Cold-active esterase from Psychrobacter sp. Ant300: gene cloning,characterization, and the effects of Gly→Pro substitution near the active site on its catalytic activity and stability

The gene encoding an esterase (PsyEst) of Psychrobacter sp. Ant300, a psychrophilic bacterium isolated from Antarctic soil, was cloned, sequenced, and expressed in Escherichia coli. PsyEst, which is a member of hormone-sensitive lipase (HSL) group of the lipase/esterase family, is a cold-active, themolabile enzyme with high catalytic activity at low temperatures (5–25 °C), low activation energy (e.g., 4.6 kcal/mol for hydrolysis of p-nitrophenyl butyrate), and a t_1/2 value of 16 min for thermal inactivation during incubation at 40 °C and pH 7.9. A three-dimensional structural model of PsyEst predicted that Gly²⁴⁴ was located in the loop near the active site of PsyEst and that substitution of this amino-acid residue by proline should potentially rigidify the active-site environment of the enzyme. Thus, we introduced the Gly²⁴⁴→Pro substitution into the enzyme. Stability studies showed that the t_1/2 value for thermal inactivation of the mutant during incubation at 40 °C and pH 7.9 was 11.6 h, which was significantly greater than that of the wild-type enzyme. The k_cat/K_m value of the mutant was lower for all substrates examined than the value of the wild type. Moreover, this amino-acid substitution caused a shift of the acyl-chain length specificity of the enzyme toward higher preference for short-chain fatty acid esters. All of these observations could be explained in terms of a decrease in active-site flexibility brought about by the mutation and were consistent with the hypothesis that cold activity and thermolability arise from local flexibility around the active site of the enzyme.     

Delivery of adeno-associated virus vectors to the fetal retina: impact of viral capsid proteins on retinal neuronal progenitor transduction

The development of fetal ocular gene transfer may be useful as a therapeutic tool for the prevention of retinal genetic disorders with congenital or early clinical manifestations. In this study we explored the neural progenitor transduction patterns of adeno-associated virus (AAV) vectors following delivery to the developing retina. Recombinant vectors with the same genome carrying the enhanced green fluorescent protein (EGFP) transgene packaged in capsids of differing serotypes (serotypes 1, 2, and 5, termed AAV2/1, AAV2/2, and AAV2/5, respectively) were created. Delivery of the AAV vectors during early retinal development resulted in efficient and stable transduction of retinal progenitors. Vector surface proteins and the developmental status of the retina profoundly affected viral tropism and transgene distribution. The procedure is not detrimental to retinal development and function and therefore provides a safe delivery vehicle for potential therapeutic applications and a means of assessing the mechanisms of retina development and disease.     

Study on two unrecorded genera,Barycnemis Förster and Phradis Förster (Hymenoptera: Ichneumonidae: Tersilochinae), from South Korea

Six genera of the subfamily Tersilochinae (Barycnemis, Diaparsis, Gelanes, Phradis, Probles and Tersilochus) are recognized in South Korea. Two genera, Diaparsis and Tersilochus, were previously recognized from South Korea, whereas the other four genera are recorded from this country for the first time. All genera found in South Korea, except the almost cosmopolitan Diaparsis, are entirely or predominantly Holarctic. A key to the six genera of Tersilochine occurring in South Korea is provided. Two genera of Korean Tersilochinae (Barycnemis and Phradis) are reviewed here, and a key to the other four species (B. bellator, B. dissimilis, P. kyushuensis and P. nikishenae) is provided in this paper.     

The Epigenome of Evolving Drosophila Neo-Sex Chromosomes: Dosage Compensation and Heterochromatin Formation

Sex chromosomes originated from autosomes but have evolved a highly specialized chromatin structure. Drosophila Y chromosomes are composed entirely of silent heterochromatin, while male X chromosomes have highly accessible chromatin and are hypertranscribed as a result of dosage compensation. Here, we dissect the molecular mechanisms and functional pressures driving heterochromatin formation and dosage compensation of the recently formed neo-sex chromosomes of Drosophila miranda. We show that the onset of heterochromatin formation on the neo-Y is triggered by an accumulation of repetitive DNA. The neo-X has evolved partial dosage compensation and we find that diverse mutational paths have been utilized to establish several dozen novel binding consensus motifs for the dosage compensation complex on the neo-X, including simple point mutations at pre-binding sites, insertion and deletion mutations, microsatellite expansions, or tandem amplification of weak binding sites. Spreading of these silencing or activating chromatin modifications to adjacent regions results in massive mis-expression of neo-sex linked genes, and little correspondence between functionality of genes and their silencing on the neo-Y or dosage compensation on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those becoming heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities along the ancestral chromosome that formed the sex chromosome to adopt active or repressive chromatin configurations. Our findings have broad implications for current models of sex chromosome evolution, and demonstrate how mechanistic constraints can limit evolutionary adaptations. Our study also highlights how evolution can follow predictable genetic trajectories, by repeatedly acquiring the same 21-bp consensus motif for recruitment of the dosage compensation complex, yet utilizing a diverse array of random mutational changes to attain the same phenotypic outcome.     

Synthesis of Oligoribonucleotides Containing 2′-O-Methoxymethyl Group by the Phosphotriester Method

An effective procedure for the synthesis of ribonucleotide monomers containing a 2 ′-О-methoxymethyl-modifying group was developed. These monomers were used for the synthesis of RNA fragments by the solid-phase phosphotriester method under O-nucleophilic intramolecular catalysis. The properties of 2 ′-О-methoxymethyl-containing oligoribonucleotides were examined.     

Synthesis of Non-Nucleoside Triphosphate Analogues,a New Type of Substrates for Terminal Deoxynucleotidyl Transferase

Abstract

A series of non-nucleoside triphosphate analogues were synthesized. In place of the nucleoside fragment, substituents bearing aromatic groups were introduced; the triphosphate component was replaced at α, β, or γ-positions by phosphonates. α-[2-N-(9-Fluorenylmethoxycarbonyl)aminoethylphosphonyl]-β,γ-difluoromethylenediphosphonate (IIc) revealed the best substrate properties toward terminal deoxynucleotidyl transferase.     

T4 Phage and Its Head Surface Proteins Do Not Stimulate Inflammatory Mediator Production

Viruses are potent activators of the signal pathways leading to increased cytokine or ROS production. The effects exerted on the immune system are usually mediated by viral proteins. Complementary to the progress in phage therapy practice, advancement of knowledge about the influence of bacteriophages on mammalian immunity is necessary. Particularly, the potential ability of phage proteins to act like other viral stimulators of the immune system may have strong practical implications for the safety and efficacy of bacteriophage therapy. Here we present studies on the effect of T4 phage and its head proteins on production of inflammatory mediators and inflammation-related factors: IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-12 p40/p70, IFN-γ, TNF-α, MCP-1, MIG, RANTES, GCSF, GM-CSF and reactive oxygen species (ROS). Plasma cytokine profiles in an in vivo mouse model and in human blood cells treated with gp23*, gp24*, Hoc and Soc were evaluated by cytokine antibody arrays. Cytokine production and expression of CD40, CD80, CD86 and MHC class II molecules were also investigated in mouse bone marrow-derived dendritic cells treated with whole T4 phage particle or the same capsid proteins. The influence of T4 and gp23*, gp24*, Hoc and Soc on reactive oxygen species generation was examined in blood cells using luminol-dependent chemiluminescence assay. In all performed assays, the T4 bacteriophage and its capsid proteins gp23*, gp24*, Hoc and Soc did not affect production of inflammatory-related cytokines or ROS. These observations are of importance for any medical or veterinary application of bacteriophages.     

Modeling of in vivo Proteolytic Degradation of Hemoglobin

Abstract

Based on the amino acid sequences of endogenous peptides and X-ray spatial structure, mechanism of the in vivo proteolly degradation of bovine hemoglobin was analysed. The degradation was shown to be a multi-stage process. Its first stage is determined by the spatial organization of the native protein substrate, and the next stages—;by the distribution of the electrostatic field potential of the protein fragments formed at the earlier stage.