Alterations in voltage-sensing of the mitochondrial permeability transition pore in ANT1-deficient cells

The probability of mitochondrial permeability transition (mPT) pore opening is inversely related to the magnitude of the proton electrochemical gradient. The module conferring sensitivity of the pore to this gradient has not been identified. We investigated mPT's voltage-sensing properties elicited by calcimycin or H2O2 in human fibroblasts exhibiting partial or complete lack of ANT1 and in C2C12 myotubes with knocked-down ANT1 expression. mPT onset was assessed by measuring in situ mitochondrial volume using the 'thinness ratio' and the 'cobalt-calcein' technique. De-energization hastened calcimycin-induced swelling in control and partially-expressing ANT1 fibroblasts, but not in cells lacking ANT1, despite greater losses of mitochondrial membrane potential. Matrix Ca(2+) levels measured by X-rhod-1 or mitochondrially-targeted ratiometric biosensor 4mtD3cpv, or ADP-ATP exchange rates did not differ among cell types. ANT1-null fibroblasts were also resistant to H2O2-induced mitochondrial swelling. Permeabilized C2C12 myotubes with knocked-down ANT1 exhibited higher calcium uptake capacity and voltage-thresholds of mPT opening inferred from cytochrome c release, but intact cells showed no differences in calcimycin-induced onset of mPT, irrespective of energization and ANT1 expression, albeit the number of cells undergoing mPT increased less significantly upon chemically-induced hypoxia than control cells. We conclude that ANT1 confers sensitivity of the pore to the electrochemical gradient

GC Content Increased at CpG Flanking Positions of Fish Genes Compared with Sea Squirt Orthologs as a Mechanism for Reducing Impact of DNA Methylation

Background: Fractional DNA methylation in sea squirts evolved to global DNA methylation in fish. The impact of global DNA methylation is reflected by more CpG depletions and/or more A/T to G/C changes at CpG flanking positions due to context-dependent mutations of methylated CpG sites. Methods and Findings: In this report, we demonstrate that the sea squirt genes have undergone more CpG to TpG/CpA substitutions than the fish orthologs using homologous fragments from orthologous genes among Ciona intestinalis, Ciona savignyi, fugufish and zebrafish. To avoid premature transcription, the TGA sites derived from CGA were largely converted to TGG in sea squirt genes. By contrast, a significant increment of GC content at CpG flanking positions was shown in fish genes. The positively selected A/T to G/C substitutions, in combination with the CpG to TpG/CpA substitutions, are the sources of the extremely low CpG observed/expected ratios in vertebrates. The nonsynonymous substitutions caused by the GC content increase have resulted in frequent amino acid replacements in the directions that were not noticed previously. Conclusion: The increased GC content at CpG flanking positions can reduce CpG loss in fish genes and attenuate the impact of DNA methylation on CpG-containing codons, probably accounting for evolution towards vertebrates. © 2008 Wang, Leung.published_or_final_versio

Alterations in voltage-sensing of the mitochondrial permeability transition pore in ANT1-deficient cells

The probability of mitochondrial permeability transition (mPT) pore opening is inversely related to the magnitude of the proton electrochemical gradient. The module conferring sensitivity of the pore to this gradient has not been identified. We investigated mPT's voltage-sensing properties elicited by calcimycin or H2O2 in human fibroblasts exhibiting partial or complete lack of ANT1 and in C2C12 myotubes with knocked-down ANT1 expression. mPT onset was assessed by measuring in situ mitochondrial volume using the 'thinness ratio' and the 'cobalt-calcein' technique. De-energization hastened calcimycin-induced swelling in control and partially-expressing ANT1 fibroblasts, but not in cells lacking ANT1, despite greater losses of mitochondrial membrane potential. Matrix Ca(2+) levels measured by X-rhod-1 or mitochondrially-targeted ratiometric biosensor 4mtD3cpv, or ADP-ATP exchange rates did not differ among cell types. ANT1-null fibroblasts were also resistant to H2O2-induced mitochondrial swelling. Permeabilized C2C12 myotubes with knocked-down ANT1 exhibited higher calcium uptake capacity and voltage-thresholds of mPT opening inferred from cytochrome c release, but intact cells showed no differences in calcimycin-induced onset of mPT, irrespective of energization and ANT1 expression, albeit the number of cells undergoing mPT increased less significantly upon chemically-induced hypoxia than control cells. We conclude that ANT1 confers sensitivity of the pore to the electrochemical gradient

Comprehensive analysis of the base composition around the transcription start site in Metazoa

BACKGROUND: The transcription start site of a metazoan gene remains poorly understood, mostly because there is no clear signal present in all genes. Now that several sequenced metazoan genomes have been annotated, we have been able to compare the base composition around the transcription start site for all annotated genes across multiple genomes. RESULTS: The most prominent feature in the base compositions is a significant local variation in G+C content over a large region around the transcription start site. The change is present in all animal phyla but the extent of variation is different between distinct classes of vertebrates, and the shape of the variation is completely different between vertebrates and arthropods. Furthermore, the height of the variation correlates with CpG frequencies in vertebrates but not in invertebrates and it also correlates with gene expression, especially in mammals. We also detect GC and AT skews in all clades (where %G is not equal to %C or %A is not equal to %T respectively) but these occur in a more confined region around the transcription start site and in the coding region. CONCLUSIONS: The dramatic changes in nucleotide composition in humans are a consequence of CpG nucleotide frequencies and of gene expression, the changes in Fugu could point to primordial CpG islands, and the changes in the fly are of a totally different kind and unrelated to dinucleotide frequencies

Shape optimization for the generalized Graetz problem

We apply shape optimization tools to the generalized Graetz problem which is a convection-diffusion equation. The problem boils down to the optimization of generalized eigen values on a two phases domain. Shape sensitivity analysis is performed with respect to the evolution of the interface between the fluid and solid phase. In particular physical settings, counterexamples where there is no optimal domains are exhibited. Numerical examples of optimal domains with different physical parameters and constraints are presented. Two different numerical methods (level-set and mesh-morphing) are show-cased and compared

TRAF6 ubiquitinates TGFβ type I receptor to promote its cleavage and nuclear translocation in cancer

Transforming growth factor β (TGFβ) is a pluripotent cytokine promoting epithelial cell plasticity during morphogenesis and tumour progression. TGFβ binding to type II and type I serine/threonine kinase receptors (TβRII and TβRI) causes activation of different intracellular signaling pathways. TβRI is associated with the ubiquitin ligase tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Here we show that TGFβ, via TRAF6, causes Lys63-linked polyubiquitination of TβRI, promoting cleavage of TβRI by TNF-alpha converting enzyme (TACE), in a PKCζ-dependent manner. The liberated intracellular domain (ICD) of TβRI associates with the transcriptional regulator p300 to activate genes involved in tumour cell invasiveness, such as Snail and MMP2. Moreover, TGFβ-induced invasion of cancer cells is TACE- and PKCζ- dependent and the TβRI ICD is localized in the nuclei of different kinds of tumour cells in tissue sections. Thus, our data reveal a specific role for TβRI in TGFβ mediated tumour invasion

Imaging and Modeling Data from the Hydrogen Epoch of Reionization Array

We analyze data from the Hydrogen Epoch of Reionization Array. This is the third in a series of papers on the closure phase delay-spectrum technique designed to detect the HI 21cm emission from cosmic reionization. We present the details of the data and models employed in the power spectral analysis, and discuss limitations to the process. We compare images and visibility spectra made with HERA data, to parallel quantities generated from sky models based on the GLEAM survey, incorporating the HERA telescope model. We find reasonable agreement between images made from HERA data, with those generated from the models, down to the confusion level. For the visibility spectra, there is broad agreement between model and data across the full band of $\sim 80$MHz. However, models with only GLEAM sources do not reproduce a roughly sinusoidal spectral structure at the tens of percent level seen in the observed visibility spectra on scales $\sim 10$ MHz on 29 m baselines. We find that this structure is likely due to diffuse Galactic emission, predominantly the Galactic plane, filling the far sidelobes of the antenna primary beam. We show that our current knowledge of the frequency dependence of the diffuse sky radio emission, and the primary beam at large zenith angles, is inadequate to provide an accurate reproduction of the diffuse structure in the models. We discuss implications due to this missing structure in the models, including calibration, and in the search for the HI 21cm signal, as well as possible mitigation techniques

Cardioprotective Effect of Nicorandil, a Mitochondrial ATP-Sensitive Potassium Channel Opener, Prolongs Survival in HSPB5 R120G Transgenic Mice

BACKGROUND: Transgenic (TG) mice with overexpression of an arg120gly (R120G) missense mutation in HSPB5 display desmin-related cardiomyopathy, which is characterized by formation of aggresomes. It is also known that progressive mitochondrial abnormalities and apoptotic cell death occur in the hearts of R120G TG mice. The role of mitochondrial dysfunction and apoptosis in disease progression, however, remains uncertain. METHODS AND RESULTS: Mitochondrial abnormalities and apoptotic cell death induced by overexpression of HSPB5 R120G were analyzed in neonatal rat cardiomyocytes. Overexpression of mutant HSPB5 led to development of aggresomes with a concomitant reduction in cell viability in the myocytes. Overexpression of mutant HSPB5 induced a reduction in the cytochrome c level in the mitochondrial fraction and a corresponding increase in the cytoplasmic fraction in the myocytes. Down-regulation of BCL2 and up-regulation of BAX were detected in the myocytes expressing the mutant HSPB5. Concomitant with mitochondrial abnormality, the activation of caspase-3 and increased apoptotic cell death was observed. Cell viability was dose-dependently recovered in myocytes overexpressing HSPB5 R120G by treatment with nicorandil a mitochondrial ATP-sensitive potassium channel opener. Nicorandil treatment also inhibited the increase in BAX, the decrease in BCL2, activation of caspase-3 and apoptotic cell death by mutant HSPB5. To confirm the results of the in-vitro study, we analyzed the effect of nicorandil in HSPB5 R120G TG mice. Nicorandil treatment appeared to reduce mitochondrial impairment and apoptotic cell death and prolonged survival in HSPB5 R120G TG mice. CONCLUSIONS: Nicorandil may prolong survival in HSPB5 R120G TG mice by protecting against mitochondrial impairments