Alcohol-induced temporal transcriptome remodeling in the prefrontal cortex in a mouse model of alcohol dependence

textAlcohol dependence (alcoholism) is a complex disease influenced by both environmental factors and genetic predisposition. Mouse models have been used to study many alcohol dependence-related traits and the genetics that underlie them. Two of the most commonly used mice in alcohol research are the C57BL/6J (B6) and DBA/2J (D2) inbred strains, which diverge on several alcohol-related traits including the development of acute physical dependence. Here we utilized the B6 and D2 mice as a genetic model of acute physical dependence, coupled with mRNA Differential Display (DD) and cDNA microarray analysis, to uncover the transcriptional response of the brain to an acute dose of alcohol as a function of time. About 150 genetically divergent and alcohol-responsive genes were identified between the whole brains of B6 and D2 mice using DD and were added as additional targets to the mouse microarrays. Microarray analysis of the prefrontal cortex of B6 and D2 mice revealed strain-specific, acute alcohol-responsive transcriptome remodeling manifested as temporal patterns of gene expression. Distinct expression patterns were identified for physiologically relevant alcohol-related consequences including intoxication, withdrawal and neuroadaptation. In silico characterization of the differentially expressed genes showed genotype dependent and independent transcriptional regulation and functional classification. In addition, categorization of differentially expressed genes by their cellular profiles revealed that some of the genes were known to be more highly expressed in either excitatory or inhibitory neuronal cell types. Our results indicate that the B6 and D2 prefrontal cortices have very different cellular and molecular responses to acute alcohol exposure. The specific roles that the genes identified in this study may play in mediating the divergent alcohol-related behavior between the strains warrant further study.Cellular and Molecular Biolog

Volumetric properties of binary mixtures of 2,4,6-trimethylpyridine with 1,2-ethanediol, methanol, and water, and the association energies of the O-H...N bonded complexes

2,4,6-Trimethylpyridine forms 1:1 complexes with methanol, 1,2-ethanediol, and water due to the O–H· · ·N bonds. The association energy of the complexes was calculated usingMP2 and DFT methods. The complexes with 1,2-ethanediol and water aggregate in the liquid phase as a result of the O–H· · ·O bonds. In spite of the higher O–H· · ·N bond energy, the aggregation of the ethanediolic complexes is less pronounced than that of the aqueous ones. That is probably caused by the weaker induction effect due to the C–C chain separating the hydroxyl groups in the diol molecule. Aggregation is impossible in the methanolic system, because of the lack of proton-donating functional groups. Differences in the hydrogen bond energy and in the ability to aggregate are manifested in the volumetric properties of the mixtures

Fotofizyka i fotochemia kobalamin w świetle obliczeń metodą funkcjonałów gęstości

Homolityczne rozerwanie aksjalnego wiazania Co-C w kobalaminach jest jednym z głównych etapów wielu reakcji enzymatycznych. Dokładne poznanie mechanizmu homolizy tego wiązania może znacząco przyczynić się do lepszego zrozumienia procesów enzymatycznych zachodzących przy udziale kobalamin. Efektywność tych procesów w dużej mierze zależy od własności wiązania Co-C, a te w pierwszej kolejności zdeterminowane są rodzajem liganda aksjalnego. Eksperymentalne badania fotolitycznego rozerwania wiązania kobalt-węgiel w kobalaminach prowadzone są w celu poznania mechanizmu tego procesu, postrzeganego jako przybliżony model fizyczny homolizy enzymatycznej. O ile, badania z wykorzystaniem technik spektroskopowych są w stanie odpowiedzieć na wiele istotnych pytań odnośnie struktury elektronowej stanów wzbudzonych i fotochemii kobalamin, 0 tyle struktura stanów wzbudzonych jak i sam mechanizm procesu fotolizy w dużej mierze jest nieznany na poziomie molekularnym. Dlatego badania z zastosowaniem metod kwantowochemicznych, mogą przyczynić się do wyjaśnienia wielu dotychczas niezrozumiałych zjawisk dotyczących fotochemii kobalamin. Tematyka podjęta w pracy doktorskiej jest kontynuacją prac, zapoczątkowanych badaniami teoretycznymi nad widmem elektronowym wolnej koryny , kobal(I)aminy , metylokobalaminy i adenozylokobalaminy. Zasadniczym celem pracy jest: (a) wyjaśnienie struktury elektronowej najniższego, singletowego stanu wzbudzonego dla cyjano-, metylo- i adenozylokobalaminy, (b) wyjaśnienie mechanizmu dezaktywacji stanu Si cyjanokobalaminy, (c) zaproponowanie mechanizmu fotodysocjacji grupy metylowej i adenozylowej, odpowiednio dla metylokobalaminy i adenozylokobalaminy. Wybór przedmiotu badań, a mianowicie trzech podstawowych pochodnych kobal(III)aminy - CNCbl, MeCbl i AdoCbl, wynika z następujących przesłanek: (a) są to najczęściej występujące w procesach biologicznych pochodne kobalaminy, (b) charakteryzują się istotnymi różnicami w własnościach fotochemicznych, wiązanie aksjalne Co-C nie ulega fotolizie w przypadku cyjanokobalaminy, zachodzi natomiast w metylo- i adenozylokobalaminie

Structure of the full-length TRPV2 channel by cryo-EM.

Transient receptor potential (TRP) proteins form a superfamily Ca(2+)-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a 'minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels

Theoretical study of cobalt and nickel complexes involved in methyl transfer reactions: structures, redox potentials and methyl binding energies

Cobalamins, cobalt glyoximate complexes and nickel complexes with Triphos (bis(diphenylphosphinoethyl)phenylphosphine) and PPh2CH2CH2SEt ligands were studied with the DFT/BP86 method in connection with methyl transfer reactions. Geometries, methyl binding energies and redox potentials were determined for the studied complexes. Three- and four-coordinate structures were considered for nickel complex with PPh2CH2CH2SEt ligand, whereas four- and five-coordinate for its methyl derivative. On the basis of calculations, the possible mechanism of methyl transfer reaction between cobalt and nickel complexes was considered

Theoretical Studies of Acetyl-CoA Synthase Catalytic Mechanism

DFT calculations were performed for the A-cluster from the enzyme Acetyl-CoA synthase (ACS). The acid constants (pKa), reduction potentials, and pH-dependent reduction potential for the A-cluster with different oxidation states and ligands were calculated. Good agreement of the reduction potentials, dependent on pH in the experiment, was obtained. On the basis of the calculations, a mechanism for the methylation reaction involving two–electron reduction and protonation on the proximal nickel atom of the reduced A-cluster is proposed

Role of protein kinase C in functional selectivity for desensitization at the µ-opioid receptor: from pharmacological curiosity to therapeutic potential

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72412/1/j.1476-5381.2009.00198.x.pd

TD-DFT insight into photodissociation of the Co-C bond in coenzyme B12

Coenzyme B12 (AdoCbl) is one of the most biologically active forms of vitamin B12, and continues to be a topic of active research interest. The mechanism of Co-C bond cleavage in AdoCbl, and the corresponding enzymatic reactions are however, not well understood at the molecular level. In this work, time-dependent density functional theory (TD-DFT) has been applied to investigate the photodissociation of coenzyme B12. To reduce computational cost, while retaining the major spectroscopic features of AdoCbl, a truncated model based on ribosylcobalamin (RibCbl) was used to simulate Co-C photodissociation. Equilibrium geometries of RibCbl were obtained by optimization at the DFT/BP86/TZVP level of theory, and low-lying excited states were calculated by TD-DFT using the same functional and basis set. The calculated singlet states, and absorption spectra were simulated in both the gas phase, and water, using the polarizable continuum model (PCM). Both spectra were in reasonable agreement with experimental data, and potential energy curves based on vertical excitations were plotted to explore the nature of Co-C bond dissociation. It was found that a repulsive 3(σCo-C → σ* Co-C) triplet state became dissociative at large Co-C bond distance, similar to a previous observation for methylcobalamin (MeCbl). Furthermore, potential energy surfaces (PESs) obtained as a function of both Co-CRib and Co-NIm distances, identify the S1 state as a key intermediate generated during photoexcitation of RibCbl, attributed to a mixture of a metal-to-ligand charge transfer (MLCT) and a s bonding-ligand charge transfer (SBLCT) states

Protein Footprinting: Auxiliary Engine to Power the Structural Biology Revolution

Structural biology is entering an exciting time where many new high-resolution structures of large complexes and membrane proteins are determined regularly. These advances have been driven by over fifteen years of technology advancements, first in macromolecular crystallography, and recently in Cryo-electron microscopy. These structures are allowing detailed questions about functional mechanisms of the structures, and the biology enabled by these structures, to be addressed for the first time. At the same time, mass spectrometry technologies for protein structure analysis, “footprinting” studies, have improved their sensitivity and resolution dramatically and can provide detailed sub-peptide and residue level information for validating structures and interactions or understanding the dynamics of structures in the context of ligand binding or assembly. In this perspective, we review the use of protein footprinting to extend our understanding of macromolecular systems, particularly for systems challenging for analysis by other techniques, such as intrinsically disordered proteins, amyloidogenic proteins, and other proteins/complexes so far recalcitrant to existing methods. We also illustrate how the availability of high-resolution structural information can be a foundation for a suite of hybrid approaches to divine structure-function relationships beyond what individual techniques can deliver

Characterization of Copper(II) Interactions with Sinefungin, a Nucleoside Antibiotic: Combined Potentiometric, Spectroscopic and DFT Studies

Interactions between sinefungin and copper(II) ions were investigated. Stoichiometry and stability constants of the metal-free system and two mononuclear complexes present in solution were determined on the basis of potentiometric data analysis. The results were compared to the Cu(II)-ornithine system due to structural similarities between both molecules. Combined spectroscopic and theoretical studies allowed for determination of coordination pattern for the Cu(II)-sinefungin complexes. At acidic pH, copper is bound in “glycine-like” coordination mode, identical with that of ornithine. This involves α-amino group and the carboxyl oxygen. At higher pH, a “bis-complex” is formed by two sinefungin molecules. The second ligand binds in equatorial position displacing two water molecules, what results in the stable {2N,2O} coordination. Both axial positions are supposed to be occupied by N1 nitrogen donors of adenine moiety, what is confirmed by DFT calculations. They interact indirectly with copper(II) through water molecules as the result of dominant syn conformation of purine