA-Sequenzen und Sekundärstrukturen werden als Abbildungen aus einem Raum aller Sequenzen in einen Raum aller Strukturen aufgefaßt. Diese Abbildungen werden durch Falten aller binären Sequenzen desGC-undAU-Alphabets mit Kettenlängen bis zun=30 untersucht. Die berechneten Strukturdaten werden durch vollständiges Abzählen ausgewertet und als eine exakte Referenz zum Überprüfen analytischer Resultate aus mathematischen Modellen sowie zum Testen statistisch erhobener Proben verwendet. Einige neuartige Konzepte zur Beschreibung der Beziehungen zwischen Sequenzen und Strukturen werden eingehend untersucht, unter ihnen der Begriff derneutralen Netzwerke. Ein neutrales Netzwerk besteht aus allen Sequenzen, die eine bestimmte Struktur ausbilden. Vollständiges Abzählen ermöglicht beispielsweise die Bestimmung aller Strukturen minimaler freier Energie in Abhängigkeit von der Kettenlänge ebenso wie die Bestimmung der Häufigkeitsverteilungen der Strukturen bei konstanten Kettenlängen. Die letzteren folgen einer verallgemeinerten FormZipfschen Gesetzes.

     

P588, A SECOND RETINAL-CONTAINING PIGMENT IN HALOBACTERIUM HALOBIUM

Abstract— Halobacterium mutant strains with defects in the biosynthesis of various pigments have been isolated. One of these strains, mutant ET-15, is incapable of producing the light-driven proton pump bacteriorhodopsin and the carotenoid bacterioruberin. However, ET-15 synthesizes another photochemically active, retinal-containing pigment, P₅₈₈, which mediates light-induced proton uptake enhanced by uncouplers. P₅₈₈ and bacteriorhodopsin are simultaneously present in wild-type cells grown under normal conditions; however, they can be distinguished by the following criteria.

• 1 They can be separated by independent mutational events.
• 2 Proton ionophores such as FCCP diminish bacteriorhodopsin-driven proton translocation but enhance P₅₈₈-mediated proton flows. We define here proton translocations which can be diminished by the addition of uncouplers (e.g. FCCP) as ‘active’, others which can be enhanced by FCCP as ‘passive’.
• 3 The kinetic and spectral properties of the photocycle intermediates of bacteriorhodopsin and P₅₈₈ are different.
• 4 The action spectrum for photocycling of P₅₈₈ is red-shifted with respect to that for bacteriorhodopsin.

A comparison of action spectra for proton translocations with that for photocycling links pigment P₅₈₈ and light-driven, passive proton uptake by ET-15 envelope vesicles. When chemically bleached P₅₈₈ was regenerated with all-trans-retinal, both photochemical activity and light-induced proton inflow were restored to equal extents. This identifies P₅₈₈ with the energizer for the passive proton flows.     

Thermodynamic studies of complexation and enantiorecognition processes of monoterpenoids by alpha- and beta-cyclodextrin in gas chromatography

Gas-liquid chromatography was applied in thermodynamic investigations of processes of complexation and enantioseparation by alpha- and [-cyclodextrins of chiral monoterpenoids. The distribution constants, stability constants and thermodynamic parameters enthalpy, entropy and free energy of the complexation processes were determined. It has been found that enantioseparation of monoterpenes by alpha- and beta-cyclodextrins is the result of formation of 1:2 stoichiometric complexes. When 1:1 stoichiometric complexes are formed, enantioselectivity is not observed. All investigated processes of complexation are enthalpy-driven regardless of the stoichiometry of the formed complexes. -deltaH, -TdeltaS and -deltaG of complexation process have higher values for bicyclic than for monocyclic monoterpenoids as well as for alpha-CD than for beta-CD. The first or second step of complexation may be responsible for enantioselectivity.     

1,2-Dialkoxyacenaphthylenes and 2,3,11,12-bis-(1,2-acenaphtho)-[18]crown-6

A general synthesis of 1,2-dialkoxyacenaphthylenes by dehydrogenation of the corresponding acenaphthene derivatives with high potential quinones is described. The new crown ether, 2,3,11,12-bis(1,2-acenaphtho)-[18]crown-6, 1, is obtained by this route. The surprisingly poor complexing ability of 1 is ascribed to electronic and geometrical effects of the acenaphthylene rings as shown by spectroscopic and voltammetric data and the crystal structure of the free ligand 1.     

A new inorganic-organic polymer for the passivation of thin film capacitors

Inorganic-organic polymers with barrier properties against water vapor, excellent electrical data (3, 2, R _D>10¹⁶ cm, E _D up to 400 V/µm) and good adhesion to various substrate materials have been developed. Tailored modifications of these materials provide an excellent protective coating for thin film capacitors. Several mm thick, expensive, encapsulations could be replaced by thin coatings (up to 10 µm). The polymer coating allows the use of thin film chip capacitors in surface-mount technology. As a measure for the efficiency of the coating, the capacitance decrease under controlled humidity has been used. The influence of the material composition, the type of catalyst during sol-gel processing and the curing conditions have been studied. Adhesion and water vapor permeation properties of the polymers and rheological properties of the coating solutions have been investigated. A protective coating is developed, which increases the withstandness of capacitors against humid conditions (90°C, 100% rel. humidity) by a factor of about 30 (compared to uncoated capacitors) and shows no crack formation during thermal cycling.