Unsaturated lipid bodies as a hallmark of inflammation studied by Raman 2D and 3D microscopy.

Endothelial HMEC-1 cells incubated with pro-inflammatory cytokine TNF-α for 6 and 24 hours were studied as a model of inflammation using Raman imaging. Striking changes in distribution, composition and concentration of cellular lipids were observed after exposure to TNF-α compared to the control. In particular, 3D Raman imaging revealed a significant increase in the amount of lipid entities formed under inflammation. Lipid bodies were randomly distributed in the cytoplasm and two types of droplets were assembled: more saturated one, in spectral characteristics resembling phosphatidylcholine and saturated cholesteryl esters, observed also in the control, and highly unsaturated one, containing also cholesterols, being a hallmark of inflamed cells. The statistical analysis showed that the number of lipid bodies was significantly dependent on the exposure time to TNF-α. Overall, observed formation of unsaturated lipid droplets can be directly correlated with the increase in production of prostacyclins - endogenous inflammation mediators.

Pre-resonance enhancement of exceptional intensity in Aggregation-Induced Raman Optical Activity (AIROA) spectra of lutein derivatives.

Recently reported new phenomenon of Aggregation-Induced Raman Optical Activity is demonstrated here for the first time in the pre-resonance conditions for lutein diacetate and 3'-epi-lutein supramolecular self-assembles. We demonstrate that minor alterations in the lutein structure (e.g. acetylation of hydroxyl groups or different configuration at one of the chiral center) can lead to definitely different spectral profiles and optical properties due to formation of aggregates of different structure and type. Lutein forms only H-aggregates, lutein diacetate only J-aggregates, while 3'-epi-lutein can occur in both forms simultaneously. Variety of aggregates' structures is so large that not only the type of aggregation is different, but also their chirality. It is remarkable that even in the pre-resonance conditions, aggregation of lutein derivatives can lead to the intense ROA signal, and moreover, 3'-epi-lutein demonstrated the highest resonance ROA CID ratio that has ever been reported.

Identification of a biochemical marker for endothelial dysfunction using Raman spectroscopy.

In the present work, we propose the spectroscopic approach to identify biochemical alterations in endothelial dysfunction. The method is based on the quantification of the ratio of phenylalanine (Phe) to tyrosine (Tyr) contents in the endothelium. The synthesis of Tyr from Phe requires the presence of tetrahydrobiopterin (BH4) as a cofactor of phenylalanine hydroxylase (PAH). Limitation of BH4 availability in the endothelium is a hallmark endothelial nitric oxide synthase (eNOS) dysfunction that may also lead to PAH dysfunction and a fall in Tyr contents. Using Raman spectra, the ratio of marker bands of Tyr to Phe was calculated and the pathological state of the endothelium was detected. We provide evidence that Phe/Tyr ratio analysis by Raman spectroscopy discriminate endothelial dysfunction in ApoE/LDLR(-/-) mice as compared to control mice.

Biochemical changes of the endothelium in the murine model of NO-deficient hypertension.

The main spectral differences between the biochemical compositions of the vascular endothelium of control, hypertensive NO-deficient, and NO-deficient mice supplemented with nitrate were studied using Raman microimaging. A significantly different Raman signature of the endothelium in these three groups in the 1200-1400 cm(-1) region was assigned to the α-helix and ß-sheet alterations in the protein secondary structure upon the development of hypertension. The second pronounced biochemical marker of endothelium alterations was the lipid to protein ratio. A lower intensity of the band at 2940 cm(-1) relative to the feature at 1007 cm(-1) in the endothelium in hypertension compared to the control indicated a decrease of the lipid content relative to proteins during the progress of the pathology. The nitrate-based treatment partially reversed the effects of hypertension. The nitrate supplementation restored the lipid to protein ratio in the endothelium to the control level, while the changes in the secondary structure of proteins were irreversible upon nitrate administration.

Influence of cage confinement on the photochemistry of matrix-isolated E-ß-ionone: FT-IR and DFT study.

ß-ionone, a model compound of carotenoids ring structure, was investigated by FT-IR spectroscopy in a low-temperature argon matrix as well as using B3LYP/6-311++G(d,p) and MP2/6-311++G(d,p) quantum-chemical calculations. The spectrum of matrix-isolated E-ß-ionone was analyzed and attributed to six conformers of the compound. Then, matrix-isolated E-ß-ionone was submitted to UV irradiation using either a broadband source (with different cutoff filters) or a narrowband laser/MOPO system (at various wavelengths). Upon 240 nm narrowband irradiation, the formation of both Z-retro-γ-ionone and Z-ß-ionone was observed, the reactant and the photoproducts being in a photostationary equilibrium. Under these conditions, the matrix environment was found to hamper subsequent reactions of Z-retro-γ-ionone and Z-ß-ionone, so that this last species could be observed directly for the first time. Furthermore, the formation of Z-retro-γ-ionone was shown to occur directly via an intramolecular [1,5] H-atom shift and thereby, under the constraints imposed by the matrix confinement, the conformations assumed by this photoproduct were found to be strictly determined by those initially assumed by the reactant molecules. Broadband irradiation resulted in the completion of the reaction (disappearance of the reactant) and the sole observation of Z-retro-γ-ionone. These results imply that under these conditions the Z-ß-ionone is unstable, very likely decaying to additional conformers of Z-retro-γ-ionone, as reflected in the broader bands due to this photoproduct observed in the infrared spectra of the broadband irradiated matrix.

Oligomerization of G protein-coupled receptors: biochemical and biophysical methods.

Dimerization and oligomerization of G protein-coupled receptors (GPCRs), proposed almost 30 years ago, have crucial relevance for drug design. Indeed, formation of GPCR oligomers may affect the diversity and performance by which extracellular signals are transferred to G proteins in the process of receptor transduction. Thus, the control of oligomer assembly/disassembly and signaling will be a powerful pharmacological tool. This, however, requires (i) the determination that oligomerization takes place between particular receptors, (ii) the confirmation that the oligomer has pharmacological importance and (iii) the availability of the oligomer 3D structure. This review aims at presenting experimental methods which unveil the complexity of GPCR dimerization/oligomerization focusing on biochemical and biophysical approaches. In total, we review 22 methods, including biochemical methods (radiation inactivation technique, receptor co-expression and trans-complementation studies, cross-linking experiments, co-immunoprecipitation and immunoblotting studies and analysis of receptor mutants and chimeras) and biophysical methods (Fluorescence Resonance Energy Transfer, (FRET), including photobleaching FRET (pb-FRET) and Time-Resolved FRET (TR-FRET), Luminescence Resonance Energy Transfer (LRET), Bioluminescence Resonance Energy Transfer (BRET), Bimolecular Fluorescence Complementation (BiFC), Luminescence Fluorescence Complementation (BiLC), Fluorescence Recovery after Photobleaching (FRAP), Confocal Microscopy, Immunofluorescence Microscopy, Single Fluorescent-Molecule Imaging, Transmission Electron Microscopy, Immunoelectron Microscopy, Atomic Force Microscopy, Total Internal Reflectance Fluorescence Microscopy (TIRFM) and X-ray Crystallography). For each method the scientific basis of the approach is shortly described followed by the extensive description of its application for studying GPCR oligomers presented according to their classes and families. Based on the wealth of experimental evidence, there is no doubt about the existence of GPCR dimers, oligomers and receptor mosaics which constitute a new and highly promising group of novel drug targets for more selective and safer drugs.

Oligomerization of G protein-coupled receptors: computational methods.

Recent research has unveiled the complexity of mechanisms involved in G protein-coupled receptor (GPCR) functioning in which receptor dimerization/oligomerization may play an important role. Although the first high-resolution X-ray structure for a likely functional chemokine receptor dimer has been deposited in the Protein Data Bank, the interactions and mechanisms of dimer formation are not yet fully understood. In this respect, computational methods play a key role for predicting accurate GPCR complexes. This review outlines computational approaches focusing on sequence- and structure-based methodologies as well as discusses their advantages and limitations. Sequence-based approaches that search for possible protein-protein interfaces in GPCR complexes have been applied with success in several studies, but did not yield always consistent results. Structure-based methodologies are a potent complement to sequence-based approaches. For instance, protein-protein docking is a valuable method especially when guided by experimental constraints. Some disadvantages like limited receptor flexibility and non-consideration of the membrane environment have to be taken into account. Molecular dynamics simulation can overcome these drawbacks giving a detailed description of conformational changes in a native-like membrane. Successful prediction of GPCR complexes using computational approaches combined with experimental efforts may help to understand the role of dimeric/oligomeric GPCR complexes for fine-tuning receptor signaling. Moreover, since such GPCR complexes have attracted interest as potential drug target for diverse diseases, unveiling molecular determinants of dimerization/oligomerization can provide important implications for drug discovery.

Molecular structure of ionotropic glutamate receptors.

L-glutamate is the major excitatory neurotransmitter in the central nervous system (CNS). Although just a few glutamate receptor ligands have turned out to be clinically useful, primarily because of unfavorable psychotropic side effects, the glutamate system remains an attractive molecular target in the treatment of epilepsy, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's chorea), schizophrenia, ischemia, pain, alcoholism and mood disorders. Knowledge about the structure of ionotropic glutamate receptors (iGluRs) at atomic resolution is vital for the determination of their physiological and pathological importance and, thus, for drug design. Recently, tremendous progress has been made in structure elucidation and understanding of the functioning of iGluRs. The data about general topology and modular composition of iGluRs as well as numerous crystal structures of ligand binding domains of many iGluR subtypes has been supplemented with the first molecular models of the whole receptor protein, followed by the first crystal structures of N-terminal domains and finally by the first crystal structure of the whole tetrameric iGluR. This review summarizes experimental and computational efforts to determine iGluR molecular architecture and focus on the above listed achievements of the last years. In particular, the aspects of iGluR structure which are important for drug design, like the molecular characterstics of the ligand binding sites, are depicted in detail.

Thermally induced sigmatropic isomerization of pseudosaccharyl allylic ether.

The thermally induced sigmatropic isomerization of the pseudosaccharyl allylic ether [3-(allyloxy)-1,2-benzisothiazole 1,1-dioxide; ABID] has been investigated by a multidisciplinary approach using temperature dependent infrared spectroscopy, differential scanning calorimetry, and polarized light thermomicroscopy, complemented by theoretical methods. Migration of the allylic system from O to N occurs in the melted ABID, and the thermally obtained 2-allyl-1,2-benzisothiazol-3(2H)-one 1,1-dioxide (ABIOD) starts to be produced at ca. 150 degrees C, in a process with an activation energy of approximately 92 kJ mol(-1). From kinetic data, a concerted [3,3'] sigmatropic mechanism is proposed. In the temperature range investigated, ABIOD was found to exhibit polymorphism. Cooling of the molten compound leads to the production of a metastable crystalline form, which upon annealing at room temperature might be transformed to the stable crystalline phase. ABID shows a single crystalline variety. Assignments were proposed for the infrared spectra of the observed neat condensed phases of the two compounds.

Matrix-isolated monomeric tryptophan: electrostatic interactions as nontrivial factors stabilizing conformers.

An extensive analysis of the conformational space of tryptophan (Trp) was performed at the B3LYP/6-311++G(d,p) level and verified by comparison with the infrared spectra of the compound isolated in low-temperature argon and xenon matrixes. Different types of conformers have been unequivocally identified in the matrixes. Type I exhibits the trans arrangement of the carboxylic group and is stabilized by an O-H...N intramolecular H-bond. Types II and III have the carboxylic group in the cis conformation and feature N-H...O=C and N-H...O-C hydrogen bonds, respectively. Three individual conformers of type I were identified in the matrixes. Other conformational degrees of freedom are related with the Calpha-Cbeta-Cgamma=C and C1-Calpha-Cbeta-Cgamma angles (chi1 and chi2, respectively). In proteins, these two dihedral angles define the conformations of the amino acid residues. In monomeric Trp, chi1 adopts the "+" (ca. +90 degrees ) and "-" (ca. -90 degrees ) orientations, while average values of -67.4, 170.5, and 67.6 degrees ("a", "b", and "c", respectively) were found for chi2. Theoretical analysis revealed two important factors in stabilizing the structures of the Trp conformers: the H-bond type and electrostatic interactions. Classified by the H-bond type, the most stable are forms I, followed by II and III. Out of possible combinations of the chi1 and chi2 dihedral angles, "a+", "b+", and "c-" were theoretically found more stable than their "a-", "b-", and "c+" counterparts. Thus, the stabilizing effect of interactions involving the pyrrole ring (which are possible in Ia+, Ib+, and Ic- conformers) is considerably higher compared to those in which the phenyl ring is engaged (existing in the Ia-, Ib-, and Ic+ forms).

Conformational behavior of dimethyl 5-methyl-1H,3H-pyrrolo[1,2-c][1,3]thiazole-6,7-dicarboxylate 2,2-dioxide isolated in low-temperature matrixes.

The structure of dimethyl 5-methyl-1H,3H-pyrrolo[1,2-c][1,3]thiazole-6,7-dicarboxylate 2,2-dioxide (PTD) was investigated in low-temperature noble gas matrixes (Ar, Kr, Xe), amorphous solid, and the crystalline state by infrared spectroscopy and computational methods. The geometry of PTD conformers is defined by the orientation of two methyl ester groups, which may adopt pseudo-trans or pseudo-cis positions in relation to the pyrrolo-thiazole system. For both methyl ester groups, the latter arrangement was predicted by the calculations to be energetically the most favorable in the isolated molecule. The envelope form of the thiazolidine ring is present in all conformers, with the sulfur atom placed in the apex position, while the pyrrole ring is almost planar. Three types of conformers differing in the orientation of the methyl ester groups relative to the pyrrolo-thiazole system (cis/cis, trans/cis, cis/trans) were identified in the matrixes. The cis/cis forms were found to be the most stable ones in both gaseous state and argon matrixes. On the other hand, the more polar trans/cis forms were found to be stabilized in the more polarizable krypton and xenon matrixes as well as in the neat amorphous and crystalline phases. On the basis of annealing experiments, performed in argon and xenon matrixes up to 35 and 68 K, respectively, conformational changes preceding the aggregation of the compound are suggested.

Importance of entropy in the conformational equilibrium of phenylalanine: a matrix-isolation infrared spectroscopy and density functional theory study.

The conformational behavior and infrared spectrum of l-phenylalanine were studied by matrix-isolation infrared spectroscopy and DFT [B3LYP/6-311++G(d,p)] calculations. The fourteen most stable structures were predicted to differ in energy by less than 10 kJ mol(-1), eight of them with abundances higher than 5% at the temperature of evaporation of the compound (423 K). Experimental results suggest that six conformers contribute to the spectrum of the isolated compound, whereas two conformers (IIb(3) and IIIb(3)) relax in matrix to a more stable form (IIb(2)) due to low energy barriers for conformational isomerization (conformational cooling). The two lowest-energy conformers (Ib(1), Ia) differ only in the arrangement of the amino acid group relative to the phenyl ring; they exhibit a relatively strong stabilizing intramolecular hydrogen bond of the O-H...N type and the carboxylic group in the trans configuration (O=C-O-H dihedral angle ca. 180 degrees ). Type II conformers have a weaker H-bond of the N-H...O=C type, but they bear the more favorable cis arrangement of the carboxylic group. Being considerably more flexible, type II conformers are stabilized by entropy and the relative abundances of two conformers of this type (IIb(2) and IIc(1)) are shown to significantly increase with temperature due to entropic stabilization. At 423 K, these conformers are found to be the first and third most abundant species present in the conformational equilibrium, with relative populations of ca. 15% each, whereas their populations could be expected to be only ca. 5% if entropy effects were not taken into consideration. Indeed, phenylalanine can be considered a notable example of a molecule where entropy plays an essential role in determining the relative abundance of the possible low-energy conformational states and then, the thermodynamics of the compound, even at moderate temperatures. Upon UV irradiation (lambda > 235 nm) of the matrix-isolated compound, unimolecular photodecomposition of phenylalanine is observed with production of CO(2) and phenethylamine.