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.     

Nonplanar DNA Base Pairs

Abstract

Three-dimensional structures of a representative set of more than 30 hydrogen-bonded nucleic acids base pairs have been studied by reliable ab initio quantum mechanical methods. We show that many hydrogen-bonded nucleic acid base pairs are intrinsically nonplanar, mainly due to the partial sp³ hybridization of nitrogen atoms of their amino groups and secondary electrostatic interactions. This finding extends the variability of intermolecular interactions of DNA bases in that i) flexibility of the base pairs is larger than has been assumed before, and ii) attractive proton-proton acceptor interactions oriented out of the base pair plane are allowed. For example, all four G…A mismatch base pairs are propeller twisted, and the energy preferences for the nonplanar structures range from less than 0.1 kcal/mol to 1.8 kcal/mol. We predict that nonplanarity of the amino group of guanine in the G(anti)…A(anti) pair of the ApG step of the d(CCAAGATTGG)₂ crystal structure is an important stabilizing factor that improves the energy of this structure by almost 3 kcal/mol. Currently used empirical potentials are not accurate enough to properly cover the interactions associated with amino-group and base-pair nonplanarity.     

Overexpression of TaHSF3 in Transgenic Arabidopsis Enhances Tolerance to Extreme Temperatures

Heat shock factors (HSFs) in plants regulate heat stress response by mediating expression of a set of heat shock protein (HSP) genes. In the present study, we isolated a novel heat shock gene, TaHSF3, encoding a protein of 315 amino acids in wheat. Phylogenetic analysis showed that TaHSF3 belonged to HSF class B2. Subcellular localization analysis indicated that TaHSF3 localized in nuclei. TaHSF3 was highly expressed in wheat spikes and showed intermediate expression levels in roots, stems, and leaves under normal conditions. It was highly upregulated in wheat seedlings by heat and cold and to a lesser extent by drought and NaCl and ABA treatments. Overexpression of TaHSF3 in Arabidopsis enhanced tolerance to extreme temperatures. Frequency of survival of three TaHSF3 transgenic Arabidopsis lines was 75–91 % after heat treatment and 85–95 % after freezing treatment compared to 25 and 10 %, respectively, in wild-type plants (WT). Leaf chlorophyll contents of the transformants were higher (0.52–0.67 mg/g) than WT (0.35 mg/g) after heat treatment, and the relative electrical conductivities of the transformants after freezing treatment were lower (from 17.56 to 18.6 %) than those of WT (37.5 %). The TaHSF3 gene from wheat therefore confers tolerance to extreme temperatures in transgenic Arabidopsis by activating HSPs, such as HSP70.     

Multiscale simulations of protein folding: application to formation of secondary structures

A multiscale simulation method of protein folding is proposed, using atomic representation of protein and solvent, combing genetic algorithms to determine the key protein structures from a global view, with molecular dynamic simulations to reveal the local folding pathways, thus providing an integrated landscape of protein folding. The method is found to be superior to previously investigated global search algorithms or dynamic simulations alone. For secondary structure formation of a selected peptide, RN24, the structures and dynamics produced by this method agree well with corresponding experimental results. Three most populated conformations are observed, including hairpin, β-sheet and α-helix. The energetic barriers separating these three structures are comparable to the kinetic energy of the atoms of the peptide, implying that the transition between these states can be easily triggered by kinetic perturbations, mainly through electrostatic interactions between charged atoms. Transitions between α-helix and β-sheet should jump over at least two energy barriers and may stay in the energetic trap of hairpin. It is proposed that the structure of proteins should be jointly governed by thermodynamic and dynamic factors; free energy is not the exclusive dominant for stability of proteins.     

Explicit Solvent Molecular Dynamics Simulation of Duplex Formed by the Modified Oligonucleotide with Alternating Phosphate/Phosphonate Internucleoside Linkages and its Natural Counterpart

Abstract

Impact of the internucleoside linkage modification by inserting a methylene group on the ability of the modified oligonucleotide to hybridize with a natural DNA strand was studied by fully solvated molecular dynamics (MD) simulations. Three undecamer complexes were analyzed: natural dT₁₁.dA₁₁ duplex as a reference and two its analogs with alternating modified and natural linkages in the deoxyadenosine chain. The isopolar, non-isosteric modified linkages were of 5′-O-PO₂-CH₂-O-3′ (5′PC3′) or 5′-O-CH₂-PO₂-O-3′ (5′CP3′) type. Simulations were performed by using the AMBER 5.0 software package with the force field completed by a set of parameters needed to model the modified segments. Both modifications were found to lead to double helical complexes, in which the thymidine strand as well as deoxyriboses and unmodified linkages in the adenosine strand adopted conformations typical for the B-type structure. For each of the two conformational richer modified linkages two stable conformations were found at 300 K: the -ggt and ggt for the 5′PC3′ and ggg, tgg for the 5′CP3′, respectively. Both modified chains adopted helical conformations with heightened values of the inclination parameter but without affecting the Watson-Crick hydrogen bonds.     

Theoretical insight into the structural mechanism for the binding of vinblastine with tubulin

Vinblastine (VLB) is one of vinca alkaloids with high cytotoxicity toward cancer cells approved for clinical use. However, because of drug resistance, toxicity, and other side effects caused from the use of VLB, new vinca alkaloids with higher cytotoxicity toward cancer cells and other good qualities need to develop. One strategy is to further study and better understand the essence why VLB possesses the high cytotoxicity toward cancer cells. In present work, by using molecular simulation, molecular docking, density functional calculation, and the crystal structure of α,β-tubulin complex, we find two modes labeled in catharanthine moiety (CM) and vindoline moiety (VM) modes of VLB bound with the interface of α,β-tubulin to probe the essence why VLB has the high cytotoxicity toward cancer cells. In the CM mode, nine key residues B-Ser178, B-Asp179, B-Glu183, B-Tyr210, B-Asp226, C-Lys326, C-Asp327, C-Lys336, and C-Lys352 from the α,β-tubulin complex are determined as the active sites for the interaction of VLB with α,β-tubulin. Some of them such as B-Ser178, B-Glu183, B-Tyr210, B-Asp226, C-Lys326, C-Asp327, and C-Lys336 are newly identified as the active sites in present work. The affinity between VLB and the active pocket within the interface of α,β-tubulin is ?60.8 kJ mol^?1 in the CM mode. In the VM mode, that is a new mode established in present paper, nine similar key residues B-Lys176, B-Ser178, B-Asp179, B-Glu183, B-Tyr210, B-Asp226, C-Lys326, C-Asp327, and C-Lys336 from the α,β-tubulin complex are found as the active sites for the interaction with VLB. The difference is from one key residue C-Lys352 in the CM mode changed to the key residue B-Lys176 in the VM mode. The affinity between VLB and the active pocket within the interface of α,β-tubulin is ?96.3 kJ mol^?1 in the VM mode. Based on the results obtained in present work, and because VLB looks like two faces, composed of CM and VM both to have similar polar active groups, to interact with the active sites, we suggest double-faces sticking mechanism for the binding of VLB to the interface of α,β-tubulin. The double-faces sticking mechanism can be used to qualitatively explain high cytotoxicity toward cancer cells of vinca alkaloids including vinblastine, vincristine, vindestine, and vinorelbine approved for clinical use and vinflunine still in a phase III clinical trial. Furthermore, this mechanism will be applied to develop novel vinca alkaloids with much higher cytotoxicity toward cancer cells.     

3D-QSAR,molecular docking and molecular dynamics studies of a series of RORγt inhibitors

The discovery of clinically relevant inhibitors of retinoic acid receptor-related orphan receptor-gamma-t (RORγt) for autoimmune diseases therapy has proven to be a challenging task. In the present work, to find out the structural features required for the inhibitory activity, we show for the first time a three-dimensional quantitative structure–activity relationship (3D-QSAR), molecular docking and molecular dynamics (MD) simulations for a series of novel thiazole/thiophene ketone amides with inhibitory activity at the RORγt receptor. The optimum CoMFA and CoMSIA models, derived from ligand-based superimposition I, exhibit leave-one-out cross-validated correlation coefficient (R²_cv) of .859 and .805, respectively. Furthermore, the external predictive abilities of the models were evaluated by a test set, producing the predicted correlation coefficient (R²_pred) of .7317 and .7097, respectively. In addition, molecular docking analysis was applied to explore the binding modes between the inhibitors and the receptor. MD simulation and MM/PBSA method were also employed to study the stability and rationality of the derived conformations, and the binding free energies in detail. The QSAR models and the results of molecular docking, MD simulation, binding free energies corroborate well with each other and further provide insights regarding the development of novel RORγt inhibitors with better activity.     

Histamine synergistically promotes bFGF‐induced angiogenesis by enhancing VEGF production via H1 receptor

Histamine, a major mediator present in mast cells that is released into the extracellular milieu upon degranulation, is well known to possess a wide range of biological activities in several classic physiological and pathological processes. However, whether and how it participates in angiogenesis remains obscure. In the present study, we observed its direct and synergistic action with basic fibroblast growth factor (bFGF), an important inducer of angiogenesis, on in vitro angiogenesis models of endothelial cells. Data showed that histamine (0.1, 1, 10 µM) itself was absent of direct effects on the processes of angiogenesis, including the proliferation, migration, and tube formation of endothelial cells. Nevertheless, it could concentration‐dependently enhance bFGF‐induced angiogenesis as well as production of vascular endothelial growth factor (VEGF) from endothelial cells. The synergistic effect of histamine on VEGF production could be reversed by pretreatments with diphenhydramine (H1‐receptor antagonist), SB203580 (selective p38 mitogen‐activated protein kinase (MAPK) inhibitor) and L ‐NAME (nitric oxide synthase (NOS) inhibitor), but not with cimetidine (H2‐receptor antagonist) and indomethacin (cyclooxygenase (COX) inhibitor). Moreover, histamine could augment bFGF‐incuced phosphorylation and degradation of IκBα, a key factor accounting for the activation and translocation of nuclear factor κB (NF‐κB) in endothelial cells. These findings indicated that histamine was able to synergistically augment bFGF‐induced angiogenesis, and this action was linked to VEGF production through H1‐receptor and the activation of endothelial nitric oxide synthase (eNOS), p38 MAPK, and IκBα in endothelial cells. J. Cell. Biochem. 114: 1009–1019, 2013. © 2012 Wiley Periodicals, Inc.