Phosphoinositides are phosphatidylinositol derived, well known to be second messengers in various cell signaling pathways as well as in processes such as cell differentiation, cellular stress response, gene transcription, and chromatin remodeling. The pleckstrin homology domain of phospholipase C-delta 1 is responsible for recognizing and binding to PI(4,5)P2 and for this reason has been widely used to study this phosphoinositide as a biosensor when it is conjugated to a fluorescent tag. In this work, we modified the primary structure of pleckstrin homology domain by site-specific mutagenesis to change the specificity for phosphoinositides. We obtained 3 mutants: K30A, W36F, and W36Y with different specificity to phosphoinositides. Mutant domain K30A recognized PI(4,5)P2 , PI(3,4,5)P3 , phosphatidic acid (PA), and weakly PI(3,5)P2 . Mutant domain W36F recognized all the phosphoinositides studied and the PA. Finally, mutant domain W36Y seemed to interact with PA and all the other phosphoinositides studied, except PI(3)P. The changes in recognition argue against a simple charge and nonpolar region model for these interactions and more in favor of a specific docking region with a specific recognition site. We conducted in silico modeling that explains the mechanisms behind the observed changes and showed that aromatic amino acids appear to play more important role, than previously thought, in the specificity of phospholipids' binding domains.
- MeSH
- aminokyseliny aromatické chemie MeSH
- fosfatidylinositolfosfáty metabolismus MeSH
- fosfolipasa C delta chemie MeSH
- krysa rodu rattus MeSH
- molekulární modely MeSH
- mutageneze cílená MeSH
- mutantní proteiny chemie metabolismus MeSH
- PH-doména * MeSH
- sekvence aminokyselin MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The histopathological structure of malignant tumours involves two essential compartments - the tumour parenchyma with the actual transformed cells, and the supportive tumour stroma. The latter consists of specialized mesenchymal cells, such as fibroblasts, macrophages, lymphocytes and vascular cells, as well as of their secreted products, including components of the extracellular matrix, matrix modifying enzymes and numerous regulatory growth factors and cytokines. In consequence, the tumour stroma has the ability to influence virtually all aspects of tumour development and progression, including therapeutic response. AIM: In this article we review the current knowledge of tumor stroma interactions in urothelial carcinoma and present various experimental systems that are currently in use to unravel the biological basis of these heterotypic cell interactions. RESULTS: For urothelial carcinoma, an extensive tumour stroma is quite typical and markers of activated fibroblasts correlate significantly with clinical parameters of advanced disease. Another clinically important variable is provided by the stromal expression of syndecan-1. CONCLUSION: Integration of markers of activated stroma into clinical risk evaluation could aid to better stratification of urothelial bladder carcinoma patients. Elucidation of biological mechanisms underlying tumour-stroma interactions could provide new therapeutical targets.
- MeSH
- biologické modely MeSH
- lidé MeSH
- mezibuněčná komunikace MeSH
- nádorové mikroprostředí * MeSH
- nádorové proteiny metabolismus MeSH
- nádory močového měchýře metabolismus patologie MeSH
- urotel metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
- přehledy MeSH
Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.
- MeSH
- aktiny metabolismus MeSH
- barvení a značení metody MeSH
- buněčné jádro MeSH
- elektronová mikroskopie MeSH
- fosfatidylinositol-4,5-difosfát metabolismus MeSH
- HeLa buňky MeSH
- imunohistochemie metody MeSH
- jaderné proteiny metabolismus MeSH
- kovové nanočástice chemie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- protilátky imunologie MeSH
- ribonukleoproteiny malé jaderné metabolismus MeSH
- transportní proteiny metabolismus MeSH
- zlato chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nuclear actin and nuclear myosin I (NMI) are important players in transcription of ribosomal genes. Transcription of rDNA takes place in highly organized intranuclear compartment, the nucleolus. In this study, we characterized the localization of these two proteins within the nucleolus of HeLa cells with high structural resolution by means of electron microscopy and gold-immunolabeling. We demonstrate that both actin and NMI are localized in specific compartments within the nucleolus, and the distribution of NMI is transcription-dependent. Moreover, a pool of NMI is present in the foci containing nascent rRNA transcripts. Actin, in turn, is present both in transcriptionally active and inactive regions of the nucleolus and colocalizes with RNA polymerase I and UBF. Our data support the involvement of actin and NMI in rDNA transcription and point out to other functions of these proteins in the nucleolus, such as rRNA maturation and maintenance of nucleolar architecture.
- MeSH
- aktiny metabolismus MeSH
- buněčné jadérko metabolismus MeSH
- genetická transkripce fyziologie MeSH
- HeLa buňky MeSH
- imunohistochemie MeSH
- lidé MeSH
- myosin typu I metabolismus MeSH
- ribozomální DNA metabolismus MeSH
- RNA ribozomální metabolismus MeSH
- RNA-polymerasa I metabolismus MeSH
- transkripční iniciační komplex Pol1 - proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Actin is a well-known protein that has shown a myriad of activities in the cytoplasm. However, recent findings of actin involvement in nuclear processes are overwhelming. Actin complexes in the nucleus range from very dynamic chromatin-remodeling complexes to structural elements of the matrix with single partners known as actin-binding proteins (ABPs). This review summarizes the recent findings of actin-containing complexes in the nucleus. Particular attention is given to key processes like chromatin remodeling, transcription, DNA replication, nucleocytoplasmic transport and to actin roles in nuclear architecture. Understanding the mechanisms involving ABPs will definitely lead us to the principles of the regulation of gene expression performed via concerting nuclear and cytoplasmic processes.
- MeSH
- aktiny chemie metabolismus MeSH
- biologické modely MeSH
- buněčné jádro chemie metabolismus MeSH
- lidé MeSH
- mikrofilamentové proteiny chemie metabolismus MeSH
- oprava DNA MeSH
- replikace DNA MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- MeSH
- biologie buňky dějiny MeSH
- dějiny 19. století MeSH
- Check Tag
- dějiny 19. století MeSH
- Publikační typ
- biografie MeSH
- časopisecké články MeSH
- historické články MeSH
- Geografické názvy
- Československo MeSH
- O autorovi
- Purkyně, Jan Evangelista, 1787-1869 Autorita
