The relationships of phytosterols and oxyphytosterols in plasma and aortic valve cusps in patients with severe aortic stenosis

Phytosterols such as campesterol and sitosterol are susceptible to oxidation by reactive oxygen species. We hypothesize that the plant sterols (PS) campesterol and sitosterol and their 7-oxygenated metabolites (POPs) correlate within and between human plasma and aortic valve cusps tissues. Plasma and tissue concentrations of PS and POPs were analyzed by gas chromatography–mass spectrometry-selected ion monitoring. Prior to analysis valve cusps tissue was mechanically separated from the calcified parts. PS and POP levels per dry cusps tissue weight were significantly higher compared with the concentrations in the calcified part. Against our hypothesis we found that despite the fact that there is a high correlation between plant sterols in and between plasma and valves cusps tissue, as well as a high correlation between plant sterols and oxyphytosterols and oxyphytosterols themselves within the valve cusps tissue, there was hardly any correlation in the amount of oxyphytosterols in plasma and between plasma and valves. Because plasma samples are easily accessible for large scale population based studies, we have to understand in more detail what the analysis of POPs implies in terms of CVD risk for the future.     

Cardiac expression and atrial fibrillation-associated remodeling of K2P2.1 (TREK-1) K channels in a porcine model

Aims

Effective management of atrial fibrillation (AF) often remains an unmet need. Cardiac two-pore-domain K⁺ (K_2P) channels are implicated in action potential regulation, and their inhibition has been proposed as a novel antiarrhythmic strategy. K_2P2.1 (TREK-1) channels are expressed in the human heart. This study was designed to identify and functionally express porcine K_2P2.1 channels. In addition, we sought to analyze cardiac expression and AF-associated K_2P2.1 remodeling in a clinically relevant porcine AF model.

Main methods

Three pK_2P2.1 isoforms were identified and amplified. Currents were recorded using voltage clamp electrophysiology in the Xenopus oocyte expression system. K_2P2.1 remodeling was studied by quantitative real time PCR and Western blot in domestic pigs during AF induced by atrial burst pacing.

Key findings

Human and porcine K_2P2.1 proteins share 99% identity. Residues involved in phosphorylation or glycosylation are conserved. Porcine K_2P2.1 channels carried outwardly rectifying K⁺ currents similar to their human counterparts. In pigs, K_2P2.1 was expressed ubiquitously in the heart with predominance in the atrial tissue. AF was associated with time-dependent reduction of K_2P2.1 protein in the RA by 70% (7 days of AF) and 80% (21 days of AF) compared to control animals in sinus rhythm. K_2P2.1 expression in the left atrium, AV node, and ventricles was not affected by AF.

Significance

Similarities between porcine and human K_2P2.1 channels indicate that the pig may represent a valid model for mechanistic and preclinical studies. AF-related atrial K_2P2.1 remodeling has potential implications for arrhythmia maintenance and antiarrhythmic therapy.     

Brain-derived proteins in the CSF, do they correlate with brain pathology in CJD?

Background

Brain derived proteins such as 14-3-3, neuron-specific enolase (NSE), S 100b, tau, phosphorylated tau and Aβ_1–42 were found to be altered in the cerebrospinal fluid (CSF) in Creutzfeldt-Jakob disease (CJD) patients. The pathogenic mechanisms leading to these abnormalities are not known, but a relation to rapid neuronal damage is assumed. No systematic analysis on brain-derived proteins in the CSF and neuropathological lesion profiles has been performed.

Methods

CSF protein levels of brain-derived proteins and the degree of spongiform changes, neuronal loss and gliosis in various brain areas were analyzed in 57 CJD patients.

Results

We observed three different patterns of CSF alteration associated with the degree of cortical and subcortical changes. NSE levels increased with lesion severity of subcortical areas. Tau and 14-3-3 levels increased with minor pathological changes, a negative correlation was observed with severity of cortical lesions. Levels of the physiological form of the prion protein (PrP^c) and Aβ_1–42 levels correlated negatively with cortical pathology, most clearly with temporal and occipital lesions.

Conclusion

Our results indicate that the alteration of levels of brain-derived proteins in the CSF does not only reflect the degree of neuronal damage, but it is also modified by the localization on the brain pathology. Brain specific lesion patterns have to be considered when analyzing CSF neuronal proteins.     

Developmental activation of the lysozyme gene in chicken macrophage cells is linked to core histone acetylation at its enhancer elements

Native chromatin IP assays were used to define changes in core histone acetylation at the lysozyme locus during developmental maturation of chicken macrophages and stimulation to high-level expression by lipo-polysaccharide. In pluripotent precursors the lysozyme gene (Lys) is inactive and there is no acetylation of core histones at the gene, its promoter or at the upstream cis-control elements. In myeloblasts, where there is a very low level of Lys expression, H4 acetylation appears at the cis-control elements but not at the Lys gene or its promoter: neither H3 nor H2B become significantly acetylated in myeloblasts. In mature macrophages, Lys expression increases 5-fold: H4, H2B and H2A.Z are all acetylated at the cis-control elements but H3 remains unacetylated except at the −2.4 S silencer. Stimulation with LPS increases Lys expression a further 10-fold: this is accompanied by a rise in H3 acetylation throughout the cis-control elements; H4 and H2B acetylation remain substantial but acetylation at the Lys gene and its promoter remains low. Acetylation is thus concentrated at the cis-control elements, not at the Lys gene or its immediate promoter. H4 acetylation precedes H3 acetylation during development and H3 acetylation is most directly linked to high-level Lys expression.     

The relative magnitude of the effects of biological and physical settlement cues for cypris larvae of the acorn barnacle, Semibalanus balanoides L

Barnacle cypris larvae respond to many cues when selecting a settlement site. The settlement of over a million larvae on tiles of different textures, orientations and densities of incumbent settlers was measured on the rocky intertidal at Great Cumbrae, Scotland. Half of the tiles were replaced every tide whereas the others simultaneously accumulated settlers. Factor effects varied on each tide, and converged in the accumulating deployment. Increasing incumbent density led to net loss of settlement, which was less probable on the textures on which fastest settlment occurred ('very fine'), and more probable on those on which settlement was slowest ('smooth'). More settlement occurred on down-facing orientations during daylight and vice versa. Cue ranks were non-linear, so a path analysis model quantified the relative influence of each factor. Gregariousness was the most influential cue measured, although unmeasured factors had greater effects, highlighting the complexity of settlement influences in this species.     

Mesenchymal stem cells in regenerative medicine: Opportunities and challenges for articular cartilage and intervertebral disc tissue engineering

Defects of load‐bearing connective tissues such as articular cartilage and intervertebral disc (IVD) can result from trauma, degenerative, endocrine, or age‐related disease. Current surgical and pharmacological options for the treatment of arthritic rheumatic conditions in the joints and spine are ineffective. Cell‐based surgical therapies such as autologous chondrocyte transplantation (ACT) have been in clinical use for cartilage repair for over a decade but this approach has shown mixed results. This review focuses on the potential of mesenchymal stem cells (MSCs) as an alternative to cells derived from patient tissues in autologous transplantation and tissue engineering. Here we discuss the prospects of using MSCs in regenerative medicine and summarize the advantages and disadvantages of these cells in articular cartilage and IVD tissue engineering. We discuss the conceptual and practical difficulties associated with differentiating and pre‐conditioning MSCs for subsequent survival in a physiologically harsh extracellular matrix, an environment that will be highly hypoxic, acidic, and nutrient deprived. Implanted MSCs will be exposed to traumatic physical loads and high levels of locally produced inflammatory mediators and catabolic cytokines. We also explore the potential of culture models of MSCs, fully differentiated cells and co‐cultures as “proof of principle” ethically acceptable “3Rs” models for engineering articular cartilage and IVD in vitro for the purpose of replacing the use of animals in arthritis research. J. Cell. Physiol. 222:23–32, 2010. © 2009 Wiley‐Liss, Inc.     

Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H2-Consuming and H2-Producing Reactions in Escherichia coli

Escherichia coli uptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H₂ to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochrome b membrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H₂ evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H₂ evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H₂-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H₂ oxidation or H₂ evolution in whole cells, reversible H₂-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H₂ evolution mode. Our findings suggest that Hyd-2 switches between H₂-consuming and H₂-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H₂ evolution from glycerol requires reverse electron transport.