α-Substituted norstatines as the transition-state mimic in inhibitors of multiple digestive vacuole malaria aspartic proteases

The impact of moving the P1 side-chain from the β-position to the α-position in norstatine-containing plasmepsin inhibitors was investigated, generating two new classes of tertiary alcohol-comprising α-benzylnorstatines and α-phenylnorstatines. Twelve α-substituted norstatines were designed, synthesized and evaluated for their inhibitory potencies against plasmepsin II and the plasmepsin IV orthologues (PM4) present in the digestive vacuole of all four Plasmodium species causing malaria in man. New synthetic routes were developed for producing the desired α-substituted norstatines as pure stereoisomers. The best compounds provided K_i values in the nanomolar range for all PM4, with a best value of 110 nM in PM4 from Plasmodium ovale. In addition, excellent selectivity over the closely related human aspartic protease Cathepsin D was achieved. The loss of affinity to Plasmodium falciparum PM4, which was experienced upon the move of the P1 substituent, was rationalized by the calculation of inhibitor–protein binding affinities using the linear interaction energy method (LIE).     

Humidity levels drive reproductive modes and phylogenetic diversity of amphibians in the Brazilian Atlantic Forest

Aim The diversity of reproductive modes among amphibians constitutes a striking example of how differences in the biology of species provide important explanations for species distribution patterns on a broad scale. We hypothesize that sites with a higher humidity level will support more modes of reproduction than drier sites and will consequently exhibit a higher phylogenetic diversity. Furthermore, if there is a gradient in the tolerance of reproductive modes to desiccation, there will be a nested pattern in the composition of reproductive modes among sites. Location Twenty‐seven forest sites in the Brazilian Atlantic Forest. Methods Through a path analysis approach, we evaluated the direct and indirect effects of the humidity level on the number of reproductive modes, as well as the relative importance of both variables on amphibian phylogenetic diversity. A nestedness analysis was used to quantify the extent to which the compositions of both species and reproductive modes in drier sites correspond to subsets of those in sites with higher annual precipitation. Results We found that the reproductive modes present in drier sites are non‐random subsets of those present in sites with higher humidity levels. Because reproductive modes are phylogenetically conserved among amphibians, sites with a greater number of reproductive modes supported greater phylogenetic diversity. Sites with high precipitation throughout the year provided suitable environmental conditions for a larger number of reproductive modes, whereas sites with low precipitation and typical seasonal climates supported only those reproductive modes specialized to resist desiccation. Main conclusions Our results show that humidity‐related variables are key environmental factors related to both the richness of reproductive modes and phylogenetic diversity. Our results support the hypothesis that the higher phylogenetic diversity found in moister sites reflects differences in the tolerance to desiccation among different reproductive modes. Given that reproductive modes are associated with susceptibility to desiccation, their incorporation into explanatory models may trigger a significant advance in the understanding of the mechanisms regulating the species richness and composition of amphibian communities.     

Vernakalant activates human cardiac K2P17.1 background K channels

Atrial fibrillation (AF) contributes significantly to cardiovascular morbidity and mortality. The growing epidemic is associated with cardiac repolarization abnormalities and requires the development of more effective antiarrhythmic strategies. Two-pore-domain K⁺ channels stabilize the resting membrane potential and repolarize action potentials. Recently discovered K_2P17.1 channels are expressed in human atrium and represent potential targets for AF therapy. However, cardiac electropharmacology of K_2P17.1 channels remains to be investigated. This study was designed to elucidate human K_2P17.1 regulation by antiarrhythmic drugs.     

Competing with Lower Level Opponents Decreases Intra-Team Movement Synchronization and Time-Motion Demands during Pre-Season Soccer Matches

This study aimed to quantify the time-motion demands and intra-team movement synchronization during the pre-season matches of a professional soccer team according to the opposition level. Positional data from 20 players were captured during the first half of six pre-season matches of a Portuguese first league team. Time-motion demands were measured by the total distance covered and distance covered at different speed categories. Intra-team coordination was measured by calculating the relative phase of all pairs of outfield players. Afterwards, the percentage of time spent in the −30° to 30° bin (near-in-phase mode of coordination) was calculated for each dyad as a measure of space-time movement synchronization. Movement synchronization data were analyzed for the whole team, according to each dyad average speed and by groups of similar dyadic synchronization tendencies. Then, these data were compared according to the opponent team level (first league; second league; amateurs). Time-motion demands showed no differences in total distance covered per opposition levels, while matches opposing teams of superior level revealed more distance covered at very high intensity. Competing against superior level teams implied more time in synchronized behavior for the overall displacements and displacements at higher intensities. These findings suggest that playing against higher-level opponents (1^st league teams) increased time-motion demands at high intensities in tandem with intra-team movement synchronization tendencies.     

Characterization of in vivo recombination activities in the mouse embryo

Homologous recombination makes use of sequence homology to repair DNA and to rearrange genetic material. In mammals, these processes have mainly been characterized using cultured cell systems. We have developed an assay that allows us to quantitatively analyze homologous recombination in vivo in the mouse embryo. Transgenic mouse lines were generated by microinjection into a fertilized mouse ovum of a vector containing two homologous LINE-1 (L1) sequences arranged as a direct repeat: these sequences can recombine with each other and with endogenous L1 sequences before, during or after integration of the vector into the genome. Using a plasmid rescue procedure, we determined the composition of the integrated vector array in several transgenic mice and their descendants. Homologous recombination frequencies were found to be strikingly high, involving 70% of integrated vectors in some arrays, with homologous deletions being five times more frequent than gene conversion without crossing-over. Interestingly, non-homologous recombination was found to be much less frequent. We also found that endogenous L1 sequences could be involved in homologous recombination events in the mouse embryo, and that the integrated arrays could be modified from generation to generation by homologous recombination between the integrated L1 sequences.     

In<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>, the effect of H<Subscript>2</Subscript>O<Subscript>2</Subscript> on ATP,but not on glyceraldehyde-3-phosphate dehydrogenase,depends on the glucose concentration

As has been previously shown, Saccharomyces cerevisiae grown in 2% or 0.025% glucose uses this carbohydrate by the fermentative or oxidative pathways, respectively. Depending on the glucose concentration in the medium, the effect of the addition of H₂O₂ on the level of ATP and on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity differed. In the presence of 2% glucose, ATP and GAPDH decreased sharply during the first few minutes of treatment, whereas in the presence of 0.025% glucose, GAPDH activity decreased similarly, but the ATP level remained practically unchanged. The addition of 3 mM glutathione to the culture media prevented the depletion of ATP levels and GAPDH activity in the presence of H₂O₂. Catalase and superoxide dismutase activities did not vary significantly when yeast cells were grown either in 2% or in 0.025% glucose.     

Crystal structure of an empty capsid of turnip yellow mosaic virus

Empty capsids (artificial top component) of turnip yellow mosaic virus were co-crystallized with an encapsidation initiator RNA hairpin. No clear density was observed for the RNA, but there were clear differences in the conformation of a loop of the coat protein at the opening of the pentameric capsomer (formed by five A-subunits) protruding from the capsid, compared to the corresponding loop in the intact virus. Further differences were found at the N terminus of the A-subunit. These differences have implications for the mechanism of decapsidation of the virus, required for infection.     

Foreignness as a matter of degree: the relative immunogenicity of peptide/MHC ligands

The ability of T lymphocytes (T cells) to recognize and attack foreign invaders while leaving healthy cells unharmed is often analysed as a discrete self/non-self dichotomy, with each peptide/MHC ligand classified as either self or non-self. We argue that the ligand immunogenicity is more naturally treated as a continuous quantity, and show how to define and quantitate relative ligand immunogenicity. In our theory, self-tolerance is acquired through reduction of the relative immunogenicity of autoantigens, whereas xenoantigens, typically not presented during induction of deletional tolerance, retain a high degree of relative immunogenicity. Autoantigens that are not prominently presented in deletional tolerance likewise retain a high relative immunogenicity and remain essentially foreign. According to our analysis, any given autoantigen can attain a high level of relative immunogenicity, provided it is presented at sufficiently high levels. Our theory provides a quantitative tool to analyse the immunogenicity of tumour-associated neoantigens and the ætiology of autoimmune disease.     

Amino acid residues associated with enzymatic activities of the isomerizing phycoviolobilin-lyase PecE/F

PecE and PecF jointly catalyze the covalent attachment of phycocyanobilin to Cys-alpha84 of PecA and its concomitant isomerization to phycoviolobilin. (a) An Eschertchia coli supernatant expressing pecF has a residual activity of 6%; compared to the holoenzyme, this activity is lost upon purification. (b) Functional domains of both subunits from the cyanobacterium Mastigocladus laminosus were evaluated by mutageneses and chemical modification of amino acids. When in PecE the two motifs Y29YAAWWL and D263DLL were deleted, the holoenzyme lost its activity; it is also inactivated upon deletion of a central part (R111 to A122). The three conserved cysteines C48, C91, and C161 have only minor effects on catalysis. When in PecF the 20 C-terminal and 56 N-terminal amino acids were truncated, the lyase-isomerase activity in combination with PecE decreased to 12% and 15%, respectively, compared to the native enzyme. The catalytic efficiency (k(cat)/K(m)) decreased 16-fold when the unique four histidine residues in PecF beginning at H53 were deleted. H121 and C122 of PecF are essential for the enzyme activity; they are part of a unique stretch extending from A104 to N125 which is absent in the beta-subunit of related but nonisomerizing lyases. A single histidine and a single tryptophan are required for activity in both PecE and PecF, as judged from diethyl pyrocarbonate and N-bromosuccinimide modification and statistical analyses. Inactivation of PecE and PecF is also possible by arginine-specific reagents, while modifications of lysine, glutamate, and aspartate retained activity. (c) PecE and PecF, as well as most of the mutants, bind PCB covalently in substoichiometric amounts, as assayed by Zn(2+)-induced fluorescence on denaturing gels.     

The correlation between genomic G+C and optimal growth temperature of prokaryotes is robust: a reply to Marashi and Ghalanbor

We have recently shown that optimal growth temperature (T(opt)) is one of the factors that influence genomic GC in prokaryotes. Our results have been disputed by Marashi and Ghalanbor, who claim that the correlations we show are not "robust" because the elimination of some points (arbitrarily chosen) leads, in some families, to variations in the correlation coefficients and/or significance of correlations. Here, we test whether the correlation between T(opt) and genomic GC is robust by using two independent approaches: detection of possible outliers (using robust Mahalanobis distance) and usage of a non-parametric correlation coefficient that is not sensitive to the presence of outliers. The results presented here reinforce our previous proposal that T(opt) is correlated with genomic GC in prokaryotes.