A chemical quenching apparatus for studying rapid reactions.

A chemical quench-flow apparatus is described for studying enzymatic reactions with half-lives of 0.005 sec or longer. The syringe pistons are driven by a stepping motor which provides precise control over the volume and rate of flow of reactants. The drive mechanism also ensures a rapid approach to a steady flow velocity and thus minimizes the amount of material used per stroke. Improved mixing efficiency is accomplished by means of ball mixers which utilize the zone of turbulence in the wake of a sphere as the mixing mechanism. The instrument was used to follow the presteady state time course of phosphorylation and dephosphorylation of a microsomal preparation of (Na⁺ + K⁺)-stimulated ATPase.     

Transport defects as the physiological basis for eye color mutants of Drosophila melanogaster

Kynurenine-H ³ transport and conversion to 3-hydroxykynurenine were studied in organ culture using the Malpighian tubules and developing eyes from wild type and the eye color mutants w, st, 1td, ca, and cn of Drosophila melanogaster. Malpighian tubules from wild type have the ability to concentrate kynurenine and convert it to 3-hydroxykynurenine. The tubules from w, st, 1td, and ca are deficient in the ability to transport kynurenine, as are the eyes of the mutants w, st, and 1td. This defect in kynurenine transport provides a physiological explanation for the phenotypic properties of the mutants. The relationship of these measurements to previous observations on these eye color mutants is discussed and the transport defect hypothesis is consistently supported. We have concluded that several of the eye color mutants in Drosophila are transport mutants.     

Detection of expanded-spectrum cephalosporin hydrolysis by lateral flow immunoassay

Early detection of expanded-spectrum cephalosporin (ESC) resistance is essential not only for an effective therapy but also for the prompt implementation of infection control measures to prevent dissemination in the hospital. We have developed and validated a lateral flow immunoassay (LFIA), called LFIA-CTX test, for the detection of ESC (cefotaxime) hydrolytic activity based on structural discrimination between the intact antibiotic and its hydrolysed product. A single bacterial colony was suspended in an extraction buffer containing cefotaxime. After a 30-min incubation, the solution is loaded on the LFIA for reading within 10 min. A total of 348 well-characterized Gram-negative isolates were tested. Among them, the 38 isolates that did not express any cefotaxime-hydrolysing β-lactamase gave negative results. Of the 310 isolates expressing at least one cefotaxime-hydrolysing β-lactamase, all were tested positive, except three OXA-48-like producers, which were repeatedly detected negative. Therefore, the sensitivity was 99.1% and the specificity was 100%. The LFIA-CTX test is efficient, fast, low-cost and easy to implement in the workflow of a routine microbiology laboratory.     

A process‐based model supports an association between dispersal and the prevalence of species traits in tropical reef fish assemblages

Habitat dynamics interacting with species dispersal abilities could generate gradients in species diversity and prevalence of species traits when the latter are associated with species dispersal potential. Using a process‐based model of diversification constrained by a dispersal parameter, we simulated the interplay between reef habitat dynamics during the past 140 million years and dispersal, shaping lineage diversification history and assemblage composition globally. The emerging patterns from the simulations were compared to current prevalence of species traits related to dispersal for 6315 tropical reef fish species. We found a significant spatial congruence between the prevalence of simulated low dispersal values and areas with a large proportion of species characterized by small adult body size, narrow home range mobility behaviour, pelagic larval duration shorter than 21 days and diurnal activity. Species characterized by such traits were found predominantly in the Indo‐Australian Archipelago and the Caribbean Sea. Furthermore, the frequency distribution of the dispersal parameter was found to match empirical distributions for body size, PLD and home range mobility behaviour. Also, the dispersal parameter in the simulations was associated to diversification rates and resulted in trait frequency matching empirical distributions. Overall, our findings suggest that past habitat dynamics, in conjunction with dispersal processes, influenced diversification in tropical reef fishes, which may explain the present‐day geography of species traits.     

Development and application of a green fluorescent protein sentinel system for identification of RNA interference in Blastomyces dermatitidis illuminates the role of septin in morphogenesis and sporulation

A high-throughput strategy for testing gene function would accelerate progress in our understanding of disease pathogenesis for the dimorphic fungus Blastomyces dermatitidis, whose genome is being completed. We developed a green fluorescent protein (GFP) sentinel system of gene silencing to rapidly study genes of unknown function. Using Gateway technology to efficiently generate RNA interference plasmids, we cloned a target gene, "X," next to GFP to create one hairpin to knock down the expression of both genes so that diminished GFP reports target gene expression. To test this approach in B. dermatitidis, we first used LACZ and the virulence gene BAD1 as targets. The level of GFP reliably reported interference of their expression, leading to rapid detection of gene-silenced transformants. We next investigated a previously unstudied gene encoding septin and explored its possible role in morphogenesis and sporulation. A CDC11 septin homolog in B. dermatitidis localized to the neck of budding yeast cells. CDC11-silenced transformants identified with the sentinel system grew slowly as flat or rough colonies on agar. Microscopically, they formed ballooned, distorted yeast cells that failed to bud, and they sporulated poorly as mold. Hence, this GFP sentinel system enables rapid detection of gene silencing and has revealed a pronounced role for septin in morphogenesis, budding, and sporulation of B. dermatitidis.     

Comparative molecular and phytochemical study of the tree species Santalum austrocaledonicum (Santalaceae) distributed in the New-Caledonian archipelago

We have tried to elucidate the origin of phytochemical variation in trees by studying concomitantly the chemical and microsatellite variations in Santalum austrocaledonicum. Eight natural populations were sampled in the New-Caledonian archipelago, a total of 157 individuals being analyzed. The main components, as revealed by gas chromatography (GC), were alpha- and beta-santalol (as in other sandalwood species), although the level of (Z)-lanceol was particularly high. Most of the chemical variation was observed within populations (83.7%). With microsatellites, the variation between populations was more pronounced (32% of the total variation). Although the chemical variation between populations was small, we investigated the effects of genetic drift and migration by comparing the chemical- and molecular-differentiation patterns. The poor congruence between neighbor-joining trees, confirmed by the non-significant Mantel test between the molecular and chemical distance matrices (R=0.26, P=0.12), showed that genetic drift and migration are not the main evolutionary forces acting on chemical differentiation between populations. We could not find any effect of soil and rainfall conditions neither. Although the impact of drift and migration cannot be discounted in rationalizing between-population differentiation, the low variation among populations could result from a stabilizing selection caused by the same phytopathogen charge across the natural range.