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Buschart A.,University of Bremen | Buschart A.,Center for Infection Research | Sachs S.,University of Bremen | Chen X.,University of Bremen | And 4 more authors.
Molecular Plant-Microbe Interactions | Year: 2012

Azoarcus sp. strain BH72 is an endophytic betaproteobacterium able to colonize rice roots without induction of visible disease symptoms. BH72 possesses one polar flagellum. The genome harbors three copies of putative fliC genes, generally encoding the major structural protein flagellin. It is not clear whether, in endophytic interactions, flagella mediate endophytic competence or act as MAMPs (microbe-asscociated molecular patterns) inducing plant defense responses. Therefore, possible functions of the three FliC proteins were investigated. Only fliC3 was found to be highly expressed in pure culture and in association with rice roots and to be required for bacterial motility, suggesting that it encodes the major flagellin. Endophytic colonization of rice roots was significantly reduced in the in-frame deletion mutant, while the establishment of microcolonies on the root surface was not affected. Moreover, an elicitation of defense responses related to FliC3 was not observed. In conclusion, our data support the hypothesis that FliC3 does not play a major role as a MAMP but is required for endophytic colonization in the Azoarcus-rice interaction, most likely for spreading inside the plant. © 2012 The American Phytopathological Society.


Roth S.D.,Red Cross | Schuttrumpf J.,Red Cross | Schuttrumpf J.,Biomedical Research Institute Georg Speyer Haus | Schuttrumpf J.,Biotest AG | And 8 more authors.
PLoS ONE | Year: 2012

Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking. © 2012 Roth et al.


Brendel C.,Biomedical Research Institute Georg Speyer Haus | Hanseler W.,University of Zürich | Wohlgensinger V.,University of Zürich | Bianchi M.,University of Zürich | And 9 more authors.
Human Gene Therapy Methods | Year: 2013

Targeting transgene expression to specific hematopoietic cell lineages could contribute to the safety of retroviral vectors in gene therapeutic applications. Chronic granulomatous disease (CGD), a defect of phagocytic cells, can be managed by gene therapy, using retroviral vectors with targeted expression to myeloid cells. In this context, we analyzed the myelospecificity of the human miR223 promoter, which is known to be strongly upregulated during myeloid differentiation, to drive myeloid-restricted expression of p47 phox and gp91phox in mouse models of CGD and in primary patient-derived cells. The miR223 promoter restricted the expression of p47 phox, gp91phox, and green fluorescent protein (GFP) within self-inactivating (SIN) gamma- and lentiviral vectors to granulocytes and macrophages, with only marginal expression in lymphocytes or hematopoietic stem and progenitor cells. Furthermore, gene transfer into primary CD34+ cells derived from a p47phox patient followed by ex vivo differentiation to neutrophils resulted in restoration of Escherichia coli killing activity by miR223 promoter-mediated p47phox expression. These results indicate that the miR223 promoter as an internal promoter within SIN gene therapy vectors is able to efficiently correct the CGD phenotype with negligible activity in hematopoietic progenitors, thereby limiting the risk of insertional oncogenesis and development of clonal dominance. © Mary Ann Liebert, Inc.

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