Tissue-specific localization of gibberellins and expression of gibberellin-biosynthetic and signaling genes in wood-forming tissues in aspen

Bioactive gibberellins (GAs) are known regulators of shoot growth and development in plants. In an attempt to identify where GAs are formed, we have analyzed the expression patterns of six GA biosynthesis genes and two genes with predicted roles in GA signaling and responses in relation to measured levels of GAs. The analysis was based on tangential sections, giving tissue-specific resolution across the cambial region of aspen trees (Populus tremula). Gibberellin quantification by GC/MS-SRM showed that the bioactive GA1 and GA4 were predominantly located in the zone of expansion of xylem cells. Based on co-localization of the expression of the late GA biosynthesis gene GA 20-oxidase 1 and bioactive GAs, we suggest that de novo GA biosynthesis occurs in the expanding xylem. However, expression levels of the first committed GA biosynthesis enzyme, ent-copalyl diphosphate synthase, were high in the phloem, suggesting that a GA precursor(s) may be transported to the xylem. The expression of the GA signaling and response genes DELLA-like1 and GIP-like1 coincided well with sites of high bioactive GA levels. We therefore suggest that the main role of GA during wood formation is to regulate early stages of xylem differentiation, including cell elongation.     

In-vitro analysis of the expression of TGFbeta -superfamily-members during chondrogenic differentiation of mesenchymal stem cells and chondrocytes during dedifferentiation in cell culture

Traditional surgical methods for the reconstruction of cartilage defects rely on the transplantation of autologous and allogenous tissues. The disadvantages of these techniques are the limited availability of suitable tissues and the donor site morbidity of transplants. In addition, in cultured chondrocytes, the dedifferentiation of cells seems unavoidable during multiplication. In this study, we investigated the expression of distinct markers during the dedifferentiation of human chondrocytes (HC) and human mesenchymal stem cells (MSC) in cell culture using microarray technique, immunohistochemistry and RT-PCR. Transforming growth factor beta (TGFbeta) is a multifunctional peptide that plays play a crucial role in inducing and maintaining chondrogenic differentiation. In dedifferentiating chondrocytes, the gene for TGFbeta1 was constantly expressed, while the gene for TGFbeta2 was never expressed. The genes for TGFalpha, TGFbeta4 and TGFbetai were activated with ongoing dedifferentiation. TGFbeta-receptor 3 was constantly expressed, while the genes for the TGFbeta-receptors 1 and 2 were never expressed. Immunohistochemical staining for TGFbeta beta 3 revealed upregulation in the course of dedifferentiation. The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation, whereas the gene for LTBP3 was constantly expressed, and negative results were obtained for the gene for LTBP4. The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation. During chondrogenic differentiation, the MSCs constantly expressed TGFbeta1, beta2, beta3 and beta4. LTBP1, LTBP2 and TGFbeta-R3 were downregulated. In conclusion, TGFbeta3, TGFbeta4, TGFbetai, LTBP1 and LTBP2 may assist the process of dedifferentiation, while TGFbeta1 and beta2 might not be involved in this process. Of the TGFbeta-receptors, only type 3 might be involved in dedifferentiation.     

Plasmacytoid dendritic cells control TLR7 sensitivity of naive B cells via type I IFN

Detailed information of human B cell activation via TLR may lead to a better understanding of B cell involvement in autoimmunity and malignancy. In this study we identified a fundamental difference in the regulation of TLR7- and TLR9-mediated B cell stimulation: whereas the induction of polyclonal naive B cell proliferation by the TLR7 ligands resiquimod (R848) and loxoribine required the presence of plasmacytoid dendritic cells (PDCs), activation via the TLR9 ligand CpG was independent of PDCs. We found that PDC-derived type I IFN enhanced TLR7 sensitivity of B cells by selectively up-regulating TLR7 expression. In contrast the expression levels of TLR9 and of other TLRs studied remained unchanged. In the presence of type I IFN, TLR7 ligation triggered polyclonal B cell expansion and B cell differentiation toward Ig-producing plasma cells; notably, this occurred independently of T cell help and B cell Ag. Human B cells did not respond to ligands of other TLRs including TLR2, TLR4 and TLR6 with and without type I IFN. In conclusion, our results reveal a distinct regulation of TLR7 and TLR9 function in human B cells and highlight TLR7 and TLR9 as unique targets for therapeutic intervention in B cell-mediated immunity and disease.     

Integrating phylogenetics and environmental niche models to explore speciation mechanisms in dendrobatid frogs

We developed an approach that combines distribution data, environmental geographic information system layers, environmental niche models, and phylogenetic information to investigate speciation processes. We used Ecuadorian frogs of the family Dendrobatidae to illustrate our methodology. For dendrobatids there are several cases for which there is significant environmental divergence for allopatric and parapatric lineages. The consistent pattern that many related taxa or nodes exist in distinct environmental space reinforces Lynch and Duellman's hypothesis that differential selection likely played an important role in species differentiation of frogs in the Andes. There is also some evidence that the Río Esmeraldas basin is a geographic barrier to species distributed in low to middle elevations on the western side of the Andes. Another useful aspect of this approach is that it can point to common environmental parameters that correlate with speciation. For dendrobatids, sister clades generally segregate along temperature/elevational and/or seasonality axes. The joint analysis of environmental and geographic data for this group of dendrobatid frogs has identified potentially important speciation mechanisms and specific sister lineages that warrant intensive study to test hypotheses generated in this investigation. Further, the method outlined in this paper will be increasingly useful as knowledge of distribution and phylogeny of tropical species increases.     

A strategy for modelling dynamic responses in metabolic samples characterized by GC/MS

A multivariate strategy for studying the metabolic response over time in urinary GC/MS data is presented and exemplified by a study of drug-induced liver toxicity in the rat. The strategy includes the generation of representative data through hierarchical multivariate curve resolution (H-MCR), highlighting the importance of obtaining resolved metabolite profiles for quantification and identification of exogenous (drug related) and endogenous compounds (potential biomarkers) and for allowing reliable comparisons of multiple samples through multivariate projections. Batch modelling was used to monitor and characterize the normal (control) metabolic variation over time as well as to map the dynamic response of the drug treated animals in relation to the control. In this way treatment related metabolic responses over time could be detected and classified as being drug related or being potential biomarkers. In summary the proposed strategy uses the relatively high sensitivity and reproducibility of GC/MS in combination with efficient multivariate curve resolution and data analysis to discover individual markers of drug metabolism and drug toxicity. The presented results imply that the strategy can be of great value in drug toxicity studies for classifying metabolic markers in relation to their dynamic responses as well as for biomarker identification.     

Prolyl Hydroxylase Domain (PHD) 2 Affects Cell Migration and F-actin Formation via RhoA/Rho-associated Kinase-dependent Cofilin Phosphorylation

Cells are responding to hypoxia via prolyl-4-hydroxylase domain (PHD) enzymes, which are responsible for oxygen-dependent hydroxylation of the hypoxia-inducible factor (HIF)-1α subunit. To gain further insight into PHD function, we generated knockdown cell models for the PHD2 isoform, which is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. Induction of a PHD2 knockdown in tetracycline-inducible HeLa PHD2 knockdown cells resulted in increased F-actin formation as detected by phalloidin staining. A similar effect could be observed in the stably transfected PHD2 knockdown cell clones 1B6 and 3B7. F-actin is at least in part responsible for shaping cell morphology as well as regulating cell migration. Cell migration was impaired significantly as a consequence of PHD2 knockdown in a scratch assay. Mechanistically, PHD2 knockdown resulted in activation of the RhoA (Ras homolog gene family member A)/Rho-associated kinase pathway with subsequent phosphorylation of cofilin. Because cofilin phosphorylation impairs its actin-severing function, this may explain the F-actin phenotype, thereby providing a functional link between PHD2-dependent signaling and cell motility.     

MASQOT-GUI: spot quality assessment for the two-channel microarray platform

MASQOT-GUI provides an open-source, platform-independent software pipeline for two-channel microarray spot quality control. This includes gridding, segmentation, quantification, quality assessment and data visualization. It hosts a set of independent applications, with interactions between the tools as well as import and export support for external software. The implementation of automated multivariate quality control assessment, which is a unique feature of MASQOT-GUI, is based on the previously documented and evaluated MASQOT methodology. Further abilities of the application are outlined and illustrated. AVAILABILITY: MASQOT-GUI is Java-based and licensed under the GNU LGPL. Source code and installation files are available for download at http://masqot-gui.sourceforge.net/     

Pastoralist Responses to Floodplain Rehabilitation in North Cameroon

This paper examines the responses of mobile pastoralists to a floodplain rehabilitation program in north Cameroon. From 1993 to 1999, we measured changes in number of camps and herds, and the time they spent in the 600 km² of the Logone floodplain that was reflooded in 1994. The first year, few pastoralists anticipated the reflooding or its impact, and the increase in grazing intensity was caused by a prolonged stay of pastoralists who already used the area for transit. The following three years showed a sharp increase in the number of camps and herds, which stabilized from 1997 onwards. Overall, grazing intensity increased threefold, following the gradually recovering perennial grasslands, with no signs of overexploitation of the area. These developments closely match the ideal preemptive distribution model. We also examined how reflooding affected pastoral incursions in the Waza National Park located in the floodplain.

Identification of relevant ICF categories for indication, intervention planning and evaluation of health resort programs: a Delphi exercise

Health resort programs have a long tradition, mainly in European countries and Japan. They rely on local resources and the physical environment, physical medicine interventions and traditional medicine to optimise functioning and health. Arguably because of the long tradition, there is only a limited number of high-quality studies that examine the effectiveness of health resort programs. Specific challenges to the evaluation of health resort programs are to randomise the holistic approach with a varying number of specific interventions but also the reliance on the effect of the physical environment. Reference standards for the planning and reporting of health resort studies would be highly beneficial. With the International Classification of Functioning Disability and Health (ICF), we now have such a standard that allows us to describe body functions and structures, activities and participation and interaction with environmental factors. A major challenge when applying the ICF in practice is its length. Therefore, the objective of this project was to identify the ICF categories most relevant for health resort programs. We conducted a consensus-building, three-round, e-mail survey using the Delphi technique. Based on the consensus of the experts, it was possible to come up with an ICF Core Set that can serve as reference standards for the indication, intervention planning and evaluation of health resort programs. This preliminary ICF Core Set should be tested in different regions and in subsets of health resort visitors with varying conditions.