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.

Melatonin protects against ischemia-reperfusion injury and inhibits apoptosis in isolated working rat heart.

INTRODUCTION: Melatonin (MEL), a pineal hormone, is well known as a potent antioxidant in a variety of ischemia-reperfusion models. Recent studies have assumed a pivotal role of reactive oxygen species (ROS) in the development of apoptosis. There are few pieces of information concerning a possible protective role of MEL against apoptosis in ischemia-reperfusion injury of myocardium. METHODS: We conducted an in vitro experiment: (1) to study the effect of MEL in the model of isolated and perfused working rat heart; (2) to evaluate the antioxidant capacity of MEL by a simple fluorescence test; and (3) to analyze the extent of apoptosis inhibition by MEL. Four groups of male Wistar rat were used: (a) group 'MEL 50 muM' (n=8); (b) group 'ischemia 30 min' (n=8); (c) group 'controls' (n=8); and (d) group 'controls+MEL 50 muM' (n=8). The perfusion medium was an oxygenated Krebs-Henseleit buffer (KHB). Hearts in groups (a) and (b) underwent 30 min of global normothermic ischemia and 45 min of reperfusion; 3 min before ischemia the hearts of group (a) received KHB with MEL 50 muM (and MEL 50 muM was also present in KHB solution during reperfusion). Hearts of group (c) were only perfused by KHB, and hearts of group (d) perfused by KHB+MEL 50 muM throughout the experiment. Registered were basic hemodynamic parameters: coronary, aortic, cardiac output and heart rate. At the end of each experiment, a left ventricle samples were taken for in situ detection of apoptosis using a TUNEL in-situ detection kit (POD) and quantitative analysis was performed. Malonedialdehyde concentrations were evaluated from heart homogenate to determine the severity of oxidative damage. To study the antioxidant capacity of MEL, a fluorescence test with allophycocyanin as an indicator was performed. A peroxyl radical generator, 2,2'-azobis(2-amidinopropan)-4-hydrochloride (AAPH) was used, and the antioxidant effect of MEL was expressed in oxygen-radical absorbing capacity (ORAC) units. RESULTS: Treatment by MEL resulted in a significant improvement of hemodynamic parameters and reduction of postischemic arrhythmias during reperfusion. All hearts in group 'ischemia 30 min' developed fatal ventricular fibrillations. MEL significantly reduced the incidence of apoptotic cells (14+/-4.3%; **P<0.01) vs. group 'ischemia 30 min' (58+/-2.1%). No apoptotic cells were detected in both control groups (c) and (d). In the fluorescence test, MEL exhibited a significant dose-dependent protective effect against peroxyl radical; MEL also reduced significantly the level of lipoperoxidation (MDA; *P<0.05). Analysis of hemodynamic parameters in both control groups (c) and (d) did not show any significant differences; the presence of MEL 50 muM in KHB solution did not have any important influence on cardiac performance in this type of experiment. CONCLUSION: We confirmed the previously reported beneficial effects of MEL against ischemia-reperfusion injury, presumably via its antioxidant properties. A significant suppression of apoptosis and the peroxyl radical scavenging properties of MEL in our study could contribute to the hypothesis of a close link between oxidative stress and apoptosis promotion.

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.

Prevention of apoptosis by deferoxamine during 4 hours of cold cardioplegia and reperfusion: in vitro study of isolated working rat heart model.

INTRODUCTION: Heart transplantation is often accompanied by multiple functional alterations, especially in reperfusion period. These are probably related to the reactive oxygen species (ROS) formation catalyzed by transition metals such as iron and copper, and thus the preservation time of the donor hearts is limited. Metabolic protection of the heart grafts is a permanent objective of numerous experiments. Recently, an iron chelator deferoxamine (DFX) was proposed as antioxidant agent for storage solutions in heart grafts. Oxidative stress is also known to mediate the apoptotic cell death in different tissues during ischemia-reperfusion. METHODS: The aim of this study was to evaluate a possible role of DFX in prevention of apoptosis using in vitro model of isolated working rat heart and cold cardioplegia. Two groups of rats were evaluated: (a) group 'DFX 50 &mgr;M' (n=8) and (b) group 'controls' (n=8). Isolated rat hearts were perfused by Krebs-Henseleit buffer (KHB) for 30 min, arrested by cardioplegic solution and stored for 4 h in B21 solution at 4 degrees C. Then, the hearts were reperfused by KHB for 45 min. DFX was added to the cardioplegic and storage solutions and in KHB in reperfusion. Basic functional parameters were evaluated: coronary, aortic, cardiac outputs and heart rate. At the end of reperfusion period a tissue samples were taken from left ventricle and in situ detection of apoptotic cells was performed using an ApopTag kit. RESULTS: DFX significantly reduced the occurrence of apoptotic cells in myocardium (*P<0.05). Hearts treated by 50 &mgr;M of DFX showed also a better recovery of the cardiac output (***P<0.001). The presence of DFX in KHB, cardioplegic and storage solution reduced also the incidence of postischemic arrhythmias and fibrillation's but without statistical significance. CONCLUSIONS: Our results give evidence of the protective potential of DFX during cold ischemia and reperfusion, presumably due to its antioxidant properties. The significant decrease of apoptosis in hearts treated by DFX could be considered as an existence of close link between oxidative stress and apoptotic death promotion in ischemia-reperfusion injury.

[Effects of carvedilol, a sympatholytic, in experimental alloxan diabetes in laboratory rats]. / Studium úcinku sympatolytika carvedilolu v podmínkách experimentálního alloxanového diabetu u laboratorního potkana.

A possible effect of the sympatholytic carvedilol on alloxan-induced diabetes mellitus in the laboratory rat was examined in experiments. The animals were divided into a group treated with carvedilol in a single daily dose of 10mg/kg in 1 ml of diluting solution i.p and the control group which received only diluting solution in the pertinent amount. The values of malondialdehyde and glucose in the serum, diuresis and total losses of sugar in the urine within 24 hours were estimated and histopathological examination of the kidneys of the treated and control groups was performed. The results show an effect of the tested dose of the drug, primarily in the region the proximal renal tubule.

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.

[The effects of Carvedilol, a beta-blocker, in experimental ischemia-reperfusion kidney injury]. / Studium úcinku beta-blokátoru Carvedilolu pri ischemicko-reperfuzním poskození ledviny v experimentu.

Carvedilol is a recently introduced drug with multiple action with a non-selective beta-antiadrenergic and selective alpha1-antiadrenergic action used for treatment of mild to medium severe hypertension. The authors investigated in their experiments the protective effect of carvedilol under conditions of ischaemia-reperfusion of the kidney in the laboratory rat. The animals were divided into four groups 1. the control group was fed a diet without carvedilol for a period of two weeks. Groups 2, 3 and 4 were fed for two weeks a diet containing carvedilol, 1-3-10 mg/kg/day resp. After completed medication in all animals ischaemia of the kidney was induced (60 min.) with subsequent reperfusion (10 min.) Then the animals were sacrificed, the kidney was removed for histopathological examination, in blood the malondialdehyde (MDA) level was assessed. The conclusions of the investigation indicate a marked protective effect of the administered preparation. Carvedilol prevents the disintegration of tubular epithelia, pycnosis of the nuclei, and reduced the development of oedematous changes. These findings correlate with MDA levels.