Insight into the gas phase dissociation of CF<Subscript>3</Subscript>CH<Subscript>2</Subscript>I and its reactions with H and OH by first principles

The Arrhenius kinetic parameters of dissociation reactions and reactions of CF₃CH₂I with radicals like H, O, and OH are determined using highly accurate first principles calculations. Thermophysical properties like molar heat capacity (C_p), thermal stability index, and the bond dissociation energies are also determined for the CF₃CH₂I molecule under the PBE/DNP formalism. Since, there are no theoretical study or experimental investigation reports available regarding the dissociation reactions of CF₃CH₂I and reactions of this molecule with the H and OH radical, a parallel comparative analysis is done with similar iodoalkanes to ascertain the precision of the results obtained. The atmospheric lifetime of 0.54 years is obtained for this molecule.     

Yes-associated Protein (YAP) Promotes Cell Survival by Inhibiting Proapoptotic Dendrin Signaling

Kidney podocytes are highly specialized terminally differentiated cells that form the final barrier to urinary protein loss. Podocytes are a target for injury by metabolic, autoimmune, hereditary, inflammatory, and other stressors. Persistence of podocyte injury leads to podocyte death and loss, which results in progressive kidney damage and ultimately kidney failure. Dendrin is a dual compartment protein with proapoptotic signaling properties. Nuclear relocation of dendrin in response to glomerular injury promotes podocyte apoptosis. Here we show that Yes-associated protein (YAP), a downstream target of Hippo kinases and an inhibitor of apoptosis, is expressed in the nucleus of podocytes. The WW domains of YAP mediate the interaction with the PPXY motifs of dendrin. This interaction is functionally relevant because YAP binding to dendrin reduces dendrin-dependent, staurosporine-induced apoptosis in co-transfected HEK293 cells. Moreover gene silencing of YAP in podocytes increases adriamycin-induced podocyte apoptosis. It also increases staurosporine-induced caspase-3/7 activity, which is rescued by dendrin depletion in YAP knockdown cells. Our findings elucidate YAP binding to dendrin as a prosurvival mechanism. The antiapoptotic signaling properties of YAP in podocytes could hold significance in the quest for targeted therapeutics aimed at preventing podocyte loss.     

Unexpected Histone H3 Tail-clipping Activity of Glutamate Dehydrogenase

Clipping of histone tails has been reported in several organisms. However, the significance and regulation of histone tail clipping largely remains unclear. According to recent discoveries H3 clipping has been found to be involved in regulation of gene expression and chromatin dynamics. Earlier we had provided evidence of tissue-specific proteolytic processing of histone H3 in White Leghorn chicken liver nuclei. In this study we identify a novel activity of glutamate dehydrogenase (GDH) as a histone H3-specific protease in chicken liver tissue. This protease activity is regulated by divalent ions and thiol-disulfide conversion in vitro. GDH specifically clips H3 in its free as well as chromatin-bound form. Furthermore, we have found an inhibitor that inhibits the H3-clipping activity of GDH. Like previously reported proteases, GDH too may have the potential to regulate/modulate post-translational modifications of histone H3 by removing the N-terminal residues of the histone. In short, our findings identify an unexpected proteolytic activity of GDH specific to histone H3 that is regulated by redox state, ionic concentrations, and a cellular inhibitor in vitro.     

Evaluation of the Impacts of Formulation Variables and Excipients on the Drug Release Dynamics of a Polyamide 6,10-Based Monolithic Matrix Using Mathematical Tools

Drug release from hydrophilic matrices is regulated mainly by polymeric erosion, disentanglement, dissolution, swelling front movement, drug dissolution and diffusion through the polymeric matrix. These processes depend upon the interaction between the dissolution media, polymeric matrix and drug molecules, which can be significantly influenced by formulation variables and excipients. This study utilized mathematical parameters to evaluate the impacts of selected formulation variables and various excipients on the release performance of hydrophilic polyamide 6,10 (PA 6,10) monolithic matrix. Amitriptyline HCl and theophylline were employed as the high and low solubility model drugs, respectively. The incorporation of different excipient concentrations and changes in formulation components influenced the drug release dynamics as evidenced by computed mathematical quantities (t _x%, MDT _x%, f ₁, f ₂, k ₁, k ₂, and К _F). The effects of excipients on drug release from the PA 6,10 monolithic matrix was further elucidated using static lattice atomistic simulations wherein the component energy refinements corroborates the in vitro and in silico experimental data. Consequently, the feasibility of modulating release kinetics of drug molecules from the novel PA 6,10 monolithic matrix was well suggested.     

NMR-based structure of anticancer drug mitoxantrone stacked with terminal base pair of DNA hexamer sequence d-(ATCGAT)2

Mitoxantrone is a promising antitumor drug having considerably reduced cardiotoxicity as compared to anthracyclines. Its binding to deoxyhexanucleotides sequence d-(ATCGAT)₂ has been studied by proton and phosphorous-31 nuclear magnetic resonance spectroscopy. The stoichiometry reveals that 1:1 and 2:1 mitoxantrone-d(ATCGAT)₂ complexes are formed in solution. Significant upfield shifts in 6H/7H, 2H/3H, 11NH, and 12NH protons (～.5?ppm) of mitoxantrone and T6NH imino protons (～.3?ppm) are observed. The phosphorous resonances do not shift significantly indicating that the base pairs do not open at any nucleotide step along the sequence of hexamer. Several inter-molecular Nuclear Overhauser Enhancement connectivities between mitoxantrone and hexanucleotide protons indicate that mitoxantrone chromophore stacks with terminal A1-T6 base pair and side chains involving 12CH₂, 12NH, and 14OH protons are in close proximity of A1, T2, A5, and T6 bases. Absorption and emission spectra show red shift in wavelength maxima, which is characteristic of stacking interaction. At higher mitoxantrone to nucleic acid ratios, electrostatic interactions are dominant. The 2:1 drug/DNA stoichiometric structure obtained by restrained Molecular Dynamics simulations shows considerable distortions in backbone torsional angles and helicoidal parameters although structural fluctuations in 25?ps analysis of trajectory are found to be negligible. Mitoxantrone binds as a monomer at either or both ends of hexamer externally with side chains interacting specifically with DNA. The findings are relevant to the understanding of pharmacological action of drug.     

Detection of Polymorphism and Sequence Characterization of Toll-Like Receptor 7 Gene of Indian Goat Revealing Close Relationship Between Ruminant Species

In this study, approximately 3.4 kb nucleotide sequence of caprine TLR7 (Toll-like receptor 7) gene was generated from twelve different Indian goat breeds belonging to different geographical regions. Goat TLR7 gene ORF (Open Reading Frame) was found to be 3141 nucleotides long coding for 1046 amino acids similar to sheep. The sequence analysis at nucleotide level revealed goat TLR7 having 99.5% homology with sheep, followed by other livestock species. Simple Modular Architecture Research Tool (SMART) was used for the structural analysis of goat TLR7 that showed the presence of 22 leucine rich repeats (LRRs) along with single Toll/interleukin-1 receptor (TIR) domains. TIR domain, when compared, was found to be similar in ruminant species, goat, sheep, cattle, and buffalo. The phylogenetic analysis also revealed grouping of all ruminant species together, goat being closer to sheep followed by cattle and buffalo. A total of 22 polymorphic sites were observed in TLR7 gene of 24 goats representing 12 different breeds, out of which 19 were present within the coding region and three in 3'UTR. Out of the seven nonsynonymous SNPs, two were in ectodomains and one in TIR domain. Overall our results indicate substantial variation within goat TLR7 gene, which could be exploited for association with disease susceptibility.     

Structural Insight into DFMO Resistant Ornithine Decarboxylase from Entamoeba histolytica: An Inkling to Adaptive Evolution

Background

Polyamine biosynthetic pathway is a validated therapeutic target for large number of infectious diseases including cancer, giardiasis and African sleeping sickness, etc. α-Difluoromethylornithine (DFMO), a potent drug used for the treatment of African sleeping sickness is an irreversible inhibitor of ornithine decarboxylase (ODC), the first rate limiting enzyme of polyamine biosynthesis. The enzyme ODC of E. histolytica (EhODC) has been reported to exhibit resistance towards DFMO.

Methodology/Principal Finding

The basis for insensitivity towards DFMO was investigated by structural analysis of EhODC and conformational modifications at the active site. Here, we report cloning, purification and crystal structure determination of C-terminal truncated Entamoeba histolytica ornithine decarboxylase (EhODCΔ15). Structure was determined by molecular replacement method and refined to 2.8 Å resolution. The orthorhombic crystal exhibits P2₁2₁2₁ symmetry with unit cell parameters a = 76.66, b = 119.28, c = 179.28 Å. Functional as well as evolutionary relations of EhODC with other ODC homologs were predicted on the basis of sequence analysis, phylogeny and structure.

Conclusions/Significance

We determined the tetrameric crystal structure of EhODCΔ15, which exists as a dimer in solution. Insensitivity towards DFMO is due to substitution of key substrate binding residues in active site pocket. Additionally, a few more substitutions similar to antizyme inhibitor (AZI), a non-functional homologue of ODCs, were identified in the active site. Here, we establish the fact that EhODC sequence has conserved PLP binding residues; in contrast few substrate binding residues are mutated similar to AZI. Further sequence analysis and structural studies revealed that EhODC may represent as an evolutionary bridge between active decarboxylase and inactive AZI.     

Enzymatically active APOBEC3G is required for efficient inhibition of human immunodeficiency virus type 1

APOBEC3G (APO3G) is a cellular cytidine deaminase with potent antiviral activity. Initial studies of the function of APO3G demonstrated extensive mutation of the viral genome, suggesting a model in which APO3G's antiviral activity is due to hypermutation of the viral genome. Recent studies, however, found that deaminase-defective APO3G mutants transiently expressed in virus-producing cells exhibited significant antiviral activity, suggesting that the antiviral activity of APO3G could be dissociated from its deaminase activity. To directly compare the antiviral activities of wild-type (wt) and deaminase-defective APO3G, we used two approaches: (i) we titrated wt and deaminase-defective APO3G in transient-transfection studies to achieve similar levels of virus-associated APO3G and (ii) we constructed stable cell lines and selected clones expressing comparable amounts of wt and deaminase-defective APO3G. Viruses produced under these conditions were tested for viral infectivity. The results from the two approaches were consistent and suggested that the antiviral activity of deaminase-defective APO3G was significantly lower than that of wt APO3G. We conclude that efficient inhibition of vif-defective human immunodeficiency virus type 1 requires catalytically active APO3G.     

An Epichlorohydrin-Crosslinked Semi-Interpenetrating GG-PEO Network as a Xerogel Matrix for Sustained Release of Sulpiride

The current study involved the development of a novel sustained release crosslinked semi-IPN xerogel matrix tablet prepared by chemical crosslinking of poly(ethylene) oxide (PEO) and gellan gum (GG) employing epichlorohydrin (EPI) as crosslinker. A Box–Behnken design was employed for the statistical optimization of the matrix system to ascertain the ideal combination of native polymeric and crosslinking agents. Characterization studies were performed by employing standard polymer characterization techniques such as Fourier transform infrared spectrometry, differential scanning calorimetry, and scanning electron microscopy. Formulated matrix tablets displayed zero-order release kinetics, extending over 24 h. The mechanism of drug release was primarily by swelling and surface erosion. Crosslinked semi-IPN xerogel matrix tablets were compared to non-crosslinked polymer blends; results from the study conducted showed that the physiochemical properties of the PEO and GG were sufficiently modified to allow for sustained release of sulpiride with a 100% drug release at 24 h in a controlled manner as compared to non-crosslinked formulations which displayed further release beyond the test period. Crosslinked formulations displayed water uptake between 450 and 500% indicating a controlled rate of swelling and erosion allowing for sustained release. Surface morphology of the crosslinked system depicted a porous structure formed by interpenetrating networks of polymers, allowing for a greater degree of controlled penetration into the system affording it the ability to sustain drug release. Therefore, conclusively, based on the study performed, crosslinked PEO-GG allows for the sustained release of sulpiride from a hydrophilic semi-IPN xerogel matrix system.KEY WORDS: epichlorohydrin, matrix tablet, semi-interpenetrating polymer network, sustained release, sulpiride