Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.     

Molecular Modeling Study on Tunnel Behavior in Different Histone Deacetylase Isoforms

Histone deacetylases (HDACs) have emerged as effective therapeutic targets in the treatment of various diseases including cancers as these enzymes directly involved in the epigenetic regulation of genes. However the development of isoform-selective HDAC inhibitors has been a challenge till date since all HDAC enzymes possess conserved tunnel-like active site. In this study, using molecular dynamics simulation we have analyzed the behavior of tunnels present in HDAC8, 10, and 11 enzymes of class I, II, and IV, respectively. We have identified the equivalent tunnel forming amino acids in these three isoforms and found that they are very much conserved with subtle differences to be utilized in selective inhibitor development. One amino acid, methionine of HDAC8, among six tunnel forming residues is different in isoforms of other classes (glutamic acid (E) in HDAC10 and leucine (L) in HDAC 11) based on which mutations were introduced in HDAC11, the less studied HDAC isoform, to observe the effects of this change. The HDAC8-like (L268M) mutation in the tunnel forming residues has almost maintained the deep and narrow tunnel as present in HDAC8 whereas HDAC10-like (L268E) mutation has changed the tunnel wider and shallow as observed in HDAC10. These results explained the importance of the single change in the tunnel formation in different isoforms. The observations from this study can be utilized in the development of isoform-selective HDAC inhibitors.     

Molecular Dynamic Simulation Reveals Damaging Impact of RAC1 F28L Mutation in the Switch I Region

Ras-related C3 botulinum toxin substrate 1 (RAC1) is a plasma membrane-associated small GTPase which cycles between the active GTP-bound and inactive GDP-bound states. There is wide range of evidences indicating its active participation in inducing cancer-associated phenotypes. RAC1 F28L mutation (RAC^F28L) is a fast recycling mutation which has been implicated in several cancer associated cases. In this work we have performed molecular docking and molecular dynamics simulation (~0.3 μs) to investigate the conformational changes occurring in the mutant protein. The RMSD, RMSF and NHbonds results strongly suggested that the loss of native conformation in the Switch I region in RAC1 mutant protein could be the reason behind its oncogenic transformation. The overall results suggested that the mutant protein attained compact conformation as compared to the native. The major impact of mutation was observed in the Switch I region which might be the crucial reason behind the loss of interaction between the guanine ring and F28 residue.     

Investigations on Modulating Effect of Vanadium Supplementation on Growth and Metabolism Through Improved Immune Response,Antioxidative Profile and Endocrine Variables in Hariana heifers

Biological Trace Element Research - This study was conducted to investigate the effect of vanadium (V) supplementation on growth, metabolism, antioxidant, and immunological and endocrine variables...     

Improvement of antagonism and carbendazim tolerance in native Trichoderma isolates through ethyl methane sulfonate (EMS) mutagenesis

Genetic enhancement of TCT₄ and TCT₁₀ was aimed in the present paper. Trichoderma reesei (TCT₁₀/M₁₈) mutant isolate evolved by ethyl methane sulfonate mutations was found to exhibit altered properties compared to its parent isolates. This mutant grew well in the potato dextrose agar (PDA) medium containing carbendazim (50 ppm). RAPD-PCR results suggested the uniqueness of mutants, which was useful in differentiating mutant and wild Trichoderma isolates. These mutants established well in the rhizosphere of rough lemon seedlings. The seedlings treated with carbendazim followed by an application of carbendazim-resistant mutant (TCT₁₀/M₁₈) resulted in a better seedling emergence and a less dry root rot disease caused by Fusarium solani in nursery conditions.     

Inhibitor binding studies of Mycobacterium tuberculosis MraY (Rv2156c): Insights from molecular modeling,docking, and simulation studies

Abstract

Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis (M.tb) or tubercule bacillus, and H37Rv is the most studied clinical strain. The recent development of resistance to existing drugs is a global health-care challenge to control and cure TB. Hence, there is a critical need to discover new drug targets in M.tb. The members of peptidoglycan biosynthesis pathway are attractive target proteins for antibacterial drug development. We have performed in silico analysis of M.tb MraY (Rv2156c) integral membrane protein and constructed the three-dimensional (3D) structure model of M.tb MraY based on homology modeling method. The validated model was complexed with antibiotic muraymycin D2 (MD2) and was used to generate structure-based pharmacophore model (e-pharmacophore). High-throughput virtual screening (HTVS) of Asinex database and molecular docking of hits was performed to identify the potential inhibitors based on their mode of interactions with the key residues involved in M.tb MraY–MD2 binding. The validation of these molecules was performed using molecular dynamics (MD) simulations for two best identified hit molecules complexed with M.tb MraY in the lipid bilayer, dipalmitoylphosphatidyl-choline (DPPC) membrane. The results indicated the stability of the complexes formed and retained non-bonding interactions similar to MD2. These findings may help in the design of new inhibitors to M.tb MraY involved in peptidoglycan biosynthesis.