Time-resolved microspectrofluorimetry and fluorescence microscopy imaging-two complementary fluorescence techniques-provide important information about the intracellular distribution, level of uptake and binding/interactions inside living cell of the labeled molecule of interest. They were employed to monitor the "fate" of AS1411 aptamer labeled by ATTO 425 in human living cells. Confocal microspectrofluorimeter adapted for time-resolved intracellular fluorescence measurements by using a phase-modulation principle with homodyne data acquisition was employed to obtain emission spectra and to determine fluorescence lifetimes in U-87 MG tumor brain cells and Hs68 non-tumor foreskin cells. Acquired spectra from both the intracellular space and the reference solutions were treated to observe the aptamer localization and its interaction with biological structures inside the living cell. The emission spectra and the maximum emission wavelengths coming from the cells are practically identical, however significant lifetime lengthening was observed for tumor cell line in comparison to non-tumor one.
- MeSH
- aptamery nukleotidové metabolismus MeSH
- časové faktory MeSH
- fluorescenční mikroskopie metody MeSH
- fluorescenční spektrometrie metody MeSH
- intracelulární prostor genetika metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- oligodeoxyribonukleotidy genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
N-methyl-D-aspartátový receptor je ve vztahu ke schizofrenii v posledních dvou dekádách značně studován. Ukazuje se, že dysfunkce glutamatergního systému je komplexním jevem, a tak je namístě zavzít do studia NMDAR také další molekuly spojené s těmito receptory, jako jsou proteiny postsynaptické density (PSD). V naší práci jsme u potkanů pomocí antisense oligodeoxynukleotidů (aODN) ovlivňovali expresi NRl, NR2A a NR2B podjednotek NMDAR, a to každou zvlášť, nebo v kombinacích. U takto ovlivněných zvířat jsme hodnotili prepulzní inhibici akustické úlekové reakce (PPI) a v hipokampech pak expresi jednotlivých podjednotek NMDAR a také expresi PSD proteinů. Mezi skupinami jsme našli signifikantní rozdíl v expresi PSD-95 a NRl. Aplikace aODN (NR2A, NR2B) byla spojena s významným poklesem PSD-95. PPI a exprese NR2A, NR2B a PSD-93 se v souvislosti s aplikací aÓDN významně nelišily.
N-methyl-D-aspartate receptor is widely examined in schizophrenia-related studies for last two decades. It has been shown that the glutamater- gic system dysfunction is a complex phenomenon and so it might be important to focus the studies not only on NMDAR but also on associated molecules like proteins of postsynaptic density (PSD). In our work, using it separately or in combination, antisense oligodeox ynucleotide (aODN) for NR1, NR2A and NR2B subunit of NMDAR, we affected expression of these proteins in rat hippocampus. We assessed prepulse inhi bition of acoustic startle reaction (PPI) in rats and protein expression of NMDAR subunits and expression of PSD proteins. There were significant differences in expression of PSD-95 and NR1 between groups. Application of aODN (NR2A, NR2B) was associated with a significant decrease of PSD-95. PPI and expression of NR2A, NR2B and PSD-93 were not changed after aODN application.
- Klíčová slova
- prepulzní inhibice akustického úleku, antisence oligodeoxynukleotidy pro NMDA-NR1/NR2 podjednotky, PSD proteiny, NMDA receptor,
- MeSH
- financování organizované MeSH
- hipokampus fyziologie patofyziologie účinky léků MeSH
- interakční proteinové domény a motivy genetika účinky léků MeSH
- krysa rodu rattus MeSH
- laboratorní zvířata MeSH
- modely u zvířat MeSH
- oligodeoxyribonukleotidy genetika MeSH
- potkani Wistar MeSH
- receptory N-methyl-D-aspartátu fyziologie genetika MeSH
- schizofrenie genetika patofyziologie MeSH
- statistika jako téma MeSH
- úleková reakce fyziologie genetika účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
The p73 gene possesses an extrinsic P1 promoter and an intrinsic P2 promoter, resulting in TAp73 and DeltaNup73 isoforms, respectively. The ultimate effect of p73 in oncogenesis is thought to depend on the apoptotic TA to antiapoptotic DeltaN isoforms' ratio. This study was aimed at identifying novel transcription factors that affect TA isoform synthesis. With the use of bioinformatics tools, in vitro binding assays, and chromatin immunoprecipitation analysis, a region extending -233 to -204 bp upstream of the transcription start site of the human p73 P1 promoter, containing conserved Sp1-binding sites, was characterized. Treatment of cells with Sp1 RNAi and Sp1 inhibitor functionally suppress TAp73 expression, indicating positive regulation of P1 by the Sp1 protein. Notably Sp1 inhibition or knockdown also reduces DeltaNup73 protein levels. Therefore, Sp1 directly regulates TAp73 transcription and affects DeltaNup73 levels in lung cancer. TAp73gamma was shown to be the only TA isoform overexpressed in several lung cancer cell lines and in 26 non-small cell lung cancers, consistent with Sp1 overexpression, thereby questioning the apoptotic role of this specific p73 isoform in lung cancer.
- MeSH
- DNA vazebné proteiny * genetika metabolismus MeSH
- DNA chemie metabolismus MeSH
- down regulace genetika MeSH
- jaderné proteiny * genetika metabolismus MeSH
- konzervovaná sekvence genetika MeSH
- lidé MeSH
- malá interferující RNA genetika MeSH
- mithramycin analogy a deriváty farmakologie MeSH
- molekulární sekvence - údaje MeSH
- nádorové buněčné linie MeSH
- nádorové supresorové proteiny * genetika metabolismus MeSH
- nádory plic * metabolismus MeSH
- oligodeoxyribonukleotidy genetika metabolismus MeSH
- promotorové oblasti (genetika) * genetika MeSH
- protein - isoformy * genetika metabolismus MeSH
- retardační test MeSH
- sekvence nukleotidů genetika MeSH
- transkripční faktor Sp1 * antagonisté a inhibitory genetika metabolismus MeSH
- vaskulární endoteliální růstový faktor A metabolismus MeSH
- vazba proteinů genetika MeSH
- vazebná místa genetika MeSH
- výpočetní biologie MeSH
- Check Tag
- lidé MeSH
- MeSH
- exprese genu genetika MeSH
- financování organizované MeSH
- hipokampus chemie patofyziologie MeSH
- laboratorní zvířata MeSH
- modely u zvířat MeSH
- oligodeoxyribonukleotidy genetika MeSH
- potkani Wistar MeSH
- receptory N-methyl-D-aspartátu metabolismus nedostatek MeSH
- schizofrenie enzymologie etiologie patofyziologie MeSH
- statistika jako téma metody MeSH
- úleková reakce fyziologie MeSH
- Publikační typ
- srovnávací studie MeSH
Clinically ineffective transplatin [trans-diamminedichloridoplatinum(II)] is used in the studies of the structure-pharmacological activity relationship of platinum compounds. In addition, a number of transplatin analogs exhibit promising toxic effects in several tumor cell lines including those resistant to conventional antitumor cisplatin. Moreover, transplatin-modified oligonucleotides have been shown to be effective modulators of gene expression. Owing to these facts and because DNA is also considered the major pharmacological target of platinum complexes, interactions between transplatin and DNA are of great interest. We examined, using biophysical and biochemical methods, the stability of 1,3-GNG intrastrand cross-links (CLs) formed by transplatin in short synthetic oligodeoxyribonucleotide duplexes and natural double-helical DNA. We have found that transplatin forms in double-helical DNA 1,3-GNG intrastrand CLs, but their stability depends on the sequence context. In some sequences the 1,3-GNG intrastrand CLs formed by transplatin in double-helical DNA readily rearrange into interstrand CLs. On the other hand, in a number of other sequences these intrastrand CLs are relatively stable. We show that the stability of 1,3-GNG intrastrand CLs of transplatin correlates with the extent of conformational distortion and thermodynamic destabilization induced in double-helical DNA by this adduct.
- MeSH
- biofyzikální jevy MeSH
- cisplatina metabolismus MeSH
- DNA genetika chemie metabolismus MeSH
- financování organizované MeSH
- kalorimetrie MeSH
- konformace nukleové kyseliny MeSH
- oligodeoxyribonukleotidy genetika chemie metabolismus MeSH
- reagencia zkříženě vázaná metabolismus MeSH
- sekvence nukleotidů MeSH
- termodynamika MeSH
- MeSH
- cystická fibróza * genetika MeSH
- heteroduplexy nukleové kyseliny * genetika MeSH
- heterozygot MeSH
- lidé MeSH
- membránové proteiny * genetika MeSH
- molekulární sekvence - údaje MeSH
- mutace genetika MeSH
- oligodeoxyribonukleotidy genetika MeSH
- protein CFTR MeSH
- sekvence nukleotidů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- komentáře MeSH
- práce podpořená grantem MeSH
