Involvement of Volume-Activated Chloride Channels in H2O2 Preconditioning Against Oxidant-Induced Injury Through Modulating Cell Volume Regulation Mechanisms and Membrane Permeability in PC12 Cells

The functions of chloride channels in preconditioning-induced cell protection remain unclear. In this report, we show that the volume-activated chloride channels play a key role in hydrogen peroxide (H₂O₂) preconditioning-induced cell protection in pheochromocytoma PC12 cells. The preconditioning with 100 μM H₂O₂ for 90 min protected the cells from injury induced by long period exposure to 300 μM H₂O₂. The protective effect was attenuated by pretreatment with the chloride channel blockers, 5-nitro-2-3-phenylpropylamino benzoic acid (NPPB) and tamoxifen. H₂O₂ preconditioning directly activated a chloride current, which was moderately outward-rectified and sensitive to the chloride channel blockers and hypertonicity-induced cell shrinkage. H₂O₂ preconditioning functionally up-regulated the activities of volume-activated chloride channels and enhanced the regulatory volume decrease when exposure to extracellular hypotonic challenges. In addition, acute application of H₂O₂ showed distinctive actions on cell volume and membrane permeability in H₂O₂ preconditioned cells. In H₂O₂ preconditioned cells, acute application of 300 μM H₂O₂ first promptly induced a decrease of cell volume and enhancement of cell membrane permeability, and then, cell volume was maintained at a relatively stable level and the facilitation of membrane permeability was reduced. Conversely, in control cells, 300 μM H₂O₂ induced a slow but persistent apoptotic volume decrease (AVD) and facilitation of membrane permeability. H₂O₂ preconditioning also significantly up-regulated the expression of ClC-3 protein, the molecular candidate of the volume-activated chloride channel. These results suggest that H₂O₂ preconditioning can enhance the expression and functional activities of volume-activated chloride channels, thereby modulate cell volume and cell membrane permeability, which may contribute to neuroprotection against oxidant-induced injury.     

The regulation of the UCH-L1 gene by transcription factor NF-κB in podocytes

In kidney, the ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) is involved in podocyte injury and proteinuria but details of the mechanism underlying its regulation are not known. Activation of NF-κB is thought to be the predominant risk factor for kidney disease; therefore, it is postulated that UCH-L1 may be one of the NF-κB target genes. In this study, we investigated the involvement of NF-κB activation in the regulation of UCH-L1 expression and the function of murine podocytes. Stimulation of podocytes with the cytokines TNF-α and IL-1β up-regulated UCH-L1 expression rapidly at the mRNA and protein levels and the NF-κB-specific inhibitor pyrrolidine dithiocarbamate resulted in down-regulation. NF-κB up-regulates UCH-L1 via binding the ? 300 bp and ? 109 bp sites of its promoter, which was confirmed by the electrophoretic mobility shift assay of DNA–nuclear protein binding. In the renal biopsy from lupus nephritis patients, the expressions of NF-κB and UCH-L1 increased in immunohistochestry staining and were positively correlated. Activation of NF-κB up-regulates UCH-L1 expression following changing of other podocytes molecules, such as nephrin and snail. These results suggest that activation of the NF-κB signaling pathway could be the major pathogenesis to up-regulate UCH-L1 in podocyte injury, followed by the turnover of other molecules, which might result in morphological changes and dysfunction of podocytes. This work help us to understand the effect of NF-κB on specific target molecules of podocytes, and suggest that targeting the NF-κB–UCH-L1 interaction could be a novel therapeutic strategy for the treatment of podocyte lesions and proteinuria.     

Preparative Separation of Enantiomers Based on Functional Nucleic Acids Modified Gold Nanoparticles

The preparative‐scale separation of chiral compounds is vitally important for the pharmaceutical industry and related fields. Herein we report a simple approach for rapid preparative separation of enantiomers using functional nucleic acids modified gold nanoparticles (AuNPs). The separation of _DL‐tryptophan (_DL‐Trp) is demonstrated as an example to show the feasibility of the approach. AuNPs modified with enantioselective aptamers were added into a racemic mixture of _DL‐Trp. The aptamer‐specific enantiomer (_L‐Trp) binds to the AuNPs surface through aptamer‐_L‐Trp interaction. The separation of _DL‐Trp is then simply accomplished by centrifugation: the precipitate containing _L‐Trp bounded AuNPs is separated from the solution, while the _D‐Trp remains in the supernatant. The precipitate is then redispersed in water. The aptamer is denatured under 95 °C and a second centrifugation is then performed, resulting in the separation of AuNPs and _L‐Trp. The supernatant is finally collected to obtain pure _L‐Trp in water. The results show that the racemic mixture of _DL‐Trp is completely separated into _D‐Trp and _L‐Trp, respectively, after 5 rounds of repeated addition of fresh aptamer‐modified AuNPs to the _DL‐Trp mixture solution. Additionally, the aptamer‐modified AuNPs can be repeatedly used for at least eight times without significant loss of its binding ability because the aptamer can be easily denatured and renatured in relatively mild conditions. The proposed approach could be scaled up and extended to the separation of other enantiomers by the adoption of other enantioselective aptamers. Chirality 25:751–756, 2013. © 2013 Wiley Periodicals, Inc.     

Tetraplex Formation of d(GGGGGTTTTT): 1H NMR Study in Solution

Abstract

The oligonucleotide d(G₅T₅) can in principle form a fully matched duplex with G · T pairing and/or a tetraplex. Non-denaturing gel electrophoresis, circular dichroism and NMR experiments show that the tetraplex is exclusively formed by this oligomer in solution. In the presence of its complementary strand d(A₅C₅) at low temperature, d(G₅T₅) forms the tetraplex over the normally expected Watson-Crick duplex. However, when d(G₅T₅) and d(A₅C₅) are mixed together in equimolar amounts and heated for several minutes at 85°C, and then allowed to cool, the product was essentially the Watson-Crick duplex. The lack of resolution in the 500 MHz ¹H NMR spectra and the presence of extensive spin diffusion do not allow us to derive a quantitative structure for the tetraplex from the NMR data. However, we find good qualitative agreement between the NOESY and MINSY data and a theoretically derived stereochemically sound structure in which the G's and T's are part of a parallel tetraplex.     

Mesenchymal stem cells protect cigarette smoke‐damaged lung and pulmonary function partly via VEGF–VEGF receptors

Progressive pulmonary inflammation and emphysema have been implicated in the progression of chronic obstructive pulmonary disease (COPD), while current pharmacological treatments are not effective. Transplantation of bone marrow mesenchymal stem cells (MSCs) has been identified as one such possible strategy for treatment of lung diseases including acute lung injury (ALI) and pulmonary fibrosis. However, their role in COPD still requires further investigation. The aim of this study is to test the effect of administration of rat MSCs (rMSCs) on emphysema and pulmonary function. To accomplish this study, the rats were exposed to cigarette smoke (CS) for 11 weeks, followed by administration of rMSCs into the lungs. Here we show that rMSCs infusion mediates a down‐regulation of pro‐inflammatory mediators (TNF‐α, IL‐1β, MCP‐1, and IL‐6) and proteases (MMP9 and MMP12) in lung, an up‐regulation of vascular endothelial growth factor (VEGF), VEGF receptor 2, and transforming growth factor (TGFβ‐1), while reducing pulmonary cell apoptosis. More importantly, rMSCs administration improves emphysema and destructive pulmonary function induced by CS exposure. In vitro co‐culture system study of human umbilical endothelial vein cells (EA.hy926) and human MSCs (hMSCs) provides the evidence that hMSCs mediates an anti‐apoptosis effect, which partly depends on an up‐regulation of VEGF. These findings suggest that MSCs have a therapeutic potential in emphysematous rats by suppressing the inflammatory response, excessive protease expression, and cell apoptosis, as well as up‐regulating VEGF, VEGF receptor 2, and TGFβ‐1. J. Cell. Biochem. 114: 323–335, 2013. © 2012 Wiley Periodicals, Inc.     

HEF1 promotes epithelial mesenchymal transition and bone invasion in prostate cancer under the regulation of microRNA‐145

The principal problem arising from prostate cancer (PCa) is its propensity to metastasize to bones, and it's crucial to understand the mechanism of tumor progression to metastasis in order to develop therapies that may reduce the morbidity and mortality of PCa patients. Although we had identified that microRNA(miR)‐145 could repress bone metastasis of PCa via regulating epithelial–mesenchymal transition (EMT) in previous study, it is still unknown how miR‐145 regulated EMT. In the present study, we constructed a luciferase reporter system and identified HEF1 as a direct target of miR‐145. More importantly, HEF1 was shown to promote migration, invasion and EMT of PC‐3 cells, a human PCa cell line originated from a bone metastatic PCa specimen. And HEF1 was also shown to partially mediate miR‐145 suppression of EMT and invasion. Furthermore, inhibition of HEF1 repressed bone invasion of PC‐3 cells in vivo. Expression of HEF1 was negatively correlated with miR‐145 in primary PCa and bone metastatic specimens, but HEF1 was higher in samples which were more likely to commit to bone metastasis or those with higher free prostate‐specific antigen (fPSA) levels and Gleason scores. Taken together, these findings indicate that HEF1 promotes EMT and bone invasion in prostate cancer by directly targeted by miR‐145, and miR‐145 suppresses EMT and invasion, at least in part, through repressing HEF1. J. Cell. Biochem. 114: 1606–1615, 2013. © 2013 Wiley Periodicals, Inc.     

A mutation in a coproporphyrinogen III oxidase gene confers growth inhibition, enhanced powdery mildew resistance and powdery mildew-induced cell death in Arabidopsis

Key message

A gene encoding a coproporphyrinogen III oxidase mediates disease resistance in plants by the salicylic acid pathway.

Abstract

A number of genes that regulate powdery mildew resistance have been identified in Arabidopsis, such as ENHANCED DISEASE RESISTANCE 1 to 3 (EDR1 to 3). To further study the molecular interactions between the powdery mildew pathogen and Arabidopsis, we isolated and characterized a mutant that exhibited enhanced resistance to powdery mildew. The mutant also showed dramatic powdery mildew-induced cell death as well as growth defects and early senescence in the absence of pathogens. We identified the affected gene by map-based cloning and found that the gene encodes a coproporphyrinogen III oxidase, a key enzyme in the tetrapyrrole biosynthesis pathway, previously known as LESION INITIATION 2 (LIN2). Therefore, we designated the mutant lin2-2. Further studies revealed that the lin2-2 mutant also displayed enhanced resistance to Hyaloperonospora arabidopsidis (H.a.) Noco2. Genetic analysis showed that the lin2-2-mediated disease resistance and spontaneous cell death were dependent on PHYTOALEXIN DEFICIENT 4 (PAD4), SALICYLIC ACID INDUCTION-DEFICIENT 2 (SID2), and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), which are all involved in salicylic acid signaling. Furthermore, the relative expression levels of defense-related genes were induced after powdery mildew infection in the lin2-2 mutant. These data indicated that LIN2 plays an important role in cell death control and defense responses in plants.     

Mitochondrial DNA genetic polymorphism in thirteen rice cytoplasmic male sterile lines

Key message

Thirteen rice CMS lines derived from different cytoplasms were classified into eight groups by PCR amplification on mtDNA. The orf79 gene, which causes Boro II CMS, possibly results in Dian1-CMS.

Abstract

Thirteen rice cytoplasmic male sterile (CMS) lines derived from different cytoplasms are widely used for hybrid rice breeding. Based on 27 loci on mitochondrial DNA, including single nucleotide polymorphisms and segmental sequence variations between typical indica and japonica as well as high-polymorphism segmental sequence variations and single nucleotide polymorphisms among rice CMS lines, the 13 rice CMS lines were classified into eight groups: (I) wild-abortive CMS, Indonesian Shuitiangu CMS, K-CMS, Gang CMS, D-CMS and dwarf abortive CMS; (II) Maxie-CMS; (III) Honglian CMS; (IV) Boro II CMS; (V) Dian1-CMS; (VI) Liao-CMS; (VII) Lead CMS; and (VIII) Chinese wild rice CMS. According to their pollen abortion phenotypes, groups I and II (including 7 CMS lines) were classified as sporophytic CMS lines, the cytoplasmic genetic relationships among which were very close. They could have originated from similar, or even the same, cytoplasm donors. Groups III–VIII (including 6 CMS lines) were categorized as gametophytic CMS lines, the cytoplasms of which differed from one another, with some having relatively far genetic relationships. Dian1-CMS was found to harbor the orf79 gene, which causes Boro II CMS, whereas Liao-CMS had an orf79 structure that does not result in Lead CMS. Therefore, we speculated that orf79 is associated with Dian1-CMS but not with Liao-CMS. The atp6–orf79 structure related to sterility was also found to experience multiple evolutionary turnovers. All sporophytic CMS lines were indica-like. Except the Honglian CMS line, which was indica-like, all gametophytic CMS lines were japonica-like.