Antagonistic and cooperative AGO2-PUM interactions in regulating mRNAs.

Approximately 1500 RNA-binding proteins (RBPs) profoundly impact mammalian cellular function by controlling distinct sets of transcripts, often using sequence-specific binding to 3' untranslated regions (UTRs) to regulate mRNA stability and translation. Aside from their individual effects, higher-order combinatorial interactions between RBPs on specific mRNAs have been proposed to underpin the regulatory network. To assess the extent of such co-regulatory control, we took a global experimental approach followed by targeted validation to examine interactions between two well-characterized and highly conserved RBPs, Argonaute2 (AGO2) and Pumilio (PUM1 and PUM2). Transcriptome-wide changes in AGO2-mRNA binding upon PUM knockdown were quantified by CLIP-seq, and the presence of PUM binding on the same 3'UTR corresponded with cooperative and antagonistic effects on AGO2 occupancy. In addition, PUM binding sites that overlap with AGO2 showed differential, weakened binding profiles upon abrogation of AGO2 association, indicative of cooperative interactions. In luciferase reporter validation of candidate 3'UTR sites where AGO2 and PUM colocalized, three sites were identified to host antagonistic interactions, where PUM counteracts miRNA-guided repression. Interestingly, the binding sites for the two proteins are too far for potential antagonism due to steric hindrance, suggesting an alternate mechanism. Our data experimentally confirms the combinatorial regulatory model and indicates that the mostly repressive PUM proteins can change their behavior in a context-dependent manner. Overall, the approach underscores the importance of further elucidation of complex interactions between RBPs and their transcriptome-wide extent

Molecular Cloning of a Bovine Immunoglobulin Lambda Chain cDNA

A cDNA library of the bovine mammary gland constructed in pBR322 was screened by mRNA hybrid-selected translation and by differential hybridization. Several immunoglobulin (Ig) λ light-chain clones were identified and sequenced. Nucleotide sequence comparison of bovine and human Ig λ chains showed a high degree of homology for constant regions and for J regions. The amino acid (aa) sequence encoded by the constant region of the bovine Ig λ chain cDNA contains 107 aa with differences at 24 aa positions from the human Ig A chain. Three complementarity-determining regions (CDR1,2,3) characteristic of the variable region of bovine Ig λ chain cDNA can be distinguished. The bovine and human sequences display good homology in the framework region 3 (FR3) but only patches of homology throughout the FR2 region. The 5′ end of the bovine Ig λ chain cDNA fragment of clone 1-14E contains five stop codons: two in CDR1, one in FR1 and two in the hydrophobic prepeptide region. These data suggest that the Igλ mRNA of clone 1-14E is transcribed from the Vλ pseudogene

Пример синтеза управляющих воздействий для шестиногого шагающего робота при передвижении по неровной поверхности

Control actions are provided on the basis of inverse kinematic problem. Now there is a set of methods to solve this task.This article considers an example of the author’s approach application to the inverse kinematic problem.The main idea of approach is as follows:1. The limited set of the joints necessary to implement the chosen gait is selected from all joints of the robot. For these joints a strict sequence of the movement within each step and restriction of changing generalized coordinates are specified. 2. The joints non-involved in implementing the chosen gait are disabled, with no calculations performed for them.Thus, the sources of basic data for the inverse kinematic problem are the kinematic scheme of the executive mechanism of the walking robot and the chosen gait.To use the offered approach it is necessary:1. To number the legs and their joints.2. To choose joints to be involved in realization of the chosen gait.3. To appoint a sequence of the change of supporting legs when moving by the chosen gait.4. To specify a motion sequence of the chosen joints within a step for each leg.5. To specify restrictions of changes of the generalized coordinates in the chosen joints.The inverse kinematic problem process consists in gradual approach to the solution by change (increase or decrease) of the generalized coordinates in the same order in which the joints of a leg corresponding to these coordinates move within a step by the chosen gait when walking.Criterion of completing calculations is the limits reached or the fact that a leg is fixed on a supporting plane by a contact sensor (or a condition in the modeling program). Changes of generalized coordinates are within a cycle; each generalized coordinate changes by a certain value at each of iterations of a cycle. The total time of a cycle corresponds to the estimated time of a step to be done.Advantages of the approach are following: unambiguity of the received solution, possibility to consider degenerate configurations of the executive mechanism of the walking robot, possibility to calculate in the conditions of kinematic redundancy, and high-speed calculations.The possibility to consider degenerate configurations is owing to the fact that, when calculating, the device of matrix transformations is not used (in particular, calculation of the return matrix of Jacobi).The possibility to calculate in the conditions of kinematic redundancy, unambiguity of the received solution and high speed are caused by fact that the sequence of the joints movement initially defines a certain desirable leg configuration in space and, in fact, specifies a solution direction.There is no need to verify the received solution as kinematic restrictions has been are already taken into consideration in the course of calculations.One more important advantage of the approach is the fact that robots can have any number of legs, and joints in legs.A lack of possibility to create universal algorithms for the walking robots with essentially different kinematic schemes (for example, anthropomorphous and arachnoid) because of constructive differences in executive mechanisms can be the shortcoming of the approach. But other heuristic methods, such as FABRIK, FTL or CCD also have the same shortcoming. The approach is akin to these methods by the fact that there is no certain formula which use allows us to receive the solution with these methods at once. There are only algorithms of search checking a set of the specified conditions, which are criteria of correction or end of the solution when calculating.В статье изложен новый подход к решению обратной задачи кинематики. Показан пример применения подхода при синтезе управляющих воздействий для приводов шагающих роботов. Идея подхода заключается в следующем: из всех сочленений робота выбирается ограниченный набор сочленений, необходимых для реализации избранной походки. Для этих сочленений назначается строгая последовательность движения в течение каждого шага и ограничения изменений обобщенных координат. Сочленения, не участвующие в реализации избранной походки, блокируются, для них расчеты не проводятся. Достоинствами подхода являются: однозначность получаемого решения, возможность рассмотрения вырожденных конфигураций исполнительного механизма шагающего робота, возможность вычислений в условиях кинематической избыточности, высокая скорость вычислений. Указанные достоинства обусловлены тем, что последовательность движения сочленений изначально устанавливает определенную желаемую конфигурацию ноги в пространстве и, по сути, задает направление решения. Получение управляющих воздействий показано на примере шестиногого шагающего робота, имеющего по шесть сочленений в каждой ноге. DOI: 10.7463/aplts.0315.078245

Designed Azolopyridinium Salts Block Protective Antigen Pores In Vitro and Protect Cells from Anthrax Toxin

Background:Several intracellular acting bacterial protein toxins of the AB-type, which are known to enter cells by endocytosis, are shown to produce channels. This holds true for protective antigen (PA), the binding component of the tripartite anthrax-toxin of Bacillus anthracis. Evidence has been presented that translocation of the enzymatic components of anthrax-toxin across the endosomal membrane of target cells and channel formation by the heptameric/octameric PA63 binding/translocation component are related phenomena. Chloroquine and some 4-aminoquinolones, known as potent drugs against Plasmodium falciparium infection of humans, block efficiently the PA63-channel in a dose dependent way.Methodology/Principal Findings:Here we demonstrate that related positively charged heterocyclic azolopyridinium salts block the PA63-channel in the μM range, when both, inhibitor and PA63 are added to the same side of the membrane, the cis-side, which corresponds to the lumen of acidified endosomal vesicles of target cells. Noise-analysis allowed the study of the kinetics of the plug formation by the heterocycles. In vivo experiments using J774A.1 macrophages demonstrated that the inhibitors of PA63-channel function also efficiently block intoxication of the cells by the combination lethal factor and PA63 in the same concentration range as they block the channels in vitro.Conclusions/Significance:These results strongly argue in favor of a transport of lethal factor through the PA63-channel and suggest that the heterocycles used in this study could represent attractive candidates for development of novel therapeutic strategies against anthrax. © 2013 Beitzinger et al

Nucleotide sequence of the Galleria mellonella nuclear polyhedrosis virus origin of DNA replication

AbstractThe initiation sites of the Galleria mellonella L. nuclear polyhedrosis virus (G.m. NPV) DNA replication were revealed. For this purpose SCLd 135 cells permitting the G.m. NPV productive reproduction were transformed by the recombinant plasmids containing the viral genome individual fragments in pRSF 2124 and pBR 322 vectors. It was revealed that 2 of the 32 recombinant plasmids can autonomously replicate in the eucaryotic cells. According to the Maxam-Gilbert method the DNA G.m. NPV fragment (1300 bp) primary structure of pHBR plasmid was determined. The structure analysis revealed the typical regulator signals as in the replicons. The possible regulation mechanisms of the DNA G.m. NPV synthesis initiation was supposed

The miR-17 similar to 92 cluster collaborates with the Sonic Hedgehog pathway in medulloblastoma

Medulloblastomas (MBs) are the most common brain tumors in children. Some are thought to originate from cerebellar granule neuron progenitors (GNPs) that fail to undergo normal cell cycle exit and differentiation. Because microRNAs regulate numerous aspects of cellular physiology and development, we reasoned that alterations in miRNA expression might contribute to MB. We tested this hypothesis using 2 spontaneous mouse MB models with specific initiating mutations, Ink4c(-/-); Ptch1(+/-) and Ink4c(-/-); p53(-/-). We found that 26 miRNAs showed increased expression and 24 miRNAs showed decreased expression in proliferating mouse GNPs and MBs relative to mature mouse cerebellum, regardless of genotype. Among the 26 overexpressed miRNAs, 9 were encoded by the miR-17 similar to 92 cluster family, a group of microRNAs implicated as oncogenes in several tumor types. Analysis of human MBs demonstrated that 3 miR-17 similar to 92 cluster miRNAs (miR-92, miR-19a, and miR-20) were also overexpressed in human MBs with a constitutively activated Sonic Hedgehog (SHH) signaling pathway, but not in other forms of the disease. To test whether the miR-17 similar to 92 cluster could promote MB formation, we enforced expression of these miRNAs in GNPs isolated from cerebella of postnatal (P) day P6 Ink4c(-/-); Ptch1(+/-) mice. These, but not similarly engineered cells from Ink4c(-/-); p53(-/-) mice, formed MBs in orthotopic transplants with complete penetrance. Interestingly, orthotopic mouse tumors ectopically expressing miR-17 similar to 92 lost expression of the wild-type Ptch1 allele. Our findings suggest a functional collaboration between the miR-17 similar to 92 cluster and the SHH signaling pathway in the development of MBs in mouse and man

Cortactin and Crk cooperate to trigger actin polymerization during Shigella invasion of epithelial cells

Shigella, the causative agent of bacillary dysentery, invades epithelial cells in a process involving Src tyrosine kinase signaling. Cortactin, a ubiquitous actin-binding protein present in structures of dynamic actin assembly, is the major protein tyrosine phosphorylated during Shigella invasion. Here, we report that RNA interference silencing of cortactin expression, as does Src inhibition in cells expressing kinase-inactive Src, interferes with actin polymerization required for the formation of cellular extensions engulfing the bacteria. Shigella invasion induced the recruitment of cortactin at plasma membranes in a tyrosine phosphorylation–dependent manner. Overexpression of wild-type forms of cortactin or the adaptor protein Crk favored Shigella uptake, and Arp2/3 binding–deficient cortactin derivatives or an Src homology 2 domain Crk mutant interfered with bacterial-induced actin foci formation. Crk was shown to directly interact with tyrosine-phosphorylated cortactin and to condition cortactin-dependent actin polymerization required for Shigella uptake. These results point at a major role for a Crk–cortactin complex in actin polymerization downstream of tyrosine kinase signaling

Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers

The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers were transcribed. For family I CRISPR loci, the presence of the protospacer CC motif was shown to be important for the occurrence of deletions. The results are consistent with a low level of random dynamic recombination occurring spontaneously, either inter-genomically or intra-genomically, at the repeat regions of Sulfolobus CRISPR loci. Moreover, the relatively high incidence of single-spacer deletions observed for S. islandicus suggests that an additional more directed mechanism operates in this organism

Tailored ß-Cyclodextrin Blocks the Translocation Pores of Binary Exotoxins from C. Botulinum and C. Perfringens and Protects Cells from Intoxication

International audienceBackgroundClostridium botulinum C2 toxin and Clostridium perfringens iota toxin are binary exotoxins, which ADP-ribosylate actin in the cytosol of mammalian cells and thereby destroy the cytoskeleton. C2 and iota toxin consists of two individual proteins, an enzymatic active (A-) component and a separate receptor binding and translocation (B-) component. The latter forms a complex with the A-component on the surface of target cells and after receptor-mediated endocytosis, it mediates the translocation of the A-component from acidified endosomal vesicles into the cytosol. To this end, the B-components form heptameric pores in endosomal membranes, which serve as translocation channels for the A-components.Here we demonstrate that a 7-fold symmetrical positively charged ß-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-ß-cyclodextrin, protects cultured cells from intoxication with C2 and iota toxins in a concentration-dependent manner starting at low micromolar concentrations. We discovered that the compound inhibited the pH-dependent membrane translocation of the A-components of both toxins in intact cells. Consistently, the compound strongly blocked transmembrane channels formed by the B-components of C2 and iota toxin in planar lipid bilayers in vitro. With C2 toxin, we consecutively ruled out all other possible inhibitory mechanisms showing that the compound did not interfere with the binding of the toxin to the cells or with the enzyme activity of the A-component.Conclusions/SignificanceThe described ß-cyclodextrin derivative was previously identified as one of the most potent inhibitors of the binary lethal toxin of Bacillus anthracis both in vitro and in vivo, implying that it might represent a broad-spectrum inhibitor of binary pore-forming exotoxins from pathogenic bacteria