Molecular dynamics and principal components analysis of human telomeric quadruplex multimers.

Guanine-rich DNA repeat sequences located at the terminal ends of chromosomal DNA can fold in a sequence-dependent manner into G-quadruplex structures, notably the terminal 150–200 nucleotides at the 3' end, which occur as a single-stranded DNA overhang. The crystal structures of quadruplexes with two and four human telomeric repeats show an all-parallel-stranded topology that is readily capable of forming extended stacks of such quadruplex structures, with external TTA loops positioned to potentially interact with other macromolecules. This study reports on possible arrangements for these quadruplex dimers and tetramers, which can be formed from 8 or 16 telomeric DNA repeats, and on a methodology for modeling their interactions with small molecules. A series of computational methods including molecular dynamics, free energy calculations, and principal components analysis have been used to characterize the properties of these higher-order G-quadruplex dimers and tetramers with parallel-stranded topology. The results confirm the stability of the central G-tetrads, the individual quadruplexes, and the resulting multimers. Principal components analysis has been carried out to highlight the dominant motions in these G-quadruplex dimer and multimer structures. The TTA loop is the most flexible part of the model and the overall multimer quadruplex becoming more stable with the addition of further G-tetrads. The addition of a ligand to the model confirms the hypothesis that flat planar chromophores stabilize G-quadruplex structures by making them less flexible.

Structural change of vortex patterns in anisotropic Bose-Einstein condensates

We study the changes in the spatial distribution of vortices in a rotating Bose-Einstein condensate due to an increasing eccentricity of the trapping potential. By breaking the rotational symmetry, the vortex system undergoes a rich variety of structural changes, including the formation of zigzag and linear configurations. These spatial rearrangements are well signaled by the change in the behavior of the vortex-pattern eigenmodes against the eccentricity parameter. This behavior allows to actively control the distribution of vorticity in many-body systems and opens the possibility of studying interactions between quantum vortices over a large range of parameters.

Excess Electron Localization in Solvated DNA Bases

We present a first-principles molecular dynamics study of an excess electron in condensed phase models of solvated DNA bases. Calculations on increasingly large microsolvated clusters taken from liquid phase simulations show that adiabatic electron affinities increase systematically upon solvation, as for optimized gas-phase geometries. Dynamical simulations after vertical attachment indicate that the excess electron, which is initially found delocalized, localizes around the nucleobases within a 15 fs time scale. This transition requires small rearrangements in the geometry of the bases.

A structural analysis of G-quadruplex/ligand interactions

This focused review article discusses in detail, all available high-resolution small molecule ligand/G-quadruplex structural data derived from crystallographic and NMR based techniques, in an attempt to understand key factors in ligand binding and to highlight the biological importance of these complexes. In contrast to duplex DNA, G-quadruplexes are four-stranded nucleic acid structures folded from guanine rich repeat sequences stabilized by the stacking of guanine G-quartets and extensive Watson-Crick/Hoogsteen hydrogen bonding. Thermally stable, these topologies can play a role in telomere regulation and gene expression. The core structures of G-quadruplexes form stable scaffolds while the loops have been shown, by the addition of small molecule ligands, to be sufficiently adaptable to generate new and extended binding platforms for ligands to associate, either by extending G-quartet surfaces or by forming additional planar dinucleotide pairings. Many of these structurally characterised loop rearrangements were totally unexpected opening up new opportunities for the design of selective ligands. However these rearrangements do significantly complicate attempts to rationally design ligands against well defined but unbound topologies, as seen for the series of napthalene diimides complexes. Drawing together previous findings and with the introduction of two new crystallographic quadruplex/ligand structures we aim to expand the understanding of possible structural adaptations available to quadruplexes in the presence of ligands, thereby aiding in the design of new selective entities. (C) 2011 Elsevier Masson SAS. All rights reserved.

Sequential C-H and C-Ru bond formation and cleavage during the thermally induced rearrangement of aryl ruthenium(II) complexes with [C6H3(CH2NMe2)(2)-2,6](-) as a bidentate eta(2)-C,N coordinated ligand. The crystal structures of the isomeric pairs [RuCl{eta(6)-C10H14}{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,n}] (n = 4 or 6) and [Ru(eta(5)-C5H5){(eta(2)-C,N-C6H3(CH2NMe2)(2)-2,n} (PPH3)] (n = 4 or 6)

New air-stable ruthenium(II) complexes that contain the aryldiamine ligand [C6H3(CH2-NMe2)(2)-2,6](-) (NCN) are described. These complexes are [RuCl{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,6}(eta(6)-C10H14)] (2; C10H14 = p-cymene = C6H4Me-Pr-i-4), [Ru{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,6}(eta(5)-C5H5)(PPh3)] (5), and their isomeric forms [RuCl{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,4}(eta(6)-C10H14)] (3) and [Ru{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,4}(eta(5)-C5H5)(PPh3)] (6), respectively. Complex 2 has been prepared from the reaction of [Li(NCN)](2) with [RuCl2(eta(6)-C10H14)](2), whereas complex 5 has been prepared by the treatment of [RuCl{eta(3)-N,C,N-C6H3(CH2NMe2)(2)-2,6}(PPh3)] (4) with [Na(C5H5)](n). Both 2 and 5 are formally 18-electron ruthenium(II) complexes in which the monoanionic potentially tridentate coordinating ligand NCN is eta(2)-C,N-bonded, In solution (halocarbon solvent at room temperature or in aromatic solvents at elevated temperature), the intramolecular rearrangements of 2 and 5 afford complexes 3 and 6, respectively. This is a result of a shift of the metal-C-aryl bond from position-1 to position-3 on the aromatic ring of the NCN ligand. The mechanism of the isomerization is proposed to involve a sequence of intramolecular oxidative addition and reductive elimination reactions of both aromatic and aliphatic C-H bonds. This is based on results from deuterium labeling, spectroscopic studies, and some kinetic experiments. The mechanism is proposed to contain fully reversible steps in the case of 5, but a nonreversible step involving oxidative addition of a methyl NCH2-H bond in the case of 2. The solid-state structures of complexes 2, 3, 5, and 6 have been determined by single-crystal X-ray diffraction. A new dinuclear 1,4-phenylene-bridged bisruthenium(II) complex, [1,4-{RuCl(eta(6)-C10H14)}(2){C-6(CH2NMe2)(4)-2,3,5,6-C,N,C',N'}] (9) has also been prepared from the dianionic ligand [C-6(CH2NMe2)(4)-2,3,5,6](2-) (C2N4). The C2N4 ligand is in an eta(2)-C,N-eta(2)-C',N'-bis(bidentate) bonding mode. Compound 9 does not isomerize in solution (halocarbon solvent), presumably because of the absence of an accessible C-aryl-H bond. Complex 9 could not be isolated in an analytically pure form, probably because of its high sensitivity to air and very low solubility, which precludes recrystallization.

Isolation of Rhodococcus rhodochrous NCIMB13064 derivatives with new biodegradative abilities

Rhodococcus rhodochrous NCIMB13064 can dehalogenate and utilise a number of halogenated aliphatic compounds as sole carbon and energy source. Mutants of NCIMB13064 can be easily isolated with an enlarged range of 1-chloroalkane utilising ability. Dehalogenation of 1-chlorononane, 1-chlorodecane and short-chain 1-chloroalkanes (C-3-C-8) is encoded by the same plasmid pRTL1. However, a different genetic element(s) is required for the dehalogenation of 3-chloropropionic acid. Two derivatives (P200 and P400) of R. rhodochrous NCIMB13064 were isolated which had acquired the ability to utilise naphthalene as sole carbon and energy source. Both strains lost the ability to utilise short-chain 1-chloroalkanes and underwent some rearrangements associated with pRTL1 plasmid.

Bystander responses induced by low LET radiation

Radiation-induced bystander responses are observed when cells respond to their neighbours being irradiated. Considerable evidence is now available regarding the importance of these responses in cell and tissue models. Most studies have utilized two approaches where either a media-transferable factor has been assessed or cells have been exposed to low fluences of charged particles, where only a few percent are exposed. The development of microbeams has allowed nontargeted responses such as bystander effects to be more carefully analysed. As well as charged particle microbeams, X-ray microprobes have been developed, and several groups are also developing electron microbeams. Using the Gray Cancer Institute soft X-ray microprobe, it has been possible to follow the response of individual cells to targeted low doses of carbon-characteristic soft X-rays. Studies in human fibroblasts have shown evidence of a significant radiation quality-dependent bystander effect, measured as chromosomal damage in the form of micronuclei which is radiation quality dependent. Other studies show that even under conditions when only a single cell is targeted with soft X-rays, significant bystander-mediated cell killing is observed. The observation of bystander responses with low LET radiation suggests that these may be important in understanding radiation risk from background levels of radiation, where cells observe only single electron track traversals. Also, the indirect evidence for these responses in vivo indicates that they may have a role to play in current radiotherapy approaches and future novel strategies involving modulating nontargeted responses.

Genomic instability in Chinese hamster cells after exposure to X rays or alpha particles of different mean linear energy transfer

Evidence has accumulated that radiation induces a transmissible persistent destabilization of the genome, which mag. result in effects arising in the progeny of irradiated but surviving cells. An enhanced death rate among the progeny of cells surviving irradiation persists for many generations in the form of a reduced plating efficiency. Such delayed reproductive death is correlated with an increased occurrence of micronuclei. Since it has been suggested that radiation-induced chromosomal instability might depend on the radiation quality, we investigated the effects of alpha particles of different LET by looking at the frequency of delayed micronuclei in Chinese hamster V79 cells after cytochalasin-induced block of cell division, A dose-dependent increase in the frequency of micronuclei was found in cells assayed 1 week postirradiation or later. Also, there was a persistent increase in the frequency of dicentrics in surviving irradiated cells, Moreover, we found an increased micronucleus frequency in all of the 30 clones isolated from individual cells which had been irradiated with doses equivalent to either one, two or three alpha-particle traversals per cell nucleus, We conclude that the target for genomic instability in Chinese hamster cells must be larger than the cell nucleus. (C) 1997 by Radiation Research Society

A review of studies of ionizing radiation-induced double-strand break clustering

Underpinning current models of the mechanisms of the action of radiation is a central role for DNA damage and in particular double-strand breaks (DSBs). For radiations of different LET, there is a need to know the exact yields and distributions of DSBs in human cells. Most measurements of DSB yields within cells now rely on pulsed-field gel electrophoresis as the technique of choice. Previous measurements of DSB yields have suggested that the yields are remarkably similar for different types of radiation with RBE values less than or equal to1.0. More recent studies in mammalian cells, however, have suggested that both the yield and the spatial distribution of DSBs are influenced by radiation quality. RBE values for DSBs induced by high-LET radiations are greater than 1.0, and the distributions are nonrandom. Underlying this is the interaction of particle tracks with the higher-order chromosomal structures within cell nuclei. Further studies are needed to relate nonrandom distributions of DSBs to their rejoining kinetics. At the molecular level, we need to determine the involvement of clustering of damaged bases with strand breakage, and the relationship between higher-order clustering over sizes of kilobase pairs and above to localized clustering at the DNA level. Overall, these studies will allow us to elucidate whether the nonrandom distributions of breaks produced by high-LET particle tracks have any consequences for their repair and biological effectiveness. (C) 2001 by Radiation Research Society.

Calcium fluxes modulate the radiation-induced bystander responses in targeted glioma and fibroblast cells

Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. This study investigated the role of changes in calcium levels in bystander responses leading to chromosomal damage in nonirradiated T98G glioma cells and AG01522 fibroblasts that had been either exposed to conditioned medium from irradiated cells or co-cultured with a population where a fraction of cells were individually targeted through the nucleus or cytoplasm with a precise number of microbeam helium-3 particles. After the recipient cells were treated with conditioned medium from T98G or AG01522 cells that had been irradiated through either nucleus or cytoplasm, rapid calcium fluxes were monitored in the nonirradiated recipient cells. Their characteristics were dependent on the source of the conditioned medium but had no dependence on radiation dose. When recipient cells were co-cultured with an irradiated population of either T98G or AG01522 cells, micronuclei were induced in the nonirradiated cells, but this response was eliminated by treating the cells with calcicludine (CaC), a potent blocker of Ca2+ channels. Moreover, both the calcium fluxes and the bystander effect were inhibited when the irradiated T98G cells were treated with aminoguanidine, an inhibitor of nitric oxide synthase (NOS), and when the irradiated AG01522 cells were treated with DMSO, a scavenger of reactive oxygen species (ROS), which indicates that NO and ROS were involved in the bystander responses generated from irradiated T98G and AG01522 cells, respectively. Our findings indicate that calcium signaling may be an early response in radiation-induced bystander effects leading to chromosome damage. (c) 2006 by Radiation Research Society.

Characterization of IS2112, a new insertion sequence from Rhodococcus, and its relationship with mobile elements belonging to the IS110 family

A new insertion sequence (IS2112) was identified in the genome of the 1-haloalkane-utilizing bacterium Rhodococcus rhodochrous NCIMB 13064. The insertion element is 1415 bp long, does not contain terminal inverted repeats, and is not flanked by directly repeated sequences. IS2112 belongs to the IS110 family of transposable elements, and forms a separate subfamily, along with IS116, Two copies of IS2112 were found in R, rhodochrous NCIMB 13064 and one, two or three copies of a similar sequence were detected in five other 1-haloalkane-degrading Rhodococcus strains. There were no sequences homologous to IS2112 found in the l-haloalkane-degrading 'Pseudomonas pavonaceae' 170 and Rhodococcus sp, HA1 or in several Rhodococcus strains which do not utilize haloalkanes, IS2112 was originally found in plasmid pRTL1 of R. rhodochrous NCIMB 13064 which harbours genes encoding utilization of l-haloalkanes, and was located 5 kbp upstream of the haloalkane dehalogenase gene (dhaA), Although the second copy of IS2112 in strain NCIMB 13064 was also present on the pRTL1 plasmid, these sequences do not apparently comprise a single composite transposon encoding haloalkane utilization. An analysis of derivatives of NCIMB 13064 revealed that IS2112 was involved in genome rearrangements. IS2112 appeared to change its location as a result of transposition and as a result of other rearrangements of the NCIMB 13064 genome.

Applied aspects of Rhodococcus genetics

Eubacteria of the genus Rhodococcus are a diverse group of microorganisms commonly found in many environmental niches from soils to seawaters and as plant and animal pathogens. They exhibit a remarkable ability to degrade many organic compounds and their economic importance is becoming increasingly apparent. Although their genetic organisation is still far from understood, there have been many advances in recent years. Reviewed here is the current knowledge of rhodococci relating to gene transfer, recombination, plasmid replication and functions, cloning vectors and reporter genes, gene expression and its control, bacteriophages, insertion sequences and genomic rearrangements. Further fundamental studies of Rhodococcus genetics and the application of genetic techniques to the these bacteria will be needed for their continued biotechnological exploitation.

A novel sensor kinase-response regulator hybrid controls biofilm formation and type VI secretion system activity in Burkholderia cenocepacia

Burkholderia cenocepacia is an important opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis (CF). Adaptation of B. cenocepacia to the CF airways may play an important role in the persistence of the infection. We have identified a sensor kinase-response regulator (BCAM0379) named AtsR in B. cenocepacia K56-2 that shares 19% amino acid identity with RetS from Pseudomonas aeruginosa. atsR inactivation led to increased biofilm production and a hyperadherent phenotype in both abiotic surfaces and lung epithelial cells. Also, the atsR mutant overexpressed and hypersecreted an Hcp-like protein known to be specifically secreted by the type VI secretion system (T6SS) in other gram-negative bacteria. Amoeba plaque assays demonstrated that the atsR mutant was more resistant to Dictyostelium predation than the wild-type strain and that this phenomenon was T6SS dependent. Macrophage infection assays also demonstrated that the atsR mutant induces the formation of actin-mediated protrusions from macrophages that require a functional Hcp-like protein, suggesting that the T6SS is involved in actin rearrangements. Three B. cenocepacia transposon mutants that were found in a previous study to be impaired for survival in chronic lung infection model were mapped to the T6SS gene cluster, indicating that the T6SS is required for infection in vivo. Together, our data show that AtsR is involved in the regulation of genes required for virulence in B. cenocepacia K56-2, including genes encoding a T6SS.

Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase

The lpcA locus has been identified in Escherichia coli K12 novobiocin-supersensitive mutants that produce a short lipopolysaccharide (LPS) core which lacks glyceromannoheptose and terminal hexoses. We have characterized lpcA as a single gene mapping around 5.3 min (246 kilobases) on the E. coli K12 chromosome and encoding a 22.6-kDa cytosolic protein. Recombinant plasmids containing only lpcA restored a complete core LPS in the E. coli strain chi711. We show that this strain has an IS5-mediated chromosomal deletion of 35 kilobases that eliminates lpcA. The LpcA protein showed discrete similarities with a family of aldose/ketose isomerases and other proteins of unknown function. The isomerization of sedoheptulose 7-phosphate, into a phosphosugar presumed to be D-glycero-D-mannoheptose 7-phosphate, was detected in enzyme reactions with cell extracts of E. coli lpcA+ and of lpcA mutants containing the recombinant lpcA gene. We concluded that LpcA is the phosphoheptose isomerase used in the first step of glyceromannoheptose synthesis. We also demonstrated that lpcA is conserved among enteric bacteria, all of which contain glyceromannoheptose in the inner core LPS, indicating that LpcA is an essential component in a conserved biosynthetic pathway of inner core LPS.

Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110:identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a

We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity.