The development of an ideal vascular prosthesis represents an important challenge in terms of the treatment of cardiovascular diseases with respect to which new materials are being considered that have produced promising results following testing in animal models. This study focuses on nanofibrous polycaprolactone-based grafts assessed by means of histological techniques 10 days and 6 months following suturing as a replacement for the rat aorta. A novel stereological approach for the assessment of cellular distribution within the graft thickness was developed. The cellularization of the thickness of the graft was found to be homogeneous after 10 days and to have changed after 6 months, at which time the majority of cells was discovered in the inner layer where the regeneration of the vessel wall was found to have occurred. Six months following implantation, the endothelialization of the graft lumen was complete, and no vasa vasorum were found to be present. Newly formed tissue resembling native elastic arteries with concentric layers composed of smooth muscle cells, collagen, and elastin was found in the implanted polycaprolactone-based grafts. Moreover, the inner layer of the graft was seen to have developed structural similarities to the regular aortic wall. The grafts appeared to be well tolerated, and no severe adverse reaction was recorded with the exception of one case of cartilaginous metaplasia close to the junctional suture.

• Klíčová slova
• electrospinning, histological evaluation, polycaprolactone, stereology, vascular graft, vascular remodeling,
• Publikační typ
• časopisecké články MeSH

Phosphoinositides are glycerol-based phospholipids, and they play essential roles in cellular signalling, membrane and cytoskeletal dynamics, cell movement, and the modulation of ion channels and transporters. Phosphoinositides are also associated with fundamental nuclear processes through their nuclear protein-binding partners, even though membranes do not exist inside of the nucleus. Phosphatidylinositol 4-phosphate (PI(4)P) is one of the most abundant cellular phosphoinositides; however, its functions in the nucleus are still poorly understood. In this study, we describe PI(4)P localisation in the cell nucleus by super-resolution light and electron microscopy, and employ immunoprecipitation with a specific anti-PI(4)P antibody and subsequent mass spectrometry analysis to determine PI(4)P's interaction partners. We show that PI(4)P is present at the nuclear envelope, in nuclear lamina, in nuclear speckles and in nucleoli and also forms multiple small foci in the nucleoplasm. Nuclear PI(4)P undergoes re-localisation to the cytoplasm during cell division; it does not localise to chromosomes, nucleolar organising regions or mitotic interchromatin granules. When PI(4)P and PI(4,5)P2 are compared, they have different nuclear localisations during interphase and mitosis, pointing to their functional differences in the cell nucleus. Mass spectrometry identified hundreds of proteins, including 12 potentially novel PI(4)P interactors, most of them functioning in vital nuclear processes such as pre-mRNA splicing, transcription or nuclear transport, thus extending the current knowledge of PI(4)P's interaction partners. Based on these data, we propose that PI(4)P also plays a role in essential nuclear processes as a part of protein-lipid complexes. Altogether, these observations provide a novel insight into the role of PI(4)P in nuclear functions and provide a direction for further investigation.

• Klíčová slova
• PI(4)P, nucleus, phosphoinositides,
• MeSH
• buněčné jadérko metabolismus   ultrastruktura   MeSH
• buněčné jádro metabolismus   ultrastruktura   MeSH
• buněčný cyklus MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• jaderné proteiny metabolismus   MeSH
• jaderný obal metabolismus   ultrastruktura   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• proteom metabolismus   MeSH
• shluková analýza MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfatidylinositolfosfáty MeSH
• jaderné proteiny MeSH
• phosphatidylinositol 4-phosphate MeSH Prohlížeč
• proteom MeSH

During meiosis, homologous chromosomes undergo a dramatic movement in order to correctly align. This is a critical meiotic event but the molecular properties of this 'chromosomal dance' still remainunclear. We identified DEB-1 - an orthologue of mammalian vinculin - as a new component of the mechanistic modules responsible for attaching the chromosomes to the nuclear envelope as apart of the LINC complex. In early meiotic nuclei of C. elegans, DEB-1 is localized to the nuclear periphery and alongside the synaptonemal complex of paired homologues. Upon DEB-1 depletion, chromosomes attached to SUN-1 foci remain highly motile until late pachytene. Although the initiation of homologue pairing started normally, irregularities in the formation of the synaptonemal complex occur, and these results in meiotic defects such as increased number of univalents at diakinesis and high embryonic lethality. Our data identify DEB-1 as a new player regulating chromosome dynamics and pairing during meiotic prophase I.

• Klíčová slova
• DEB-1, LINC complex, chromosome pairing, prophase I, vinculin,
• MeSH
• Caenorhabditis elegans genetika   MeSH
• chromozomy genetika   MeSH
• meióza genetika   MeSH
• párování chromozomů genetika   MeSH
• vinkulin genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• vinkulin MeSH

In biomedical studies, the colocalization is commonly understood as the overlap between distinctive labelings in images. This term is usually associated especially with quantitative evaluation of the immunostaining in fluorescence microscopy. On the other hand, the evaluation of the immunolabeling colocalization in the electron microscopy images is still under-investigated and biased by the subjective and non-quantitative interpretation of the image data. We introduce a novel computational technique for quantifying the level of colocalization in pointed patterns. Our approach follows the idea included in the widely used Manders' colocalization coefficients in fluorescence microscopy and represents its counterpart for electron microscopy. In presented methodology, colocalization is understood as the product of the spatial interactions at the single-particle (single-molecule) level. Our approach extends the current significance testing in the immunoelectron microscopy images and establishes the descriptive colocalization coefficients. To demonstrate the performance of the proposed coefficients, we investigated the level of spatial interactions of phosphatidylinositol 4,5-bisphosphate with fibrillarin in nucleoli. We compared the electron microscopy colocalization coefficients with Manders' colocalization coefficients for confocal microscopy and super-resolution structured illumination microscopy. The similar tendency of the values obtained using different colocalization approaches suggests the biological validity of the scientific conclusions. The presented methodology represents a good basis for further development of the quantitative analysis of immunoelectron microscopy data and can be used for studying molecular interactions at the ultrastructural level. Moreover, this methodology can be applied also to the other super-resolution microscopy techniques focused on characterization of discrete pointed structures.

• Klíčová slova
• Colocalization, Immunohistochemistry, Manders’ coefficients, Pointed patterns, Quantitative analysis, Transmission electron microscopy,
• MeSH
• algoritmy * MeSH
• fluorescenční mikroskopie MeSH
• imunoelektronová mikroskopie MeSH
• konfokální mikroskopie MeSH
• počítačové zpracování obrazu * MeSH
• Publikační typ
• časopisecké články 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
• nukleofosmin MeSH
• proteiny buněčného cyklu 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
• Názvy látek
• aktiny MeSH
• fosfatidylinositol-4,5-difosfát MeSH
• jaderné proteiny MeSH
• nukleofosmin MeSH
• proteiny buněčného cyklu MeSH
• protilátky MeSH
• ribonukleoproteiny malé jaderné MeSH
• SMC2 protein, human MeSH Prohlížeč
• transportní proteiny MeSH
• zlato MeSH

Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca(2+) response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)(2) or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcεRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)(2) fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcεRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• antigeny CD9 fyziologie   MeSH
• antigeny CD98 - lehké řetězce metabolismus   MeSH
• antigeny metabolismus   MeSH
• biologické modely MeSH
• buněčná membrána metabolismus   MeSH
• chemotaxe MeSH
• cytoskelet metabolismus   MeSH
• fosfoproteiny metabolismus   MeSH
• fosforylace MeSH
• glukuronidasa metabolismus   MeSH
• imunoglobuliny - Fab fragmenty chemie   MeSH
• krysa rodu Rattus MeSH
• mastocyty cytologie   MeSH
• membránové proteiny metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• potkani Wistar MeSH
• receptory IgE metabolismus   MeSH
• transportní systém aminokyselin y+ metabolismus   MeSH
• tyrosin chemie   MeSH
• vápník metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• antigeny CD9 MeSH
• antigeny CD98 - lehké řetězce MeSH
• antigeny MeSH
• fosfoproteiny MeSH
• glukuronidasa MeSH
• imunoglobuliny - Fab fragmenty MeSH
• Lat protein, mouse MeSH Prohlížeč
• Lat protein, rat MeSH Prohlížeč
• membránové proteiny MeSH
• receptory IgE MeSH
• SLC7A8 protein, mouse MeSH Prohlížeč
• Slc7a8 protein, rat MeSH Prohlížeč
• transportní systém aminokyselin y+ MeSH
• tyrosin MeSH
• vápník 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
• Názvy látek
• aktiny MeSH
• myosin typu I MeSH
• ribozomální DNA MeSH
• RNA ribozomální MeSH
• RNA-polymerasa I MeSH
• transcription factor UBF MeSH Prohlížeč
• transkripční iniciační komplex Pol1 - proteiny MeSH

The earliest known biochemical step that occurs after ligand binding to the multichain immune recognition receptor is tyrosine phosphorylation of the receptor subunits. In mast cells and basophils activated by multivalent antigen-IgE complexes, this step is mediated by Src family kinase Lyn, which phosphorylates the high affinity IgE receptor (Fc epsilonRI). However, the exact molecular mechanism of this phosphorylation step is incompletely understood. In this study, we tested the hypothesis that changes in activity and/or topography of protein-tyrosine phosphatases (PTPs) could play a major role in the Fc epsilonRI triggering. We found that exposure of rat basophilic leukemia cells or mouse bone marrow-derived mast cells to PTP inhibitors, H(2)O(2) or pervanadate, induced phosphorylation of the Fc epsilonRI subunits, similarly as Fc epsilonRI triggering. Interestingly, and in sharp contrast to antigen-induced activation, neither H(2)O(2) nor pervanadate induced any changes in the association of Fc epsilonRI with detergent-resistant membranes and in the topography of Fc epsilonRI detectable by electron microscopy on isolated plasma membrane sheets. In cells stimulated with pervanadate, H(2)O(2) or antigen, enhanced oxidation of active site cysteine of several PTPs was detected. Unexpectedly, most of oxidized phosphatases bound to the plasma membrane were associated with the actin cytoskeleton. Several PTPs (SHP-1, SHP-2, hematopoietic PTP, and PTP-MEG2) showed changes in their enzymatic activity and/or oxidation state during activation. Based on these and other data, we propose that down-regulation of enzymatic activity of PTPs and/or changes in their accessibility to the substrates play a key role in initial tyrosine phosphorylation of the Fc epsilonRI and other multichain immune receptors.

• MeSH
• aktivace enzymů účinky léků   genetika   imunologie   MeSH
• antigeny imunologie   metabolismus   farmakologie   MeSH
• fosforylace účinky léků   genetika   imunologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• krysa rodu Rattus MeSH
• mastocyty imunologie   metabolismus   MeSH
• membránové mikrodomény genetika   imunologie   metabolismus   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• oxidace-redukce účinky léků   MeSH
• oxidancia farmakologie   MeSH
• peroxid vodíku farmakologie   MeSH
• receptory IgE genetika   imunologie   metabolismus   MeSH
• skupina kinas odvozených od src-genu genetika   imunologie   metabolismus   MeSH
• transport proteinů účinky léků   genetika   imunologie   MeSH
• tyrosinfosfatasy antagonisté a inhibitory   genetika   imunologie   metabolismus   MeSH
• vanadáty farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny MeSH
• inhibitory enzymů MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• oxidancia MeSH
• peroxid vodíku MeSH
• pervanadate MeSH Prohlížeč
• receptory IgE MeSH
• skupina kinas odvozených od src-genu MeSH
• tyrosinfosfatasy MeSH
• vanadáty MeSH

Nuclear actin plays an important role in such processes as chromatin remodeling, transcriptional regulation, RNA processing, and nuclear export. Recent research has demonstrated that actin in the nucleus probably exists in dynamic equilibrium between monomeric and polymeric forms, and some of the actin-binding proteins, known to regulate actin dynamics in cytoplasm, have been also shown to be present in the nucleus. In this paper, we present ultrastructural data on distribution of actin and various actin-binding proteins (alpha-actinin, filamin, p190RhoGAP, paxillin, spectrin, and tropomyosin) in nuclei of HeLa cells and resting human lymphocytes. Probing extracts of HeLa cells for the presence of actin-binding proteins also confirmed their presence in nuclei. We report for the first time the presence of tropomyosin and p190RhoGAP in the cell nucleus, and the spatial colocalization of actin with spectrin, paxillin, and alpha-actinin in the nucleolus.

• MeSH
• aktinin MeSH
• aktiny analýza   MeSH
• buněčné jádro chemie   ultrastruktura   MeSH
• HeLa buňky MeSH
• jaderné proteiny analýza   MeSH
• lidé MeSH
• lymfocyty chemie   ultrastruktura   MeSH
• mikrofilamentové proteiny analýza   MeSH
• paxilin MeSH
• proteiny aktivující GTPasu MeSH
• spektrin MeSH
• tropomyosin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktinin MeSH
• aktiny MeSH
• jaderné proteiny MeSH
• mikrofilamentové proteiny MeSH
• paxilin MeSH
• proteiny aktivující GTPasu MeSH
• rho GTPase-activating protein MeSH Prohlížeč
• spektrin MeSH
• tropomyosin MeSH

Most DNA synthesis in HeLa cell nucleus is concentrated in discrete foci. These synthetic sites can be identified by electron microscopy after allowing permeabilized cells to elongate nascent DNA in the presence of biotin-dUTP. Biotin incorporated into nascent DNA can be then immunolabeled with gold particles. Two types of DNA synthetic sites/replication factories can be distinguished at ultrastructural level: (1) electron-dense structures--replication bodies (RB), and (2) focal replication sites with no distinct underlying structure--replication foci (RF). The protein composition of these synthetic sites was studied using double immunogold labeling. We have found that both structures contain (a) proteins involved in DNA replication (DNA polymerase alpha, PCNA), (b) regulators of the cell cycle (cyclin A, cdk2), and (c) RNA processing components like Sm and SS-B/La auto antigens, p80-coilin, hnRNPs A1 and C1/C2. However, at least four regulatory and structural proteins (Cdk1, cyclin B1, PML and lamin B1) differ in their presence in RB and RF. Moreover, in contrast to RF, RB have structural organization. For example, while DNA polymerase alpha, PCNA and hnRNP A1 were diffusely spread throughout RB, hnRNP C1/C2 was found only at the very outside. Surprisingly, RB contained only small amounts of DNA. In conclusion, synthetic sites of both types contain similar but not the same sets of proteins. RB, however, have more developed microarchitecture, apparently with specific functional zones. This data suggest possible differences in genome regions replicated by these two types of replication factories.

• MeSH
• buněčné jádro metabolismus   ultrastruktura   MeSH
• cyklin B genetika   MeSH
• cyklin B1 MeSH
• DNA-polymerasa I metabolismus   MeSH
• DNA chemie   genetika   MeSH
• HeLa buňky MeSH
• imunoelektronová mikroskopie MeSH
• imunohistochemie MeSH
• lamin typ B genetika   MeSH
• lidé MeSH
• monoklonální protilátky MeSH
• proteinkinasa CDC2 genetika   MeSH
• proteiny buněčného cyklu fyziologie   MeSH
• replikace DNA fyziologie   MeSH
• RNA biosyntéza   metabolismus   MeSH
• zalévání tkání MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CCNB1 protein, human MeSH Prohlížeč
• cyklin B MeSH
• cyklin B1 MeSH
• DNA-polymerasa I MeSH
• DNA MeSH
• lamin typ B MeSH
• monoklonální protilátky MeSH
• proteinkinasa CDC2 MeSH
• proteiny buněčného cyklu MeSH
• RNA MeSH