The fusion protein SS18-SSX1 employs core Wnt pathway transcription factors to induce a partial Wnt signature in synovial sarcoma.

Expression of the SS18/SYT-SSX fusion protein is believed to underlie the pathogenesis of synovial sarcoma (SS). Recent evidence suggests that deregulation of the Wnt pathway may play an important role in SS but the mechanisms whereby SS18-SSX might affect Wnt signaling remain to be elucidated. Here, we show that SS18/SSX tightly regulates the elevated expression of the key Wnt target AXIN2 in primary SS. SS18-SSX is shown to interact with TCF/LEF, TLE and HDAC but not β-catenin in vivo and to induce Wnt target gene expression by forming a complex containing promoter-bound TCF/LEF and HDAC but lacking β-catenin. Our observations provide a tumor-specific mechanistic basis for Wnt target gene induction in SS that can occur in the absence of Wnt ligand stimulation.

Analysis of viral and cellular parameters which affect the fusion process of influenza viruses

In the present investigation we studied the fusogenic process developed by influenza A, B and C viruses on cell surfaces and different factors associated with virus and cell membrane structures. The biological activity of purified virus strains was evaluated in hemagglutination, sialidase and fusion assays. Hemolysis by influenza A, B and C viruses ranging from 77.4 to 97.2%, from 20.0 to 65.0%, from 0.2 to 93.7% and from 9.0 to 76.1% was observed when human, chicken, rabbit and monkey erythrocytes, respectively, were tested at pH 5.5. At this pH, low hemolysis indexes for influenza A, B and C viruses were observed if horse erythrocytes were used as target cells for the fusion process, which could be explained by an inefficient receptor binding activity of influenza on N-glycolyl sialic acids. Differences in hemagglutinin receptor binding activity due to its specificity to N-acetyl or N-glycolyl cell surface oligosaccharides, density of these cellular receptors and level of negative charges on the cell surface may possibly explain these results, showing influence on the sialidase activity and the fusogenic process. Comparative analysis showed a lack of dependence between the sialidase and fusion activities developed by influenza B viruses. Influenza A viruses at low sialidase titers (<2) also exhibited clearly low hemolysis at pH 5.5 (15.8%), while influenza B viruses with similarly low sialidase titers showed highly variable hemolysis indexes (0.2 to 78.0%). These results support the idea that different virus and cell-associated factors such as those presented above have a significant effect on the multifactorial fusion process

Cell lineage relationship in the stomach of normal and genetically manipulated mice

The oxyntic mucosa of the mouse stomach is lined with a heterogeneous population of cells that form numerous short pits continuous with long tubular glands. Tritiated thymidine radioautography has made it possible to pinpoint the origin of all cell types and to follow the differentiation/migration of different cell lineages along the pit-gland unit. The proliferating multipotent stem cells functionally anchored in the upper glandular region, the isthmus, give rise to three main lineage precursors: 1) pre-pit cells, which migrate upward to the pit while differentiating into mucus-producing pit cells; 2) pre-neck cells, which migrate downward to the glandular neck while differentiating into mucus-producing neck cells that, by approaching the glandular base, gradually change their phenotype into pepsinogen- and intrinsic factor-producing zymogenic cells; 3) pre-parietal cells, which differentiate into acid-producing parietal cells in the isthmus and then undergo bipolar migration towards the pit and the glandular base. Thus, parietal cells are the only cells that complete their differentiation in the isthmus and then migrate to be scattered throughout the pit-gland unit. To determine whether parietal cells play a role in controlling decisions about cell fate within the pit-gland unit, the gastric epithelium has been examined in transgenic mice expressing the H,K-ATPase ß-subunit-1035 to +24/simian virus 40 large T antigen fusion gene. The blockade in parietal cell differentiation in these mice produces an amplification of lineage precursors, a marked depletion of zymogenic cells and an increase in pit cell census. Ablation of parietal cells in another transgenic mouse model expressing the H,K-ATPase ß-subunit-1035 to +24/diphtheria toxin fragment A fusion gene also produces amplification of lineage precursors, and similar effects on zymogenic and pit cell census. These findings strongly suggest that parietal cells produce regulatory signals that control the cellular differentiation program of both pit and zymogenic cell lineages, and would hopefully improve our ability to identify the cellular pathways leading to malignant transformation

A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

Caractérisation cytogénétique et clinique des gènes de fusion impliquant MLL dans les leucémies

Le gène MLL (Mixed-Lineage Leukemia), un homologue du gène trithorax de la Drosophile, localisé à la bande chromosomique 11q23, est fréquemment réarrangé dans plusieurs types de leucémies, essentiellement suite à des translocations chromosomiques. Dans les différentes translocations chromosomiques, la partie N-terminale de MLL est fusionnée avec les séquences d’un gène partenaire. Malgré le grand nombre de partenaires de fusion rapportés, peu de fusions MLL ont été bien caractérisées sur le plan moléculaire. De plus, l’impact pronostique de plusieurs fusions moins fréquentes n’est pas bien établi. L’objectif de mon projet est de caractériser plusieurs translocations MLL qui ont été détectées dans 39 spécimens leucémiques collectés par la Banque de cellules leucémiques du Québec (www.bclq.gouv.qc.ca), et d’établir une corrélation entre les résultats de la cytogénétique et différents paramètres biologiques et cliniques des leucémies respectives. L’identification des gènes partenaires de fusion (GPF) dans notre série (30 échantillons étudiés), a révélé la fusion de MLL à un gène partenaire très récurrent dans 26 leucémies: MLLT3(AF9), AFF1(AF4), MLLT4(AF6), MLLT1(ENL), ELL; à un GPF modérément commun dans 1 leucémie : MLLT6(AF17); et à un partenaire rare de MLL dans 3 leucémies : GAS7 et AF15/CASC5 (2 cas). Nous avons poursuivi notre travail avec la caractérisation des points de cassure de deux fusions, soit MLL-ELL associée à un syndrome myéloprolifératif (une association rare), et MLL-GAS7 (une fusion rare de MLL), associée à une leucémie aiguë myéloïde. L’analyse des transcrits de fusion par RT-PCR et séquençage a révélé respectivement la fusion de l’exon 9 de MLL à l’exon 2 de ELL et des exons 7 ou 8 de MLL (deux transcrits) à l’exon 2 de GAS7. Ce travail permettra d’effectuer des études fonctionnelles et des projets de recherche translationnelle en utilisant ces spécimens de leucémies avec différents réarrangements de MLL, bien caractérisés sur le plan clinique et moléculaire.

The postfusion structure of baculovirus gp64 supports a unified view of viral fusion machines

Viral fusion proteins mediate the merger of host and viral membranes during cell entry for all enveloped viruses. Baculovirus glycoprotein gp64 (gp64) is unusual in promoting entry into both insect and mammalian cells and is distinct from established class I and class II fusion proteins. We report the crystal structure of its postfusion form, which explains a number of gp64's biological properties including its cellular promiscuity, identifies the fusion peptides and shows it to be the third representative of a new class (III) of fusion proteins with unexpected structural homology with vesicular stomatitis virus G and herpes simplex virus type 1 gB proteins. We show that domains of class III proteins have counterparts in both class I and II proteins, suggesting that all these viral fusion machines are structurally more related than previously thought.

Cell entry by enveloped viruses: redox considerations for HIV and SARS-coronavirus

For enveloped viruses, genome entry into the target cell involves two major steps: virion binding to the cell-surface receptor and fusion of the virion and cell membranes. Virus-cell membrane fusion is mediated by the virus envelope complex, and its fusogenicity is the result of an active virus-cell interaction process that induces conformation changes within the envelope. For some viruses, such as influenza, exposure to an acidic milieu within the cell during the early steps of infection triggers the necessary structural changes. However, for other pathogens which are not exposed to such environmental stress, activation of fusogenicity can result from precise thiol/disulfide rearrangements mediated by either an endogenous redox autocatalytic isomerase or a cell-associated oxidoreductase. Study of the activation of HIV envelope fusogenicity has revealed new knowledge about how redox changes within a viral envelope trigger fusion. We discuss these findings and their implication for anti-HIV therapy. In addition, to compare and contrast the situation outlined for HIV with an enveloped virus that can fuse with the cell plasma membrane independent of the redox status of its envelope protein, we review parallel data obtained on SARS coronavirus entry.

Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell

Coronavirus nucleoproteins (N proteins) localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. The nucleolus is the site of ribosome biogenesis and sequesters cell cycle regulatory complexes. Two of the major components of the nucleolus are fibrillarin and nucleolin. These proteins are involved in nucleolar assembly and ribosome biogenesis and act as chaperones for the import of proteins into the nucleolus. We have found that fibrillarin is reorganized in primary cells infected with the avian coronavirus infectious bronchitis virus (IBV) and in continuous cell lines that express either IBV or mouse hepatitis virus N protein. Both N protein and a fibrillarin-green fluorescent protein fusion protein colocalized to the perinuclear region and the nucleolus. Pull-down assays demonstrated that IBV N protein interacted with nucleolin and therefore provided a possible explanation as to how coronavirus N proteins localize to the nucleolus. Nucleoli, and proteins that localize to the nucleolus, have been implicated in cell growth-cell cycle regulation. Comparison of cells expressing IBV N protein with controls indicated that cells expressing N protein had delayed cellular growth. This result could not to be attributed to apoptosis. Morphological analysis of these cells indicated that cytokinesis was disrupted, an observation subsequently found in primary cells infected with IBV. Coronaviruses might therefore delay the cell cycle in interphase, where maximum translation of viral mRNAs can occur.

Assisted remote viewing of a teleoperation work cell

Intelligent viewing systems are required if efficient and productive teleoperation is to be applied to dynamic manufacturing environments. These systems must automatically provide remote views to an operator which assist in the completion of the task. This assistance increases the productivity of the teleoperation task if the robot controller is responsive to the unpredictable dynamic evolution of the workcell. Behavioral controllers can be utilized to give reactive 'intelligence.' The inherent complex structure of current systems, however, places considerable time overheads on any redesign of the emergent behavior. In industry, where the remote environment and task frequently change, this continual redesign process becomes inefficient. We introduce a novel behavioral controller, based on an 'ego-behavior' architecture, to command an active camera (a camera mounted on a robot) within a remote workcell. Using this ego-behavioral architecture the responses from individual behaviors are rapidly combined to produce an 'intelligent' responsive viewing system. The architecture is single-layered, each behavior being autonomous with no explicit knowledge of the number, description or activity of other behaviors present (if any). This lack of imposed structure decreases the development time as it allows each behavior to be designed and tested independently before insertion into the architecture. The fusion mechanism for the behaviors provides the ability for each behavior to compete and/or co-operate with other behaviors for full or partial control of the viewing active camera. Each behavior continually reassesses this degree of competition or co-operation by measuring its own success in controlling the active camera against pre-defined constraints. The ego-behavioral architecture is demonstrated through simulation and experimentation.

Improving promiscuous mammalian cell entry by the baculovirus AcMNPV

The insect baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) enters many mammalian cell lines, prompting its application as a general eukaryotic gene delivery agent, but the basis of entry is poorly understood. For adherent mammalian cells we show that entry is favoured by low pH and increasing the available cell surface area through transient release from the substratum. Low pH also stimulated baculovirus entry into mammalian cells grown in suspension which, optimally, could reach 90% of the transduced population. The basic loop, residues 268-281, of the viral surface glycoprotein gp64 was required for entry and a tetra mutant with increasing basicity increased entry into a range of mammalian cells. The same mutant failed to plaque in Sf9 cells, instead showing individual cell entry and minimal cell to cell spread, consistent with an altered fusion phenotype. Viruses grown in different insect cells showed different mammalian cell entry efficiencies suggesting additional factors also govern entry.

Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.

Maternally expressed gene1 is a novel maize endosperm transfer cell-specific gene with a maternal parent-of-origin pattern of expression

Growth of the maize (Zea mays) endosperm is tightly regulated by maternal zygotic and sporophytic genes, some of which are subject to a parent-of-origin effect. We report here a novel gene, maternally expressed gene1 (meg1), which shows a maternal parent-of-origin expression pattern during early stages of endosperm development but biallelic expression at later stages. Interestingly, a stable reporter fusion containing the meg1 promoter exhibits a similar pattern of expression. meg1 is exclusively expressed in the basal transfer region of the endosperm. Further, we show that the putatively processed MEG1 protein is glycosylated and subsequently localized to the labyrinthine ingrowths of the transfer cell walls. Hence, the discovery of a parent-of-origin gene expressed solely in the basal transfer region opens the door to epigenetic mechanisms operating in the endosperm to regulate certain aspects of nutrient trafficking from the maternal tissue into the developing seed.

Mycolactone-dependent depletion of endothelial cell thrombomodulin is strongly associated with fibrin deposition in buruli ulcer lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin’s substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells’ ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone’s effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.

Evaluation of the contribution of the placentomal fusion during gestation in cattle

The placenta of mammals is a structure formed by the juxtaposition of the fetal membranes and the maternal tissues. The main function of the placenta is to regulate the physiological interchange between the fetus and the mother as well as to operate as an important endocrine organ during the gestation. The placentomal fusions were characterized throughout gestation of cattle using macroscopic, histological and flow cytometry analyses. Analyzing the cell cycle phases with a flow cytometry, a balance between the G2M phase and apoptosis was observed, suggesting that the placentomal fusions do not interfere in the placentary maturation process, which is a pre-requirement for the fetal-maternal disconnection and the release of fetal membrane. (C) 2011 Elsevier B.V. All rights reserved.