Computational mapping reveals dramatic effect of Hoogsteen breathing on duplex DNA reactivity with formaldehyde

Formaldehyde has long been recognized as a hazardous environmental agent highly reactive with DNA. Recently, it has been realized that due to the activity of histone demethylation enzymes within the cell nucleus, formaldehyde is produced endogenously, in direct vicinity of genomic DNA. Should it lead to extensive DNA damage? We address this question with the aid of a computational mapping method, analogous to X-ray and nuclear magnetic resonance techniques for observing weakly specific interactions of small organic compounds with a macromolecule in order to establish important functional sites. We concentrate on the leading reaction of formaldehyde with free bases: hydroxymethylation of cytosine amino groups. Our results show that in B-DNA, cytosine amino groups are totally inaccessible for the formaldehyde attack. Then, we explore the effect of recently discovered transient flipping of Watson–Crick (WC) pairs into Hoogsteen (HG) pairs (HG breathing). Our results show that the HG base pair formation dramatically affects the accessibility for formaldehyde of cytosine amino nitrogens within WC base pairs adjacent to HG base pairs. The extensive literature on DNA interaction with formaldehyde is analyzed in light of the new findings. The obtained data emphasize the significance of DNA HG breathing.     

Higher muscle performance in adolescents compared with adults after a resistance training session with different rest intervals

The aim of the present study was to compare the effect of 3 different rest intervals between sets on the total training volume, number of repetitions, ratings of perceived exertion (RPE), and resistance to fatigue in adolescents and adults during a resistance training session in the isoinertial chest press exercise. Fifteen male adolescents (15.2 ± 1.2 years; 20.7 ± 2.0 kg·m(-2); Tanner -4; 61.5 ± 8.9, 10 repetition maximum [RM]) and 15 adults (22.2 ± 2.7 years; 23.3 ± 2.0 kg·m(-2); Tanner -5; 84.3 ± 13.5, 10RM) without previous experience with resistance training participated in the study. After 10RM test-retest on 3 different occasions, participants were randomly assigned to a resistance training protocol with 30-, 60-, and 120-second rest interval between sets. The protocol consisted of 3 sets with 10RM. In all studied variables, with exception to total training volume and RPE, adolescents presented superior results as compared with adults (p < 0.001). On the other hand, both adults and adolescents exhibited a higher resistance to fatigue, total training volume, and number of repetitions with a longer rest interval (120 > 60 > 30 seconds) (p < 0.01). Thus, these results indicate that adolescents present a higher recovery capacity between sets in a resistance training session than adults and a longer rest interval results in a higher number of repetitions completed, total training volume, and resistance to fatigue.     

Exonic polymorphism vs intronic simple repeat hypervariability in MHC-DRB genes

Gene products encoded by the major histocompatibility complex often exhibit a high degree of polymorphism. In humans the HLA-DR polymorphism is due to more than 50 alleles with varying exon 2 sequences. Each group of DRB alleles contains a certain form of the basic simple repeat motif (gt)_n(ga)_m in intron 2. Identical alleles can be differentiated on the basis of the hypervariable repeat. In this study focused on cattle (Bos taurus) we identified different Bota-DRB alleles in a limited survey by amplification via polymerase chain reaction and sequencing. In addition DRB exon 2 sequences were also obtained from eight additional hoofed animal species (seven horned artiodactyls and one pig) revealing artiodactyl-specific polymorphic and nonpolymorphic substitutions. In the genus Bos the intronic simple repeat variability was compared with exonic DRB polymorphism. As in humans all Bota-DRB exons were always associated with specifically organized basic simple repeat structures. Yet the extent of simple repeat variability was lower in cattle compared to humans. Selective breeding in the process of domestication might be responsible for the diminished intronic hypervariability. Nevertheless, the hypermutable simple repeat sequences have been preserved in the same position and with the same principal structure for at least 70 × 10⁶ years of evolution. Unexpectedly, the rate of intronic simple repeat and exonic changes appear quite similar.     

Computational solvent mapping reveals the importance of local conformational changes for broad substrate specificity in mammalian cytochromes P450

Computational solvent mapping moves small organic molecules as probes around a protein surface, finds favorable binding positions, clusters the conformations, and ranks the clusters on the basis of their average free energy. Prior mapping studies of enzymes, crystallized in either substrate-free or substrate-bound form, have shown that the largest number of solvent probe clusters invariably overlaps in the active site. We have applied this method to five cytochromes P450. As expected, the mapping of two bacterial P450s, P450 cam (CYP101) and P450 BM-3 (CYP102), identified the substrate-binding sites in both ligand-bound and ligand-free P450 structures. However, the mapping finds the active site only in the ligand-bound structures of the three mammalian P450s, 2C5, 2C9, and 2B4. Thus, despite the large cavities seen in the unbound structures of these enzymes, the features required for binding small molecules are formed only in the process of substrate binding. The ability of adjusting their binding sites to substrates that differ in size, shape, and polarity is likely to be responsible for the broad substrate specificity of these mammalian P450s. Similar behavior was seen at "hot spots" of protein-protein interfaces that can also bind small molecules in grooves created by induced fit. In addition, the binding of S-warfarin to P450 2C9 creates a high-affinity site for a second ligand, which may help to explain the prevalence of drug-drug interactions involving this and other mammalian P450s.     

Genome sequence of Fusobacterium nucleatum subspecies polymorphum - a genetically tractable fusobacterium

Fusobacterium nucleatum is a prominent member of the oral microbiota and is a common cause of human infection. F. nucleatum includes five subspecies: polymorphum, nucleatum, vincentii, fusiforme, and animalis. F. nucleatum subsp. polymorphum ATCC 10953 has been well characterized phenotypically and, in contrast to previously sequenced strains, is amenable to gene transfer. We sequenced and annotated the 2,429,698 bp genome of F. nucleatum subsp. polymorphum ATCC 10953. Plasmid pFN3 from the strain was also sequenced and analyzed. When compared to the other two available fusobacterial genomes (F. nucleatum subsp. nucleatum, and F. nucleatum subsp. vincentii) 627 open reading frames unique to F. nucleatum subsp. polymorphum ATCC 10953 were identified. A large percentage of these mapped within one of 28 regions or islands containing five or more genes. Seventeen percent of the clustered proteins that demonstrated similarity were most similar to proteins from the clostridia, with others being most similar to proteins from other gram-positive organisms such as Bacillus and Streptococcus. A ten kilobase region homologous to the Salmonella typhimurium propanediol utilization locus was identified, as was a prophage and integrated conjugal plasmid. The genome contains five composite ribozyme/transposons, similar to the CdISt IStrons described in Clostridium difficile. IStrons are not present in the other fusobacterial genomes. These findings indicate that F. nucleatum subsp. polymorphum is proficient at horizontal gene transfer and that exchange with the Firmicutes, particularly the Clostridia, is common.     

Enhanced ryanodine receptor-mediated calcium leak determines reduced sarcoplasmic reticulum calcium content in chronic canine heart failure

In this study, we investigated the role of elevated sarcoplasmic reticulum (SR) Ca²⁺ leak through ryanodine receptors (RyR2s) in heart failure (HF)-related abnormalities of intracellular Ca²⁺ handling, using a canine model of chronic HF. The cytosolic Ca²⁺ transients were reduced in amplitude and slowed in duration in HF myocytes compared with control, changes paralleled by a dramatic reduction in the total SR Ca²⁺ content. Direct measurements of [Ca²⁺]_SR in both intact and permeabilized cardiac myocytes demonstrated that SR luminal [Ca²⁺] is markedly lowered in HF, suggesting that alterations in Ca²⁺ transport rather than fractional SR volume reduction accounts for the diminished Ca²⁺ release capacity of SR in HF. SR Ca²⁺ ATPase (SERCA2)-mediated SR Ca²⁺ uptake rate was not significantly altered, and Na⁺/Ca²⁺ exchange activity was accelerated in HF myocytes. At the same time, SR Ca²⁺ leak, measured directly as a loss of [Ca²⁺]_SR after inhibition of SERCA2 by thapsigargin, was markedly enhanced in HF myocytes. Moreover, the reduced [Ca²⁺]_SR in HF myocytes could be nearly completely restored by the RyR2 channel blocker ruthenium red. The effects of HF on cytosolic and SR luminal Ca²⁺ signals could be reasonably well mimicked by the RyR2 channel agonist caffeine. Taken together, these results suggest that RyR2-mediated SR Ca²⁺ leak is a major factor in the abnormal intracellular Ca²⁺ handling that critically contributes to the reduced SR Ca²⁺ content of failing cardiomyocytes.     

Equine leukocyte antigens: Relationships with sarcoid tumors and laminitis in two pure breeds

Frequencies of equine leukocyte antigen distribution were determined by complement-mediated cytotoxicity testing among populations of Thoroughbred and Standardbred horses, including animals affected with equine sarcoid and laminitis. A highly significant association is described between the presence or history of sarcoid lesions in Thoroughbreds and the expression of the major histocompatibility complex (MHC)-encoded antigens, W3 and B1. No association was found between antigenic expression frequencies and laminitis in either breed. These findings suggest that a strong relationship exists between the equine MHC and a predisposition to sarcoid.     

Accelerating and focusing protein-protein docking correlations using multi-dimensional rotational FFT generating functions

MOTIVATION: Predicting how proteins interact at the molecular level is a computationally intensive task. Many protein docking algorithms begin by using fast Fourier transform (FFT) correlation techniques to find putative rigid body docking orientations. Most such approaches use 3D Cartesian grids and are therefore limited to computing three dimensional (3D) translational correlations. However, translational FFTs can speed up the calculation in only three of the six rigid body degrees of freedom, and they cannot easily incorporate prior knowledge about a complex to focus and hence further accelerate the calculation. Furthemore, several groups have developed multi-term interaction potentials and others use multi-copy approaches to simulate protein flexibility, which both add to the computational cost of FFT-based docking algorithms. Hence there is a need to develop more powerful and more versatile FFT docking techniques. RESULTS: This article presents a closed-form 6D spherical polar Fourier correlation expression from which arbitrary multi-dimensional multi-property multi-resolution FFT correlations may be generated. The approach is demonstrated by calculating 1D, 3D and 5D rotational correlations of 3D shape and electrostatic expansions up to polynomial order L=30 on a 2 GB personal computer. As expected, 3D correlations are found to be considerably faster than 1D correlations but, surprisingly, 5D correlations are often slower than 3D correlations. Nonetheless, we show that 5D correlations will be advantageous when calculating multi-term knowledge-based interaction potentials. When docking the 84 complexes of the Protein Docking Benchmark, blind 3D shape plus electrostatic correlations take around 30 minutes on a contemporary personal computer and find acceptable solutions within the top 20 in 16 cases. Applying a simple angular constraint to focus the calculation around the receptor binding site produces acceptable solutions within the top 20 in 28 cases. Further constraining the search to the ligand binding site gives up to 48 solutions within the top 20, with calculation times of just a few minutes per complex. Hence the approach described provides a practical and fast tool for rigid body protein-protein docking, especially when prior knowledge about one or both binding sites is available.