Tyrosine Nitration of Voltage-dependent Anion Channels in Cardiac Ischemia-reperfusion: Reduction by Peroxynitrite Scavenging

Excess superoxide (O2−) and nitric oxide (NO) forms peroxynitrite (ONOO−) during cardiac ischemia reperfusion (IR) injury, which in turn induces protein tyrosine nitration (tyr-N). Mitochondria are both a source of and target for ONOO−. Our aim was to identify specific mitochondrial proteins that display enhanced tyr-N after cardiac IR injury, and to explore whether inhibiting O2−/ONOO− during IR decreases mitochondrial protein tyr-N and consequently improves cardiac function. We show here that IR increased tyr-N of 35 and 15 kDa mitochondrial proteins using Western blot analysis with 3-nitrotyrosine antibody. Immunoprecipitation (IP) followed by LC–MS/MS identified 13 protein candidates for tyr-N. IP and Western blot identified and confirmed that the 35 kDa tyr-N protein is the voltage-dependent anion channel (VDAC). Tyr-N of native cardiac VDAC with IR was verified on recombinant (r) VDAC with exogenous ONOO−. We also found that ONOO− directly enhanced rVDAC channel activity, and rVDAC tyr-N induced by ONOO− formed oligomers. Resveratrol (RES), a scavenger of O2−/ONOO−, reduced the tyr-N levels of both native and recombinant VDAC, while L-NAME, which inhibits NO generation, only reduced tyr-N levels of native VDAC. O2− and ONOO− levels were reduced in perfused hearts during IR by RES and L-NAME and this was accompanied by improved cardiac function. These results identify tyr-N of VDAC and show that reducing ONOO− during cardiac IR injury can attenuate tyr-N of VDAC and improve cardiac function

A Cobalt-Containing Eukaryotic Nitrile Hydratase

Nitrile hydratase (NHase), an industrially important enzyme that catalyzes the hydration of nitriles to their corresponding amides, has only been characterized from prokaryotic microbes. The putative NHase from the eukaryotic unicellular choanoflagellate organism Monosiga brevicollis (MbNHase) was heterologously expressed in Escherichia coli. The resulting enzyme expressed as a single polypeptide with fused α- and β-subunits linked by a seventeen-histidine region. Size-exclusion chromatography indicated that MbNHase exists primarily as an (αβ)2 homodimer in solution, analogous to the α2β2 homotetramer architecture observed for prokaryotic NHases. The NHase enzyme contained its full complement of Co(III) and was fully functional without the co-expression of an activator protein or E. coli GroES/EL molecular chaperones. The homology model of MbNHase was developed identifying Cys400, Cys403, and Cys405 as active site ligands. The results presented here provide the first experimental data for a mature and active eukaryotic NHase with fused subunits. Since this new member of the NHase family is expressed from a single gene without the requirement of an activator protein, it represents an alternative biocatalyst for industrial syntheses of important amide compounds

Expression patterns of chondrocyte genes cloned by differential display in tibial dyschondroplasia

AbstractTibial dyschondroplasia (TD) appears to involve a failure of the growth plate chondrocytes within growing long bones to differentiate fully to the hypertrophic stage, resulting in a mass of prehypertrophic chondrocytes which form the avascular TD lesion. Many biochemical and molecular markers of chondrocyte hypertrophy are absent from the lesion, or show reduced expression, but the cause of the disorder remains to be identified. As differentiation to the hypertrophic state is impaired in TD, we hypothesised that chondrocyte genes that are differentially expressed in the growth plate should show altered expression in TD. Using differential display, four genes, B-cadherin, EF2, HT7 and Ex-FABP were cloned from chondrocytes stimulated to differentiate to the hypertrophic stage in vitro, and their differential expression confirmed in vivo. Using semi-quantitative RT-PCR, the expression patterns of these genes were compared in chondrocytes from normal and TD growth plates. Surprisingly, none of these genes showed the pattern of expression that might be expected in TD lesion chondrocytes, and two of them, B-cadherin and Ex-FABP, were upregulated in the lesion. This indicates that the TD phenotype does not merely reflect the absence of hypertrophic marker genes, but may be influenced by more complex developmental mechanisms/defects than previously thought

Epigenetic control of EMT/MET dynamics: HNF4α impacts DNMT3s through miRs-29

Background and aims: Epithelial-to-mesenchymal transition (EMT) and the reverse mesenchymal-to-epithelial transition (MET) are manifestations of cellular plasticity that imply a dynamic and profound gene expression reprogramming. While a major epigenetic code controlling the coordinated regulation of a whole transcriptional profile is guaranteed by DNA methylation, DNA methyltransferase (DNMT) activities in EMT/MET dynamics are still largely unexplored. Here, we investigated the molecular mechanisms directly linking HNF4α, the master effector of MET, to the regulation of both de novo of DNMT 3A and 3B. Methods: Correlation among EMT/MET markers, microRNA29 and DNMT3s expression was evaluated by RT-qPCR, Western blotting and immunocytochemical analysis. Functional roles of microRNAs and DNMT3s were tested by anti-miRs, microRNA precursors and chemical inhibitors. ChIP was utilized for investigating HNF4α DNA binding activity. Results: HNF4α silencing was sufficient to induce positive modulation of DNMT3B, in in vitro differentiated hepatocytes as well as in vivo hepatocyte-specific Hnf4α knockout mice, and DNMT3A, in vitro, but not DNMT1. In exploring the molecular mechanisms underlying these observations, evidence have been gathered for (i) the inverse correlation between DNMT3 levels and the expression of their regulators miR-29a and miR- 29b and (ii) the role of HNF4α as a direct regulator of miR-29a-b transcription. Notably, during TGFβ-induced EMT, DNMT3s' pivotal function has been proved, thus suggesting the need for the repression of these DNMTs in the maintenance of a differentiated phenotype. Conclusions: HNF4α maintains hepatocyte identity by regulating miR-29a and -29b expression, which in turn control epigenetic modifications by limiting DNMT3A and DNMT3B levels

Light Adaptation in Phycobilisome antennas: Influence on the Rod Length and Structural Arrangement

Phycobilisomes, the light-harvesting antennas of cyanobacteria, can adapt to a wide range of environments thanks to a composition and function response to stress conditions. We study how structural changes influence excitation transfer in these super-complexes. Specifically, we show the influence of the rod length on the photon absorption and subsequent excitation transport to the core. Despite the fact that the efficiency of individual disks on the rod decreases with increasing rod length, we find an optimal length for which the average rod efficiency is maximal. Combining this study with experimental structural measurements, we propose models for the arrangement of the phycobiliproteins inside the thylakoid membranes, evaluate the importance of rod length, and predict the corresponding transport properties for different cyanobacterial species. This analysis, which links the functional and structural properties of full phycobilisome complexes, thus provides further rationals to help resolving their exact structure.Comment: 7 pages, 7 figures, 2 pages supplementary materia

Blue-fluorescence of NADPH as an indicator of marine primary production

Nicotinamide Adenine Dinucleotide Phosphate (NADPH) is the primary product of photosynthesisand can therefore serve as an indicator of biomass and photosynthetic activity. Pure NADPH whichis the reduced form of NADP shows an absorption maximum at 340 nm and a maximum of emissionat 460 nm. NADPH concentrations in terrestrial vegetation have already been studied since1957 in great detail with optical methods. However, its potential as a biomass parameter of oceanicphytoplankton which can be assessed in situ and remotely with fluorescence spectroscopy has notyet been investigated.In this paper, we report on laboratory investigations of the blue-fluorescence spectrum in algalsuspensions of Chlorella and Thalassiosira when excited with UV-A light. It is shown that cell densitiesof about 106 per litre as they are typically found under natural conditions are too low for precisedetection of NADPH fluorescence, while concentrated samples with 108-1010 cells per litre exhibitsignificant blue-fluorescence which can be related to NADPH. Inhibition of photosynthetic activityby addition of DCMU decreases the strength of blue-fluorescence remarkably. Since NADPHis an end product of photosynthesis, changes of PAR illumination levels should directly affect itsconcentration and hence the intensity of blue-fluorescence. However, no effect of illumination onblue-fluorescence could be observed in our study. Possible reasons of these observations are discussed,and perspectives for practical applications of the method used are proposed

Implications for oxidative stress and astrocytes following 26S proteasomal depletion in mouse forebrain neurones

Neurodegenerative diseases are characterized by progressive degeneration of selective neurones in the nervous system, but the underlying mechanisms involved in neuroprotection and neurodegeneration remain unclear. Dysfunction of the ubiquitin proteasome system is one of the proposed hypotheses for the cause and progression of neuronal loss. We have performed quantitative two-dimensional fluorescence difference in-gel electrophoresis combined with peptide mass fingerprinting to reveal proteome changes associated with neurodegeneration following 26S proteasomal depletion in mouse forebrain neurones. Differentially expressed proteins were validated by Western blotting, biochemical assays and immunohistochemistry. Of significance was increased expression of the antioxidant enzyme peroxiredoxin 6 (PRDX6) in astrocytes, associated with oxidative stress. Interestingly, PRDX6 is a bifunctional enzyme with antioxidant peroxidase and phospholipase A2 (PLA2) activities. The PLA2 activity of PRDX6 was also increased following 26S proteasomal depletion and may be involved in neuroprotective or neurodegenerative mechanisms. This is the first in vivo report of oxidative stress caused directly by neuronal proteasome dysfunction in the mammalian brain. The results contribute to understanding neuronal–glial interactions in disease pathogenesis, provide an in vivo link between prominent disease hypotheses and importantly, are of relevance to a heterogeneous spectrum of neurodegenerative diseases

Lens ER-stress response during cataract development in Mip-mutant mice

AbstractMajor intrinsic protein (MIP) is a functional water-channel (AQP0) that also plays a key role in establishing lens fiber cell architecture. Genetic variants of MIP have been associated with inherited and age-related forms of cataract; however, the underlying pathogenic mechanisms are unclear. Here we have used lens transcriptome profiling by microarray-hybridization and qPCR to identify pathogenic changes during cataract development in Mip-mutant (Lop/+) mice. In postnatal Lop/+ lenses (P7) 99 genes were up-regulated and 75 were down-regulated (>2-fold, p=<0.05) when compared with wild-type. A pathway analysis of up-regulated genes in the Lop/+ lens (P7) was consistent with endoplasmic reticulum (ER)-stress and activation of the unfolded protein response (UPR). The most up-regulated UPR genes (>4-fold) in the Lop/+ lens included Chac1>Ddit3>Atf3>Trib3>Xbp1 and the most down-regulated genes (>5-fold) included two anti-oxidant genes, Hspb1 and Hmox1. Lop/+ lenses were further characterized by abundant TUNEL-positive nuclei within central degenerating fiber cells, glutathione depletion, free-radical overproduction, and calpain hyper-activation. These data suggest that Lop/+ lenses undergo proteotoxic ER-stress induced cell-death resulting from prolonged activation of the Eif2ak3/Perk-Atf4-Ddit3-Chac1 branch of the UPR coupled with severe oxidative-stress

Yeast cytochrome c oxidase: a model system to study mitochondrial forms of the haem-copper oxidase superfamily.

The known subunits of yeast mitochondrial cytochrome c oxidase are reviewed. The structures of all eleven of its subunits are explored by building homology models based on the published structures of the homologous bovine subunits and similarities and differences are highlighted, particularly of the core functional subunit I. Yeast genetic techniques to enable introduction of mutations into the three core mitochondrially-encoded subunits are reviewed