Die Rolle von CpG-Dinukleotiden bei der Regulation der Transgenexpression am Beispiel verschiedener Reportergene

DNA-basierte therapeutische Strategien wie z. B. DNA-Vakzinierung oder Gentherapie werden häufig durch ein Silencen der Antigenexpression nachhaltig beeinträchtigt, da der rekombinante Wirkstoff nachfolgend nicht mehr in physiologisch ausreichender Konzentration vorliegt. So ist bekannt, dass die Methylierung von CpG-Dinukleotiden in promotornahen Bereichen einen epigenetischen Regulationsmechanismus darstellt, der in vielen Fällen zu einem Abschalten der Genexpression führt. CpG-freie Genvarianten sollten folglich vor Methylierung und einer damit verbundenen Repression der Transkription geschützt sein. Anders als erwartet wurde in dieser Arbeit für eine CpG-freie Version verschiedener Transgene in vitro jedoch eine schwächere Expressionsleistung als für das CpG-haltige Ausgangskonstrukt detektiert. Darüber hinaus erreichte ein CpG-maximiertes Reportergen gegenüber dem Ausgangskonstrukt eine gesteigerte Proteinexpression. Dieser positive Zusammenhang von CpG-Gehalt im Transgen und resultierender Reporteraktivität manifestierte sich auch auf RNA-Ebene. Dabei konnte eine für das CpG-depletierte Konstrukt beobachtete Verringerung transgenspezifischer RNA-Transkripte weder auf differentielle Kernexportraten noch auf RNA-Instabilität oder das Vorliegen alternativer Spleissprodukte zurückgeführt werden. Vielmehr wurde im Verlauf der Arbeit nachgewiesen, dass sich CpG-freie Transgene bereits durch geringere Mengen de novo synthetisierter RNA-Transkripte auszeichnen. Das beschriebene CpG-Phänomen wurde somit für virale sowie nicht-virale Transgene unabhängig von Zelltyp und Expressionsdauer nachgewiesen, was einen generellen Mechanismus der CpG-basierten Genaktivierung auf transkriptioneller Ebene impliziert. Darüber hinaus wurde in dieser Arbeit eine partiell CpG-reduzierte Vektorplattform mit modularem Aufbau etabliert. In transienten sowie in stabilen Expressionsexperimenten zeigte sich für die CpG-reduzierten Plasmide im Vergleich zu den CpG-haltigen Referenzkonstrukten eine erhöhte Expressionsleistung. Somit stellt die Modifizierung des CpG-Gehaltes in Vektorrückgrat und Transgen einen Ansatzpunkt zur Verbesserung der Expressionseffizienz dar. Folglich sollte diese Strategie bei der rationalen Konzeption DNA-basierter Therapeutika sowie der effektiven Produktion rekombinanter Proteine in Säugerzellen in Betracht gezogen werden

Using Cone Index Data to Explain Yield Variation Within a Field

Rosana G. Moreira, Editor-in-Chief; Texas A&M UniversityThis is a Technical Paper from International Commission of Agricultural Engineering (CIGR, Commission Internationale du Genie Rural) E-Journal Volume 4 (2002): N. Isaac, R. Taylor, S. Staggenborg, M. Schrock, and D. Leikam. Using Cone Index Data to Explain Yield Variation Within a Field. Vol. IV. December 2002

In vitro evidence for senescent multinucleated melanocytes as a source for tumor-initiating cells

Oncogenic signaling in melanocytes results in oncogene-induced senescence (OIS), a stable cell-cycle arrest frequently characterized by a bi- or multinuclear phenotype that is considered as a barrier to cancer progression. However, the long-sustained conviction that senescence is a truly irreversible process has recently been challenged. Still, it is not known whether cells driven into OIS can progress to cancer and thereby pose a potential threat. Here, we show that prolonged expression of the melanoma oncogene N-RAS61K in pigment cells overcomes OIS by triggering the emergence of tumor-initiating mononucleated stem-like cells from senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis-resistant and induces fast growing, metastatic tumors. Our data describe that differentiated cells, which are driven into senescence by an oncogene, use this senescence state as trigger for tumor transformation, giving rise to highly aggressive tumor-initiating cells. These observations provide the first experimental in vitro evidence for the evasion of OIS on the cellular level and ensuing transformation

Dietary Salt Accelerates Orthodontic Tooth Movement by Increased Osteoclast Activity

Dietary salt uptake and inflammation promote sodium accumulation in tissues, thereby modulating cells like macrophages and fibroblasts. Previous studies showed salt effects on periodontal ligament fibroblasts and on bone metabolism by expression of nuclear factor of activated T-cells-5 (NFAT-5). Here, we investigated the impact of salt and NFAT-5 on osteoclast activity and orthodontic tooth movement (OTM). After treatment of osteoclasts without (NS) or with additional salt (HS), we analyzed gene expression and the release of tartrate-resistant acid phosphatase and calcium phosphate resorption. We kept wild-type mice and mice lacking NFAT-5 in myeloid cells either on a low, normal or high salt diet and inserted an elastic band between the first and second molar to induce OTM. We analyzed the expression of genes involved in bone metabolism, periodontal bone loss, OTM and bone density. Osteoclast activity was increased upon HS treatment. HS promoted periodontal bone loss and OTM and was associated with reduced bone density. Deletion of NFAT-5 led to increased osteoclast activity with NS, whereas we detected impaired OTM in mice. Dietary salt uptake seems to accelerate OTM and induce periodontal bone loss due to reduced bone density, which may be attributed to enhanced osteoclast activity. NFAT-5 influences this reaction to HS, as we detected impaired OTM and osteoclast activity upon deletion

A study on <i>Maruca vitrata</i> infestation of Yard-long beans <i>(Vigna unguiculata </i>subspecies <i>sesquipedalis</i>)

AbstractGlobally, Maruca vitrata (Geyer) is a serious yield constraint on food legumes including Yard-long bean (Vigna unguiculata subspecies sesquipedalis). However, there is a dearth of information on its damage potential, distribution and population dynamics in Yard-long beans. In the present study, the level of M. vitrata larval infestation on flowers and pods of Yard-long beans in Sri Lanka was determined with respect to three consecutive cropping seasons, Yala, Off and Maha. Results indicated that larval infestation and abundance varied with developmental stage of flowers and pods, cropping season and their combined interactive effects. Flowers of Yard-long beans were more prone to M. vitrata larval attack compared to pods. Abundance and level of infestation of M. vitrata varied with plant parts, having a ranking of flower buds (highest)>open flowers>mature pods>immature pods (lowest). Peak infestation was observed six and eight weeks after planting on flowers and pods, respectively. Among the three cropping seasons, M. vitrata infestation was found to be higher during Maha and Off seasons compared to Yala. The findings of this study contribute to the identified knowledge gap regarding the field biology of an acknowledged important pest, M. vitrata, in a previously understudied crop in Sri Lanka

Impact of salt and the osmoprotective transcription factor NFAT-5 on macrophages during mechanical strain

Myeloid cells regulate bone density in response to increased salt (NaCl) intake via the osmoprotective transcription factor, nuclear factor of activated T cells-5 (NFAT-5). Because orthodontic tooth movement (OTM) is a pseudoinflammatory immunological process, we investigated the influence of NaCl and NFAT-5 on the expression pattern of macrophages in a model of simulated OTM. RAW264.7 macrophages were exposed for 4 h to 2 ⁻²g cm compressive or 16% tensile or no mechanical strain (control), with or without the addition of 40 mM NaCl. We analyzed the expression of inflammatory genes and proteins [tumor necrosis factor (TNF), interleukin (IL)-6 and prostaglandin endoperoxide synthase-2 (Ptgs-2)/prostaglandin E2 (PG-E2)] by real-time-quantitative PCR and ELISA. To investigate the role of NFAT-5 in these responses, NFAT-5 was both constitutively expressed and silenced. Salt and compressive strain, but not tensile strain increased the expression of NFAT-5 and most tested inflammatory factors in macrophages. NaCl induced the expression of Ptgs-2/PG-E2 and TNF, whereas secretion of IL-6 was inhibited. Similarly, a constitutive expression of NFAT-5 reduced IL-6 expression, while increasing Ptgs-2/PG-E2 and TNF expression. Silencing of NFAT-5 upregulated IL-6 and reduced Ptgs-2/PG-E2 and TNF expression. Salt had an impact on the expression profile of macrophages as a reaction to compressive and tensile strain that occur during OTM. This was mediated via NFAT-5, which surprisingly also seems to play a regulatory role in mechanotransduction of compressive strain. Sodium accumulation in the periodontal ligament caused by dietary salt consumption might propagate local osteoclastogenesis via increased local inflammation and thus OTM velocity, but possibly also entail side effects such as dental root resorptions or periodontal bone loss

Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

<p>Abstract</p> <p>Background</p> <p>Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the <it>Xiphophorus </it>melanoma model system, a mutated version of the EGF receptor Xmrk (<it>Xiphophorus </it>melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation.</p> <p>Methods</p> <p>Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene <it>FOSL1 </it>was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated.</p> <p>Results</p> <p>Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (<it>Fosl1</it>), early growth response 1 (<it>Egr1</it>), osteopontin (<it>Opn</it>), insulin-like growth factor binding protein 3 (<it>Igfbp3</it>), dual-specificity phosphatase 4 (<it>Dusp4</it>), and tumor-associated antigen L6 (<it>Taal6</it>). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that <it>FOSL1</it>, <it>OPN</it>, <it>IGFBP3</it>, <it>DUSP4</it>, and <it>TAAL6 </it>also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of <it>FOSL1 </it>in human melanoma cell lines reduced their proliferation and migration.</p> <p>Conclusion</p> <p>Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development. Specifically, a role of FOSL1 in melanomagenic processes is demonstrated. These data are the basis for future detailed analyses of the investigated target genes.</p

Conserved Expression Signatures between Medaka and Human Pigment Cell Tumors

Aberrations in gene expression are a hallmark of cancer cells. Differential tumor-specific transcript levels of single genes or whole sets of genes may be critical for the neoplastic phenotype and important for therapeutic considerations or useful as biomarkers. As an approach to filter out such relevant expression differences from the plethora of changes noted in global expression profiling studies, we searched for changes of gene expression levels that are conserved. Transcriptomes from massive parallel sequencing of different types of melanoma from medaka were generated and compared to microarray datasets from zebrafish and human melanoma. This revealed molecular conservation at various levels between fish models and human tumors providing a useful strategy for identifying expression signatures strongly associated with disease phenotypes and uncovering new melanoma molecules