interphase
Dotaz
Zobrazit nápovědu
The nuclear lamina represents a multifunctional platform involved in such diverse yet interconnected processes as spatial organization of the genome, maintenance of mechanical stability of the nucleus, regulation of transcription and replication. Most of lamina activities are exerted through tethering of lamina-associated chromatin domains (LADs) to the nuclear periphery. Yet, the lamina is a dynamic structure demonstrating considerable expansion during the cell cycle to accommodate increased number of LADs formed during DNA replication. We analyzed dynamics of nuclear growth during interphase and changes in lamina structure as a function of cell cycle progression. The nuclear lamina demonstrates steady growth from G1 till G2, while quantitative analysis of lamina meshwork by super-resolution microscopy revealed that microdomain organization of the lamina is maintained, with lamin A and lamin B microdomain periodicity and interdomain gap sizes unchanged. FRAP analysis, in contrast, demonstrated differences in lamin A and B1 exchange rates; the latter showing higher recovery rate in S-phase cells. In order to further analyze the mechanism of lamina growth in interphase, we generated a lamina-free nuclear envelope in living interphase cells by reversible hypotonic shock. The nuclear envelope in nuclear buds formed after such a treatment initially lacked lamins, and analysis of lamina formation revealed striking difference in lamin A and B1 assembly: lamin A reassembled within 30 min post-treatment, whereas lamin B1 did not incorporate into the newly formed lamina at all. We suggest that in somatic cells lamin B1 meshwork growth is coordinated with replication of LADs, and lamin A meshwork assembly seems to be chromatin-independent process.
- MeSH
- Cricetulus MeSH
- interfáze * MeSH
- jaderná lamina chemie metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- myši MeSH
- prasata MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To study 3D nuclear distributions of epigenetic histone modifications such as H3(K9) acetylation, H3(K4) dimethylation, H3(K9) dimethylation, and H3(K27) trimethylation, and of histone methyltransferase Suv39H1, we used advanced image analysis methods, combined with Nipkow disk confocal microscopy. Total fluorescence intensity and distributions of fluorescently labelled proteins were analyzed in formaldehyde-fixed interphase nuclei. Our data showed reduced fluorescent signals of H3(K9) acetylation and H3(K4) dimethylation (di-me) at the nuclear periphery, while dimeH3( K9) was also abundant in chromatin regions closely associated with the nuclear envelope. Little overlapping (intermingling) was observed for di-meH3(K4) and H3(K27) trimethylation (tri-me), and for di-meH3(K9) and Suv39H1. The histone modifications studied were absent in the nucleolar compartment with the exception of H3(K9) dimethylation that was closely associated with perinucleolar regions which are formed by centromeres of acrocentric chromosomes. Using immunocytochemistry, no di-meH3(K4) but only dense di-meH3(K9) was found for the human acrocentric chromosomes 14 and 22. The active X chromosome was observed to be partially acetylated, while the inactive X was more condensed, located in a very peripheral part of the interphase nuclei, and lacked H3(K9) acetylation. Our results confirmed specific interphase patterns of histone modifications within the interphase nuclei as well as within their chromosome territories.
- MeSH
- acetylace účinky léků MeSH
- centromera genetika metabolismus MeSH
- chromozom X genetika metabolismus MeSH
- financování organizované využití MeSH
- histony metabolismus MeSH
- interfáze fyziologie genetika MeSH
- interpretace obrazu počítačem metody využití MeSH
- konfokální mikroskopie metody využití MeSH
- metylace účinky léků MeSH
The arrangement of chromatin within interphase nuclei seems to be caused by topological constraints and related to gene expression depending on tissue and developmental stage. In yeast and animals it was found that homologous and heterologous chromatin association are required to realize faithful expression and DNA repair. To test whether such associations are present in plants we analyzed Arabidopsis thaliana interphase nuclei by FISH using probes from different chromosomes. We found that chromatin fiber movement and variable associations, although in general relatively seldom, may occur between euchromatin segments along chromosomes, sometimes even over large distances. The combination of euchromatin segments bearing high or low co-expressing genes did not reveal different association frequencies probably due to adjacent genes of deviating expression patterns. Based on previous data and on FISH analyses presented here, we conclude that the global interphase chromatin organization in A. thaliana is relatively stable, due to the location of its 10 centromeres at the nuclear periphery and of the telomeres mainly at the centrally localized nucleolus. Nevertheless, chromatin movement enables a flexible spatial genome arrangement in plant nuclei.
- Publikační typ
- časopisecké články MeSH
The results of repeated interphase fluorescence in-situ hybridization (I-FISH, FISH) examination of 97 CLL patients and correlation of these findings with IgVH hypermutation status, ZAP-70 and CD38 expression are presented. The appearance of new, FISH-detectable, genomic aberrations during disease course, described as clonal evolution (CE), was observed in 26% of patients. The most frequent newly acquired cytogenetic abnormality was 13q deletion in 64% (16/25). In contrast to earlier studies, there was no correlation found between CE and either one of single negative prognostic factors (unmutated IgVH; CD38 positivity; ZAP-70 positivity). However, the combination of all three negative factors correlated with CE highly significantly (p=0.005) and moreover, also with a shift from lower to higher FISH risk category (p=0.010). As the prognostic data were known in all patients, this study represents the complete insight on the association of CE and other risk parameters in CLL.
- MeSH
- antigeny CD38 analýza MeSH
- chromozomální aberace MeSH
- chronická lymfatická leukemie genetika MeSH
- dospělí MeSH
- hybridizace in situ fluorescenční metody MeSH
- interfáze MeSH
- lidé středního věku MeSH
- lidé MeSH
- protein-tyrosinkináza ZAP-70 analýza MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
Východisko. Klasickým cytogenetickým vyšetřením buněk kostní dřeně nebo periferních B-lymfocytů bylo zjištěno, že nejčastější změnou chromozómů u chronické lymfatické leukémie je trizómie 12. Molekulárně cytoge- netická metoda tzv. interfázická in situ hybridizace, kterou je možné vyšetřovat i nedělící se buňky, však dokázala, že chromozómových změn u CLL je daleko víc a že mají přímý vztah ke vzniku a průběhu onemocnění. Metody a výsledky. Během posledních dvou let vyšetřujeme buňky kostní dřeně nemocných s CLL kromě klasickou cytogenetickou metodou G-pruhováním také fluorescenční in situ hybridizací. Pomocí centromerické a-satelitní sondy pro chromozóm 12 sledujeme početní odchylky v dělících i nedělících se buňkách, dále kosmido- vými sondami hledáme drobné delece v oblasti 13q14 (Rb gen) a 17p13 (p53 protein). Tyto geny jsou zodpovědné za buněčné dělení a jsou dávány do přímé souvislosti se vznikem nádorových procesů. U CLL řešíme v současnosti otázku, zda početní (trizómie 12) a strukturní (delece) chromozómové změny jsou primární či sekundární změnou a jaký je jejich vztah k průběhu onemocnění. Sondou CEP12 jsme vyšetřili 93 nemocných, u 24 z nich (tj. 25,8 %) jsme zjistili existenci buněčného klonu s trizómií 12. Zastoupení klonu bylo 2,5-75,5 % buněk a klasickou cytogenetikou byla jeho existence prokázána jen u 2 nemocných. Delece v oblasti 13q14 byla prokázána sondou LSI D13S319 u 24 nemocných ze 73 (32,8 %), rozsah klonu 2,5-80,0 % buněk, klasickou cytogenetikou s potvrzením FISH byla delece 13q14 nalezena jen u 1 nemocného. Deleci 17p13 jsme u 61 pacientů vyšetřených sondou LSI p53 zjistili u 14 (22,9 %), a to vždy jen metodou FISH. Rozsah klonu 2,5-34,0 % buněk. Deleci del(11)(q23) jsme nezjistili u žádného z 12 vyšetřených pacientů. Všechny sondy jsou od firmy VYSIS™. Závěry. Interfázická FISH je velmi citlivou metodou, kterou lze u vysokého procenta pacientů s CLL prokázat chromozómové odchylky. Náš soubor nemocných s CLL dále rozšiřujeme a hledáme korelace mezi molekulárně- cytogenetickými, imunologickými, morfologickými a prognostickými ukazateli.
Background. Trisomy 12 was found to be the most frequent chromosomal aberration identified by conventional cytogenetic studies of bone marrow cells and peripheral lymphocytes of patients with CLL. Molecular-cytogenetic techniques which enable examination of dividing and/or non-diving interphase nuclei (I-FISH), proved existence of other chromosomal abnormalities, mainly deletions, which could have in CLL patients relation to the origin, course and prognosis of the disease. Methods and Results. During the last two years bone marrow chromosomes of all patients with CLL were examined by G-banding and by I-FISH. The numerical changes of chromosome 12 were followed by centromeric DNA probe in dividing and non-dividing cells. The small deletions were ascertained by locus specific probes for 13q14 (Rb gene), 17p13 (p53 protein) and 11q23 (MLL gene). These genes are responsible for cell division and their function is probably in connection with neoplastic process. It is of interest whether numerical and structural chromosomal rearrangements are primary or secondary changes and what is their impact on etiology of CLL. 93 patients were examined by DNA prove CEP12 and trisomy 12 was found in 24 of them (25.8 %), the range of the clone was 2.5-75.5 % of the screened cells. Deletion del(13)(q14) was examined by probe D13S319 in 73 patients and proved in 24 of them (32.8 %), pathological clone ranged 2.5-80.0 % of the cells. Deletion del(17)(p13) was found in 14 patients out of 61 examined by probe LSI p53 (22.9 %). The extent of the clone was 2.5-34.0 % of examined cells. Deletion 11q23 was not ascertained in any of 11 patients by means of probe LSI 11q23 (MLL). All probes used for FISH were manufactured by VYSIS™. Conclusions. FISH is very sensitive method, suitable for molecular-cytogenetic examination of leukemic patients. With I-FISH the deletion of 13q14 was ascertained as the most frequent chromosomal aberration in series of 73 patients with CLL. We continue to increase the number of patients screened by I-FISH with all eligible DNA probes and start the prospective study on patients with chromosomal pathology. We will correlate the immunophe- notype, morphology, clinical course and prognosis with karyotypic findings.
- MeSH
- B-lymfocyty genetika MeSH
- chronická nemoc MeSH
- delece genu MeSH
- DNA sondy diagnostické užití MeSH
- dospělí MeSH
- finanční podpora výzkumu jako téma MeSH
- hybridizace in situ fluorescenční MeSH
- lidé středního věku MeSH
- lidé MeSH
- lymfoidní leukemie diagnóza genetika MeSH
- prognóza MeSH
- senioři MeSH
- trizomie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
Papillary renal cell carcinoma (RCC) is subclassified in type 1 displaying cells with scanty pale cytoplasm arranged in a single layer and in type 2 showing pseudostratified cells with eosinophilic cytoplasm. However, the existence of more variants of papillary RCC may be inferred by the recognition of few cases with different morphological features. We report the clinicopathologic, immunohistochemical, ultrastructural, and interphase cytogenetic features of 12 papillary RCC composed by oncocytes. Ten patients were males and their median age was 67 years. The tumors were well demarcated and their median diameter was 7.1 cm. Solid oncocytoma-like areas occurred in 11 cases. The cytoplasm of the neoplastic cells was filled by mitochondria with lamellar cristae. All cases were positive for the antimitochondrial antigen and racemase and showed variable immunoreactivity for cytokeratins (AE1/AE3, CK8-18, CK7, CK19), EMA, CD10, vimentin, and parvalbumin. MIB1 was detected in 0 to 6 cells per 1 high-power field. Fluorescent in situ hybridization analysis on formalin-fixed paraffin-embedded tissue showed three or more signals for chromosome 7 and 17 (for both > or =30% of nuclei in 7 of 12 neoplasms). In males, signals of chromosome Y were absent in more than 80% of the neoplastic nuclei. One patient died of metastases. Interphase cytogenetic analysis by fluorescent in situ hybridization can be a diagnostic tool in cases mimicking an oncocytoma.
- MeSH
- aneuploidie MeSH
- chromozomální delece MeSH
- cytoplazma ultrasonografie ultrastruktura MeSH
- diferenciální diagnóza MeSH
- dospělí MeSH
- financování organizované MeSH
- hybridizace in situ fluorescenční MeSH
- interfáze genetika MeSH
- karcinom z renálních buněk genetika chemie patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- lidské chromozomy, pár 17 MeSH
- lidské chromozomy, pár 7 MeSH
- lidský chromozom Y genetika MeSH
- nádorové biomarkery analýza MeSH
- nádory ledvin genetika chemie patologie MeSH
- oxyfilní adenom diagnóza MeSH
- oxyfilní buňky patologie MeSH
- papilární karcinom genetika chemie patologie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
V předkládané práci uvádíme příklad využití imunofl uorescenčního značení nádorových plazmatických buněk a techniky tříbarevné interfázní fl uorescenční in situ hybridizace se specifi ckými DNA sondami pro chromozómy 5, 9 a 15 k detekci hyperdiploidních karyotypů u nemocných s mnohočetným myelomem.
We focus on the applications of immunofl uorescence labeling of tumor plasma cells and three-color interphase in situ hybridization with specifi c DNA probes for chromosome 5, 9, and 15 for the detection of hyperdiploidy in multiple myeloma.
- MeSH
- chromozomální aberace * MeSH
- cytogenetické vyšetření metody přístrojové vybavení MeSH
- hybridizace in situ fluorescenční * metody využití MeSH
- karyotyp MeSH
- lidé středního věku MeSH
- lidé MeSH
- mnohočetný myelom * diagnóza genetika patologie MeSH
- plazmatické buňky cytologie MeSH
- senioři MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- delece genu MeSH
- dospělí MeSH
- finanční podpora výzkumu jako téma MeSH
- hybridizace in situ fluorescenční metody MeSH
- imunomagnetická separace MeSH
- kostní dřeň MeSH
- lidé MeSH
- lidské chromozomy, pár 13 genetika MeSH
- mnohočetný myelom diagnóza genetika MeSH
- pruhování chromozomů MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
