ATM pathway is essential for ionizing radiation-induced autophagy

BackgroundATM plays an important role in response to DNA damage, while the roles of ATM in radiation-induced autophagy are still unclear in cervical cancer cells.MethodsHuman cervical cancer cells, Hela, were used, and cell models with ATM^?/? and MAPK14^?/? were established by gene engineering. Western blot was implemented to detect protein expression. MDC staining and GFP-LC3 relocalization were used to detect autophagy. CCK-8 was used to detect cell viability. Radiosensitivity was analyzed by colony formation assays. Co-immunoprecipitation was used to detect the interaction between different proteins, and apoptosis was detected by flow cytometry.ResultsAfter radiation autophagy was induced, illustrated by the increase of MAPLC3-II/MAPLC3-I ratio and decrease of p62, and phosphorylation of ATM simultaneously increased. ATM^?/? cells displayed hypersensitivity but had no influence on IR-induced apoptosis. Then inhibitor of ATM, KU55933, ATM and MAPK14 silencing were used, and autophagy was induced by IR more than 200% in control, and only by 35.72%, 53.18% and 24.76% in KU55933-treated cells, ATM^?/? and MAPK14^?/? cells, respectively. KU55933 inhibited IR-induced autophagy by activating mTOR pathways. ATM silencing decreased the expression of MAPK14 and mTOR signals significantly. Beclin's bond to PI3KIII and their interaction increased after IR, while in ATM^?/? and MAPK14^?/? cells this interaction decreased after IR. Both ATM and MAPK14 interacted with Beclin, while ATM^?/? and MAPK14^?/? cells showed no interaction.ConclusionsATM could promote IR-induced autophagy via the MAPK14 pathway, the mTOR pathway, and Beclin/PI3KIII complexes, which contributed to the effect of ATM on radiosensitivity.     

Moderate expression of Wnt signaling genes is essential for porcine parthenogenetic embryo development

Parthenogenetic embryos are invariably lost in mid-gestation, possibly due to the lack of the paternal genome and the consequent induction of aberrant gene expression. Wnt signaling is essential for embryonic development; however, the studies of this pathway in porcine parthenogenetic embryos have been limited. Here, the role of Wnt signaling in porcine parthenogenetic embryos was studied. In vivo embryos were used as controls. Single cell quantitative real-time PCR showed that Wnt signaling was down-regulated in porcine parthenogenetic embryos. Furthermore, immunofluorescence staining and real-time PCR demonstrated that porcine parthenogenetic embryo development was largely unaffected by the inhibition of Wnt signaling with IWP-2, but blastocyst hatching and trophectoderm development was blocked. In addition, parthenogenetic blastocyst hatching was improved by the activation of Wnt signaling by BIO. However, the developmental competency of porcine embryos, including blastocyst hatching, was impaired and apoptosis was induced upon the excessive activation of Wnt signaling. These findings constitute novel evidence that Wnt signaling is important for porcine pre-implantation development and that its down-regulation may lead to the low hatching rate of porcine parthenogenetic blastocysts.     

DNA methylation and MeCP2 regulation of PTCH1 expression during rats hepatic fibrosis

Hepatic stellate cell (HSC) activation plays an important role in liver fibrogenesis. Transdifferentiation of quiescent hepatic stellate cells into myofibroblastic-HSCs is a key event in liver fibrosis. The methyl-CpG-binding protein MeCP2 which promotes repressed chromatin structure is selectively detected in myofibroblasts of diseased liver. MeCP2 binds to methylated CpG dinucleotides, which are abundant in the promoters of many genes. Treatment of HSCs with DNA methylation inhibitor 5-aza-2′- deoxycytidine (5-azadC) prevented proliferation and activation. Treatment with 5-azadC prevented loss of Patched (PTCH1) expression that occurred during HSCs activation. In a search for underlying molecular medchanisms, we investigated whether the targeting of epigenetic silencing mechanisms could be useful in the treatment of PTCH1-associated fibrogenesis. It was indicated that hypermethylation of PTCH1 is associated with the perpetuation of fibroblast activation and fibrosis in the liver. siRNA knockdown of MeCP2 increased the expressions of PTCH1 mRNA and protein in hepatic myofibroblasts. These data suggest that DNA methylation and MeCP2 may provide molecular mechanisms for silencing of PTCH1.     

Enantioselective Uptake and Translocation of a Novel Chiral Neonicotinoid Insecticide Cycloxaprid in Youdonger (Brassica campestris subsp. Chinensis)

For a novel potential commercial chiral pesticide, an independent study on the fate characteristics and residues of each stereoisomer is essential if the application rates for the pesticide and human exposure are to be reduced. The absorption and translocation behavior of a chiral insecticide, cycloxaprid, in plants treated by root immersion and blade smearing was studied using ¹⁴C‐labeling tracer techniques. With the root treatment, total absorption of (1R;8S)‐cycloxaprid (RS) (12.39%) was much greater than that of (1S;8R)‐cycloxaprid (SR) (3.31%) at 192 h after treatment (HAT). The mass concentrations ( RS / SR ) of cycloxaprid in the roots, cotyledons, leaf 1, leaf 2, and leaf 3 were 37.0/16.8, 8.3/2.8, 11.7/6.5, 5.1/4.8, and 8.0/4.7 mg kg^‐1 (fresh weight), respectively, at 192 HAT at an initial concentration 1.6 mg kg^‐1. With the foliar application treatment, no significant difference was observed between the total absorption of RS (3.11%) and SR (4.03%) at the end of the treatment. Both acropetal and basipetal transport of absorbed ¹⁴C occurred and more than 71.83% of absorbed RS and 82.42% of SR remained in the treated leaf. Stereoselective absorption was observed during root uptake but not during foliar absorption. Chirality 25:686–691, 2013. © 2013 Wiley Periodicals, Inc.     

Optimization of Cross-Linked Lexitropsins

Abstract

In attempts to optimize the cross-linked lexitropsin design, a number of cross-linked dimers composed of two tris(N-methylpyrrolecarboxamide) strands were synthesized and their binding interactions with poly d(A)? poly d(T) and poly d(A-T)?poly d(A-T) were characterized by circular dichroism and ethidium fluorometry. While all alkanediyl-linked dimers showed a similar binding behavior to the homo AT polymer, particularly at low ligand concentrations, the decanediyl linker was found to be the optimal linker permitting the bidentate anti parallel side-by-side binding of the corresponding dimer to the alternating AT polymer. Thus, in comparison with the monomer, the decanediyl-linked dimer has a binding strength enhancement of about 1400 times in the I: I binding mode. Moreover, the hydrophilicity of the linker has a significant effect on the bidentate binding strength. The (3,6)-dioxaoctanediyl-linked dimer has a further binding strength enhancement of 10 times over the decanediyl-linked dimer. Overall, the best optimized dimer has a binding strength enhancement of over 14,000 times in comparison with the monomer in the I: I binding mode. This binding enhancement parallels that observed in the best optimized bisintercalators. Distance-restrained molecular modeling provides support for the experimental results. Dimers of longer linkers can readily accommodate a bidentate anti parallel side-by-side binding mode but those of shorter linkers necessitate marked structural distortions in the bound ligand molecules. It is further observed that the binding strength enhancement to the alternating AT polymer is not always accompanied by the binding specificity improvement. Our analysis suggests that the non-specific appendage-DNA backbone interaction is a key factor that controls the specificity improvement.     

Effect of Selective Substitution of 5-Bromocytosine on Conformation of DNA Triple Helices

Abstract

Three triplex DNAs containing 5-bromocytosine[^BrC] were studied by vibrational spectroscopy and molecular modelling. Firstly, three oligodeoxypyrimidines of 5′-(TC)3- T4-(^BrCT)3 [C^BrC], 5′-(T^BrC)3-T4-(CT)3 pCC] and 5′-(T^BC)3-T4-(^BrCT)3 [^BrC^BC] were synthesized and then reacted with an oligodeoxypurine of 5′-(AG)₃ at pH=4.5 in phosphate buffer respectively to form three comparative hairpin triplex named CY,YC and YY. The results of FT-Raman and IR revealed that YY is almost in A-like form, CY and YC are combinations of A-like form and B-like form, but A-form dominates in CY while B-form is equivalent as A-form in YC. The result is consistent with the theoretical analysis.     

Two Different Prunus SFB Alleles Have the Same Function in the Self-incompatibility Reaction

Many species in the families of Rosaceae, Solanaceae, and Scrophulariaceae exhibit gametophytic self-incompatibility, a phenomenon controlled by two polymorphic genes at the S-locus, style-S (S-RNase) and pollen-S (SFB). Sequences of both genes show high levels of diversity, characteristic of genes involved in recognition of self-incompatibility systems in plants. In this study, S ₂₄ -RNase and SFB ₂₄ alleles were cloned from Prunus armeniaca cv. Chuanzhihong (Chinese apricot). Sequence comparisons of deduced amino acid sequences revealed that the P. armeniaca S ₂₄ -haplotype has different SFB alleles, but shares a single S-RNase allele with P. armeniaca S ₄ -haplotype. Moreover, P. armeniaca S ₂₄ -RNase haplotype has a single and three different alleles with S ₁ -RNase of P. tenella (dwarf almond) and S ₁ -RNase of P. mira (smooth pit peach), respectively. The functionalities of SFB ₂₄ and SFB ₄ have been evaluated by pollen tube growth and controlled field tests of P. tenella and P. mira. Genetic analysis of the two intercrosses showed that progenies segregated 1:1 into two S-genotype classes, which is consistent with the expected ratio for semi-compatibility. These findings imply that the allelic function of the S ₂₄ -haplotype is identical to that of the S ₄ -haplotype in a self-incompatibility reaction. Thus, these two Prunus S-haplotypes are in fact two neutral variants of the same S-haplotype. The evolution of the S-allele is also discussed in terms of both functions and differences between S ₂₄ - and S ₄ -haplotypes in Prunus.