D6 is a promoter/enhancer of the mDach1 gene that is involved in the development of the neocortex and hippocampus. It is expressed by proliferating neural stem/progenitor cells (NSPCs) of the cortex at early stages of neurogenesis. The differentiation potential of NSPCs isolated from embryonic day 12 mouse embryos, in which the expression of green fluorescent protein (GFP) is driven by the D6 promoter/enhancer, has been studied in vitro and after transplantation into the intact adult rat brain as well as into the site of a photochemical lesion. The electrophysiological properties of D6/GFP-derived cells were studied using the whole-cell patch-clamp technique, and immunohistochemical analyses were carried out. D6/GFP-derived neurospheres expressed markers of radial glia and gave rise predominantly to immature neurons and GFAP-positive cells during in vitro differentiation. One week after transplantation into the intact brain or into the site of a photochemical lesion, transplanted cells expressed only neuronal markers. D6/GFP-derived neurons were characterised by the expression of tetrodotoxin-sensitive Na(+)-currents and K (A)- and K (DR) currents sensitive to 4-aminopyridine. They were able to fire repetitive action potentials and responded to the application of GABA. Our results indicate that after transplantation into the site of a photochemical lesion, D6/GFP-derived NSPCs survive and differentiate into neurons, and their membrane properties are comparable to those transplanted into the non-injured cortex. Therefore, region-specific D6/GFP-derived NSPCs represent a promising tool for studying neurogenesis and cell replacement in a damaged cellular environment.

• MeSH
• bikukulin metabolismus   MeSH
• biologické markery metabolismus   MeSH
• blokátory draslíkových kanálů metabolismus   MeSH
• buněčná diferenciace fyziologie   MeSH
• embryo savčí anatomie a histologie   fyziologie   MeSH
• GABA antagonisté metabolismus   MeSH
• GABA metabolismus   MeSH
• kmenové buňky cytologie   fyziologie   MeSH
• krysa rodu rattus MeSH
• metoda terčíkového zámku MeSH
• myši MeSH
• neurony cytologie   fyziologie   MeSH
• promotorové oblasti (genetika) MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• telencefalon cytologie   patologie   fyziologie   MeSH
• transplantace kmenových buněk MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Transcription factors are prominent regulators of gene expression that execute responses to various intracellular and extracellular stimuli. Recombinant transcription reporter systems can be conveniently used to study the DNA binding preferences and regulatory activity of a transcription factor under a range of conditions. Several reporter genes have been used to study transcription regulation in the fission yeast Schizosaccharomyces pombe. Each of these reporters has distinct advantages, such as high sensitivity or ease of use, and limitations, such as prohibitive costs or use of hazardous substances. To combine the strengths and mitigate the weaknesses of individual reporter genes, we have created pREPORT, a flexible multi-readout transcription reporter vector for fission yeast that employs an enhanced GFP-lacZ fusion and a customizable minimal promoter. With pREPORT, gene expression driven by the transcription factor of interest can be quantified in a number of ways, both in live cells and in vitro, using a single reporter construct.

• MeSH
• beta-galaktosidasa analýza   MeSH
• genetické vektory MeSH
• regulace genové exprese u hub * MeSH
• reportérové geny * MeSH
• Schizosaccharomyces pombe - proteiny genetika   metabolismus   MeSH
• Schizosaccharomyces genetika   MeSH
• stanovení celkové genové exprese metody   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• zelené fluorescenční proteiny analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Arabidopsisthaliana pathogenesis-related 1 (PR1) is an important defense protein, so far it has only been detected in extracellular space and its subcellular sorting and transport remain unexplained. Using a green fluorescent protein (GFP) tagged full length, as well as a C-terminus truncated version of PR1, we observed that when expressed ectopically in Nicotiana benthamiana leaves, PR1 co-localizes only partially with Golgi markers, and much more prominently with the late endosome (LE)/multivesicular body (MVB) FYVE marker. The C-truncated version PR1ΔC predominantly localized to the endoplasmic reticulum (ER). The same localizations were found for stable Arabidopsis transformants with expression of PR1 and PR1ΔC driven by the native promoter. We conclude that the A. thaliana PR1 (AtPR1) undergoes an unconventional secretion pathway, starting from the C-terminus-dependent sorting from the ER, and utilizing further transportation via phosphatidyl-inositol-3-phosphate (PI(3)P) positive LE/MVB-like vesicles. The homology model of the PR1 structure shows that the cluster of positively charged amino acid residues (arginines 60, 67, 137, and lysine 135) could indeed interact with negatively charged phospholipids of cellular membranes. It remains to be resolved whether Golgi and LE/MVB localization reflects an alternative sorting or trafficking succession, and what the role of lipid interactions in it will be.

• MeSH
• Arabidopsis metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• endozomy metabolismus   MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• Golgiho aparát metabolismus   MeSH
• konfokální mikroskopie MeSH
• listy rostlin metabolismus   MeSH
• promotorové oblasti (genetika) MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny biosyntéza   genetika   MeSH
• tabák metabolismus   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH