Engineering of lipase-catalyzed transesterification reaction media using water and diethylamine

The objective of presented study was to maximize yields of 2-methylbutyl esters, derived by transesterification reactions mediated by sn-1,3-specific lipases, through engineering of reaction medium. Effects of water and diethylamine (DEA) concentrations on the efficiency of plant oils 2-methylbutanolysis, catalyzed by either mycelium-bound Mucor circinelloides lipase (powder) or commercial immobilized lipase Lipozyme TL IM, were determined. Water content monitoring in reaction mixtures enabled to optimize the initial water content in terms of preventing the dehydration of enzyme’s microenvironment and increasing 2-methylbutyl esters yields. These yields were found to be increased by addition of either suitable amounts of water (0.5–1.5%) or diethylamine (10–30?mM) to the mixture of substrates. The presented results suggest that at low concentrations, diethylamine molecules contribute to retaining water in the microenvironment of enzyme that gives rise to increased transesterification yields and significantly reduced amounts of residual mono- and 1,2-diacyl-glycerols.     

Analysis of lymphocytes reactive to histocompatibility antigens. I. A quantitative titration assay for mixed lymphocyte interactions in the rat.

This paper describes a method for quantitating relative potencies of mixed lymphocyte interactions (MLI) in the rat by parallel line analysis of responder cell titrations. The MLI titration assay is based upon, and has certain advantages over, the popliteal lymph node graft-versus-host assay for investigations of allogeneic interactions determined by the major histocompatibility complex. Log-log plots of dose of responder cells versus cpm of [³H]thymidine incorporated were linear in the range 1 × 10⁴ to 2 × 10⁵ responder cells per microtiter well. Dilution experiments indicated that the overriding influence on the potency of any allogeneic interaction was the initial frequency of reactive cells. We have defined the limits within which the MLI titration assay behaved ideally and suggested explanations for the nonideal behavior seen when reactions of widely differing potency were compared.     

Metal contamination and human health risk associated with the consumption of Labeo rosae from the Olifants River system,South Africa

The Olifants River, a tributary of the Limpopo River system, is one of the most polluted rivers in South Africa. In May 2011 the concentrations of metals in fish muscle tissue from two impoundments, Loskop and Flag Boshielo dams, on the Olifants River were measured and a human health risk assessment conducted to investigate whether it was safe to consume Labeo rosae from these impoundments. Labeo rosae is one of the most common pan fish in these impoundments and is readily available to rural communities. Metals are accumulating in the muscle tissue of L. rosae even although the fish populations appear to be healthy. At Loskop Dam all L. rosae analysed exceeded the recommended hazard quotient (HQ) of 1 for antimony, and less than 50% exceeded that for lead. At Flag Boshielo Dam, the recommended HQ was exceeded for lead in less than 50% of L. rosae analysed, and more than 50% exceeded that for antimony. The weekly consumption of 150?g of L. rosae muscle tissue from these impoundments may pose an unacceptable health risk to rural communities.     

Influence of the linker on the biodistribution and catabolism of actinium-225 self-immolative tumor-targeted isotope generators

Current limitations to applications of monoclonal antibody (mAb) targeted isotope generators in radioimmunotherapy include the low mAb labeling yields and the nonspecific radiation of normal tissues by nontargeted radioimmunoconjugates (RIC). Radiotoxicity occurs in normal organs that metabolize radiolabeled proteins and peptides, primarily liver and kidneys, or in radiosensitive organs with prolonged exposure to the isotope from the blood, such as the bone marrow. Actinium-225 nanogenerators also have the problem of released agar-emitting daughters. We developed two new bifunctional chelating agents (BCA) in order to address these issues. Thiol-maleimide conjugation chemistry was employed to increase the efficiency of the mAb radiolabelings by up to 8-fold. In addition, one bifunctional chelating agent incorporated a cleavable linker to alter the catabolism of the alpha-particle-emitting mAb conjugate. This linker was designed to be sensitive to cathepsins to allow release and clearance of the chelated radiometal after internalization of the radioimmunoconjugate into the cell. We compared the properties of the cleavable conjugate (mAb-DOTA-G3FC) to noncleavable constructs (mAb-DOTA-NCS and mAb-DOTA-SH). The cleavable RIC was able to release 80% of its radioactive payload when incubated with purified cathepsin B. The catabolism of the constructs mAb-DOTA-G3FC and mAb-DOTA-NCS was investigated in vitro and in vivo. RIC integrity was retained at 85% over a period of 136 h in mouse serum in vivo. Both conjugates were degraded over time inside HL-60 cells after internalization and in mouse liver in vivo. While we found that the rates of degradation of the two RICs in those conditions were similar, the amounts of the radiolabeled product residues were different. The cleavable mAb-DOTA-G3FC conjugate yielded a larger proportion of fragments below 6kDa in size in mouse liver in vivo after 12 h than the DOTA-NCS conjugate. Biodistribution studies in mice showed that the mAb-DOTA-G3FC construct yielded a higher liver dose and prolonged liver retention of radioactivity compared to the mAb-DOTA-NCS conjugate. The accumulation in the liver seemed to be in part caused by the maleimide functionalization of the antibody, since the noncleavable mAb-DOTA-SH maleimide-functionalized control conjugate displayed the same biodistribution pattern. These results provide an insight into the catabolism of RICs, by demonstrating that the release of the radioisotope from a RIC is not a sufficient condition to allow the radioactive moiety to clear from the body. The excretion mechanisms of radiolabeled fragments seem to constitute a major limiting step in the chain of events leading to their clearance.     

Self-transmissibility of the integrative and conjugative element ICEPm1 between clinical isolates requires a functional integrase, relaxase, and type IV secretion system

Integrative and conjugative elements (ICEs), which are chromosomal mobile elements, can conjugatively transfer between bacteria. Recently, we identified a genomic island of Proteus mirabilis, a common agent of catheter-associated urinary tract infection (UTI), that possesses all the properties consistent with an ICE. This element, designated ICEPm1, is highly conserved in other causative agents of UTI, suggesting its mobility. We demonstrate that ICEPm1 can actively excise from the chromosome in a clonal population of bacteria and that this excision is integrase dependent. Although in P. mirabilis HI4320, ICEPm1 is annotated as integrated into the phenylalanine tRNA gene pheV, we show that ICEPm1 can integrate into either pheV or pheU. We determined that ICEPm1 transfers at a frequency of 1.35 × 10(-5) transconjugants/donor to ICEPm1-deficient P. mirabilis using plate mating assays with clinical isolates. Insertional inactivation of a putative integrase gene on ICEPm1 decreased transfer frequencies of ICEPm1 to below the limit of detection. Mutation of the relaxase of ICEPm1 also eliminates transfer and demonstrates that this element is indeed self-transmissible and not transferred in trans, as are some mobilizable genomic islands. Together, these findings clearly demonstrate that ICEPm1 can actively excise from the chromosome in an integrase-dependent manner, dynamically integrate into both phenylalanine tRNA genes, and transfer into clinical strains using its own conjugation machinery.     

Identification of benzofuran-4,5-diones as novel and selective non-hydroxamic acid, non-peptidomimetic based inhibitors of human peptide deformylase

Selective inhibitors of human peptide deformylase (HsPDF) are predicted to constitute a new class of antitumor agents. We report the identification of benzofuran-4,5-diones as the first known selective HsPDF inhibitors and we describe their selectivity profile in a panel of metalloproteases. We characterize their structure-activity relationships for antitumor activity in a panel of cancer cell lines, and we assess their in vivo efficacy in a mouse xenograft model. Our results demonstrate that selective HsPDF inhibitors based on the benzofuran-4,5-dione scaffold constitute a novel class of antitumor agents that are potent in vitro and in vivo.     

Genomic organization and chromosomal localization of a new repeat MS7, specific for heterochromatin of sex chromosomes in Microtus species voles of the arvalis group

The tandemly arranged MS4 repeat with monomeric units of 4.1 kb is species-specifically distributed in heterochromatin of sex chromosomes of four common vole species of genus Microtus, group arvalis [1, 2]. In this work, we studied the genomic organization of the MS4 homolog in euchromatin of the X chromosome of M. arvalis. It has been shown by analyzing the phage genomic clones that one MS4 copy makes a part of a monomeric unit exceeding 8.5 kb that also includes a new MS7 repeat and, possibly, LINE fragments. MS7 is located together with MS4 in heterochromatin of common vole sex chromosomes, but in a substantially lesser amount. Probably, as a result of an evolutionary transition of an original repeat from euchromatin of the X chromosome to heterochromatin of the Y chromosome, MS4 underwent multiple amplification, and MS7 spread throughout heterochromatin, being surrounded by the MS4 tandem arrays.     

Sex differences in impaling behaviour of Great Grey Shrike Lanius excubitor: Do males have better impaling skills than females?

Prey impaling in shrikes Laniidae is considered to be a feeding adaptation to dismember and consume large prey and is unique among food-storing animals. However, other exaptations of this behaviour were recorded, including signals in mate choice, where cache size is a sign of male quality. Thus, due to a strong sexual selection, male and female birds might differ in their behavioural patterns of impaling behaviour. We examined sex differences in impaling behaviour of the Great Grey Shrike Lanius excubitor - one of the species where caches are known to be sexual signals. Data were collected in western Poland during breeding seasons in the years 2006-2010. In the studied population, we recorded several sex-specific differences in impaling behaviour. Males impaled prey, invertebrates as well as vertebrates, faster and with fewer attempts per impaling event than females. Sexes differed in the location of impaled prey; males selected more visible places, especially during the mating and courtship phase, whereas females impaled prey in concealed locations. Males also had slightly better impaling success compared to females. We suggest that sex differences in impaling behaviour may be due to different uses of impaled prey, and the better impaling skills of males may be the result of better experience in impaling which is forced by sexual selection in this species. We also discuss other factors which might trigger sex-specific differences in food caching by shrikes.     

First comprehensive low-density horse linkage map based on two 3-generation, full-sibling, cross-bred horse reference families

Two 3-generation full-sibling reference families have been produced and form a unique resource for genetic linkage mapping studies in the horse. The F(2) generations, now comprising 61 individuals, consist of 28- to 32-day-old embryos removed nonsurgically from two pairs of identical twin mares. The same stallion sired all F(2)s such that the two full-sibling families are half-sibling with respect to each other. The families are crossbred to maximize levels of heterozygosity and include Arabian, Thoroughbred, Welsh Cob, and Icelandic Horse breeds. Milligram quantities of DNA have been isolated from each embryo and from blood samples of the parents and grandparents. The families have been genotyped with 353 equine microsatellites and 6 biallelic markers, and 42 linkage groups were formed. In addition, the physical location of 85 of the markers is known, and this has allowed 37 linkage groups to be anchored to the physical map. The inclusion of dams in the genotyping analysis has allowed the generation of a genetic map of the X chromosome. Markers have been assigned to all 31 autosomes and the X chromosome. The average interval between markers on the map is 10.5 cM, and the linkage groups collectively span 1780 cM. The results demonstrate the benefits for horse linkage mapping studies of genotyping on these unique full-sibling families, which comprise relatively few individuals, by the generation of a comprehensive low-density map of the horse genome.     

RNAlyzer—novel approach for quality analysis of RNA structural models

The continuously increasing amount of RNA sequence and experimentally determined 3D structure data drives the development of computational methods supporting exploration of these data. Contemporary functional analysis of RNA molecules, such as ribozymes or riboswitches, covers various issues, among which tertiary structure modeling becomes more and more important. A growing number of tools to model and predict RNA structure calls for an evaluation of these tools and the quality of outcomes their produce. Thus, the development of reliable methods designed to meet this need is relevant in the context of RNA tertiary structure analysis and can highly influence the quality and usefulness of RNA tertiary structure prediction in the nearest future. Here, we present RNAlyzer—a computational method for comparison of RNA 3D models with the reference structure and for discrimination between the correct and incorrect models. Our approach is based on the idea of local neighborhood, defined as a set of atoms included in the sphere centered around a user-defined atom. A unique feature of the RNAlyzer is the simultaneous visualization of the model-reference structure distance at different levels of detail, from the individual residues to the entire molecules.