Mechanistic and functional analysis of Slit-Robo proteins

Slits are large secreted proteins which mediate their functions by binding to single pass transmembrane receptors called Robo. This signalling axis was first identified as a pivotal guidance mechanism in the development of the nervous system. A repulsive activity of Slit proteins controls the projection and movement of Robo expressing neurons in Drosophila and vertebrates. Slit and Robo expression is not limited to the nervous system or development only, they were found to be expressed in many different tissues both in embryo and adult organism. Correspondingly, the Slit-Robo signalling axis now is implicated in many other biological and pathological processes such as angiogenesis, cancer and tissue remodelling. This study sought to investigate two different aspects of the Slit-Robo signalling system: Robo1 transmembrane signalling mechanism and possible Slit-Robo roles within the immune system. Robos and other type I transmembrane receptors are unable to transmit signal across the membrane within a single molecule because a single transmembrane α-helix restricts propagation of conformational changes between extracellular and intracellular parts of the protein. Therefore, the type I receptors usually act as homo- or heterooligomers, formation or dissociation of which is controlled by ligand binding. Based on indirect evidence a similar transmembrane signalling mechanism was suggested for Robo receptors. It was hypothesized that Slit binding induces changes in oligomeric state of Robo which in turn initiates downstream signalling events. In order to test this hypothesis and determine details of the transmembrane signalling mechanism, the oligomeric state of Robo1 receptor was assessed in live cells using two different fluorescence resonance energy transfer (FRET) based methods. A strong FRET signal was observed between differently tagged Robo1 proteins, indicating that the receptor forms oligomers in the resting state. However, contrary to the initial hypothesis, the addition of Slit2 protein did not have an observable effect on the FRET signal and thus on receptor oligomeric state. Moreover, Robo1 proteins were found to form higher density domains within the cell membrane. This property could be abolished by removing the intracellular part of the protein indicating constant Robo1 association with intracellular structures. These data show that the Robo1 transmembrane signalling mechanism might be more complicated than initially expected and likely involves structural changes within the Robo1 oligomer or Robo1 complexes with other proteins. The Slit-Robo signalling axis was linked to the immune system when it was demonstrated that Slit2 is able to inhibit chemotactic leukocyte migration and reduce inflammatory responses in mice. However, the precise role of these proteins remained unknown. In order to gain further insight into possible Slit-Robo functions within the immune system promoter analysis was performed with the aim to identify transcription factors responsible for the control of these proteins. Among many putative transcription factors discovered, Foxn1 was the most prominent as its binding site was identified both in Slit2 and Slit3 promoters. Development and functions of thymus, a primary lymphoid organ responsible for T cell development, is regulated by Foxn1 and since thymic activity includes active and abundant leukocyte movement into, out-of and within the organ, a hypothesis was suggested that Slits contribute to regulation of these processes. Quantitative Slit2-Slit3 expression studies and assessment of embryonic thymus cellular composition in Slit2 knockout mouse were employed to test this hypothesis. Quantitative PCR revealed that both Slit2 and Slit3 are downregulated four-fold in thymic epithelial cells starting embryonic stage E13, however no differences in cellular composition of embryonic thymi were detected between wild type and Slit2 knockout animals using flow cytometry. Possible Slit2 and Slit3 redundancy might be the reason for the lack of observable effects, unfortunately a Slit3 knockout model was unavailable for these studies. Interestingly, flow cytometry also revealed that in addition to thymic epithelial cells, Slit2 is expressed by pericyte type cells surrounding the thymic vasculature. It seems that Slit proteins have intricate and regulated expression patterns within the thymus, however their precise role remains an open question. In summary, data collected during this study illuminates two different aspects of the Slit-Robo signalling system. Both of them, despite Slits being secreted proteins, hint at short-range, possibly even juxtacrine, ligand – receptor interactions. Given Robo receptor evolutionary connections to cell adhesion molecules and Slit affinity to heparan sulphate it is not a completely unexpected finding

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A novel mechanism for the scission of double-stranded DNA: BfiI cuts both 3′–5′ and 5′–3′ strands by rotating a single active site

Metal-dependent nucleases that generate double-strand breaks in DNA often possess two symmetrically-equivalent subunits, arranged so that the active sites from each subunit act on opposite DNA strands. Restriction endonuclease BfiI belongs to the phospholipase D (PLD) superfamily and does not require metal ions for DNA cleavage. It exists as a dimer but has at its subunit interface a single active site that acts sequentially on both DNA strands. The active site contains two identical histidines related by 2-fold symmetry, one from each subunit. This symmetrical arrangement raises two questions: first, what is the role and the contribution to catalysis of each His residue; secondly, how does a nuclease with a single active site cut two DNA strands of opposite polarities to generate a double-strand break. In this study, the roles of active-site histidines in catalysis were dissected by analysing heterodimeric variants of BfiI lacking the histidine in one subunit. These variants revealed a novel mechanism for the scission of double-stranded DNA, one that requires a single active site to not only switch between strands but also to switch its orientation on the DNA

Associirovannoe lechenie trikhomonadoza y zhenshhin pri pomoshhi preparatov tiofastil' i aminitrozol' "Pol'fa"

At the Ist Clinic of Obstetrics and Gynecology of the Silesian Medical Academy in Zabrze 40 women with the symptoms of acute and subacute trichomonadosis of the uro-genital tract were subjected to tile treatment with Thiofastil and Aminitrozol; the former was administered in tablets by intravaginal route, 1 daily for 10 days; Aminitrozol was given paralelly in tablets per os in the amount of 0.1 g 3X a day, for 10 days. If needed, the combined treatment was repeated increasing the dosis of Thiofastil up to 20 tablets (1 tablet twice daily for 10 days). In 22 cases, Aminitrozol was also employed to treat the sexual partners. The percentage of cures was 62.5%. We have considered the patients to be entirely cured when repeatedly checked discharges from the urethra, vagina and uterine cervix did not show the presence of T. vaginalis in the saline drop, as well as in the smears stained according to Giemsa. In non-cured patients there was a considerable improvement: diminution in the vaginal discharge, disappearance of vulvar oedema and of pruritus and burning sensations. Microscopic picture was consistent with this condition, showing a reduced number of leucocytes and T. vaginalis. The percentage of cures obtained by this combined method is higher as compared with that observed after the separate use of Thiofastil and Aminitrozol in two groups of women

Genetic variability of Euglena agilis [Euglenophyceae]

The results of the internal transcribed spacer (ITS2) of extrachromosomal rDNA and the chloroplast SSU rDNA sequence analysis presented here confirmed elevated genetic polymorphism revealed earlier by RFLP and RAPD for seven clones of the cosmopolitan species - Euglena agilis Carter. High diversity among these clonal strains was not reflected by morphological criteria, with the exception of the only one character - the ability of the cell in its non-motile dividing states (palmella) to produce mucus and form a slimy envelope. Evolutionary adaptation as formation of slimy envelope may be attributed to different survival strategy of the species by which it adapts to life in a highly variable environment