5-Keto-3-cyano-2,4-diaminothiophenes as selective maternal embryonic leucine zipper kinase inhibitors

Maternal embryonic leucine zipper kinase (MELK) is involved in several key cellular processes and displays increased levels of expression in numerous cancer classes (colon, breast, brain, ovary, prostate and lung). Although no selective MELK inhibitors have yet been approved, increasing evidence suggest that inhibition of MELK would constitute a promising approach for cancer therapy. A weak high-throughput screening hit (17, IC₅₀?≈?5?μM) with lead-like properties was optimized for MELK inhibition. The early identification of a plausible binding mode by molecular modeling offered guidance in the choice of modifications towards compound 52 which displayed a 98?nM IC₅₀. A good selectivity profile was achieved for a representative member of the series (29) in a 486 protein kinase panel. Future elaboration of 52 has the potential to deliver compounds for further development with chemotherapeutic aims.     

It's in the eye of the beholder: visual lateralisation in response to the social environment in poeciliids

The social environment offers fish complex information about the quality, performance, personality and other cues of potential mates and competitors simultaneously. It is likely, therefore, that the environmental information regarding the context of mate choice is perceived and processed differently in species and sexes in respect to lateralisation. The present study comparatively assessed visual lateralisation behaviour in response to different social or sexual stimuli in three closely related poeciliid species (P. latipinna, P. mexicana, P. formosa) in comparison to a more distantly related species (P. reticulata). Individuals were presented with four different social or sexual stimuli that were tested against a control stimulus; (a) a conspecific male, (b) a conspecific female, (c) a heterosexual conspecific pair, (d) three conspecific females (shoal). In order to approach a target stimulus, focal fish had to perform detours to the right or left of a vertically straight-shaped barrier. The three closely related poeciliid species, P. latipinna, P. mexicana, P. formosa, appeared to have a general tendency to turn right (i.e., left-eye preference), whereas the more distantly related P. reticulata males and females showed an overall bias to the left (i.e., right-eye preference) in response to various social–sexual incitements. Moreover, body size seemed to significantly influence especially the males’ detour behaviour, with smaller males acting in opposition to their larger conspecifics in response to certain social stimuli. In this case, smaller and larger Poecilia spp. males responded in the same way as smaller and larger males of the other three poeciliid species. Therefore, results possibly point to differences in the degree of general social behaviour between closely and more distantly related species and mating motivation amongst larger and smaller individuals when placed in a novel social environment. Hence, present results possibly suggest a sex-specific functional lateralisation for the analysis of visual information and seem to support the closer ancestral relationships between the Poecilia spp. tested in this study and the more distantly related guppies in terms of their left–right lateralisation. Generally, present results suggest that functional asymmetries in behaviour could be widespread among vertebrates, thus supporting the hypothesis of an early evolution of lateralisation in brain and behaviour.     

Direct quantitative estimation of several iodothyronines in rat bile by radioimmunoassay and basal data on their biliary excretion

A method was developed for the hydrolysis of conjugated iodothyronines in bile with the aid of β-glucuronidase / arylsulfatase and for subsequent direct estimation of total and free iodothyronines with the aid of specific radioimmunoassay. The amount of conjugated fraction could then be calculated from the difference. Thus, basal biliary excretion of several iodothyronines was measured in 31 normal, fed rats in which the bile duct was drained with polyethylene tubing under pentobarbiturate anesthesia and the bile was collected for 2 h. The free fraction of thyroxine, 3,5,3′-triiodothyronine and 3,3′-diiodothyronine was approx. 30% of total content, while that of 3,3′,5′-triiodothyronine and 3,5-diiodothyronine was approx. 20% and that of 3′,5′-diiodothyronine was less than 10%. This suggests some considerable diffrences in the conjugation of individual iodothyronines was about 3–8 times higher than in plasma. This shows close interrelations between the iodothyronine deiodinating pathway in liver cells in vivo and the spectrum of iodothyronine in bile. The average ratio of T₃/rT₃ as found in bile was about 4.     

Impact of Prosopis invasion on a keystone tree species in the Kalahari Desert

Several Prosopis species were introduced into South Africa in the last century. Since then two species, Prosopis glandulosa var. torreyana and Prosopis velutina have invaded large parts of arid southern Africa. Here, we examine the extent to which increased mortality of Acacia erioloba, a keystone species in the Kalahari Desert, can be attributed to competition for water with Prosopis. We do this for A. erioloba and Prosopis sp. at invaded, as well as cleared sites through a determination of species abundance, canopy vitality, plant water stress and plant water source. Our stable isotope results show that in the riparian zone both A. erioloba and Prosopis are using the same water source. Our results also show that there is a 50 % increase in canopy dieback of A. erioloba in the invaded river plots relative to the cleared river plots. This dieback cannot be related to changes in rainfall and temperature as there were no adverse fluctuations (drought) in the weather in the 10 years preceding our study. We speculate that because A. erioloba is more water stressed in the invaded river plot this increase in mortality and dieback is related to plant moisture stress that is not related to climate but to competition for water with Prosopis. Our study gives strong support for the eradication of Prosopis from rivers in arid parts of Southern Africa.     

Heligmosomoides polygyrus antigens inhibit the intrinsic pathway of apoptosis by overexpression of survivin and Bcl-2 protein in CD4 T cells

Many laboratory studies and epidemiological observations confirm that nematodes prevent some immune-mediated diseases. The development of immunologically well-defined laboratory models of intestinal nematode infection has allowed significant advances to be made in understanding the immunological basis of effector mechanisms operating during infection under controlled laboratory conditions. The Heligmosomoides polygyrus- mouse system is used for studies of parasite immunomodulation. H. polygyrus causes a chronic, asymptomatic intestinal infection and effectively maintains both local and systemic tolerance to reduce allergic and autoimmune inflammation. However, exposure of mice to H. polygyrus antigen reduced spontaneous and glucocorticoid-induced apoptosis of CD4- positive T cells in mesenteric lymph node (MLN). In this study we evaluate the proliferation, cytokine secretion, cell cycle progression and expression of apoptosis related genes in MLN CD4 T cells of uninfected and H. polygyrus infected mice ex vivo and in vitro after restimulation with parasite excretory secretory antigen (ESAg), somatic antigen (SAg) and fraction 9 (F9Ag) of somatic antigen. For the first time we explain the influence of H. polygyrus antigens on the intrinsic pathway of apoptosis. We found that the proliferation provoked by fraction 9 and inhibition of apoptosis was dependent on a low Bax/Bcl-2 ratio, dramatical upregulation of survivin, D1 cyclin, P-glycoprotein, and loss of p27Kip1 protein with inhibition of active caspase-3 but not caspase- 8.     

Cell Survival Following Radiation Exposure Requires miR-525-3p Mediated Suppression of ARRB1 and TXN1

Background

microRNAs (miRNAs) are non-coding RNAs that alter the stability and translation efficiency of messenger RNAs. Ionizing radiation (IR) induces rapid and selective changes in miRNA expression. Depletion of the miRNA processing enzymes Dicer or Ago2 reduces the capacity of cells to survive radiation exposure. Elucidation of critical radiation-regulated miRNAs and their target proteins offers a promising approach to identify new targets to increase the therapeutic effectiveness of the radiation treatment of cancer.

Principal Findings

Expression of miR-525-3p is rapidly up-regulated in response to radiation. Manipulation of miR-525-3p expression in irradiated cells confirmed that this miRNA mediates the radiosensitivity of a variety of non-transformed (RPE, HUVEC) and tumor-derived cell lines (HeLa, U2-Os, EA.hy926) cell lines. Thus, anti-miR-525-3p mediated inhibition of the increase in miR-525-3p elevated radiosensitivity, while overexpression of precursor miR-525-3p conferred radioresistance. Using a proteomic approach we identified 21 radiation-regulated proteins, of which 14 were found to be candidate targets for miR-525-3p-mediated repression. Luciferase reporter assays confirmed that nine of these were indeed direct targets of miR-525-3p repression. Individual analysis of these direct targets by RNAi-mediated knockdown established that ARRB1, TXN1 and HSPA9 are essential miR-525-3p-dependent regulators of radiation sensitivity.

Conclusion

The transient up-regulation of miR-525-3p, and the resultant repression of its direct targets ARRB1, TXN1 and HSPA9, is required for cell survival following irradiation. The conserved function of miR-525-3p across several cell types makes this microRNA pathway a promising target for modifying the efficacy of radiotherapy.     

The application of genome-wide SNP genotyping methods in studies on livestock genomes

Animal genomics is currently undergoing dynamic development, which is driven by the flourishing of high-throughput genome analysis methods. Recently, a large number of animals has been genotyped with the use of whole-genome genotyping assays in the course of genomic selection programmes. The results of such genotyping can also be used for studies on different aspects of livestock genome functioning and diversity. In this article, we review the recent literature concentrating on various aspects of animal genomics, including studies on linkage disequilibrium, runs of homozygosity, selection signatures, copy number variation and genetic differentiation of animal populations. Our work is aimed at providing insight into certain achievements of animal genomics and to arouse interest in basic research on the complexity and structure of the genomes of livestock.     

Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus

The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24) of the thermophilic and radiotolerant bacterium Rubrobacter xylanophilus (RxPAL) was identified by screening the genomes of bacteria for members of the phenylalanine ammonia-lyase family. A synthetic gene encoding the RxPAL protein was cloned and overexpressed in Escherichia coli TOP 10 in a soluble form with an N-terminal His₆-tag and the recombinant RxPAL protein was purified by Ni-NTA affinity chromatography. The activity assay of RxPAL with l-phenylalanine at various pH values exhibited a local maximum at pH 8.5 and a global maximum at pH 11.5. Circular dichroism (CD) studies showed that RxPAL is associated with an extensive α-helical character (far UV CD) and two distinctive near-UV CD peaks. These structural characteristics were well preserved up to pH 11.0. The extremely high pH optimum of RxPAL can be rationalized by a three-dimensional homology model indicating possible disulfide bridges, extensive salt-bridge formation and an excess of negative electrostatic potential on the surface. Due to these properties, RxPAL may be a candidate as biocatalyst in synthetic biotransformations leading to unnatural l- or d-amino acids or as therapeutic enzyme in treatment of phenylketonuria or leukemia.