Hybrid MC/QC simulations of water-assisted proton transfer in nucleosides. Guanosine and its analog acyclovir

To provide an in-depth insight into the molecular basis of spontaneous tautomerism in DNA and RNA base pairs, a hybrid Monte Carlo (MC)–quantum chemical (QC) methodology is implemented to map two-dimensional potential energy surfaces along the reaction coordinates of solvent-assisted proton transfer processes in guanosine and its analog acyclovir in aqueous solution. The solvent effects were simulated by explicit inclusion of water molecules that model the relevant part of the first hydration shell around the solute. The position of these water molecules was estimated by carrying out a classical Metropolis Monte Carlo simulation of dilute water solutions of the guanosine (Gs) and acyclovir (ACV) and subsequently analyzing solute–solvent intermolecular interactions in the statistically-independent MC-generated configurations. The solvent-assisted proton transfer processes were further investigated using two different ab initio MP2 quantum chemical approaches. In the first one, potential energy surfaces of the ‘bare’ finite solute–solvent clusters containing Gs/ACV and four water molecules (MP2/6-31+G(d,p) level) were explored, while within the second approach, these clusters were embedded in ‘bulk’ solvent treated as polarizable continuum (C-PCM/MP2/6-31+G(d,p) level of theory). It was found that in the gas phase and in water solution, the most stable tautomer for guanosine and acyclovir is the 1H-2-amino-6-oxo form followed by the 2-amino-6-(sZ)-hydroxy form. The energy barriers of the water-assisted proton transfer reaction in guanosine and in acyclovir are found to be very similar – 11.74 kcal mol^?1 for guanosine and 11.16 kcal mol^?1 for acyclovir, and the respective rate constants (k = 1.5?×?10¹ s^?1, guanosine and k = 4.09?×?10¹ s^?1, acyclovir), are sufficiently large to generate the 2-amino-6-(sZ)-hydroxy tautomer. The analysis of the reaction profiles in both compounds shows that the proton transfer processes occur through the asynchronous concerted mechanism.     

Understanding weakly coordinating anions: tetrakis(pentafluorophenyl)borate paired with inorganic and organic cations

Efficient design of ionic compounds requires a systematic understanding of cation–anion interactions. Weakening of electrostatic attraction is essential to increase the liquid range of the ionic compound and decrease its melting point. Here, we report simulations of the closest-approach cation–anion distances in a variety of ion pairs containing the tetrakis(pentafluorophenyl)borate (TFPB^–) anion. Small alkali cations (Li⁺, Na⁺) penetrate the TFPB^– core, whereas K⁺ and larger organic cations do not. In the latter case, the shortest possible distance from the cations to the boron atom of TFPB^– ranges from 0.50 nm to 0.63 nm. TFPB^– was shown to be substantially rigid, providing a steric hindrance to thermodynamically efficient cation–anion coordination. Our results prove that TFPB^– is more efficient for electrostatic charge confinement than the tetraoctylammonium cation, whereas the perfluorophenyl group is more efficient than linear alkyl chains. These simulations will motivate development of TFPB^–-based ionic liquids with low phase transition points.

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Graphical Abstract Ionic configuration of the equilibrated “TFPB + K”system

     

Larval interspecific competition in two flea species parasitic on the same rodent host

Abstract.  1. The fleas Xenopsylla conformis and Xenopsylla ramesis exploit the same rodent host, Meriones crassus , and replace each other between two different habitats situated at the opposite sides of a steep precipitation gradient. It was hypothesised that the reason for this paratopic distribution is competition between larvae of the two species for food resources.
2. This hypothesis was tested by studying the performance of larvae of the two species in terms of their developmental success in mixed-species and single-species treatments under different air temperatures, relative humidities, substrate textures, and food abundance.
3. The number of individuals of X. conformis that survived until emergence depended significantly on the presence of competing species, being, in general, lower in mixed-species compared with single-species treatments. The decrease in developmental success of X. conformis in mixed-species treatments was found mainly during food shortage. In contrast, presence of the competitor did not affect the number of X. ramesis that survived until emergence. No effect of the presence of the competitor on duration of development or sex ratio was found in either species.
4. The results of this study, together with the results of our previous studies, provide an explanation for the paratopic distribution of X. conformis and X. ramesis that exploit the same host species.     

Phylogeny and systematics of the genus Lophozia s. str. (Dumort.) Dumort. (Hepaticae) and related taxa from nuclear ITS1-2 and chloroplast trnL-F sequences

Nuclear ITS1-2 and chloroplast trnL-F were sequenced for 21 taxa of Lophozia s. str., two species of Protolophozia, five species of Schistochilopsis, three species of Barbilophozia and Obtusifolium obtusum. The topologies of phylogenetic trees for 49 taxa constructed from combined sequences of these regions by maximum parsimony, maximum likelihood and Bayesian methods are similar. The species of Lophozia s. str., excluding Lophozia sudetica, combine into two main clades and these contradict subdivisions of Lophozia s. str. based on morphology. The species status of Lophozia lantratoviae is confirmed, whereas Lophozia austro-sibirica is almost identical to Lophozia ventricosa var. guttulata. The genus Schistochilopsis is paraphyletic and occupies basal position to Lophozia s. str., while O. obtusum is clearly separated from Schistochilopsis. A low level of divergence was found between L. sudetica and Protolophozia debiliformes, which are closer to Barbilophozia than to Lophozia s. str. Molecular divergence between geographically remote populations of L. sudetica, Lophozia silvicoloides and Protolophozia debiliformis are low as opposed to those of Lophozia polaris, Lophozia pellucida or Lophozia excisa. Consideration of the trnL intron P8 region indels alone can adequately assign some clades revealed by tree building. A consensus secondary structure of the trnL intron P8 region could not be inferred for taxa studied mainly due to high sequence length diversity originated from deletions.     

Pretreatment with heat does not affect double-strand breaks DNA rejoining in Chlamydomonas reinhardtii

We aimed to clarify if heat pretreatment could protect Chlamydomonas reinhardtii cells from gamma rays DNA damaging action. It was studied whether: (1) heat pretreatment could accelerate DNA DSB rejoining; (2) chloroplast chaperones (HSP70B, HSP90C) could be involved in protection from radiation-induced DNA DSB.It was obtained that heat pretreatment (37–42 °C) induced minor DNA DSB levels which might be insufficient as signals for DNA DSB repair induction. No correlation between chaperones overproduction and DNA DSB rejoining was shown. These are probably the first data that HSP70B and HSP90C do not protect DNA against radiation-induced damage in a plant model system.     

Reversible and irreversible modifications of skeletal muscle proteins in a rat model of acute oxidative stress

Oxidative stress caused by an imbalance of the production of “reactive oxygen species” (ROS) and cellular scavenging systems is known to a play a key role in the development of various diseases and aging processes. Such elevated ROS levels can damage all components of cells, including proteins, lipids and DNA. Here, we study the influence of highly reactive ROS species on skeletal muscle proteins in a rat model of acute oxidative stress caused by X-ray irradiation at different time points. Protein preparations depleted for functional actin by polymerization were separated by gel electrophoresis in two dimensions by applying first non-reductive and then reductive conditions in SDS-PAGE. This diagonal redox SDS-PAGE revealed significant alterations to intra- and inter-molecular disulfide bridges for several proteins, but especially actin, creatine kinase and different isoforms of the myosin light chain. Though the levels of these reversible modifications were increased by oxidative stress, all proteins followed different kinetics. Moreover, a significant degree of protein was irreversibly oxidized (carbonylated), as revealed by western blot analyses performed at different time points.     

Comparison of genomes of eight species of sections <Emphasis Type="Italic">Linum</Emphasis> and <Emphasis Type="Italic">Adenolinum</Emphasis> from the genus<Emphasis Type="Italic"> Linum</Emphasis> based on chromosome banding,molecular markers and RAPD analysis

Karyotypes of species sects. Linum and Adenolinum have been studied using C/DAPI-banding, Ag-NOR staining, FISH with 5S and 26S rDNA and RAPD analysis. C/DAPI-banding patterns enabled identification of all homologous chromosome pairs in the studied karyotypes. The revealed high similarity between species L. grandiflorum (2n = 16) and L. decumbens by chromosome and molecular markers proved their close genome relationship and identified the chromosome number in L. decumbens as 2n = 16. The similarity found for C/DAPI-banding patterns between species with the same chromosome numbers corresponds with the results obtained by RAPD-analysis, showing clusterization of 16-, 18- and 30-chromosome species into three separate groups. 5S rDNA and 26S rDNA were co-localized in NOR-chromosome 1 in the genomes of all species investigated. In 30-chromosome species, there were three separate 5S rDNA sites in chromosomes 3, 8 and 13. In 16-chromosome species, a separate 5S rDNA site was also located in chromosome 3, whereas in 18-chromosome species it was found in the long arm of NOR-chromosome 1. Thus, the difference in localization of rDNA sites in species with 2n = 16, 2n = 30 and 2n = 18 confirms taxonomists opinion, who attributed these species to different sects. Linum and Adenolinum, respectively. The obtained results suggest that species with 2n = 16, 2n = 18 and 2n = 30 originated from a 16-chromosome ancestor.     

Efficiency of transfer of essential polyunsaturated fatty acids versus organic carbon from producers to consumers in a eutrophic reservoir

One of the central paradigms of ecology is that only about 10% of organic carbon production of one trophic level is incorporated into new biomass of organisms of the next trophic level. Many of energy-yielding compounds of carbon are designated as ‘essential’, because they cannot be synthesized de novo by consumers and must be obtained with food, while they play important structural and regulatory functions. The question arises: are the essential compounds transferred through trophic chains with the same efficiency as bulk carbon? To answer this question, we measured gross primary production of phytoplankton and secondary production of zooplankton and content of organic carbon and essential polyunsaturated fatty acids of ω-3 family with 18–22 carbon atoms (PUFA) in the biomass of phytoplankton and zooplankton in a small eutrophic reservoir during two summers. Transfer efficiency between the two trophic levels, phytoplankton (producers) and zooplankton (consumers), was calculated as ratio of the primary production versus the secondary (zooplankton) production for both carbon and PUFA. We found that the essential PUFA were transferred from the producers to the primary consumers with about twice higher efficiency than bulk carbon. In contrast, polyunsaturated fatty acids with 16 carbon atoms, which are synthesized exclusively by phytoplankton, but are not essential for animals, had significantly lower transfer efficiency than both bulk carbon, and essential PUFA. Thus, the trophic pyramid concept, which implicitly implies that all the energy-yielding compounds of carbon are transferred from one trophic level to the next with the same efficiency of about on average 10%, should be specified for different carbon compounds.     

c-Src inactivation reduces renal epithelial cell-matrix adhesion, proliferation, and cyst formation

c-Src is a non-receptor tyrosine kinase whose activity is induced by phosphorylation at Y418 and translocation from the cytoplasm to the cell membrane. Increased activity of c-Src has been associated with cell proliferation, matrix adhesion, motility, and apoptosis in tumors. Immunohistochemistry suggested that activated (pY(418))-Src activity is increased in cyst-lining autosomal dominant polycystic kidney disease (ADPKD) epithelial cells in human and mouse ADPKD. Western blot analysis showed that SKI-606 (Wyeth) is a specific inhibitor of pY(418)-Src without demonstrable effects on epidermal growth factor receptor or ErbB2 activity in renal epithelia. In vitro studies on mouse inner medullary collecting duct (mIMCD) cells and human ADPKD cyst-lining epithelial cells showed that SKI-606 inhibited epithelial cell proliferation over a 24-h time frame. In addition, SKI-606 treatment caused a striking statistically significant decrease in adhesion of mIMCD and human ADPKD to extracellular collagen matrix. Retained viability of unattached cells was consistent with a primary effect on epithelial cell anchorage dependence mediated by the loss of extracellular matrix (ECM)-attachment due to α(2)β(1)-integrin function. SKI-606-mediated attenuation of the human ADPKD hyperproliferative and hyper-ECM-adhesive epithelial cell phenotype in vitro was paralleled by retardation of the renal cystic phenotype of Pkd1 orthologous ADPKD heterozygous mice in vivo. This suggests that SKI-606 has dual effects on cystic epithelial cell proliferation and ECM adhesion and may have therapeutic potential for ADPKD patients.     

Association of adaptor protein TRIP8b with clathrin

TPR-containing Rab8b-interacting protein (TRIP8b) is a brain-specific hydrophilic cytosolic protein that contains tetratricopeptide repeats (TPRs). Previous studies revealed interaction of this protein via its TPR-containing domain with Rab8b small GTPase, hyperpolarization-activated cyclic nucleotide-regulated channel (HCN) channels and G protein-coupled receptor calcium-independent receptor of α-latrotoxin. We identified clathrin as a major component of eluates from the TRIP8b affinity matrix. In the present study, by in vitro-binding analysis we demonstrate a direct interaction between clathrin and TRIP8b. The clathrin-binding site was localized in the N-terminal (non-TPR containing) part of the TRIP8b molecule that contains two short motifs involved in the clathrin binding. In transfected HEK293 cells, co-expression of HCN1 with TRIP8b resulted in translocation of the channels from the cell surface to large intracellular puncta where both TRIP8b and clathrin were concentrated. These puncta co-localized partially with an early endosome marker and strongly overlapped with lysosome staining reagent. When HCN1 was co-expressed with a clathrin-non-binding mutant of TRIP8b, clathrin did not translocate to HCN1 and TRIP8b-containing puncta, suggesting that TRIP8b interacts with HCN and clathrin independently. We found TRIP8b present in the fraction of clathrin-coated vesicles purified from brain tissues. Stripping the clathrin coat proteins from the vesicles with Tris alkaline buffer resulted in concomitant release of TRIP8b. Our data suggest complex regulatory functions of TRIP8b in neuronal endocytosis through independent interaction with membrane proteins and components of the clathrin coat.