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Selected AbstractsA novel ultra-sensitive method for the quantification of glycosaminoglycan disaccharides using an automated DNA sequencerELECTROPHORESIS, Issue 7 2006Kay Vogel Abstract Analysis of glycosaminoglycans (GAGs) is of increasing importance concerning alterations in extracellular matrix composition and selectivity of glomerular basement membrane. In this report we describe the analysis of chondroitin sulfate disaccharides as an example of GAG ,disaccharide analysis using standard DNA sequencing equipment (DNA sequencer-assisted GAG disaccharide separation, DSA-GAGS). The presented methodology allows nanomolar quantification of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-derived GAG disaccharides. In comparison to RP-HPLC the established method is much more sensitive, showing detection limits of 38,fmol/,L. Variation coefficients were approximately 10%, enabling exact quantifications after run times of 17,min at 30°C and an electrophoresis voltage of 15,kV; using a capillary DNA sequencer, available in many molecular laboratories, presented advantages like automated sample injection, opportunity of high-throughput analyses, separation of even sulfated disaccharide epimers, and the possibility of using APTS-derived fucose as an internal standard. Furthermore, highly reproducible retention times rendered easy identification of specific signals (SD,0.02). With regard to these results, the described method is a useful tool for the quantification of GAG disaccharides in low amounts, indicating advantages of obverse RP-HPLC and slab gel polyacrylamide electrophoresis in sensitivity, error-proneness, automation, and handling. [source] Simple relative space,time scaling of electrical and electromagnetic depth sounding arrays: implications for electrical static shift removal and joint DC-TEM data inversion with the most-squares criterionGEOPHYSICAL PROSPECTING, Issue 4 2005Max A. Meju ABSTRACT A simple scaling relationship is shown to facilitate comparison, correlation and integration of data recorded using the common experimental configurations in electrical and electromagnetic depth sounding. Applications of the scheme to field data from typical geological and landfill environments show that it is robust and, where transient electromagnetic (TEM) data are available, enables easy identification and quantification of electrical static shift (galvanic distortion) in magnetotelluric and direct current (DC) sounding curves. TEM-based procedures are suggested for both the direct removal of static shift in DC sounding curves and effective joint data inversion with the most-squares criterion in the presence of static shift. A case study of aquifer characterization using sounding data from borehole sites in the Vale of York in England shows that static shift is a common problem in this glacial-covered terrain and demonstrates the effectiveness of the proposed joint DC-TEM inversion strategy in handling distorted soundings. [source] A Green's function-based method for the transient analysis of plane waves obliquely incident on lossy and dispersive planar layersINTERNATIONAL JOURNAL OF NUMERICAL MODELLING: ELECTRONIC NETWORKS, DEVICES AND FIELDS, Issue 6 2008Giulio Antonini Abstract This paper presents a new methodology for the transient analysis of plane waves obliquely incident on a planar lossy and dispersive layer. The proposed model is based on the Sturm,Liouville problem associated with the propagation equations. Green's function is calculated in a series form and the open-end impedance matrix is obtained as the sum of infinite rational functions. This form permits an easy identification of poles and residues. Furthermore, the knowledge of poles leads to the development of a model order reduction technique by selecting only the dominant poles of the system. The pole,residue representation is converted into a state-space model that can be easily interfaced with ordinary differential equation solvers. The numerical results confirm the effectiveness of the proposed modeling technique. Copyright © 2008 John Wiley & Sons, Ltd. [source] The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5,-untranslated part of mRNAMICROBIAL BIOTECHNOLOGY, Issue 3 2009Laila Berg Summary Secondary structures and the short Shine,Dalgarno sequence in the 5,-untranslated region of bacterial mRNAs (UTR) are known to affect gene expression at the level of translation. Here we report the use of random combinatorial DNA sequence libraries to study UTR function, applying the strong, ,32/,38 -dependent, and positively regulated Pm promoter as a model. All mutations in the libraries are located at least 8 bp downstream of the transcriptional start site. The libraries were screened using the ampicillin-resistance gene (bla) as reporter, allowing easy identification of UTR mutants that display high levels of expression (up to 20-fold increase relative to the wild-type at the protein level). Studies of the two UTR mutants identified by a modified screening procedure showed that their expression is stimulated to a similar extent at both the transcript and protein product levels. For one such mutant a model analysis of the transcription kinetics showed significant evidence of a difference in the transcription rate (about 18-fold higher than the wild type), while there was no evidence of a difference in transcript stability. The two UTR sequences also stimulated expression from a constitutive ,70 -dependent promoter (P1/Panti-tet), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription. [source] Development of a strategy for transgenic studies and monitoring of transgene expression in two closely related Moricandia species possessing a C3 or C3,C4 intermediate photosynthetic phenotypePHYSIOLOGIA PLANTARUM, Issue 1 2003Vera Thole In order to establish a model system for comparative studies of C3 and C3,C4 intermediate photosynthesis, the development of efficient transformation systems and the monitoring of transgene behaviour and stability were carried out in two closely related Moricandia species (Brassicaceae): the C3,C4 photosynthetic intermediate species M. arvensis and the C3 species M. moricandioides. In this study the green fluorescent protein (gfp) reporter gene was used as a vital marker gene while the use of the , -glucuronidase (gusA) gene was based on the highly sensitive detection of its activity. For Agrobacterium -mediated transformation of leaf explants, a cauliflower mosaic virus 35S promoter-driven, modified version of gfp, the mgfp5-ER gene and the gusA gene, respectively, were introduced into the new dual binary transformation vector system pGreen/pSoup (Hellens et al. 2000, Plant Mol Bio 42: 819,832). GFP5 produced bright-green fluorescence in transformed tissues that was distinctly detected 5,12 days following transformation in developing calli of the two species. Visual screening, combined with antibiotic selection, enabled early and easy identification of transformation events and contributed to improvements in the transformation strategies. Transgene integration studies demonstrated that mgfp5-ER was inserted with low copy number in the M. arvensis plant lines and the transgene was transmitted in a Mendelian fashion to T1 and T2 progenies. GFP5 expression levels in a population of 100 independent primary transformed M. arvensis plant lines (T0) showed great variation between transformation events (coefficient of variation of 108%). The mgfp5-ER or gusA reporter genes were expressed in 90,95% of the kanamycin-resistant M. arvensis plant lines and in up to 98% of the independent M. moricandioides plant lines. [source] Redefining the boundaries of the hippocampal CA2 subfield in the mouse using gene expression and 3-dimensional reconstructionTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 1 2005Edward S. Lein Abstract The morphology of neurons in the main divisions of the hippocampal complex allow the easy identification of granule cells in the dentate gyrus and pyramidal cells in the CA1 and CA3 regions of Ammon's horn. However, neurons in the CA2 subfield have been much more difficult to reliably identify. We have recently identified a set of genes whose expression is restricted to either the dentate gyrus, CA1, CA2, or CA3. Here we show that these genes have an essentially nonoverlapping distribution throughout the entire septotemporal extent of the hippocampus. 3-Dimensional reconstruction of serial sections processed for in situ hybridization of mannosidase 1, alpha (CA1), bcl-2-related ovarian killer protein (CA3), and Purkinje cell protein 4 (dentate gyrus + CA2) was used to define the boundaries of each subregion throughout the entire hippocampus. The boundaries observed for these three genes are recapitulated across a much larger set of genes similarly enriched in specific hippocampal subregions. The extent of CA2 defined on the basis of gene expression is somewhat larger than that previously described on the basis of structural anatomical criteria, particularly at the rostral pole of the hippocampus. These results indicate that, at least at the molecular level, there are robust, consistent genetic boundaries between hippocampal subregions CA1, CA2, CA3, and the dentate gyrus, allowing a redefinition of their boundaries in order to facilitate functional studies of different neuronal subtypes in the hippocampus. J. Comp. Neurol. 485:1,10, 2005. © 2005 Wiley-Liss, Inc. [source] | ||||||||||