A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity

We have devised a simple high-throughput screening compatible fluorescence polarization-based assay that can be used to detect the elongation activity of nucleic acid polymerase enzymes. The assay uses a 5' end-labeled template strand and relies on an increase in the polarization signal from the fluorescent label as it is drawn in toward the active site by the action of the enzyme. If the oligonucleotide is sufficiently short, the fluorescence polarization signal can also be used to detect binding prior to elongation activity. We refer to the nucleic acid substrate as a polymerase elongation template element (PETE) and demonstrate the utility of this PETE assay in a microtiter plate format using the RNA-dependent RNA polymerase from poliovirus to extend a self-priming hairpin RNA. The PETE assay provides an efficient method for screening compounds that may inhibit the nucleic acid binding or elongation activities of polymerases.     

Carbonic anhydrase inhibitors. Inhibition of isoforms I, II, IV, VA, VII, IX, and XIV with sulfonamides incorporating fructopyranose-thioureido tails

A series of aromatic/heterocyclic sulfonamides incorporating 2,3:4,5-bis-O-(isopropylidene)-beta-d-fructopyranosyl-thioureido moieties has been synthesized and assayed for the inhibition of seven human isoforms of the zinc enzyme carbonic anhydrase (hCA, EC 4.2.1.1). The new derivatives behaved as weak hCA I inhibitors (K(I)s of 9.4 -13.3microM), were efficient hCA II inhibitors (K(I)s of 6-750nM), and slightly inhibited isoforms hCA IV and hCA VA. Only the sulfanilamide derivative showed efficient and selective inhibition of hCA IV (K(I) of 10nM). These derivatives also showed excellent hCA VII inhibitory activity (K(I)s of 10-79nM), being less efficient as inhibitors of the transmembrane isoforms hCA IX (K(I)s of 10-4500nM) and hCA XIV (K(I)s of 21-3500nM). Two of the new compounds showed anticonvulsant action in a maximal electroshock seizure test in mice, with the fluorosulfanilamide derivative being a more efficient anticonvulsant than the antiepileptic drug topiramate.     

Immunolocalization of Non-Symbiotic Hemoglobins During Somatic Embryogenesis in Chicory

Hemoglobins are ancient O₂-binding proteins, ubiquitously found in eukaryotes. They have been categorized as symbiotic, nonsymbiotic and truncated hemoglobins. We have investigated the cellular localization of nonsymbiotic hemoglobin proteins during somatic embryogenesis in Cichorium hybrid leaves (Cichorium intybus L. var. sativum × C. endivia var. latifolia) using immunolocalization technique. These proteins were detected during the two steps of culture: induction and expression. In leaves, hemoglobins colocalised with plastids, which were dispersed in the parietal cytoplasm as well as in the two guard cells of a stomata, but not in epidermis cells. Upon induction of embryogenesis, in the dark, this pattern disappeared. During the induction phase, where competent cells reinitiate the cell cycle and prepare for mitosis, hemoglobins appeared initially near chloroplasts, and then in the vicinity of vascular vessels especially in the phloem and in cells surrounding the xylem vessels. When leaf fragments were transferred to another medium for the expression phase, hemoglobins were observed in the majority of the leaf blade cells and in small young embryos but not in the older ones. Hemoglobins were also detected in other leaves cells or tissues all along the process. The role of these nonsymbiotic hemoglobins during somatic embryogenesis is discussed.Key Words: chicory, immunolocalization, nonsymbiotic hemoglobin, somatic embryogenesis     

Tuning of the enzyme ratio in a neutral redox convergent cascade: A key approach for an efficient one-pot/two-step biocatalytic whole-cell system

The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer–Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε-caprolactone and 3,4-dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi-enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter-balanced by a higher concentration. Using a preconception-free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1-indanol (7.5 mM) at a 0.5-L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε-caprolactone, for the productivity of 244 mg·L ⁻¹·h ⁻¹) and that for 1-indanol 60% (0.3 g 3,4-dihydrocoumarin, for the productivity of 140 mg·L ⁻¹·h ⁻¹).     

Coxiella burnetii infection in C57BL/6 mice aged 1 or 14 months

The objective of this study was to investigate the effects of age on infection with Coxiella burnetii, the agent of Q fever. Bacterial burden and granuloma number were increased in the spleens of 14-month-old as compared with 1-month-old mice. This increase was not the result of an anti-inflammatory macrophage response, because inflammatory and anti-inflammatory cytokines were induced in macrophages from young mice but were repressed in mature mice. In addition, macrophage microbicidal competence was similar in mature and young mice. These results suggest the importance of individual host factors in the pathophysiology of an infectious disease such as Q fever.     

From Aggregation to Dispersion: How Habitat Fragmentation Prevents the Emergence of Consensual Decision Making in a Group

In fragmented landscape, individuals have to cope with the fragmentation level in order to aggregate in the same patch and take advantage of group-living. Aggregation results from responses to environmental heterogeneities and/or positive influence of the presence of congeners. In this context, the fragmentation of resting sites highlights how individuals make a compromise between two individual preferences: (1) being aggregated with conspecifics and (2) having access to these resting sites. As in previous studies, when the carrying capacity of available resting sites is large enough to contain the entire group, a single aggregation site is collectively selected. In this study, we have uncoupled fragmentation and habitat loss: the population size and total surface of the resting sites are maintained at a constant value, an increase in fragmentation implies a decrease in the carrying capacity of each shelter. For our model organism, Blattella germanica, our experimental and theoretical approach shows that, for low fragmentation level, a single resting site is collectively selected. However, for higher level of fragmentation, individuals are randomly distributed between fragments and the total sheltered population decreases. In the latter case, social amplification process is not activated and consequently, consensual decision making cannot emerge and the distribution of individuals among sites is only driven by their individual propensity to find a site. This intimate relation between aggregation pattern and landscape patchiness described in our theoretical model is generic for several gregarious species. We expect that any group-living species showing the same structure of interactions should present the same type of dispersion-aggregation response to fragmentation regardless of their level of social complexity.     

Opposite regulation of endothelial NO synthase by HSP90 and caveolin in liver and lungs of rats with hepatopulmonary syndrome

The hepatopulmonary syndrome is a complication of cirrhosis that associates an overproduction of nitric oxide (NO) in lungs and a NO defect in the liver. Because endothelial NO synthase (eNOS) is regulated by caveolin that decreases and heat shock protein 90 (HSP90) that increases NO production, we hypothesized that an opposite regulation of eNOS by caveolin and HSP90 might explain the opposite NO production in both organs. Cirrhosis was induced by a chronic bile duct ligation (CBDL) performed 15, 30, and 60 days before sample collection and pharmacological tests. eNOS, caveolin, and HSP90 expression were measured in hepatic and lung tissues. Pharmacological tests to assess NO released by shear stress and by acetylcholine were performed in livers (n = 28) and lungs (n = 28) isolated from normal and CBDL rats. In lungs from CBDL rats, indirect evidence of high NO production induced by shear stress was associated with a high binding of HSP90 and a low binding of caveolin to eNOS. Opposite results were observed in livers from CBDL rats. Our study shows an opposite posttranslational regulation of eNOS by HSP90 and caveolin in lungs and liver from rats with CBDL. Such opposite posttranslational regulation of eNOS by regulatory proteins may explain in part the pulmonary overproduction of NO and the hepatic NO defect in rats with hepatopulmonary syndrome.     

A new mouse limb mutation identifies a <Emphasis Type="Italic">Twist</Emphasis> allele that requires interacting loci on Chromosome 4 for its phenotypic expression

Pluridigite (Pdt) is a semi-dominant mutation obtained after a mutagenesis experiment with ethyl-nitroso-urea (ENU). The mutant exhibits abnormal skeletal pattern formation characterized by the formation of extra digits (polydactyly) in the preaxial (anterior) part of the hindlimbs. The phenotype shows incomplete penetrance, depending on the genetic background. In an F₂ cross with C57BL/6, the phenotype could not be associated with a single locus. Strong linkage was observed with markers located on Chromosome (Chr) 12, in a 2-cM interval between D12Mit136 and D12Mit153. This region contains the Twist gene, and we show that the [Pdt] phenotype is dependent upon a new allele of Twist. We further identified that the whole Chr 4 is associated with the [Pdt] phenotype. The Pluridigite phenotype thus results from the combination of a Twist mutant allele and at least two additional loci.