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Blot Experiments (blot + experiment)
Kinds of Blot Experiments Selected AbstractsRANKL Treatment Releases the Negative Regulation of the Poly(ADP-Ribose) Polymerase-1 on Tcirg1 Gene Expression During Osteoclastogenesis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2006Guillaume E Beranger Abstract The Tcirg1 gene encodes the osteoclast-specific a3 isoform of the V-ATPase a subunit. Using the mouse osteoclastic model RAW264.7 cells, we studied Tcirg1 gene expression, and we identified PARP-1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis. Introduction: The TCIRG1 gene encodes the a3 isoform of the V-ATPase a subunit, and mutations at this locus account for ,60% of infantile malignant osteopetrosis cases. Using RAW264.7 cells as an osteoclastic differentiation model, we undertook a transcriptional study of the mouse Tcirg1 gene focused on the 4-kb region upstream of the transcription starting point. Materials and Methods: The promoter activity of serial-deletion fragments of the Tcirg1 gene promoter was monitored throughout the RAW264.7 cell differentiation process. We next performed EMSA, UV cross-linking, affinity purification, mass spectrometry analysis, gel supershift, and siRNA transfection experiments to identify the factor(s) interacting with the promoter. Results: The ,3946/+113 region of the mouse Tcirg1 gene displayed a high basal promoter activity, which was enhanced by RANKL treatment of RAW264.7 cells. Constructs deleted up to ,1589 retained this response to RANKL. A deletion up to ,1402 induced a 3-fold enhancement of the basal activity, whereas RANKL response was not affected. EMSA experiments led us to identify within the ,1589/,1402 region, a 10-nucleotide sequence, which bound a nuclear protein present in nondifferentiated RAW264.7 cells. This interaction was lost using nuclear extracts derived from RANKL-treated cells. Affinity purification followed by mass spectrometry analysis and gel supershift assay allowed the identification of poly(ADP-ribose) polymerase-1 (PARP-1) as this transcriptional repressor, whereas Western blot experiments revealed the cleavage of the DNA-binding domain of PARP-1 on RANKL treatment. Finally, both PARP-1 depletion after siRNA transfection and RAW264.7 cell treatment by an inhibitor of PARP-1 activity induced an increase of a3 mRNA expression. Conclusions: We provide evidence that the basal transcription activity of the Tcirg1 gene is negatively regulated by the binding of PARP-1 protein to its promoter region in mouse pre-osteoclast. On RANKL treatment, PARP-1 protein is cleaved and loses its repression effect, allowing an increase of Tcirg1 gene expression that is critical for osteoclast function. [source] A role for the transcription factor HEY1 in glioblastomaJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 1 2009Esther Hulleman Abstract Glioblastoma multiforme (GBM), the highest-grade glioma, is the most frequent tumour of the brain with a very poor prognosis and limited therapeutic options. Although little is known about the molecular mechanisms that underlie glioblastoma formation, a number of signal transduction routes, such as the Notch and Ras signalling pathways, seem to play an important role in the formation of GBM. In the present study, we show by in situ hybridization on primary tumour material that the transcription factor HEY1, a target of the Notch signalling pathway, is specifically up-regulated in glioma and that expression of HEY1 in GBM correlates with tumour-grade and survival. In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways. Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture. Together, these data imply a role for HEY1 in the progression of GBM, and therefore we propose that HEY1 may be a therapeutic target for glioblastoma patients. Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk. [source] Histamine induces neural stem cell proliferation and neuronal differentiation by activation of distinct histamine receptorsJOURNAL OF NEUROCHEMISTRY, Issue 2 2008Anayansi Molina-Hernández Abstract Histamine has neurotransmitter/neuromodulator functions in the adult brain, but its role during CNS development has been elusive. We studied histamine effects on proliferation, cell death and differentiation of neuroepithelial stem cells from rat cerebral cortex in vitro. RT-PCR and Western blot experiments showed that proliferating and differentiated cells express histamine H1, H2 and H3 receptors. Treatments with histamine concentrations (100 nM,1 mM) caused significant increases in cell numbers without affecting Nestin expression. Cell proliferation was evaluated by BrdU incorporation; histamine caused a significant increase dependent on H2 receptor activation. Apoptotic cell death during proliferation was significantly decreased at all histamine concentrations, and cell death was promoted in a concentration-dependent manner by histamine in differentiated cells. Immunocytochemistry studies showed that histamine increased 3-fold the number of neurons after differentiation, mainly by activation of H1 receptor, and also significantly decreased the glial (astrocytic) cell proportion, when compared to control conditions. In summary, histamine increases cell number during proliferative conditions, and has a neuronal-differentiating action on neural stem cells, suggesting that the elevated histamine concentration reported during development might play a role in cerebrocortical neurogenesis, by activation of H2 receptors to promote proliferation of neural precursors, and favoring neuronal fate by H1 -mediated stimulation. [source] rRNA PROBES FOR IDENTIFICATION AND CHARACTERIZATION OF MARINE PHYTOPLANKTON: THEIR POTENTIAL APPLICATION FOR DNA MICROCHIPSJOURNAL OF PHYCOLOGY, Issue 2001Article first published online: 24 SEP 200 Groben R., Lange, M. & Medlin, L. K. Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany A fast and reliable identification of nano- and picoplankton by light microscopy is often difficult because of the lack of usable morphological characteristics, whereas electron microscopy and biochemical methods are very time consuming. Identification of toxic algae also requires a great deal of taxonomic experrtise so that false positives are not recorded. One solution is to use taxon specific rRNA probes. For this purpose we designed probes for phytoplankton taxa, including toxic algae. These probes were either labelled with Digoxigenin (DIG) and used in DNA dot blot experiments, or labelled with fluorochromes and used in whole-cell hybridisations with fluorescence microscopy or flow cytometric detection. Specific probes could be used over a broad taxonomic range from higher groups (i.e. the class of dinoflagellates) to species level (i.e. Prorocentrum lima). These probes were be used in the EU MAST project AIMS for the development of an automated identification system for marine phytoplankton in combination with flow cytometry and artificial neural networks (ANNs), in the EU MAST DETAL and in the German national project (TEPS) for the development of an early warning system for harmful algal blooms. Results using Digoxigenin (DIG)-labelled probes on picoplankton samples taken from several water bodies indicate that hierarchial re-probing of spotted samples can be achieved and this suggests that probes can be adapted to DNA microchips. Preliminary field results for a hand-held DNA microchip reader are presented. This work was supported by the German BMBF TEPS 03F0161 and the EU AIMS MAS3-CT97-0080 and EU DETAL Q5RS-2000-30778 projects. [source] Detection of the human GPR50 orphan seven transmembrane protein by polyclonal antibodies mapping different epitopesJOURNAL OF PINEAL RESEARCH, Issue 1 2007Hassina Ould Hamouda Abstract:, GPR50 is an orphan seven transmembrane protein related to the melatonin receptor subfamily comprising MT1 and MT2 receptors. In the absence of any known ligand for GPR50, other tools are critical for the characterization of this protein. Here, we describe the generation, purification and characterization of the first rabbit polyclonal antibodies generated against peptides corresponding to the N-terminus, C-terminus and two additional regions within the intracellular tail of GPR50. Immune sera were purified on peptide-antigen affinity columns. Antibodies specifically recognized a GPR50-YFP fusion protein on the plasma membrane of HEK 293 cells in immunofluorescence experiments. In Western blot experiments, the monomeric and dimeric forms of GPR50 were detected as proteins of 66 and 130 kDa, respectively. In addition, these new antibodies were sufficiently sensitive to detect GPR50 in brain slices of the rat pituitary and human hippocampus. In conclusion, we successfully produced antibodies against the orphan GPR50 protein that will become valuable tools for functional studies of this protein. [source] Molecular analysis of resistance mechanisms to Orobanche cumana in sunflowerPLANT PATHOLOGY, Issue 3 2007P. Letousey Resistance to the dicotyledenous parasite Orobanche cumana in sunflower is characterized by a low number of parasitic attachments and a confinement of the parasite in host tissues leading to its necrosis. To help understand what determines such resistance mechanisms, molecular, biochemical and histological approaches were employed before (early response) and after (late response) attachment of the broomrape parasite to susceptible (2603) and resistant (LR1) sunflower genotypes. The expression patterns of 11 defence-related genes known to be involved in different metabolic pathways (phenylpropanoids, jasmonate, ethylene) and/or in resistance mechanisms against microorganisms were investigated. RT-PCR and cDNA blot experiments revealed that the resistant genotype exhibited a stronger overall defence response against O. cumana than the susceptible one, involving marker genes of the jasmonate (JA) and salicylic acid (SA) pathways. Among them, the SA-responsive gene, def. (defensin), appeared to be characteristic of LR1 sunflower resistance. However, no JA accumulation and similar SA contents (250,300 ng g,1 FW) were measured by GC/MS in both genotypes, parasitized or not. In addition, three cDNAs, isolated by a suppression-subtractive hybridization, were shown to be strongly induced only in the resistant genotype 8 days post-inoculation, when the first O. cumana attachments occurred. These genes, putatively encoding a methionine synthase, a glutathione S-transferase and a quinone oxidoreductase, might be involved in detoxification of reactive oxygen species, suggesting the occurrence of an oxidative burst during the incompatible interaction. Finally, host cell-wall modifications leading to parasite-confinement were correlated with more intense callose depositions in the resistant genotype, concomitant with over-expression of the callose synthase cDNA HaGSL1. [source] Deregulation of Aurora kinase gene expression in human testicular germ cell tumoursANDROLOGIA, Issue 4 2010E. Baldini Summary The Aurora kinases regulate chromosome segregation and cytokinesis, and alterations in their expression associate with cell malignant transformation. In this study, we demonstrated by qRT-PCR analysis of 14 seminomas that Aurora-A mRNA was, with respect to control tissues, augmented in five of 14 tumour tissues by 2.17 ± 0.30 fold (P < 0.05) and reduced in 9 to 0.38 ± 0.10 (P < 0.01). Aurora-B mRNA was increased in 11 tumour tissues by 4.33 ± 0.82 fold (P < 0.01) and reduced in 3 to 0.41 ± 0.11 fold. Aurora-C mRNA was reduced to 0.20 ± 0.32 fold (P < 0.01) in 13 seminomas and up-regulated in one case. Western blot experiments, performed on protein extracts of nine seminomas and six normal testes, showed an up-regulation of Aurora-B protein by 10.14 ± 3.51 fold (P < 0.05), while Aurora-A protein was found increased in four seminomas by 2.16 ± 0.43 (P < 0.05), unchanged in three and reduced in two tumour tissues. Aurora-C protein was increased by 9.2 ± 2.90 fold (P < 0.05), suggesting that post-transcriptional mechanisms modulate its expression. In conclusion, we demonstrated that expression of Aurora kinases is deregulated in seminomas, suggesting that they may play a role in the progression of testicular cancers. [source] Enhanced Secretion of Heterologous Proteins in Pichia pastoris Following Overexpression of Saccharomyces cerevisiae Chaperone ProteinsBIOTECHNOLOGY PROGRESS, Issue 4 2006Wei Zhang In Pichia pastoris, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). However, upon introduction of foreign proteins, heterologous proteins are often retained in the cytoplasm or in the ER as a result of suboptimal folding conditions, leading to protein aggregation. The Hsp70 and Hsp40 chaperone families in the cytoplasm or in ER importantly regulate the folding and secretion of heterologous proteins. However, it is not clear which single chaperone is most important or which combination optimally cooperates in this process. In the present study we evaluated the role of the chaperones Kar2p, Sec63, YDJ1p, Ssa1p, and PDI from Saccharomyces cerevisiae. We found that the introduction of Kar2p, Ssa1p, or PDI improves protein secretion 4,7 times. In addition, we found that the combination chaperones of YDJ1p/PDI, YDJ1p/Sec63, and Kar2p/PDI synergistically increase secretion levels 8.7, 7.6, and 6.5 times, respectively. Therefore, additional integration of chaperone genes can improve the secretory expression of the heterologous protein. Western blot experiments revealed that the chaperones partly relieved the secretion bottleneck resulting from foreign protein introduction in P. pastoris. Therefore, the findings from the present study demonstrate the presence of a network of chaperones in vivo, which may act synergistically to increase recombinant protein yields. [source] | |||||||||||||