The downregulation of the miniature gene does not replicate miniature loss-of-function phenotypes in Drosophila melanogaster wing to the full extent.

During maturation Drosophila wing epithelial cells undergo number of changes due to processes, which take place in the wing of the newly emerged fly, among which epithelial-to-mesenchymal transition (EMT) and apoptosis are pivotal. It is considered that neurohormone bursicon is responsible for their triggering. In turn, extracellular matrix protein Miniature is also essential for proper progress of apoptosis and, presumably, EMT. In accordance with our previously proposed hypothesis, Miniature and bursicon form stabilizing/accumulative complexes, which are able to diffuse freely within Drosophila wing, in such a way constitutively promoting enough concentrations of the maturation triggering signal. Here we tried to come to confirmation of our hypothesis from the other side, using UAS/GAL4 system and RNAi-silencing techniques.

The eye of Drosophila as a model system for studying intracellular signaling in ontogenesis and pathogenesis.

Many human diseases are caused by malfunction of basic types of cellular activity such as proliferation, differentiation, apoptosis, cell polarization, and migration. In turn, these processes are associated with different routes of intracellular signal transduction. A number of model systems have been designed to study normal and abnormal cellular and molecular processes associated with pathogenesis. The developing eye of the fruit fly Drosophila melanogaster is one of these systems. The sequential development of compound eyes of this insect makes it possible to model human neurodegenerative diseases and mechanisms of carcinogenesis. In this paper we overview the program of the eye development in Drosophila, with emphasis on intracellular signaling pathways that regulate this complex process. We discuss in detail the roles of the Notch, Hedgehog, TGFß, Wnt, and receptor tyrosine kinase signaling pathways in Drosophila eye development and human pathology. We also briefly describe the modern methods of experimentation with this model organism to analyze the function of human pathogenic proteins.

The Wnt/Frizzled GPCR signaling pathway.

G protein-coupled receptors (GPCRs) represent the biggest transmembrane receptor family. The Frizzled group of GPCRs is evolutionarily conserved and serves to transduce signals from the Wnt-type lipoglycoprotein growth factors. The Wnt/Frizzled signaling cascades are repeatedly used during animal development and are mostly silent in the adult. Improper activation of these cascades, e.g. through somatic mutation, underlies cancer development in various tissues. Our research over the past years has identified the trimeric G proteins as crucial transducers of the Wnt/Frizzled cascades in insect and mammalian cells. The current mini-review summarizes our findings on the role of G proteins in Wnt/Frizzled signaling, as well as on identification of other signaling intermediates in this physiologically and pathologically important type of intracellular signal transduction.

Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation.

Phosphoinositide 3-kinase (PI3K) activity is crucial for leukocyte function, but the roles of the four receptor-activated isoforms are unclear. Mice lacking heterotrimeric guanine nucleotide-binding protein (G protein)-coupled PI3Kgamma were viable and had fully differentiated neutrophils and macrophages. Chemoattractant-stimulated PI3Kgamma-/- neutrophils did not produce phosphatidylinositol 3,4,5-trisphosphate, did not activate protein kinase B, and displayed impaired respiratory burst and motility. Peritoneal PI3Kgamma-null macrophages showed a reduced migration toward a wide range of chemotactic stimuli and a severely defective accumulation in a septic peritonitis model. These results demonstrate that PI3Kgamma is a crucial signaling molecule required for macrophage accumulation in inflammation.

Information Theory: New Look at Oncogenic Signaling Pathways.

Sustained pro-proliferative signaling is one of the hallmarks of cancer. Although it is generally understood that the oncogenic signaling pathways are overactivated, or at least abnormally activated, in cancer cells, important mechanistic details of such abnormal activation remain unresolved. Among these details are such aspects of signaling as robustness, redundancy, signal amplification, and others, which touch upon the domain of information theory - the field of mathematics and engineering dealing with properties of information storage, encoding, and transmission. Information theory only recently has started to be applied to intracellular signaling. Here, we overview the recent advances provided by the information theory, focusing on the nuclear factor (NF)-κB, extracellular signal-regulated kinase (ERK), and G-protein-coupled receptor (GPCR) pathways, which are frequently hijacked in cancer. Furthermore, we show how viewing previously untouched mechanics of oncogenic signaling through information theory applications may evolve into novel ways of anticancer drug discovery.

Formation of bacteriophage MS2 infectious units in a cell-free translation system.

We show that a simple cell-free translation system from Escherichia coli, programmed with phage MS2 RNA, is able to infect F+ E. coli cells. The plaques appearing on the E. coli host strain are morphologically indistinguishable from those derived from normal phage MS2 infection. This effect is strictly translation-dependent, since an incomplete translation system or the system inhibited by antibiotics leads to no infection. The cell-free based infection is maximal under conditions favouring the highest synthesis of maturation protein (one of the four phage-encoded proteins). The infection is abolished when RNase A or trypsin treatment is included before addition of cells. Similarly, due to RNA and maturation protein degradation, the continued incubation of the translation mixture under protein synthesis conditions significantly decreases infectivity. These findings suggest the formation of 'minimal infectious units', simple complexes of MS2 RNA and maturation protein. Here we describe the first example of bacteriophage infectious unit formation directly performed in a cell-free translation system. A possible application of this phenomenon might be the construction of newly designed RNA vector delivery systems and, moreover, could be an approach for molecular evolution studies.

Viral Q beta RNA as a high expression vector for mRNA translation in a cell-free system.

Dihydrofolate reductase (DHFR) mRNA was inserted into Q beta phage RNA instead of its coat protein cistron. Translation of this recombinant mRNA in the Escherichia coli cell-free system resulted in the synthesis of DHFR, which was two orders of magnitude higher than that in the case of translation of the control DHFR mRNA. Additionally, it resulted in a significantly enhanced synthesis of Q beta replicase as compared with its synthesis when the original Q beta RNA was used.

Anti-leprosy drug clofazimine inhibits growth of triple-negative breast cancer cells via inhibition of canonical Wnt signaling.

Research on existing drugs often discovers novel mechanisms of their action and leads to the expansion of their therapeutic scope and subsequent remarketing. The Wnt signaling pathway is of the immediate therapeutic relevance, as it plays critical roles in cancer development and progression. However, drugs which disrupt this pathway are unavailable despite the high demand. Here we report an attempt to identify antagonists of the Wnt-FZD interaction among the library of the FDA-approved drugs. We performed an in silico screening which brought up several potential antagonists of the ligand-receptor interaction. 14 of these substances were tested using the TopFlash luciferase reporter assay and four of them identified as active and specific inhibitors of the Wnt3a-induced signaling. However, further analysis through GTP-binding and ß-catenin stabilization assays showed that the compounds do not target the Wnt-FZD pair, but inhibit the signaling at downstream levels. We further describe the previously unknown inhibitory activity of an anti-leprosy drug clofazimine in the Wnt pathway and provide data demonstrating its efficiency in suppressing growth of Wnt-dependent triple-negative breast cancer cells. These data provide a basis for further investigations of the efficiency of clofazimine in treatment of Wnt-dependent cancers.

Signal transduction in neutrophil chemotaxis.

This review discusses current knowledge on signal transduction pathways controlling chemotaxis of neutrophils and similar cells. Most neutrophil chemoattractants bind to seven-transmembrane-helix receptors. These receptors activate trimeric G proteins of the Gi class in neutrophils to initiate chemotaxis. Phospholipases Cbeta, phosphoinositide 3-kinase gamma, and PH domain-containing proteins play various roles in signaling further downstream. The actin cytoskeleton is crucial for cell motility, and is controlled by Rho family GTP-binding proteins. PIP 5-kinase, LIM kinase, myosin light chain kinase and phosphatase, or WASP-like proteins may be important links between Rho GTPases and actin during chemotaxis. Newly emerging ideas on the regulation of the "compass" of chemotaxing cells, which may involve Cdc42 and certain PH domain-containing proteins, are also presented.

GTPgammaS-induced actin polymerisation in vitro: ATP- and phosphoinositide-independent signalling via Rho-family proteins and a plasma membrane-associated guanine nucleotide exchange factor.

In a cell-free system from neutrophil cytosol GTP(&ggr ;)S can induce an increase in the number of free filament barbed ends and massive actin polymerisation and cross-linking. GTP(&ggr ;)S stimulation was susceptible to an excess of GDP, but not Bordetella pertussis toxin and could not be mimicked by aluminium fluoride, myristoylated GTPgammaS.Gialpha2 or Gbeta1gamma2 subunits of trimeric G proteins. In contrast, RhoGDI and Clostridium difficile toxin B (inactivating Rho family proteins) completely abrogated the effect of GTPgammaS. When recombinant, constitutively activated and GTPgammaS-loaded Rac1, RhoA, or Cdc42 proteins alone or in combination were probed at concentrations >100 times the endogenous, however, they were ineffective. Purified Cdc42/Rac-interactive binding (CRIB) domain of WASP or C3 transferase did not prevent actin polymerisation by GTPgammaS. The action of GTPgammaS was blocked by mM [Mg2+], unless a heat- and trypsin-sensitive component present in neutrophil plasma membrane was added. Liberation of barbed ends seems therefore to be mediated by a toxin B-sensitive cytosolic Rho-family protein, requiring a membrane-associated guanine nucleotide exchange factor (GEF) for its activation by GTPgammaS under physiologic conditions. The inefficiency of various protein kinase and phosphatase inhibitors (staurosporine, genistein, wortmannin, okadaic acid and vanadate) and removal of ATP by apyrase, suggests that phosphate transfer reactions are not required for the downstream propagation of the GTPgammaS signal. Moreover, exogenously added phosphoinositides failed to induce actin polymerisation and a PtdIns(4,5)P2-binding peptide did not interfere with the response to GTPgammaS. The speed and simplicity of the presented assay applicable to protein purification techniques will facilitate the further elucidation of the molecular partners involved in actin polymerisation.

Microquantification of cellular and in vitro F-actin by rhodamine phalloidin fluorescence enhancement.

Based on the enhancement of rhodamine phalloidin fluorescence after its binding to actin filaments we have developed a technique to quantify F-actin, drastically (>> 100 times) reducing consumption of the expensive fluorescent dye and sample material in comparison to previous methods. Depolymerization of F-actin is prevented by utilizing short incubation times and stabilization of the filaments by actin-binding proteins or formaldehyde. Equilibrium and kinetic mathematical models relating rhodamine fluorescence with F-actin concentrations were used to predict the optimal assay conditions. The method has been applied to measure relative and absolute F-actin concentrations in cytosolic fractions and stimulus-induced actin polymerization in neutrophils. The cells were lysed with octy1-beta-D-glucopyranoside, which is compatible with the assay due to its high critical micelle concentration. As the assay takes less than 1 h and eliminates all previously required washing or extraction steps, it is faster and much simpler than any other presented up to now for quantification of filamentous actin. Moreover, the method is unique for reliable and easy F-actin measurements in cell-free systems.