Asymmetric hydrolysis of dimethyl 3-phenylglutarate catalyzed by Lecitase Ultra: Effect of the immobilization protocol on its catalytic properties

The asymmetric hydrolysis of dimethyl 3-phenylglutarate (1) by different immobilized preparations of a phospholipase A₁ (Lecitase Ultra (LECI)) at pH 7 and 25 °C has been studied. Agarose beads coated with octyl, cyanogen bromide (CNBr), polyethylenimine (PEI) or glyoxyl groups were used as supports for the immobilization of LECI. The different derivatives behaved very differently in terms of activity, discrimination between 1 and methyl 3-phenylglutarate (2) resulting from the hydrolysis of 1, enantioselectivity (in the hydrolysis of 1 to produce R or S-2) and enantiospecificity in the hydrolysis of R-2 and S-2. Using 1 mM of 1, CNBr-LECI showed the highest activity (13 × 10⁻³ μmol/min mg protein) while octyl-LECI was about 20 times less active. All the enzyme preparations mainly produced (S)-2, but with different enantioselectivity. CNBr-Lecitase was the most enantioselective, producing the S-2 10 fold more rapidly than the R-2, while octyl-Lecitase gave only half of that difference.LECI adsorbed on octyl-agarose allowed to get a yield up to 99% of S-2 (ee was 66%). The reaction stopped in the monoester and no isomer of this compound was further hydrolyzed by the enzyme. However, when the reaction was catalyzed by the other immobilized LECI preparations, the enzyme was able to hydrolyze mainly the minority isomer, permitting to improve the ee of the remaining S-2. The best results were obtained using CNBr-LECI, which gave (S)-methyl-3-phenylglutarate with a yield of 80% and an ee exceeding 99%.     

Méthodologies et générations d’aérosol innovants appliqués à la vectorisation pulmonaire en pédiatrie

No aerosol system on the market is perfectly suited for aerosol therapy in infants. The RESPIRE project aims to answer this need by developing an innovative piezoelectric microperforated vibrating membrane nebulizer, the Baby Nimbus?; efficient, fast, portable and silent. Consisting of three complementary partners, the project was divided into three phases: technology development by the company Télémaq (Sophia Antipolis), in vitro and in vivo test performed on baboons by Inserm U618 (Tours), clinical trials in infants led by AP–HM (Marseille). Studies conducted by Inserm U618 and AP–HM focused on the influence of particle size on lung deposition in baboons and the clinical score in infants. Three devices of different droplet sizes were compared in the in vivo study, the Pari LC Sprint SP^® (Pari, Germany, MMAD 4.5μm – flow 0.18 mL/min), the E-Flow Rapid^® (Pari, Germany, MMAD 4.8μm – flow 0.35 mL/min) and Baby Nimbus? (Télémaq, France). Four versions of Baby Nimbus? were needed to achieve the MMAD of 2.4 μm and flowrate of 0.4 mL/min (Spraytec^®, Malvern, England). Preliminary in vivo results show that the amount of the initial charge deposited in the lungs is 0.9% for the Pari LC Sprint SP^®, 1.2% for E-Flow Rapid^® and 2.2% for the Baby Nimbus?. Additional in vivo tests and clinical trials are under way to rule definitively on the efficiency provided by the Baby Nimbus?.     

Phylogeny of the Drosophila obscura group as inferred from one- and two-dimensional protein electrophoresis

The phylogenetic relationships of 15 species of the obscura group of Drosophila were analysed by use of one- and two-dimensional electrophoresis. Genetic distances based on two-dimensional data are five times smaller than those based on native proteins. From the data, it is proposed that the species radiation of the obscura group happened in two evolutionary bursts, the first one giving rise to at least four palearctic proto-lineages (bifasciata, obscura (including D. subsilvestris), subobscura, and microlabis) and one or two proto-nearctic lineages (affinis, pseudoobscura), and the second, more recent burst giving rise to the current speciation within lineages.     

Conducting Studies with Fathers: Challenges and Opportunities

Traditionally, researchers interested in understanding father involvement in the lives of young children have relied on mothers as proxy respondents for fathers, yet recent research has made noteworthy strides in collecting data from fathers themselves yielding an unprecedented wealth of data on fathers' involvement in their children's lives. Despite this progress, there remain many methodological challenges in conducting studies with fathers and their children. Therefore, this article highlights several methodological challenges, including the identification of fathers, recruitment of fathers as participants, and retention of participants in small-scale studies with longitudinal designs, and discusses the advantages and disadvantages of several strategies our research team and others have used to collect data from fathers. The paper concludes with a set of suggestions for improving methodological approaches in fatherhood research, as well as remaining challenges in this area of study.     

Differential effects on enzyme stability and kinetic parameters of mutants related to human triosephosphate isomerase deficiency

Human triosephosphate isomerase (TIM) deficiency is a very rare disease, but there are several mutations reported to be causing the illness. In this work, we produced nine recombinant human triosephosphate isomerases which have the mutations reported to produce TIM deficiency. These enzymes were characterized biophysically and biochemically to determine their kinetic and stability parameters, and also to substitute TIM activity in supporting the growth of an Escherichia coli strain lacking the tim gene. Our results allowed us to rate the deleteriousness of the human TIM mutants based on the type and severity of the alterations observed, to classify four “unknown severity mutants” with altered residues in positions 62, 72, 122 and 154 and to explain in structural terms the mutation V231M, the most affected mutant from the kinetic point of view and the only homozygous mutation reported besides E104D.