Sodium arsenite-induced DAPK promoter hypermethylation and autophagy via ERK1/2 phosphorylation in human uroepithelial cells

Arsenic compounds or arsenicals are well-known toxic and carcinogenic agents. The toxic effects of arsenic that are of most concern to humans are those that occur from chronic, low-level exposure, and are associated with various human malignancies, including skin, lung and bladder cancers. In addition, arsenic could induce cell death, including apoptosis or autophagy in malignant cells. Previously, we have demonstrated that arsenite can induce autophagy and death-associated protein kinase (DAPK) promoter hypermethylation in the SV-40 immortalized human uroepithelial cell line (SV-HUC-1). However, the underlying mechanism of arsenite-induced autophagy is still unclear. In the present study, we demonstrate that arsenite can activate the extracellular signaling-regulated protein kinase 1/2 (ERK1/2) signaling pathway after treatment in SV-HUC-1 cells by using immunocytochemistry and Western blotting. In addition, our results also show an increase of autophagosomes was produced in arsenite-treated SV-HUC-1 cells by using electron microscopy. We found that, by incrementally increasing the dosages, microtubule-associated protein light chain 3B (LC3B) and Beclin-1 are important regulators for the formation of autophagosomes, in a dose-dependent manner. When the cells were pretreated with inhibitors 5-aza-CdR or U0126 for 24 h, the effect of arsenite on ERK1/2, LC3B, Beclin-1 and DAPK proteins expression is suppressed. Furthermore, our results support the notion that arsenite can induce the ERK1/2 signaling pathway to stimulate autophagy and DAPK promoter hypermethylation in human uroepithelial SV-HUC-1 cells. These findings may contribute to a better understanding of the carcinogenesis of arsenite.     

Lack of direct androgen regulation of PDE5 expression

It has been reported that penile PDE5 expression was under androgen regulation. However it remained unknown whether the observed change in PDE5 expression in castrated animals was under direct androgen regulation or due to changes in smooth muscle content. In the present study we showed that castration of rats caused a reduction of penile size and cavernous smooth muscle content. Immunostaining detected concomitant reduction of PDE5 and alpha smooth muscle actin (α-SMA) expression in the corpus cavernosum of castrated rats. Real-time PCR and Western blotting detected no change of PDE5 expression when normalized with α−SMA expression in castrated rats. Androgen receptor (AR) expression was increased while PDE5 expression remained unchanged in DHT-treated rat cavernous smooth muscle cells (CSMC). Prostate specific antigen (PSA) promoter activity was upregulated while PDE5A promoter activity remained unchanged in DHT-treated CSMC. Thus, PDE5 expression was not under direct androgen regulation.     

Avian reovirus influences phosphorylation of several factors involved in host protein translation including eukaryotic translation elongation factor 2 (eEF2) in Vero cells

Viral infection usually influences cellular protein synthesis either actively or passively via modification of various translation initiation factors. Here we demonstrated that infection with avian reovirus (ARV) interfered with cellular protein synthesis. This study demonstrated for the first time that ARV influenced the phosphorylation of translation initiation factors including eIF4E and eIF-4G. Interestingly, ARV also induced phosphorylation of eukaryotic translation elongation factor (eEF2) in a time- and dose-dependent manner. Inhibition of mTOR by rapamycin notably increased the level of phosphorylated eEF2 in infected cells. However, rapamycin did not show any negative effects on ARV replication, suggesting that phosphorylation of eEF2 in infected cells did not reduce ARV propagation. These results demonstrated for the first time that ARV promotes phosphorylation of eEF2 which in turn influenced host protein production not simply by modulating the function of translation initiation factors but also by regulating elongation factor eEF2.     

Both the RGS Domain and the Six C-Terminal Amino Acids of Mouse Axin Are Required for Normal Embryogenesis

Axin is a negative regulator of canonical Wnt signaling, which promotes the degradation of β-catenin, the major effector in this signaling cascade. While many protein-binding domains of Axin have been identified, their significance has not been evaluated in vivo. Here, we report the generation and analysis of mice carrying modified Axin alleles in which either the RGS domain or the six C-terminal amino acids (C6 motif) were deleted. The RGS domain is required for APC-binding, while the C6 motif has been implicated in the activation of c-Jun N-terminal kinase, but is not required for the effects of Axin on the Wnt/β-catenin pathway, in vitro. Both mutant Axin alleles caused recessive embryonic lethality at E9.5–E10.5, with defects indistinguishable from those caused by a null allele. As Axin-ΔRGS protein was produced at normal levels, its inability to support embryogenesis confirms the importance of interactions between Axin and APC. In contrast, Axin-ΔC6 protein was expressed at only 25–30% of the normal level, which may account for the recessive lethality of this allele. Furthermore, many Axin^ΔC6/ΔC6 embryos that were heterozygous for a β-catenin null mutation survived to term, demonstrating that early lethality was due to failure to negatively regulate β-catenin.     

Transient response of sap flow to wind speed

Transient responses of sap flow to step changes in wind speedwere experimentally investigated in a wind tunnel. A Granier-typesap flow sensor was calibrated and tested in a cylindrical tubefor analysis of its transient time response. Then the sensorwas used to measure the transient response of a well-wateredPachira macrocarpa plant to wind speed variations. The transientresponse of sap flow was described using the resistance–capacitancemodel. The steady sap flow rate increased as the wind speedincreased at low wind speeds. Once the wind speed exceeded 8.0m s^–1, the steady sap flow rate did not increase further.The transpiration rate, measured gravimetrically, showed a similartrend. The response of nocturnal sap flow to wind speed variationwas also measured and compared with the results in the daytime.Under the same wind speed, the steady sap flow rate was smallerthan that in the daytime, indicating differences between diurnaland nocturnal hydraulic function, and incomplete stomatal closureat night. In addition, it was found that the temporal responseof the Granier sensor is fast enough to resolve the transientbehaviour of water flux in plant tissue. Key words: Nocturnal transpiration, sap flow, transient response, wind speed Received 31 July 2008; Revised 6 October 2008 Accepted 20 October 2008     

Chondrocyte phenotype and ectopic ossification in collagenase-induced tendon degeneration

We report chondrocyte phenotype and ectopic ossification in a collagenase-induced patellar tendon injury model. Collagenase or saline was injected intratendinously in one limb. The patella tendon was harvested for assessment at different times. There was an increase in cellularity, vascularity, and loss of matrix organization with time after collagenase injection. The tendon did not heal histologically until week 32. Ectopic mineralization as indicated by von Kossa staining started from week 8. Tendon calcification was mediated by endochondral ossification, as shown by expression of type X collagen. viva CT imaging and polarization microscopy showed characteristic bony porous structures and collagen fiber arrangement, respectively, in the calcific regions. Marrow-like cells and blood vessels were observed inside calcific deposits. Chondrocyte-like cells as indicated by morphology, expression of type II collagen, and sox 9 were seen around and embedded inside the calcific deposits. Fibroblast-like cells expressed type II collagen and sox 9 at earlier times, suggesting that erroneous differentiation of healing tendon fibroblasts may account for failed healing and ossification in collagenase-induced tendon degeneration. Because this animal model replicates key histopathological changes in calcific tendinopathy, it can be used as a model for the study of its pathogenesis at the patellar tendon.