Isoprenoid cytokinins play a number of crucial roles in the regulation of plant growth and development. To study cytokinin receptor properties in plants, we designed and prepared fluorescent derivatives of 6-[(3-methylbut-2-en-1-yl)amino]purine (N6-isopentenyladenine, iP) with several fluorescent labels attached to the C2 or N9 atom of the purine moiety via a 2- or 6-carbon linker. The fluorescent labels included dansyl (DS), fluorescein (FC), 7-nitrobenzofurazan (NBD), rhodamine B (RhoB), coumarin (Cou), 7-(diethylamino)coumarin (DEAC) and cyanine 5 dye (Cy5). All prepared compounds were screened for affinity for the Arabidopsis thaliana cytokinin receptor (CRE1/AHK4). Although the attachment of the fluorescent labels to iP via the linkers mostly disrupted binding to the receptor, several fluorescent derivatives interacted well. For this reason, three derivatives, two rhodamine B and one 4-chloro-7-nitrobenzofurazan labeled iP were tested for their interaction with CRE1/AHK4 and Zea mays cytokinin receptors in detail. We further showed that the three derivatives were able to activate transcription of cytokinin response regulator ARR5 in Arabidopsis seedlings. The activity of fluorescently labeled cytokinins was compared with corresponding 6-dimethylaminopurine fluorescently labeled negative controls. Selected rhodamine B C2-labeled compounds 17, 18 and 4-chloro-7-nitrobenzofurazan N9-labeled compound 28 and their respective negative controls (19, 20 and 29, respectively) were used for in planta staining experiments in Arabidopsis thaliana cell suspension culture using live cell confocal microscopy.
- MeSH
- 4-chlor-7-nitrobenzofurazan farmakologie MeSH
- adenin analogy a deriváty chemie MeSH
- Arabidopsis metabolismus MeSH
- barvicí látky chemie MeSH
- cytokininy chemie farmakologie MeSH
- fluorescenční barviva chemie MeSH
- isopentenyladenosin chemická syntéza chemie farmakologie MeSH
- karbocyaniny chemie MeSH
- konfokální mikroskopie MeSH
- kukuřice setá metabolismus MeSH
- molekulární struktura MeSH
- proteiny huseníčku metabolismus MeSH
- puriny chemie MeSH
- receptory cytokinové antagonisté a inhibitory chemie MeSH
- regulace genové exprese u rostlin MeSH
- regulátory růstu rostlin metabolismus MeSH
- rhodaminy chemie MeSH
- semenáček metabolismus MeSH
- terpeny metabolismus MeSH
- vývoj rostlin MeSH
- Publikační typ
- časopisecké články MeSH
HEK293 cells stably expressing δ-opioid receptor were labeled first with fluorescent analog of cholesterol, 22-NBD-cholesterol, exposed to cholesterol-depleting agent β-cyclodextrin (β-CDX) and analyzed by fluorescence lifetime imaging microscopy (FLIM). In accordance with chemical analysis of cholesterol level, the total cellular signal of this probe was decreased to half. Distribution of lifetime (τtot) values of 22-NBD-cholesterol, however, when screened over the whole cell area indicated no significant difference between control (τtot=4.9±0.1 ns) and β-CDX-treated (τtot=4.8±0.1 ns) cells. On the contrary, comparison of control (τtot=5.1±0.1 ns) and β-CDX-treated (τtot=4.4±0.1 ns) cells by analysis of 25-NBD-cholesterol fluorescence implied highly significant decrease of lifetime values of this probe. The observation that 22-NBD-cholesterol appears to be indifferent to the changes in the membrane packing in living cells is in agreement with previous studies in model membranes. However, our data indicate that the alternation of plasma membrane structure induced by decrease of cholesterol level by β-CDX makes the membrane environment of NBD moiety of 25-NBD-cholesterol probe a significantly more hydrated. This finding not only encourages using 25-NBD-cholesterol in living cells, but also demonstrates that previously drawn discouraging conclusions on the use of 25-NBD-cholesterol in model membranes are not valid for living cells.
- MeSH
- 4-chlor-7-nitrobenzofurazan analogy a deriváty analýza metabolismus MeSH
- beta-cyklodextriny metabolismus MeSH
- buněčná membrána chemie metabolismus MeSH
- cholesterol analogy a deriváty analýza metabolismus MeSH
- fluorescenční barviva analýza metabolismus MeSH
- fluorescenční mikroskopie MeSH
- HEK293 buňky chemie metabolismus MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Topical skin lipid supplementation may provide opportunities for controlling ceramide (Cer) deficiency in skin diseases such as atopic dermatitis or psoriasis. Here we describe the synthesis of a long-chain 7-nitrobenzo[c][1,2,5]oxadiazol-4-yl (NBD)-labeled Cer and its different penetration through human skin compared to widely used short-chain fluorescent Cer tools.
- MeSH
- 4-chlor-7-nitrobenzofurazan chemická syntéza metabolismus MeSH
- atopická dermatitida metabolismus MeSH
- bisbenzimidazol metabolismus MeSH
- ceramidy chemická syntéza metabolismus MeSH
- fluorescence MeSH
- fluorescenční barviva chemická syntéza metabolismus MeSH
- kůže metabolismus MeSH
- lidé MeSH
- permeabilita MeSH
- psoriáza metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
