Saccharomyces cerevisiae Grx6 and Grx7 are monothiol glutaredoxins associated with the early secretory pathway

Saccharomyces cerevisiae Grx6 and Grx7 are two monothiol glutaredoxins whose active-site sequences (CSYS and CPYS, respectively) are reminiscent of the CPYC active-site sequence of classical dithiol glutaredoxins. Both proteins contain an N-terminal transmembrane domain which is responsible for their association to membranes of the early secretory pathway vesicles, facing the luminal side. Thus, Grx6 localizes at the endoplasmic reticulum and Golgi compartments, while Grx7 is mostly at the Golgi. Expression of GRX6 is modestly upregulated by several stresses (calcium, sodium, and peroxides) in a manner dependent on the Crz1-calcineurin pathway. Some of these stresses also upregulate GRX7 expression under the control of the Msn2/4 transcription factor. The N glycosylation inhibitor tunicamycin induces the expression of both genes along with protein accumulation. Mutants lacking both glutaredoxins display reduced sensitivity to tunicamycin, although the drug is still able to manifest its inhibitory effect on a reporter glycoprotein. Grx6 and Grx7 have measurable oxidoreductase activity in vivo, which is increased in the presence of tunicamycin. Both glutaredoxins could be responsible for the regulation of the sulfhydryl oxidative state at the oxidant conditions of the early secretory pathway vesicles. However, the differences in location and expression responses against stresses suggest that their functions are not totally overlapping.

GD3 Synthase Overexpression Sensitizes Hepatocarcinoma Cells to Hypoxia and Reduces Tumor Growth by Suppressing the cSrc/NF-κB Survival Pathway

Abstract Background: Hypoxia-mediated HIF-1a stabilization and NF-kB activation play a key role in carcinogenesis by fostering cancer cell survival, angiogenesis and tumor invasion. Gangliosides are integral components of biological membranes with an increasingly recognized role as signaling intermediates. In particular, ganglioside GD3 has been characterized as a proapoptotic lipid effector by promoting cell death signaling and suppression of survival pathways. Thus, our aim was to analyze the role of GD3 in hypoxia susceptibility of hepatocarcinoma cells and in vivo tumor growth. Methodology/Principal Findings: We generated and characterized a human hepatocarcinoma cell line stably expressing GD3 synthase (Hep3B-GD3), which catalyzes the synthesis of GD3 from GM3. Despite increased GD3 levels (2-3 fold), no significant changes in cell morphology or growth were observed in Hep3B-GD3 cells compared to wild type Hep3B cells under normoxia. However, exposure of Hep3B-GD3 cells to hypoxia (2% O2) enhanced reactive oxygen species (ROS) generation, resulting in decreased cell survival, with similar findings observed in Hep3B cells exposed to increasing doses of exogenous GD3. In addition, hypoxia-induced c-Src phosphorylation at tyrosine residues, NF-kB activation and subsequent expression of Mn-SOD were observed in Hep3B cells but not in Hep3B-GD3 cells. Moreover, MnTBAP, an antioxidant with predominant SOD mimetic activity, reduced ROS generation, protecting Hep3B-GD3 cells from hypoxia-induced death. Finally, lower tumor growth, higher cell death and reduced Mn-SOD expression were observed in Hep3B-GD3 compared to Hep3B tumor xenografts. Conclusion: These findings underscore a role for GD3 in hypoxia susceptibility by disabling the c-Src/NF-kB survival pathway resulting in lower Mn-SOD expression, which may be of relevance in hepatocellular carcinoma therapy.

Controlling the spin of metal atoms adsorbed on oxide surfaces: Ni on regular and defective sites of the MgO(001) surface

We have analyzed the relative energy of nonmagnetic and magnetic low-lying electronic states of Ni atoms adsorbed on regular and defective sites of the MgO(001) surface. To this end cluster and periodic surface models are used within density functional theory. For Ni atoms adsorbed on oxygen vacancies at low coverage, the interaction energy between the metal and the support is much larger than on regular sites. Strong bonding results in a diamagnetic adsorbed species and the energy required to reach the high-spin state increases. Moreover, a correlation appears between the low-spin to high-spin energy difference and the interaction energy hypothesizing that it is possible to prepare the surface to tune the high-spin to low-spin energy difference. Magnetic properties of adsorbed thin films obtained upon increasing coverage are more difficult to interpret. This is because the metallic bond is readily formed and dominates over the effect of the atoms directly bound to the vacancy.

Hepatocyte nuclear factor-4 alpha regulates the human apolipoprotein AV gene: Identification of a novel response element and involvement in the control by peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, AMP-activated protein kinase, and mitogen-activated protein kinase pathway

The recently discovered apolipoprotein AV (apoAV) gene has been reported to be a key player in modulating plasma triglyceride levels. Here we identify the hepatocyte nuclear factor-4 (HNF-4 ) as a novel regulator of human apoAV gene. Inhibition of HNF-4 expression by small interfering RNA resulted in down-regulation of apoAV. Deletion, mutagenesis, and binding assays revealed that HNF-4 directly regulates human apoAV promoter through DR1 [a direct repeat separated by one nucleotide (nt)], and via a novel element for HNF-4 consisting of an inverted repeat separated by 8 nt (IR8). In addition, we show that the coactivator peroxisome proliferator-activated receptor- coactivator-1 was capable of stimulating the HNF-4 -dependent transactivation of apoAV promoter. Furthermore, analyses in human hepatic cells demonstrated that AMP-activated protein kinase (AMPK) and the MAPK signaling pathway regulate human apoAV expression and suggested that this regulation may be mediated, at least in part, by changes in HNF-4 . Intriguingly, EMSAs and mice with a liver-specific disruption of the HNF-4 gene revealed a species-distinct regulation of apoAV by HNF-4 , which resembles that of a subset of HNF-4 target genes. Taken together, our data provide new insights into the binding properties and the modulation of HNF-4 and underscore the role of HNF-4 in regulating triglyceride metabolism.

Lipid phosphate phosphatase 3 participates in transport carrier formation and protein trafficking in the early secretory pathway

The inhibition of phosphatidic acid phosphatase (PAP) activity by propanolol indicates that diacylglycerol (DAG) is required for the formation of transport carriers at the Golgi and for retrograde trafficking to the ER. Here we report that the PAP2 family member lipid phosphate phosphatase 3 (LPP3, also known as PAP2b) localizes in compartments of the secretory pathway from ER export sites to the Golgi complex. The depletion of human LPP3: (i) reduces the number of tubules generated from the ER-Golgi intermediate compartment and the Golgi, with those formed from the Golgi being longer in LPP3-silenced cells than in control cells; (ii) impairs the Rab6-dependent retrograde transport of Shiga toxin subunit B from the Golgi to the ER, but not the anterograde transport of VSV-G or ssDsRed; and (iii) induces a high accumulation of Golgi-associated membrane buds. LPP3 depletion also reduces levels of de novo synthesized DAG and the Golgi-associated DAG contents. Remarkably, overexpression of a catalytically inactive form of LPP3 mimics the effects of LPP3 knockdown on Rab6-dependent retrograde transport. We conclude that LPP3 participates in the formation of retrograde transport carriers at the ER-Golgi interface, where it transitorily cycles, and during its route to the plasma membrane.

Pint lincRNA connects the p53 pathway with epigenetic silencing by the Polycomb repressive complex 2

BACKGROUND: The p53 transcription factor is located at the core of a complex wiring of signaling pathways that are critical for the preservation of cellular homeostasis. Only recently it has become clear that p53 regulates the expression of several long intergenic noncoding RNAs (lincRNAs). However, relatively little is known about the role that lincRNAs play in this pathway. RESULTS: Here we characterize a lincRNA named Pint (p53 induced noncoding transcript). We show that Pint is a ubiquitously expressed lincRNA that is finely regulated by p53. In mouse cells, Pint promotes cell proliferation and survival by regulating the expression of genes of the TGF-β, MAPK and p53 pathways. Pint is a nuclear lincRNA that directly interacts with the Polycomb repressive complex 2 (PRC2), and is required for PRC2 targeting of specific genes for H3K27 tri-methylation and repression. Furthermore, Pint functional activity is highly dependent on PRC2 expression. We have also identified Pint human ortholog (PINT), which presents suggestive analogies with the murine lincRNA. PINT is similarly regulated by p53, and its expression significantly correlates with the same cellular pathways as the mouse ortholog, including the p53 pathway. Interestingly, PINT is downregulated in colon primary tumors, while its overexpression inhibits the proliferation of tumor cells, suggesting a possible role as tumor suppressor. CONCLUSIONS: Our results reveal a p53 autoregulatory negative mechanism where a lincRNA connects p53 activation with epigenetic silencing by PRC2. Additionally, we show analogies and differences between the murine and human orthologs, identifying a novel tumor suppressor candidate lincRNA.

Three-step pathway engineering results in more incidence rate and higher emission of nerolidol and improved attraction of Diadegma semiclausum

The concentration and ratio of terpenoids in the headspace volatile blend of plants have a fundamental role in the communication of plants and insects. The sesquiterpene (E)-nerolidol is one of the important volatiles with effect on beneficial carnivores for biologic pest management in the field. To optimize de novo biosynthesis and reliable and uniform emission of (E)-nerolidol, we engineered different steps of the (E)-nerolidol biosynthesis pathway in Arabidopsis thaliana. Introduction of a mitochondrial nerolidol synthase gene mediates de novo emission of (E)-nerolidol and linalool. Co-expression of the mitochondrial FPS1 and cytosolic HMGR1 increased the number of emitting transgenic plants (incidence rate) and the emission rate of both volatiles. No association between the emission rate of transgenic volatiles and their growth inhibitory effect could be established. (E)-Nerolidol was to a large extent metabolized to non-volatile conjugates.

Compositional influence on the electrical performance of zinc indium tin oxide transparent thin-film transistors

In this work, zinc indium tin oxide layers with different compositions are used as the active layer of thin film transistors. This multicomponent transparent conductive oxide is gaining great interest due to its reduced content of the scarce indium element. Experimental data indicate that the incorporation of zinc promotes the creation of oxygen vacancies. In thin-film transistors this effect leads to a higher threshold voltage values. The field-effect mobility is also strongly degraded, probably due to coulomb scattering by ionized defects. A post deposition annealing in air reduces the density of oxygen vacancies and improves the fieldeffect mobility by orders of magnitude. Finally, the electrical characteristics of the fabricated thin-film transistors have been analyzed to estimate the density of states in the gap of the active layers. These measurements reveal a clear peak located at 0.3 eV from the conduction band edge that could be attributed to oxygen vacancies.

Genetic Variation in the TP53 Pathway and Bladder Cancer Risk. A Comprehensive Analysis

Introduction: Germline variants in TP63 have been consistently associated with several tumors, including bladder cancer, indicating the importance of TP53 pathway in cancer genetic susceptibility. However, variants in other related genes, including TP53 rs1042522 (Arg72Pro), still present controversial results. We carried out an in depth assessment of associations between common germline variants in the TP53 pathway and bladder cancer risk. Material and Methods: We investigated 184 tagSNPs from 18 genes in 1,058 cases and 1,138 controls from the Spanish Bladder Cancer/EPICURO Study. Cases were newly-diagnosed bladder cancer patients during 1998–2001. Hospital controls were age-gender, and area matched to cases. SNPs were genotyped in blood DNA using Illumina Golden Gate and TaqMan assays. Cases were subphenotyped according to stage/grade and tumor p53 expression. We applied classical tests to assess individual SNP associations and the Least Absolute Shrinkage and Selection Operator (LASSO)-penalized logistic regression analysis to assess multiple SNPs simultaneously. Results: Based on classical analyses, SNPs in BAK1 (1), IGF1R (5), P53AIP1 (1), PMAIP1 (2), SERINPB5 (3), TP63 (3), and TP73 (1) showed significant associations at p-value#0.05. However, no evidence of association, either with overall risk or with specific disease subtypes, was observed after correction for multiple testing (p-value$0.8). LASSO selected the SNP rs6567355 in SERPINB5 with 83% of reproducibility. This SNP provided an OR = 1.21, 95%CI 1.05–1.38, p-value = 0.006, and a corrected p-value = 0.5 when controlling for over-estimation. Discussion: We found no strong evidence that common variants in the TP53 pathway are associated with bladder cancer susceptibility. Our study suggests that it is unlikely that TP53 Arg72Pro is implicated in the UCB in white Europeans. SERPINB5 and TP63 variation deserve further exploration in extended studies.

Correlation between charge transport and electroluminescence properties of Si-rich oxide/nitride/oxide-based light emitting capacitors.

The electrical and electroluminescence (EL) properties at room and high temperatures of oxide/ nitride/oxide (ONO)-based light emitting capacitors are studied. The ONO multidielectric layer is enriched with silicon by means of ion implantation. The exceeding silicon distribution follows a Gaussian profile with a maximum of 19%, centered close to the lower oxide/nitride interface. The electrical measurements performed at room and high temperatures allowed to unambiguously identify variable range hopping (VRH) as the dominant electrical conduction mechanism at low voltages, whereas at moderate and high voltages, a hybrid conduction formed by means of variable range hopping and space charge-limited current enhanced by Poole-Frenkel effect predominates. The EL spectra at different temperatures are also recorded, and the correlation between charge transport mechanisms and EL properties is discussed.

Comment on 'The Molecular Evolutionary Patterns of the Insulin/FOXO Signaling Pathway'

Letter to the Editor on Wang M, Wang Q, Wang Z, Zhang X, Pan Y. The molecular evolutionary patterns of the insulin/FOXO signaling pathway

The Electronic Structure of Co-Sputtered Zinc Indium Tin Oxide Thin Films

Zinc indium tin oxide (ZITO) transparent conductive oxide layers were deposited via radio frequency (RF) magnetron co-sputtering at room temperature. A series of samples with gradually varying zinc content was investigated. The samples were characterized with x-ray and ultraviolet photoemission spectroscopy (XPS, UPS) to determine the electronic structure of the surface. Valence and conduction bands maxima (VBM, CBM), and work function were determined. The experiments indicate that increasing Zn content results in films with a higher defect rate at the surface leading to the formation of a degenerately doped surface layer if the Zn content surpasses 50%. Furthermore, the experiments demonstrate that ZITO is susceptible to ultraviolet light induced work function reduction, similar to what was earlier observed on ITO and TiO2 films.

GdBaCo2O5+x layered perovskite as an intermediate temperature solid oxide fuel cell cathode

GdBaCo2O5+x (GBCO) was evaluated as a cathode for intermediate-temperature solid oxide fuel cells. A porous layer of GBCO was deposited on an anode-supported fuel cell consisting of a 15m thick electrolyte of yttria-stabilized zirconia (YSZ) prepared by dense screen-printing anda Ni–YSZ cermet as an anode (Ni–YSZ/YSZ/GBCO). Values of power density of 150 mW cm−2 at 700◦C and ca. 250 mW cm−2 at 800◦C are reported for this standard configuration using 5% of H2 in nitrogen as fuel. An intermediate porous layer of YSZ was introduced between the electrolyte and the cathode improving the performance of the cell. Values for power density of 300 mW cm−2 at 700◦C and ca. 500 mW cm−2 at 800◦C in this configuration were achieved.

The p38/MK2/Hsp25 pathway is required for BMP-2-induced cell migration.

Background: Bone morphogenetic proteins (BMPs) have been shown to participate in the patterning and specification of several tissues and organs during development and to regulate cell growth, differentiation and migration in different cell types. BMP-mediated cell migration requires activation of the small GTPase Cdc42 and LIMK1 activities. In our earlier report we showed that activation of LIMK1 also requires the activation of PAKs through Cdc42 and PI3K. However, the requirement of additional signaling is not clearly known. Methodology/Principal Findings: Activation of p38 MAPK has been shown to be relevant for a number of BMP-2¿s physiological effects. We report here that BMP-2 regulation of cell migration and actin cytoskeleton remodelling are dependent on p38 activity. BMP-2 treatment of mesenchymal cells results in activation of the p38/MK2/Hsp25 signaling pathway downstream from the BMP receptors. Moreover, chemical inhibition of p38 signaling or genetic ablation of either p38¿ or MK2 blocks the ability to activate the downstream effectors of the pathway and abolishes BMP-2-induction of cell migration. These signaling effects on p38/MK2/Hsp25 do not require the activity of either Cdc42 or PAK, whereas p38/MK2 activities do not significantly modify the BMP-2-dependent activation of LIMK1, measured by either kinase activity or with an antibody raised against phospho-threonine 508 at its activation loop. Finally, phosphorylated Hsp25 colocalizes with the BMP receptor complexes in lamellipodia and overexpression of a phosphorylation mutant form of Hsp25 is able to abolish the migration of cells in response to BMP-2. Conclusions: These results indicate that Cdc42/PAK/LIMK1 and p38/MK2/Hsp25 pathways, acting in parallel and modulating specific actin regulatory proteins, play a critical role in integrating responses during BMP-induced actin reorganization and cell migration.