Three positive regulators of leaf senescence in Arabidopsis, ORE1, ORE3 and ORE9, play roles in crosstalk among multiple hormone-mediated senescence pathways

Leaf senescence is a developmentally programmed event, but the initiation and progression of leaf senescence are affected by a range of plant hormones including abscisic acid (ABA), ethylene and methyl jasmonate (MeJA). To investigate plant hormone crosstalk during leaf senescence, hormone-induced senescence phenotypes were analyzed in three leaf senescence mutants [ore1 (oresara1), ore3 and ore9] showing delayed senescence phenotypes in age-dependent and dark-induced senescence. The ore mutants exhibited delayed leaf senescence phenotypes following treatment with ABA, ACC (aminocyclo-propane-1-carboxylic acid) or MeJA. After each hormone treatment, the photochemical efficiency of photosystem II and chlorophyll content were significantly higher in the ore mutant leaves than in the wild-type leaves. The expression of CAB2 and SEN4 in the wild-type was rapidly altered following each hormone treatment. However, the decrease in CAB2 expression and the induction of SEN4 expression in the mutants were less affected by ABA, ACC or MeJA treatment. It is suggested that ORE1, ORE3 and ORE9 are required for the proper progression of leaf senescence mediated by ABA, ethylene and MeJA. This implies that ORE1, ORE3 and ORE9 may be linked to the crosstalk among senescence pathways induced by ABA, ethylene and MeJA, as well as age and darkness.     

Detrimental effects of environmental tobacco smoke in relation to asthma severity

Background

Environmental tobacco smoke (ETS) has adverse effects on the health of asthmatics, however the harmful consequences of ETS in relation to asthma severity are unknown.

Methods

In a multicenter study of severe asthma, we assessed the impact of ETS exposure on morbidity, health care utilization and lung functions; and activity of systemic superoxide dismutase (SOD), a potential oxidative target of ETS that is negatively associated with asthma severity.

Findings

From 2002–2006, 654 asthmatics (non-severe 366, severe 288) were enrolled, among whom 109 non-severe and 67 severe asthmatics were routinely exposed to ETS as ascertained by history and validated by urine cotinine levels. ETS-exposure was associated with lower quality of life scores; greater rescue inhaler use; lower lung function; greater bronchodilator responsiveness; and greater risk for emergency room visits, hospitalization and intensive care unit admission. ETS-exposure was associated with lower levels of serum SOD activity, particularly in asthmatic women of African heritage.

Interpretation

ETS-exposure of asthmatic individuals is associated with worse lung function, higher acuity of exacerbations, more health care utilization, and greater bronchial hyperreactivity. The association of diminished systemic SOD activity to ETS exposure provides for the first time a specific oxidant mechanism by which ETS may adversely affect patients with asthma.     

Downregulation of protein kinase CK2 activity facilitates tumor necrosis factor-α-mediated chondrocyte death through apoptosis and autophagy

Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF)-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA) model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence versus a cause of the degeneration in vivo.     

Proline-rich tyrosine kinase 2 (Pyk2) promotes cell motility of hepatocellular carcinoma through induction of epithelial to mesenchymal transition

Aims

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase of the focal adhesion kinase (FAK) family, is up-regulated in more than 60% of the tumors of hepatocellular carcinoma (HCC) patients. Forced overexpression of Pyk2 can promote the proliferation and invasion of HCC cells. In this study, we aimed to explore the underlying molecular mechanism of Pyk2-mediated cell migration of HCC cells.

Methodology/Principal Findings

We demonstrated that Pyk2 transformed the epithelial HCC cell line Hep3B into a mesenchymal phenotype via the induction of epithelial to mesenchymal transition (EMT), signified by the up-regulation of membrane ruffle formation, activation of Rac/Rho GTPases, down-regulation of epithelial genes E-cadherin and cytokeratin as well as promotion of cell motility in presence of lysophosphatidic acid (LPA). Suppression of Pyk2 by overexpression of dominant negative PRNK domain in the metastatic HCC cell line MHCC97L transformed its fibroblastoid phenotype to an epithelial phenotype with up-regulation of epithelial genes, down-regulation of mesenchymal genes N-cadherin and STAT5b, and reduction of LPA-induced membrane ruffle formation and cell motility. Moreover, overexpression of Pyk2 in Hep3B cells promoted the phosphorylation and localization of mesenchymal gene Hic-5 onto cell membrane while suppression of Pyk2 in MHCC97L cells attenuated its phosphorylation and localization.

Conclusion

These data provided new evidence of the underlying mechanism of Pyk2 in controlling cell motility of HCC cells through regulation of genes associated with EMT.     

The spatial organization of proton and lactate transport in a rat brain tumor

Tumors create a heterogeneous acidic microenvironment which assists their growth and which must be taken into account in the design of drugs and their delivery. In addition, the acidic extracellular pH (pHe) is itself exploited in several experimental techniques for drug delivery. The way the acidity is created is not clear. We report here the spatial organization of key proton-handling proteins in C6 gliomas in rat brain. The mean profiles across the tumor rim of the Na+/H+ exchanger NHE1, and the lactate-H+ cotransporter MCT1, both showed peaks. NHE1, which is important for extension and migration of cells in vitro, showed a peak 1.55 times higher than in extratumoural tissue at 0.33 mm from the edge. MCT1 had a broader peak, further into the tumor (maximum 1.76 fold at 1.0 mm from the edge). In contrast, MCT4 and the carbonic anhydrase CAIX, which are associated with hypoxia, were not significantly upregulated in the rim. The spatial distribution of MCT4 was highly correlated with that of CAIX, suggesting that their expression is regulated by the same factors. Since protons extruded by NHE1 diffuse away through extracellular clefts, NHE1 requires a continuous source of intracellular protons. From the stoichiometries of metabolic pathways that produce or consume H+, and the greater availability of glucose compared to oxygen in most parts of a tumor, we support the classic view that most of the net proton efflux from C6 gliomas originates in glycolytic formation of lactate and H+ inside the tumor, but add that some lactate is taken up into cells in the rim on MCT1, and some lactate diffuses away, leaving its associated protons available to re-enter cells for extrusion on NHE1. Therapeutic inhibition of NHE1, MCT1 or CAIX is predicted to affect different parts of a tumor.     

The genetic effect of copy number variations on the risk of type 2 diabetes in a Korean population

Background

Unlike Caucasian populations, genetic factors contributing to the risk of type 2 diabetes mellitus (T2DM) are not well studied in Asian populations. In light of this, and the fact that copy number variation (CNV) is emerging as a new way to understand human genomic variation, the objective of this study was to identify type 2 diabetes–associated CNV in a Korean cohort.

Methodology/Principal Findings

Using the Illumina HumanHap300 BeadChip (317,503 markers), genome-wide genotyping was performed to obtain signal and allelic intensities from 275 patients with type 2 diabetes mellitus (T2DM) and 496 nondiabetic subjects (Total n = 771). To increase the sensitivity of CNV identification, we incorporated multiple factors using PennCNV, a program that is based on the hidden Markov model (HMM). To assess the genetic effect of CNV on T2DM, a multivariate logistic regression model controlling for age and gender was used. We identified a total of 7,478 CNVs (average of 9.7 CNVs per individual) and 2,554 CNV regions (CNVRs; 164 common CNVRs for frequency>1%) in this study. Although we failed to demonstrate robust associations between CNVs and the risk of T2DM, our results revealed a putative association between several CNVRs including chr15:45994758–45999227 (P = 8.6E-04, P^corr = 0.01) and the risk of T2DM. The identified CNVs in this study were validated using overlapping analysis with the Database of Genomic Variants (DGV; 71.7% overlap), and quantitative PCR (qPCR). The identified variations, which encompassed functional genes, were significantly enriched in the cellular part, in the membrane-bound organelle, in the development process, in cell communication, in signal transduction, and in biological regulation.

Conclusion/Significance

We expect that the methods and findings in this study will contribute in particular to genome studies of Asian populations.     

Caveolin-1 plays a crucial role in inhibiting neuronal differentiation of neural stem/progenitor cells via VEGF signaling-dependent pathway

In the present study, we aim to elucidate the roles of caveolin-1(Cav-1), a 22 kDa protein in plasma membrane invaginations, in modulating neuronal differentiation of neural progenitor cells (NPCs). In the hippocampal dentate gyrus, we found that Cav-1 knockout mice revealed remarkably higher levels of vascular endothelial growth factor (VEGF) and the more abundant formation of newborn neurons than wild type mice. We then studied the potential mechanisms of Cav-1 in modulating VEGF signaling and neuronal differentiation in isolated cultured NPCs under normoxic and hypoxic conditions. Hypoxic embryonic rat NPCs were exposed to 1% O₂ for 24 h and then switched to 21% O₂ for 1, 3, 7 and 14 days whereas normoxic NPCs were continuously cultured with 21% O₂. Compared with normoxic NPCs, hypoxic NPCs had down-regulated expression of Cav-1 and up-regulated VEGF expression and p44/42MAPK phosphorylation, and enhanced neuronal differentiation. We further studied the roles of Cav-1 in inhibiting neuronal differentiation by using Cav-1 scaffolding domain peptide and Cav-1-specific small interfering RNA. In both normoxic and hypoxic NPCs, Cav-1 peptide markedly down-regulated the expressions of VEGF and flk1, decreased the phosphorylations of p44/42MAPK, Akt and Stat3, and inhibited neuronal differentiation, whereas the knockdown of Cav-1 promoted the expression of VEGF, phosphorylations of p44/42MAPK, Akt and Stat3, and stimulated neuronal differentiation. Moreover, the enhanced phosphorylations of p44/42MAPK, Akt and Stat3, and neuronal differentiation were abolished by co-treatment of VEGF inhibitor V1. These results provide strong evidence to prove that Cav-1 can inhibit neuronal differentiation via down-regulations of VEGF, p44/42MAPK, Akt and Stat3 signaling pathways, and that VEGF signaling is a crucial target of Cav-1. The hypoxia-induced down-regulation of Cav-1 contributes to enhanced neuronal differentiation in NPCs.     

Enhancement of differentiation efficiency of hESCs into vascular lineage cells in hypoxia via a paracrine mechanism

Hypoxia is one way of inducing differentiation due to the activation of the key regulatory factor, Hypoxia-inducible factor 1 alpha (HIF-1α). However, the action of HIF-1α on the differentiation of hESCs was unclear until now. To investigate the effect of hypoxia on the differentiation of hESCs, we compared the differentiation efficacy into vascular lineage cells under normoxic and hypoxic conditions. We observed HIF-1α expression and the related expression of pro-angiogenic factors VEGF, bFGF, Ang-1 and PDGF in hEBs cultured under hypoxic conditions. Along with this, differentiation efficacy into vascular lineage cells was improved under hypoxic conditions. When HIF-1α was blocked by echinomycin, both angiogenic factors and the differentiation efficacy were down-regulated, suggesting that the enhancement of differentiation efficacy was caused by intrinsic up-regulation of HIF-1α and these pro-angiogenic factors under hypoxic condition. This response might be primarily regulated by the HIF-1α signal pathway, and hypoxia might be the key to improving the differentiation of hESCs into vascular lineage cells. Therefore, this study demonstrated that microenvironmental changes (i.e., hypoxia) can improve differentiation efficacy of hESCs into a vascular lineage without exogenous factors via cell-intrinsic up-regulation of angiogenic factors. These facts will contribute to the regulation of stem cell fate.     

Proteome analysis of Thermococcus onnurineus NA1 reveals the expression of hydrogen gene cluster under carboxydotrophic growth

Thermococcus onnurineus NA1 is a typical sulfur-reducing hyperthermophilic archaeon. Genome sequence analysis has shown that T. onnurineus NA1 retains the metabolic pathways necessary not only for organotrophic, but also for carboxydotrophic, growth. T. onnurineus NA1 carboxydotrophic growth may result in hydrogen production, as this archaeon produces hydrogen during oxidizing formate. In this study, we profiled the proteome of T. onnurineus NA1 cultured under carboxydotrophic conditions using CO as an electron donor by the SDS-PAGE/LC-MS/MS method. A total of 1395 proteins were identified by two independent proteomic analyses, which corresponds to ~71% of the total predicted open reading frames. To our knowledge, this level of identification coverage exceeds those of other global proteome profiling studies in Archaea. Furthermore, the biological functions of the identified proteins were predicted and cognate enzymes were mapped to the appropriate metabolic pathways. More than 90% of the genes belonging to hydrogenase gene clusters such as Mbx, Sulf-I, Mbh, Hyg4-I, Hyg4-II, and Hyf4-III were expressed during CO culture. This means that hydrogenases induced under carboxydotrophic conditions surpass those induced under an organotrophic condition (yeast extract-peptone-sulfur). Our data suggest that hydrogen production is caused by the expression and functional assembly of T. onnurineus NA1 hydrogenase genes during culture in the presence of CO.