TY - JOUR A1 - Yin, Jun A1 - Brocher, Jan A1 - Fischer, Utz A1 - Winkler, Christoph T1 - Mutant Prpf31 causes pre-mRNA splicing defects and rod photoreceptor cell degeneration in a zebrafish model for Retinitis pigmentosa JF - Molecular neurodegeneration N2 - Background: Retinitis pigmentosa (RP) is an inherited eye disease characterized by the progressive degeneration of rod photoreceptor cells. Mutations in pre-mRNA splicing factors including PRPF31 have been identified as cause for RP, raising the question how mutations in general factors lead to tissue specific defects. Results: We have recently shown that the zebrafish serves as an excellent model allowing the recapitulation of key events of RP. Here we use this model to investigate two pathogenic mutations in PRPF31, SP117 and AD5, causing the autosomal dominant form of RP. We show that SP117 leads to an unstable protein that is mislocalized to the rod cytoplasm. Importantly, its overexpression does not result in photoreceptor degeneration suggesting haploinsufficiency as the underlying cause in human RP patients carrying SP117. In contrast, overexpression of AD5 results in embryonic lethality, which can be rescued by wild-type Prpf31. Transgenic retina-specific expression of AD5 reveals that stable AD5 protein is initially localized in the nucleus but later found in the cytoplasm concurrent with progressing rod outer segment degeneration and apoptosis. Importantly, we show for the first time in vivo that retinal transcripts are wrongly spliced in adult transgenic retinas expressing AD5 and exhibiting increased apoptosis in rod photoreceptors. Conclusion: Our data suggest that distinct mutations in Prpf31 can lead to photoreceptor degeneration through different mechanisms, by haploinsufficiency or dominant-negative effects. Analyzing the AD5 effects in our animal model in vivo, our data imply that aberrant splicing of distinct retinal transcripts contributes to the observed retina defects. KW - Factor gene PRPF31 KW - TRI-SNRNP KW - Transgenic zebrafish KW - Homebox gene KW - Chinese family KW - Mutations KW - RP11 KW - Expression KW - Disease KW - Protein KW - Retinitis pigmentosa (RP) KW - PRPF31 KW - AD5 mutation KW - SP117 mutation KW - haploinsufficiency KW - dominant-negative KW - rod degeneration KW - apoptosis KW - splicing defect Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-141090 VL - 6 IS - 56 ER - TY - JOUR A1 - Wedel, Steffen A1 - Hudak, Lukasz A1 - Seibel, Jens-Michael A1 - Makarevic, Jasmina A1 - Juengel, Eva A1 - Tsaur, Igor A1 - Waaga-Gasser, Ana A1 - Haferkamp, Axel A1 - Blaheta, Roman A. T1 - Molecular targeting of prostate cancer cells by a triple drug combination down-regulates integrin driven adhesion processes, delays cell cycle progression and interferes with the cdk-cyclin axis JF - BMC Cancer N2 - Background: Single drug use has not achieved satisfactory results in the treatment of prostate cancer, despite application of increasingly widespread targeted therapeutics. In the present study, the combined impact of the mammalian target of rapamycin (mTOR)-inhibitor RAD001, the dual EGFr and VGEFr tyrosine kinase inhibitor AEE788 and the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on prostate cancer growth and adhesion in vitro was investigated. Methods: PC-3, DU-145 and LNCaP cells were treated with RAD001, AEE788 or VPA or with a RAD-AEE-VPA combination. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT-assay, flow cytometry and western blotting, respectively. Furthermore, tumor cell adhesion to vascular endothelium or to immobilized extracellular matrix proteins as well as migratory properties of the cells was evaluated, and integrin alpha and beta subtypes were analyzed. Finally, effects of drug treatment on cell signaling pathways were determined. Results: All drugs, separately applied, reduced tumor cell adhesion, migration and growth. A much stronger anticancer effect was evoked by the triple drug combination. Particularly, cdk1, 2 and 4 and cyclin B were reduced, whereas p27 was elevated. In addition, simultaneous application of RAD001, AEE788 and VPA altered the membranous, cytoplasmic and gene expression pattern of various integrin alpha and beta subtypes, reduced integrin-linked kinase (ILK) and deactivated focal adhesion kinase (FAK). Signaling analysis revealed that EGFr and the downstream target Akt, as well as p70S6k was distinctly modified in the presence of the drug combination. Conclusions: Simultaneous targeting of several key proteins in prostate cancer cells provides an advantage over targeting a single pathway. Since strong anti-tumor properties became evident with respect to cell growth and adhesion dynamics, the triple drug combination might provide progress in the treatment of advanced prostate cancer. KW - Growth-factor receptor KW - Mammalian target KW - Radical prostatectomy KW - Up-regulation KW - C-MYC KW - Pathway KW - Expression KW - Activation KW - Inhibition KW - Apoptosis Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-141075 VL - 11 IS - 375 ER - TY - JOUR A1 - Song, Ning-Ning A1 - Xiu, Jian-Bo A1 - Huang, Ying A1 - Chen, Jia-Yin A1 - Zhang, Lei A1 - Gutknecht, Lise A1 - Lesch, Klaus Peter A1 - Li, He A1 - Ding, Yu-Qiang T1 - Adult Raphe-Specific Deletion of Lmx1b Leads to Central Serotonin Deficiency JF - PLoS ONE N2 - The transcription factor Lmx1b is essential for the differentiation and survival of central serotonergic (5-HTergic) neurons during embryonic development. However, the role of Lmx1b in adult 5-HTergic neurons is unknown. We used an inducible Cre-LoxP system to selectively inactivate Lmx1b expression in the raphe nuclei of adult mice. Pet1-CreER(T2) mice were generated and crossed with Lmx1b(flox/flox) mice to obtain Pet1-CreER(T2); Lmx1b(flox/flox) mice (which termed as Lmx1b iCKO). After administration of tamoxifen, the level of 5-HT in the brain of Lmx1b iCKO mice was reduced to 60% of that in control mice, and the expression of tryptophan hydroxylase 2 (Tph2), serotonin transporter (Sert) and vesicular monoamine transporter 2 (Vmat2) was greatly down-regulated. On the other hand, the expression of dopamine and norepinephrine as well as aromatic L-amino acid decarboxylase (Aadc) and Pet1 was unchanged. Our results reveal that Lmx1b is required for the biosynthesis of 5-HT in adult mouse brain, and it may be involved in maintaining normal functions of central 5-HTergic neurons by regulating the expression of Tph2, Sert and Vmat2. KW - Molecular-genetics KW - Sonic hedgehog KW - Neurons KW - Mice KW - Brain KW - Expression KW - System KW - PET-1 KW - Transporter KW - Disorders Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133581 VL - 6 IS - 1 ER - TY - JOUR A1 - Schierack, Peter A1 - Kleta, Sylvia A1 - Tedin, Karsten A1 - Babila, Julius Tachu A1 - Oswald, Sibylle A1 - Oelschlaeger, Tobias A. A1 - Hiemann, Rico A1 - Paetzold, Susanne A1 - Wieler, Lothar H. T1 - E. coli Nissle 1917 Affects Salmonella Adhesion to Porcine Intestinal Epithelial Cells JF - PLoS ONE N2 - Background: The probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known. Methodology/Principal Findings: In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra-and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN. Conclusions: We propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion. KW - Nonpathogenic Escherichia-coli KW - Enterica serovar typhimurium KW - Strain nissle-1917 KW - In-vitro KW - Invasion genes KW - Diarrhea KW - Growth KW - Expression KW - Infection KW - PPGPP Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135298 VL - 6 IS - 2 ER - TY - JOUR A1 - Rauert, H. A1 - Stühmer, T. A1 - Bargou, R. A1 - Wajant, H. A1 - Siegmund, D. T1 - TNFR1 and TNFR2 regulate the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms JF - Cell Death and Disease N2 - The huge majority of myeloma cell lines express TNFR2 while a substantial subset of them failed to show TNFR1 expression. Stimulation of TNFR1 in the TNFR1-expressing subset of MM cell lines had no or only a very mild effect on cellular viability. Surprisingly, however, TNF stimulation enhanced cell death induction by CD95L and attenuated the apoptotic effect of TRAIL. The contrasting regulation of TRAIL- and CD95L-induced cell death by TNF could be traced back to the concomitant NFjBmediated upregulation of CD95 and the antiapoptotic FLIP protein. It appeared that CD95 induction, due to its strength, overcompensated a rather moderate upregulation of FLIP so that the net effect of TNF-induced NFjB activation in the context of CD95 signaling is pro-apoptotic. TRAIL-induced cell death, however, was antagonized in response to TNF because in this context only the induction of FLIP is relevant. Stimulation of TNFR2 in myeloma cells leads to TRAF2 depletion. In line with this, we observed cell death induction in TNFR1-TNFR2-costimulated JJN3 cells. Our studies revealed that the TNF-TNF receptor system adjusts the responsiveness of the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms that generate a highly context-dependent net effect on myeloma cell survival KW - apoptosis KW - CD95 KW - multiple myeloma KW - NFkB KW - TNF KW - TRAIL KW - NF-Kappa-B KW - Tumor-necrosis-factor KW - Factor receptor KW - Factor-alpha KW - Activation KW - Polymorphisms KW - Inhibitor KW - Promoter KW - Transcription KW - Expression Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133486 VL - 2 ER - TY - JOUR A1 - Haddad, Dana A1 - Chen, Nanhai G. A1 - Zhang, Qian A1 - Chen, Chun-Hao A1 - Yu, Yong A. A1 - Gonzalez, Lorena A1 - Carpenter, Susanne G. A1 - Carson, Joshua A1 - Au, Joyce A1 - Mittra, Arjun A1 - Gonen, Mithat A1 - Zanzonico, Pat B. A1 - Fong, Yuman A1 - Szalay, Aladar A. T1 - Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus JF - Journal of Translational Medicine N2 - Introduction: Oncolytic viruses show promise for treating cancer. However, to assess therapeutic efficacy and potential toxicity, a noninvasive imaging modality is needed. This study aimed to determine if insertion of the human sodium iodide symporter (hNIS) cDNA as a marker for non-invasive imaging of virotherapy alters the replication and oncolytic capability of a novel vaccinia virus, GLV-1h153. Methods: GLV-1h153 was modified from parental vaccinia virus GLV-1h68 to carry hNIS via homologous recombination. GLV-1h153 was tested against human pancreatic cancer cell line PANC-1 for replication via viral plaque assays and flow cytometry. Expression and transportation of hNIS in infected cells was evaluated using Westernblot and immunofluorescence. Intracellular uptake of radioiodide was assessed using radiouptake assays. Viral cytotoxicity and tumor regression of treated PANC-1tumor xenografts in nude mice was also determined. Finally, tumor radiouptake in xenografts was assessed via positron emission tomography (PET) utilizing carrier-free (124)I radiotracer. Results: GLV-1h153 infected, replicated within, and killed PANC-1 cells as efficiently as GLV-1h68. GLV-1h153 provided dose-dependent levels of hNIS expression in infected cells. Immunofluorescence detected transport of the protein to the cell membrane prior to cell lysis, enhancing hNIS-specific radiouptake (P < 0.001). In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts (P < 0.001). Finally, intratumoral injection of GLV-1h153 facilitated imaging of virus replication in tumors via (124)I-PET. Conclusion: Insertion of the hNIS gene does not hinder replication or oncolytic capability of GLV-1h153, rendering this novel virus a promising new candidate for the noninvasive imaging and tracking of oncolytic viral therapy. KW - Human Sodium/Iodide symporter KW - Reporter gene KW - NA+/I-symporter KW - Nude-mice KW - Cancer KW - In-Vivo KW - Expression KW - Therapy KW - Transporter KW - GLV-1H68 Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-140847 VL - 9 IS - 36 ER - TY - JOUR A1 - Enjuanes, Anna A1 - Fernandez, Veronica A1 - Hernandez, Luis A1 - Navarro, Alba A1 - Bea, Silvia A1 - Pinyol, Magda A1 - Lopez-Guillermo, Armando A1 - Rosenwald, Andreas A1 - Ott, German A1 - Campo, Elias A1 - Jares, Pedro T1 - Identification of Methylated Genes Associated with Aggressive Clinicopathological Features in Mantle Cell Lymphoma JF - PLoS ONE N2 - Background: Mantle cell lymphoma (MCL) is genetically characterized by the t(11; 14)(q13; q32) translocation and a high number of secondary chromosomal alterations. The contribution of DNA methylation to MCL lymphomagenesis is not well known. We sought to identify epigenetically silenced genes in these tumours that might have clinical relevance. Methodology/Principal Findings: To identify potential methylated genes in MCL we initially investigated seven MCL cell lines treated with epigenetic drugs and gene expression microarray profiling. The methylation status of selected candidate genes was validated by a quantitative assay and subsequently analyzed in a series of primary MCL (n = 38). After pharmacological reversion we identified 252 potentially methylated genes. The methylation analysis of a subset of these genes (n = 25) in the MCL cell lines and normal B lymphocytes confirmed that 80% of them were methylated in the cell lines but not in normal lymphocytes. The subsequent analysis in primary MCL identified five genes (SOX9, HOXA9, AHR, NR2F2, and ROBO1) frequently methylated in these tumours. The gene methylation events tended to occur in the same primary neoplasms and correlated with higher proliferation, increased number of chromosomal abnormalities, and shorter survival of the patients. Conclusions: We have identified a set of genes whose methylation degree and gene expression levels correlate with aggressive clinicopathological features of MCL. Our findings also suggest that a subset of MCL might show a CpG island methylator phenotype (CIMP) that may influence the behaviour of the tumours. KW - Histone deacetylase inhibition KW - Genome wide analysis KW - Molecular pathogenesis KW - DNA hypermethylation KW - Breast-cancer KW - Lung-cancer KW - Promoter KW - Expression KW - Targets KW - Sox9 Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-140632 VL - 6 IS - 5 ER - TY - JOUR A1 - Binder, Andreas A1 - May, Denisa A1 - Baron, Ralf A1 - Maier, Christoph A1 - Tölle, Thomas R. A1 - Treede, Rolf-Detlef A1 - Berthele, Achim A1 - Faltraco, Frank A1 - Flor, Herta A1 - Gierthmühlen, Janne A1 - Haenisch, Sierk A1 - Huge, Volker A1 - Magerl, Walter A1 - Maihöfner, Christian A1 - Richter, Helmut A1 - Rolke, Roman A1 - Scherens, Andrea A1 - Üçeyler, Nurcan A1 - Ufer, Mike A1 - Wasner, Gunnar A1 - Zhu, Jihong A1 - Cascorbi, Ingolf T1 - Transient Receptor Potential Channel Polymorphisms Are Associated with the Somatosensory Function in Neuropathic Pain Patients JF - PLoS ONE N2 - Transient receptor potential channels are important mediators of thermal and mechanical stimuli and play an important role in neuropathic pain. The contribution of hereditary variants in the genes of transient receptor potential channels to neuropathic pain is unknown. We investigated the frequency of transient receptor potential ankyrin 1, transient receptor potential melastin 8 and transient receptor potential vanilloid 1 single nucleotide polymorphisms and their impact on somatosensory abnormalities in neuropathic pain patients. Within the German Research Network on Neuropathic Pain (Deutscher Forscbungsverbund Neuropathischer Schmerz) 371 neuropathic pain patients were phenotypically characterized using standardized quantitative sensory testing. Pyrosequencing was employed to determine a total of eleven single nucleotide polymorphisms in transient receptor potential channel genes of the neuropathic pain patients and a cohort of 253 German healthy volunteers. Associations of quantitative sensory testing parameters and single nucleotide polymorphisms between and within groups and subgroups, based on sensory phenotypes, were analyzed. Single nucleotide polymorphisms frequencies did not differ between both the cohorts. However, in neuropathic pain patients transient receptor potential ankyrin 1 710G>A (rs920829, E179K) was associated with the presence of paradoxical heat sensation (p=0.03), and transient receptor potential vanilloid 1 1911A>G (rs8065080, I585V) with cold hypoalgesia (p=0.0035). Two main subgroups characterized by preserved (1) and impaired (2) sensory function were identified. In subgroup 1 transient receptor potential vanilloid 1 1911A>G led to significantly less heat hyperalgesia, pinprick hyperalgesia and mechanical hypaesthesia (p=0.006, p=0.005 and p<0.001) and transient receptor potential vanilloid 1 1103C>G (rs222747, M315I) to cold hypaesthesia (p=0.002), but there was absence of associations in subgroup 2. In this study we found no evidence that genetic variants of transient receptor potential channels are involved in the expression of neuropathic pain, but transient receptor potential channel polymorphisms contributed significantly to the somatosensory abnormalities of neuropathic pain patients. KW - Paradoxical heat sensation KW - Neurogenic inflammation KW - Capsaicin receptor KW - TRP Channels KW - Cold KW - Mechanisms KW - Hyperalgesia KW - Sensitivity KW - Expression KW - Stimuli Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-142782 VL - 6 IS - 3 ER - TY - JOUR A1 - Bergmiller, Tobias A1 - Pena-Miller, Rafael A1 - Boehm, Alexander A1 - Ackermann, Martin T1 - Single-cell time-lapse analysis of depletion of the universally conserved essential protein YgjD JF - BMC Microbiology N2 - Background: The essential Escherichia coli gene ygjD belongs to a universally conserved group of genes whose function has been the focus of a number of recent studies. Here, we put ygjD under control of an inducible promoter, and used time-lapse microscopy and single cell analysis to investigate the phenotypic consequences of the depletion of YgjD protein from growing cells. Results: We show that loss of YgjD leads to a marked decrease in cell size and termination of cell division. The transition towards smaller size occurs in a controlled manner: cell elongation and cell division remain coupled, but cell size at division decreases. We also find evidence that depletion of YgjD leads to the synthesis of the intracellular signaling molecule (p) ppGpp, inducing a cellular reaction resembling the stringent response. Concomitant deletion of the relA and spoT genes - leading to a strain that is uncapable of synthesizing (p) ppGpp abrogates the decrease in cell size, but does not prevent termination of cell division upon YgjD depletion. Conclusions: Depletion of YgjD protein from growing cells leads to a decrease in cell size that is contingent on (p) ppGpp, and to a termination of cell division. The combination of single-cell time-lapse microscopy and statistical analysis can give detailed insights into the phenotypic consequences of the loss of essential genes, and can thus serve as a new tool to study the function of essential genes. KW - Transfer-RNA modification KW - Escherichia-coli K-12 KW - Gene KW - Division KW - Expression KW - Inactivation KW - Maintenance KW - Growth KW - Level KW - Ftsz Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-142324 VL - 11 IS - 118 ER -