Mesenchymal state of intimal cells may explain higher propensity to ascending aortic aneurysm in bicuspid aortic valves.

Individuals with a bicuspid aortic valve (BAV) are at significantly higher risk of developing aortic complications than individuals with tricuspid aortic valves (TAV) and defective signaling during the embryonic development and/or life time exposure to abnormal hemodynamic have been proposed as underlying factors. However, an explanation for the molecular mechanisms of aortopathy in BAV has not yet been provided. We combined proteomics, RNA analyses, immunohistochemistry, and electron microscopy to identify molecular differences in samples of non-dilated ascending aortas from BAV (N = 62) and TAV (N = 54) patients. Proteomic analysis was also performed for dilated aortas (N = 6 BAV and N = 5 TAV) to gain further insight into the aortopathy of BAV. Our results collectively showed the molecular signature of an endothelial/epithelial-mesenchymal (EndMT/EMT) transition-like process, associated with instability of intimal cell junctions and activation of RHOA pathway in the intima and media layers of ascending aorta in BAV patients. We propose that an improper regulation of EndMT/EMT during the spatiotemporally related embryogenesis of semilunar valves and ascending aorta in BAV individuals may result in aortic immaturity and instability prior to dilation. Exasperation of EndMT/EMT state in post embryonic life and/or exposure to non-physiological hemodynamic could lead to the aneurysm of ascending aorta in BAV individuals.

A combined proteomic and transcriptomic approach shows diverging molecular mechanisms in thoracic aortic aneurysm development in patients with tricuspid- and bicuspid aortic valve.

Thoracic aortic aneurysm is a pathological local dilatation of the aorta, potentially leading to aortic rupture or dissection. The disease is a common complication of patients with bicuspid aortic valve, a congenital disorder present in 1-2% of the population. Using two dimensional fluorescence difference gel electrophoresis proteomics followed by mRNA expression, and alternative splicing analysis of the identified proteins, differences in dilated and nondilated aorta tissues between 44 patients with bicuspid and tricuspid valves was examined. The pattern of protein expression was successfully validated with LC-MS/MS. A multivariate analysis of protein expression data revealed diverging protein expression fingerprints in patients with tricuspid compared with the patients with bicuspid aortic valves. From 302 protein spots included in the analysis, 69 and 38 spots were differentially expressed between dilated and nondilated aorta specifically in patients with tricuspid and bicuspid aortic valve, respectively. 92 protein spots were differentially expressed between dilated and nondilated aorta in both phenotypes. Similarly, mRNA expression together with alternative splicing analysis of the identified proteins also showed diverging fingerprints in the two patient groups. Differential splicing was abundant but the expression levels of differentially spliced mRNA transcripts were low compared with the wild type transcript and there was no correlation between splicing and the number of spots. Therefore, the different spots are likely to represent post-translational modifications. The identification of differentially expressed proteins suggests that dilatation in patients with a tricuspid aortic valve involves inflammatory processes whereas aortic aneurysm in patients with BAV may be the consequence of impaired repair capacity. The results imply that aortic aneurysm formation in patients with bicuspid and tricuspid aortic valves involve different biological pathways leading to the same phenotype.

De novo sequencing of a 21-kDa cytochrome c4 from Thiocapsa roseopersicina by nanoelectrospray ionization ion-trap and Fourier-transform ion-cyclotron resonance mass spectrometry.

We have determined the primary structure of cytochrome c(4) from Thiocapsa roseopersicina by de novo protein sequencing using the 'bottom up' approach. Three different enzymes (trypsin, endoproteinase Lys-C, and endoproteinase Glu-C) were employed to prepare four different sets of proteolytic digests. The digestion strategy was designed to permit a gradual buildup of smaller peptides into larger ones that were overlapped to yield the complete protein sequence. In this way we countered the main problem: peptides larger than about 1500 Da were difficult to sequence fully by tandem mass spectrometry. Direct infusion and online liquid chromatography were used on a linear ion trap Fourier-transform ion-cyclotron resonance hybrid instrument. The high resolving power, high mass accuracy and the availability of electron capture dissociation and collision-induced dissociation were essential to achieve full sequence coverage. The software DeNovoX complemented by manual interpretation was used to generate sequence information from tandem mass spectra. The predominantly automated nature of data acquisition and handling allowed for a relatively straightforward and fast procedure, which could compete with the mainstream alternative of nucleotide sequence determination.

Cross-crystallization method used for the crystallization and preliminary diffraction analysis of a novel di-haem cytochrome c4.

The newly discovered di-haem cytochrome c4 from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina is the first cytochrome c4 to be crystallized from an anaerobic organism. It was crystallized using the addition of metal-ion salts to the standard vapour-diffusion method. Coloured well shaped three-dimensional crystals with dimensions of approximately 0.6 x 0.05 x 0.02 mm grew within 3-4 d at pH 5 and diffracted to 1.72 angstroms without radiation damage. Cytochrome c4 crystallized in space group P4(1)2(1)2 as a primitive tetragonal system with unit-cell parameters a = b = 75.29, c = 37.12 angstroms, alpha = beta = gamma = 90 degrees.

Theoretical calculations on hydrogenase kinetics: explanation of the lag phase and the enzyme concentration dependence of the activity of hydrogenase uptake.

Two models of the hydrogenase reaction cycle were investigated by means of theoretical calculations and model simulations. The first model is the widely accepted triangular hydrogenase reaction cycle with minor modifications; the second is a modified triangular model, where we have introduced an autocatalytic step into the reaction cycle. Both models include a one-step activation reaction. The theoretical calculations and model simulations corroborate the assumed autocatalytic reaction step concluded from the experimental characteristics of the hydrogenase reaction.