Synthesis of gatifloxacin derivatives and their biological activities against Mycobacterium leprae and Mycobacterium tuberculosis

Novel 3′-piperazinyl derivatives of the 8-hydrogeno and 8-methoxy-6-fluoro-1-cyclopropyl-4-quinolone-3-carboxylic acid scaffolds were designed, synthesized and characterized by ¹H, ¹³C and ¹⁹F NMR, and HRMS. The activity of these derivatives against pathogenic mycobacteria (M. leprae and M. tuberculosis), wild-type (WT) strains or strains harboring mutations implicated in quinolone resistance, were determined by measuring drug concentrations inhibiting cell growth (MIC) and/or DNA supercoiling by DNA gyrase (IC₅₀), or inducing 25% DNA cleavage by DNA gyrase (CC₂₅). Compound 4 (with a methoxy in R₈ and a secondary carbamate in R₃′) and compound 5 (with a hydrogen in R₈ and an ethyl ester in R₃′) displayed biological activities close to those of ofloxacin but inferior to those of gatifloxacin and moxifloxacin against M. tuberculosis and M. leprae WT DNA gyrases, whereas all of the compounds were less active in inhibiting M. tuberculosis growth and M. leprae mutant DNA gyrases. Since R₃′ substitutions have been poorly investigated previously, our results may help to design new quinolone derivatives in the future.     

Superacid synthesis of halogen containing N-substituted-4-aminobenzene sulfonamides: New selective tumor-associated carbonic anhydrase inhibitors

A series of new, halogen containing N-substituted 4-aminobenzenesulfonamides were synthesized by using superacid HF/SbF₅ chemistry and investigated as inhibitors of several human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, that is, the cytosolic hCA I and II and, the tumor-associated transmembrane isoforms hCA IX and XII. Despite the substitution of the sulfonamide function, the presence of fluorine atom(s) in β position of the sulfonamide function strongly favors hCA inhibition. A similar effect of the β-fluorinated alkyl substitution on the amino function has been also observed. Among the tested compounds, several chlorinated derivatives have been identified as selective nanomolar, tumor-associated isoforms inhibitors. These non-primary sulfonamides probably bind in the coumarin-binding site, at the entrance of the cavity, and not to the metal ion as the primary sulfonamide inhibitors.     

Genome-wide interval mapping using SNPs identifies new QTL for growth,body composition and several physiological variables in an F2 intercross between fat and lean chicken lines

Background

For decades, genetic improvement based on measuring growth and body composition traits has been successfully applied in the production of meat-type chickens. However, this conventional approach is hindered by antagonistic genetic correlations between some traits and the high cost of measuring body composition traits. Marker-assisted selection should overcome these problems by selecting loci that have effects on either one trait only or on more than one trait but with a favorable genetic correlation. In the present study, identification of such loci was done by genotyping an F₂ intercross between fat and lean lines divergently selected for abdominal fatness genotyped with a medium-density genetic map (120 microsatellites and 1302 single nucleotide polymorphisms). Genome scan linkage analyses were performed for growth (body weight at 1, 3, 5, and 7 weeks, and shank length and diameter at 9 weeks), body composition at 9 weeks (abdominal fat weight and percentage, breast muscle weight and percentage, and thigh weight and percentage), and for several physiological measurements at 7 weeks in the fasting state, i.e. body temperature and plasma levels of IGF-I, NEFA and glucose. Interval mapping analyses were performed with the QTLMap software, including single-trait analyses with single and multiple QTL on the same chromosome.

Results

Sixty-seven QTL were detected, most of which had never been described before. Of these 67 QTL, 47 were detected by single-QTL analyses and 20 by multiple-QTL analyses, which underlines the importance of using different statistical models. Close analysis of the genes located in the defined intervals identified several relevant functional candidates, such as ACACA for abdominal fatness, GHSR and GAS1 for breast muscle weight, DCRX and ASPSCR1 for plasma glucose content, and ChEBP for shank diameter.

Conclusions

The medium-density genetic map enabled us to genotype new regions of the chicken genome (including micro-chromosomes) that influenced the traits investigated. With this marker density, confidence intervals were sufficiently small (14 cM on average) to search for candidate genes. Altogether, this new information provides a valuable starting point for the identification of causative genes responsible for important QTL controlling growth, body composition and metabolic traits in the broiler chicken.     

Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins

The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D₂O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d₁₀. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.     

Barth syndrome: Cellular compensation of mitochondrial dysfunction and apoptosis inhibition due to changes in cardiolipin remodeling linked to tafazzin (TAZ) gene mutation

Cardiolipin is a mitochondrion-specific phospholipid that stabilizes the assembly of respiratory chain complexes, favoring full-yield operation. It also mediates key steps in apoptosis. In Barth syndrome, an X chromosome-linked cardiomyopathy caused by tafazzin mutations, cardiolipins display acyl chain modifications and are present at abnormally low concentrations, whereas monolysocardiolipin accumulates. Using immortalized lymphoblasts from Barth syndrome patients, we showed that the production of abnormal cardiolipin led to mitochondrial alterations. Indeed, the lack of normal cardiolipin led to changes in electron transport chain stability, resulting in cellular defects. We found a destabilization of the supercomplex (respirasome) I + III₂ + IV_n but also decreased amounts of individual complexes I and IV and supercomplexes I + III and III + IV. No changes were observed in the amounts of individual complex III and complex II. We also found decreased levels of complex V. This complex is not part of the supercomplex suggesting that cardiolipin is required not only for the association/stabilization of the complexes into supercomplexes but also for the modulation of the amount of individual respiratory chain complexes. However, these alterations were compensated by an increase in mitochondrial mass, as demonstrated by electron microscopy and measurements of citrate synthase activity. We suggest that this compensatory increase in mitochondrial content prevents a decrease in mitochondrial respiration and ATP synthesis in the cells. We also show, by extensive flow cytometry analysis, that the type II apoptosis pathway was blocked at the mitochondrial level and that the mitochondria of patients with Barth syndrome cannot bind active caspase-8. Signal transduction is thus blocked before any mitochondrial event can occur. Remarkably, basal levels of superoxide anion production were slightly higher in patients' cells than in control cells as previously evidenced via an increased protein carbonylation in the taz1Δ mutant in the yeast. This may be deleterious to cells in the long term. The consequences of mitochondrial dysfunction and alterations to apoptosis signal transduction are considered in light of the potential for the development of future treatments.     

One‐year experiment on the physiological response of the Mediterranean crustose coralline alga,Lithophyllum cabiochae,to elevated pCO2 and temperature

The response of respiration, photosynthesis, and calcification to elevated pCO₂ and temperature was investigated in isolation and in combination in the Mediterranean crustose coralline alga Lithophyllum cabiochae. Algae were maintained in aquaria during 1 year at near‐ambient conditions of irradiance, at ambient or elevated temperature (+3°C), and at ambient (ca. 400 μatm) or elevated pCO₂ (ca. 700 μatm). Respiration, photosynthesis, and net calcification showed a strong seasonal pattern following the seasonal variations of temperature and irradiance, with higher rates in summer than in winter. Respiration was unaffected by pCO₂ but showed a general trend of increase at elevated temperature at all seasons, except in summer under elevated pCO₂. Conversely, photosynthesis was strongly affected by pCO₂ with a decline under elevated pCO₂ in summer, autumn, and winter. In particular, photosynthetic efficiency was reduced under elevated pCO₂. Net calcification showed different responses depending on the season. In summer, net calcification increased with rising temperature under ambient pCO₂ but decreased with rising temperature under elevated pCO₂. Surprisingly, the highest rates in summer were found under elevated pCO₂ and ambient temperature. In autumn, winter, and spring, net calcification exhibited a positive or no response at elevated temperature but was unaffected by pCO₂. The rate of calcification of L. cabiochae was thus maintained or even enhanced under increased pCO₂. However, there is likely a trade‐off with other physiological processes. For example, photosynthesis declines in response to increased pCO₂ under ambient irradiance. The present study reports only on the physiological response of healthy specimens to ocean warming and acidification, however, these environmental changes may affect the vulnerability of coralline algae to other stresses such as pathogens and necroses that can cause major dissolution, which would have critical consequence for the sustainability of coralligenous habitats and the budgets of carbon and calcium carbonate in coastal Mediterranean ecosystems.     

A Systematic Approach for the Genetic Dissection of Protein Complexes in Living Cells

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Global marine bacterial diversity peaks at high latitudes in winter

Genomic approaches to characterizing bacterial communities are revealing significant differences in diversity and composition between environments. But bacterial distributions have not been mapped at a global scale. Although current community surveys are way too sparse to map global diversity patterns directly, there is now sufficient data to fit accurate models of how bacterial distributions vary across different environments and to make global scale maps from these models. We apply this approach to map the global distributions of bacteria in marine surface waters. Our spatially and temporally explicit predictions suggest that bacterial diversity peaks in temperate latitudes across the world''s oceans. These global peaks are seasonal, occurring 6 months apart in the two hemispheres, in the boreal and austral winters. This pattern is quite different from the tropical, seasonally consistent diversity patterns observed for most macroorganisms. However, like other marine organisms, surface water bacteria are particularly diverse in regions of high human environmental impacts on the oceans. Our maps provide the first picture of bacterial distributions at a global scale and suggest important differences between the diversity patterns of bacteria compared with other organisms.     

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 August 2012 – 30 September 2012

This article documents the addition of 83 microsatellite marker loci and 96 pairs of single‐nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Bembidion lampros, Inimicus japonicus, Lymnaea stagnalis, Panopea abbreviata, Pentadesma butyracea, Sycoscapter hirticola and Thanatephorus cucumeris (anamorph: Rhizoctonia solani). These loci were cross‐tested on the following species: Pentadesma grandifolia and Pentadesma reyndersii. This article also documents the addition of 96 sequencing primer pairs and 88 allele‐specific primers or probes for Plutella xylostella.