Biomedical subjects

M Horký

Publications and source records attributed to M Horký.

6 recordsLinked to original sources

PubMed2004

Immunohistochemical detection of acetylation and phosphorylation of histone H3 in cervical smears.

OBJECTIVE: Histones bind in a sequence-independent manner to form chromatin. The aminoterminal tails of histones are targets for both phosphorylation and acetylation events. These modifications are thought to fundamentally regulate chromatin structure to accommodate transcription, DNA replication, mitosis and DNA repair. Regeneration of squamous epithelium is accompanied by marked cellular atypia, nuclear and nucleolar pleomorphism which could be confused with neoplasia. The aim of the study was to detect phosphorylated and acetylated forms of histone H3 in cytological smears. DESIGN: Translational research. SETTING: Department of Experimental Oncology, Masaryk Memorial Cancer Institute, Department of Pathological Physiology, Medical Faculty and Department of Obstetrics and Gynecology, Masaryk University, Czech Republic. METHODS: Smears from women aged between 20 to 46 years were selected. The specimens comprised 10 squamous metaplasia, 20 CIN I, 12 CIN II, and 14 CIN III. The smears were stained with polyclonal antibodies against phosphorylated and acetylated forms of histone H3. RESULTS: We found that nuclear positivity for phosphorylated (P) and acetylated (A) forms of histone H3 in CIN II (23% P, 33% A) and CIN III (25% P, 44% A) was higher in comparison with CIN I (8% P, 15% A) and metaplasia (11% P, 12% A). CONCLUSION: We revealed a marked association of histone H3 modifications with the progression of CIN II, CIN III in comparison with CIN I and metaplasia. Our results are in agreement with recent findings: 1. staining of cells with anti-phospho-histone H3 antibodies therefore provides a highly specific marker for mitosis. 2. acetylation of nucleosomal histones correlates with localised transcriptional activity.

Acetylation↗

PubMed2003

The onset of apoptosis of neurons induced by ischemia-reperfusion injury is delayed by transient period of hypertension in rats.

We investigated the potential neuroprotective effect of transient hypertension on neuronal cell death induced by ischemia-reperfusion. Recovery of neurons, terminally differentiated cells, is almost entirely dependent upon active transcription and repair of DNA damage. We focused on the histochemical detection of distribution of NOR (argyrophylic nucleolar proteins) reflecting nucleolar integrity, immunohistochemical detection of PARP-1 (poly(ADP-ribose) polymerase-1), MADD (mitogen-activated death domain), a protein accumulated in nucleoli upon stimulation by ischemia, the active form of caspase-3, a universal proteolytic enzyme of apoptosis. The terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick-end-labeling method (TUNEL) proved the presence of in situ DNA fragmentation. We used the model of transient focal cerebral ischemia in rats with occlusion of middle cerebral artery. In experimental group of rats, the transient hypertension was induced by constriction of the abdominal aorta. The period of ischemia lasted 15, 30, 60 and 120 min followed by 48 h of reperfusion. We examined the frontal lobe of the ipsilateral hemisphere for apoptosis of neurons and compared it with the intact brain tissue. In normotensive rats with transient focal cerebral ischemia, we found disintegrated nucleoli of cortical as well as subcortical neurons at all investigated periods of ischemia, whereas the neurons of intact animals showed compact nucleoli with a few satellites. Nuclear positivity for MADD and PARP-1 was apparent in the neocortex after 15 min and peaked after 30 min of ischemia. On the other hand, the subcortical neurons showed nuclear positivity after 60 and 120 min. The immunohistochemical reaction for active caspase 3 was apparent after 30 min onwards predominantly in the cortex. The TUNEL staining was distinct after 60 and 120 min. In hypertensive rats, we found nucleolar disintegration, positivity for MADD, PARP-1 and caspase 3 after 30 min cortically and subcortically, followed by TUNEL positive staining of cortical neurons after 60 and 120 min. In summary, we detected delayed activation of neuronal apoptosis in transiently hypertensive rats with focal cerebral ischemia compared to normotensive animals. The apoptotic phenotype was confirmed by a panel of complementary methods showing rapid proteolysis-nucleolar segregation, MADD, PARP-1 and caspase-3 positivity as well as ultimate DNA fragmentation proved by the TUNEL assay.

Animals↗

PubMed2002

Nucleolus and apoptosis.

The nucleolus represents a highly dynamic nuclear compartment of the interphase nucleus. It plays a key role in ribosome biogenesis. The number of nucleoli, their size, and their activity increase in exponentially growing cells; therefore these parameters reflect the proliferating activity of the cells. A variety of staining techniques have been employed to vizualize nucleolar changes in malignant cells. Staining of so-called nucleolar organizer regions (NORs), based upon a strong avidity of nucleolar proteins to bind silver ions, represents the technique most frequently used by pathologists. Nucleolar changes and pleomorphism associated with overt proliferation of tumor cells have also been documented by immunohistochemical and ultrastructural studies. Contrary to cell proliferation, cytostatics-induced changes of nucleolar phenotype in malignant cells point to a potential role of nucleolar components in the execution of active cell death. Recent studies have provided direct clues that so-called death domains and other apoptosis-related proteins are accumulated in nucleoli upon induction of active cell death. It can be concluded that the plurifunctionality of nucleoli regarding cell proliferation and apoptosis could open new vistas toward understanding dysregulation in malignant cells.

Apoptosis↗

PubMed2000

The role of tumour suppressors and viral oncoproteins in cervical carcinogenesis.

This article summarizes current knowledge of the cervical carcinogenesis with a special focus on the molecular mechanisms involving the interaction of cellular tumour suppressors (p53, RB, p73) with viral oncoproteins (E6, E7). The E6-induced degradation of p53 protein results in the inhibition of apoptosis, inability to repair DNA and fixation of mutations. The p53-dependent tumourigenesis is influenced by interaction not only with E6/HPV 16, 18 but also with MDM2, bcl-2 and RB protein. The polymorphism of p53 seems to contribute to malignant transformation of cervix. On contrary, there are experimental data showing that p53 may not be the only factor playing role in malignant transformation in cervical cancer. It has been generally agreed that viral oncoprotein E6 is a critical step in the onset of malignant transformation of cervix. There is a vast number of experimental and clinical studies confirming the validity of E6 induced cervical cancer including alteration of the genotype and phenotypic characteristics of the transforming cells. The modern tools of molecular biology offer an exact diagnosis as well as relevant targets for gene therapy of the cervical cancer.

Female↗

PubMed1997

Different onset of nucleolar activation in endocardial endothelial cells and cardiomyocytes following pressure overload in rat heart.

We evaluated the time course of activation of endocardial endothelial cells and cardiomyocytes in adult rats with pressure overload induced by the abdominal aortic constriction. The silver staining technique for nucleolar organizer region was used to mark an increased transcriptional activity of the cells. An increased number of nucleoli was already detected in the endocardial endothelium of the left ventricle 5 h post-operatively, while the response of cardiomyocytes was still absent. Twenty-four h after constriction, the activation of the endocardial endothelium and cardiomyocytes was evident in both ventricles. A similar delay was observed between the nucleolar activation in vascular endothelium and smooth muscle cells of the abdominal aorta. In contrast, these cells of the thoracic aorta did not exhibit any significant increase of the transcriptional activity. The sequential stimulation of the transcriptional activity of the left ventricular endocardial endothelial cells and the subjacent cardiac myocytes due to pressure overload is in accord with the view that the endocardium may serve as a mechanotransducer which converts the hemodynamic changes into the signals that influence the growth of the cardiac myocytes.

Animals↗

PubMed1997

Induction of cell-cycle inhibitor p21 in rat ventricular myocytes during early postnatal transition from hyperplasia to hypertrophy.

To examine a possible involvement of p21 protein, an inhibitor of cyclin-dependent kinases (CDKs), in the transition from hyperplastic to hypertrophic growth of rat ventricular myocytes during the first postnatal week, we analysed day-by-day changes in the number of p21 positive cells using specific antibodies against this protein. Paraffin-embedded sections of the left ventricular myocardium were examined by means of immunoperoxidase technique and hematoxylin-eosin counterstaining. While during the first three postnatal days, the positive reaction for p21 was detected only in a small fraction of myocytes (12-20%), a sudden increase in positivity occurred on day 4 (54%) and continued till day 6 when the fraction of cells expressing p21 reached 87%. Our results show that the induction of CDK inhibitor p21 in rat ventricular myocytes is developmentally regulated. Moreover, the fact that the sudden increase in p21 positivity occurred at the same stage when the myocyte proliferation rapidly ceases, suggests that this protein is likely to be involved in mediating this key event of cardiac development.

Aging↗