Role of TRPM2 in H2O2-induced cell apoptosis in endothelial cells

Melastatin-like transient receptor potential channel 2 (TRPM2) is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H(2)O(2))-induced cytosolic Ca(2+) ([Ca(2+)](i)) elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca(2+)](i) rise and whole-cell current change in response to H(2)O(2). Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H(2)O(2)-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect against H(2)O(2)-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-alpha-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca(2+)](i) and whole-cell currents to H(2)O(2). TRPM2 overexpression also aggravated the H(2)O(2)-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca(2+) overload in response to H(2)O(2) and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.published_or_final_versio

Cloning, purification, and functional characterization of Carocin S2, a ribonuclease bacteriocin produced by Pectobacterium carotovorum

<p>Abstract</p> <p>Background</p> <p>Most isolates of <it>Pectobacterium carotovorum subsp. carotovorum </it>(Pcc) produce bacteriocins. In this study, we have determined that Pcc strain F-rif-18 has a chromosomal gene encoding the low-molecular-weight bacteriocin, Carocin S2, and that this bacteriocin inhibits the growth of a closely related strain. Carocin S2 is inducible by ultraviolet radiation but not by mutagenic agents such as mitomycin C.</p> <p>Results</p> <p>A carocin S2-defective mutant, TF1-2, was obtained by Tn5 insertional mutagenesis using F-rif-18. A 5706-bp DNA fragment was detected by Southern blotting, selected from a genomic DNA library, and cloned to the vector, pMS2KI. Two adjacent complete open reading frames within pMS2KI were sequenced, characterized, and identified as caroS2K and caroS2I, which respectively encode the killing protein and immunity protein. Notably, carocin S2 could be expressed not only in the mutant TF1-2 but also in <it>Escherichia coli </it>DH5α after entry of the plasmid pMS2KI. Furthermore, the C-terminal domain of CaroS2K was homologous to the nuclease domains of colicin D and klebicin D. Moreover, SDS-PAGE analysis showed that the relative mass of CaroS2K was 85 kDa and that of CaroS2I was 10 kDa.</p> <p>Conclusion</p> <p>This study shown that another nuclease type of bacteriocin was found in <it>Pectobacterium carotovorum</it>. This new type of bacteriocin, Carocin S2, has the ribonuclease activity of CaroS2K and the immunity protein activity of CaroS2I.</p

AMiBA: scaling relations between the integrated Compton-y and X-ray derived temperature, mass, and luminosity

We investigate the scaling relations between the X-ray and the thermal Sunyaev-Zel'dovich Effect (SZE) properties of clusters of galaxies, using data taken during 2007 by the Y.T. Lee Array for Microwave Background Anisotropy (AMiBA) at 94 GHz for the six clusters A1689, A1995, A2142, A2163, A2261, and A2390. The scaling relations relate the integrated Compton-y parameter Y_{2500} to the X-ray derived gas temperature T_{e}, total mass M_{2500}, and bolometric luminosity L_X within r_{2500}. Our results for the power-law index and normalization are both consistent with the self-similar model and other studies in the literature except for the Y_{2500}-L_X relation, for which a physical explanation is given though further investigation may be still needed. Our results not only provide confidence for the AMiBA project but also support our understanding of galaxy clusters.Comment: Accepted by ApJ; 8 pages, 3 figures, 5 table

Generation of induced pluripotent stem cell lines from 3 distinct laminopathies bearing heterogeneous mutations in lamin A/C

The term laminopathies defines a group of genetic disorders caused by defects in the nuclear envelope, mostly the lamins. Lamins are the main constituents of the nuclear lamina, a filamentous meshwork associated with the inner nuclear membrane that provides mechanical stability and plays important roles in processes such as transcription, DNA replication and chromatin organization. More than 300 mutations in lamin A/C have been associated with diverse clinical phenotypes, understanding the molecular basis of these diseases may provide a rationale for treating them. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a patient with inherited dilated cardiomiopathy and 2 patients with distinct accelerated forms of aging, atypical Werner syndrome and Hutchinson Gilford progeria, all of which are caused by mutations in lamin A/C. These cell lines were pluripotent and displayed normal nuclear membrane morphology compared to donor fibroblasts. Their differentiated progeny reproduced the disease phenotype, reinforcing the idea that they represent excellent tools for understanding the role of lamin A/C in normal physiology and the clinical diversity associated with these diseases

AMiBA: Broadband Heterodyne CMB Interferometry

The Y. T. Lee Array for Microwave Background (AMiBA) has reported the first science results on the detection of galaxy clusters via the Sunyaev Zel'dovich effect. The science objectives required small reflectors in order to sample large scale structures (20') while interferometry provided modest resolutions (2'). With these constraints, we designed for the best sensitivity by utilizing the maximum possible continuum bandwidth matched to the atmospheric window at 86-102GHz, with dual polarizations. A novel wide-band analog correlator was designed that is easily expandable for more interferometer elements. MMIC technology was used throughout as much as possible in order to miniaturize the components and to enhance mass production. These designs will find application in other upcoming astronomy projects. AMiBA is now in operations since 2006, and we are in the process to expand the array from 7 to 13 elements.Comment: 10 pages, 6 figures, ApJ in press; a version with high resolution figures available at http://www.asiaa.sinica.edu.tw/~keiichi/upfiles/AMiBA7/mtc_highreso.pd

Confirmation of the Chinese version of the Problematic Internet Use Questionnaire Short Form (PIUQ-SF)

Although an increasing number of studies have focused on problematic Internet use, numerous measures exist with only modest investigation into their reliability and validity, both within and across different cultures (Király et al. 2015). The most frequently used questionnaire is the Internet Addiction Test (Young 1998). However, its factor structure appears to be unstable (e.g., Fernandez-Villa et al. 2015; Pontesetal. 2014). Koronczai et al. (2011) suggested that a suitable measure should fit six basic requirements. More specifically, it should be (i) comprehensive, that is examining more, possibly all, aspects of problematic Internet use; (ii) as concise as possible, in order to be able to assess the more impulsive population, and to use in time-limited surveys (or having different forms with different length). Such a measure should be (iii) reliable and valid for different methods of data collection (e.g. online, paper-and-pencil self-rating, face-to-face); as well as (iv) for different age groups (e.g. adolescents and adults), and (v) in different cultural settings. Finally, (vi) cut-off scores should be defined on the basis of clinical examination. The Problematic Internet Use Questionnaire (PIUQ) (Demetrovics et al. 2008) fulfills several of the aforementioned criteria. The 18-item PIUQ is a comprehensive measure that assesses three basic aspects of problematic Internet use (i.e., obsession, neglect, and control disorder). The 18-item and the shorter 9-item versions of the PIUQ have a reliable structure, and are suitable for time-limited surveys and have proved to be valid across various methods of data collection (i.e., online as well as paper-pencil) and age groups (Demetrovics et al. 2008; Koronczai et al. 2011). However, its implementation has so far mostly taken place in Western individualistic cultures (Kelley and Gruber 2010, 2013; Koronczai et al. 2011; Zahodne et al. 2011. To date, the PIUQ has also been applied in a survey in Iran with good Cronbach’s alpha coefficients and subscales (Mazhari 2012a, 2012b). Consequently, validation of the questionnaire in a culture that is significantly different from the Western culture is needed. Accordingly, the aim of the present study was the validation of the 9-item PIUQ among the Chinese population

Chloroplast phosphoglycerate kinase, a gluconeogenetic enzyme, is required for efficient accumulation of Bamboo mosaic virus

The tertiary structure in the 3′-untranslated region (3′-UTR) of Bamboo mosaic virus (BaMV) RNA is known to be involved in minus-strand RNA synthesis. Proteins found in the RNA-dependent RNA polymerase (RdRp) fraction of BaMV-infected leaves interact with the radio labeled 3′-UTR probe in electrophoretic mobility shift assays (EMSA). Results derived from the ultraviolet (UV) cross-linking competition assays suggested that two cellular factors, p43 and p51, interact specifically with the 3′-UTR of BaMV RNA. p43 and p51 associate with the poly(A) tail and the pseudoknot of the BaMV 3′-UTR, respectively. p51-containing extracts specifically down-regulated minus-strand RNA synthesis when added to in vitro RdRp assays. LC/MS/MS sequencing indicates that p43 is a chloroplast phosphoglycerate kinase (PGK). When the chloroplast PKG levels were knocked down in plants, using virus-induced gene silencing system, the accumulation level of BaMV coat protein was also reduced

AMiBA Wideband Analog Correlator

A wideband analog correlator has been constructed for the Yuan-Tseh Lee Array for Microwave Background Anisotropy. Lag correlators using analog multipliers provide large bandwidth and moderate frequency resolution. Broadband IF distribution, backend signal processing and control are described. Operating conditions for optimum sensitivity and linearity are discussed. From observations, a large effective bandwidth of around 10 GHz has been shown to provide sufficient sensitivity for detecting cosmic microwave background variations.Comment: 28 pages, 23 figures, ApJ in press