Prevalence of Y chromosome microdeletions in infertile men with severe oligozoospermia in Serbia.

AIM: The aim of this study was to determine the prevalence and type of microdeletions of the Y chromosome of men with severe oligozoospermia-ICSI candidates in the Serbian population and to compare our findings with those from other parts of the world. METHODS: In all patients spermiogram has been performed in order to determine the sperm concentration. Patients were subjected to detailed clinical, endocrinological and cytogenetic examinations. Microdeletion analysis was performed by polymerase chain reaction (PCR) on 203 patients with normal cytogenetic findings. The STS markers tested in each case were sY84, sY86 (AZFa); sY127, sY134 (AZFb); sY254, sY255 (AZFc). RESULTS: at least one of the STS markers was deleted in 11 of the 203 cases (5.4%). CONCLUSION: AZFc microdeletions were identified with a rather high prevalence in men with severe oligozoospermia ICSI candidates in Serbian population.

Bcl-2 expression in oral squamous cell carcinoma.

Apoptosis is a genetically regulated process involved in tissue size regulation, morphogenesis, and elimination of genetically damaged cells. A pallet of genes is involved in the control of apoptosis, such as bcl-2 family whose oncogenic potential has been demonstrated in oral tumorigenesis. Different members of bcl-2 family may promote or inhibit apoptosis by synthesizing anti- and proapoptotic proteins. One of antiapoptotic proteins, bcl-2, with a crucial role in apoptosis regulation was the object of our study. By means of immunohistochemistry we estimated the level of overexpression of bcl-2 proteins in a series of the 26 formalin fixed, paraffin-embedded samples of oral squamous cell carcinoma (OSCC). Analyzed tumors originated from different sites of oral cavity; 7/26 belonged to stage II, 14/26 to stage III, and 5/26 to stage IV. Immunoreactivity was scored according to the percentage and intensity of positive cytoplasmic bcl-2 staining. All tumors had low percentage of positively stained bcl-2 cells, with mean values for lower/higher intensity of 8.3 +/- 2.5/34.4 +/- 7, 7.5 +/- 1.1/31.9 +/- 4.3, and 8.4 +/- 5.8/31.5 +/- 5.8 within stages II, III, and IV, respectively. Low level of bcl-2 expression in our sample seems to be associated with higher survival rate: 77% for the 5-year follow-up period. Comparing clinicopathologic and risk factors data within each and between three groups of analyzed tumors (lip-tongue P = 0.58, tongue-floor of the mouth, P = 0.21, lip-floor of the mouth, P = 0.50) there was no significant difference. However, our results suggest that the level of bcl-2 expression could be a valuable predictor of tumor behavior and disease outcome.

Chromosomal assignment of loci susceptible to replication errors by radiation hybrid mapping.

Genetic instability due to a DNA mismatch repair deficiency leads to the replication error (RER) phenotype in cancer cells. Certain loci are more susceptible to replication errors than the genomic average. The mapping of such loci is important, because it could indicate chromosomal domains preferentially affected by RER. Here, we report the radiation hybrid mapping of five markers known to be highly sensitive to RER in carcinomas. They were localized in chromosomes 2q34-q36.1, 6p24.3-25.2, 7q22.1-q13.2, 16q23.2-q23.3 and Xq13.1-q21.2 near genes that could be involved in oncogenesis.

Brain inflammatory response induced by intracerebroventricular infusion of lipopolysaccharide: an immunohistochemical study.

Inflammatory processes may play a critical role in the pathogenesis of the degenerative changes associated with Alzheimer's disease (AD). In the present study, we used an animal model of brain inflammation in order to study a possible mechanism involved in AD. Lipopolysaccharide (LPS) was used to produce global microglial reactivity within the brain of young rats. Time-dependent changes in the inflammatory reaction and the participation of glial cells after acute injection of LPS (50 or 100 microg) into the lateral ventricle or the fourth ventricle were compared with the chronic infusion of LPS (0.15, 0.5, 1.5 or 5.0 microg/h) into the fourth ventricle (14 days). Several immunohistochemical markers were used to characterize the microglial response. Acute and chronic exposure to LPS induced major histocompatibility complex class II (MHC II) antigen expression, detected with OX-6 antibody, in a sub-population of microglial cells in defined brain areas. The morphological features and distribution of OX-6 positive cells observed in the proximity of the cannula track after LPS injection into the lateral ventricle suggested the recruitment of monocytes/macrophages from the periphery. The activation of the resident microglial cells was delayed and mainly concentrated within the temporal lobe regions and the limbic system. Chronic infusion to LPS into the fourth ventricle induced a comparable activation of microglial cells. Quantitative analysis of OX-6 positive cells showed a dose-dependent response to LPS exposure.

MDL 74,180 reduces cerebral infarction and free radical concentrations in rats subjected to ischaemia and reperfusion.

The protective effect of MDL 74,180 (2,3-dihydro-2,2,4,6, 7-pentamethyl-3-(4-methylpiperazino)-methyl-1-benzofuran-5-ol dihydrochloride) and alpha-tocopherol analogue free radical scavenger, against cerebral ischaemia and reperfusion in conscious rats has been demonstrated. Tissue damage following middle cerebral artery occlusion (2 h) and reperfusion (8 days) was decreased by MDL 74,180 (0.1 and 1.0 mg/kg per h) infusion beginning 15 min before the onset of reperfusion and continuing for 2 h into the reperfusion period, in a dose-related manner. Nitroxide radical adducts, characterized and quantified by electron spin resonance spectroscopy, were formed on the addition of spin traps to homogenized rat brain tissue previously subjected to global ischaemia and reperfusion. The primary oxidative chain free radicals form diamagnetic intermediates whose slow homolytic decomposition subsequently yields the observed stable spin adducts. Infusion of MDL 74,180 (1-10 mg/kg per h) beginning 15 min before the induction of global cerebral ischaemia (20 min) until the end of reperfusion (5 min), led to a dose-dependent reduction in the final concentration of spin adducts.

Receptor density in the rat heart following ischaemia and prolonged reperfusion.

1. The free radical scavenger MDL 73,404 decreases infarct size and improves heart performance after myocardial ischaemia and prolonged reperfusion. In the present study the possibility that changes in receptor density might contribute to the MDL 73,404-induced increase in contractility has been investigated in rats subjected to myocardial ischaemia and 8 days of reperfusion. 2. Both in saline- and MDL 73,404-treated rats a significant decrease in beta1- and increase in beta2-adrenoceptor densities was apparent in the infarcted tissue. 3. In non-infarcted septum and right ventricle tissue ischaemia followed by reperfusion tended to increase the density of beta2-adrenoceptors which was significantly increased after MDL 73,404 treatment. 4. The increased density of beta2-receptors in the non-infarcted tissue may contribute to the MDL 73,404 induced improvement of cardiac performance.

Myocardial protection by a cardioselective free radical scavenger.

Oxygen-derived free radicals are involved in myocardial reperfusion injury. In the present study MDL 74,405 (S-(-)-3,4-dihydro-6-hydroxy-N,N,N-2,5,7,8-heptamethyl-2H-1-benzo pyran-2-ethanaminium 4-methylbenzenesulfonate), a hydrophilic derivative of alpha-tocopherol, has been shown to inhibit lipid peroxidation in rat brain homogenate, ex vivo lipid peroxidation in mouse heart and to accumulate in myocardial tissue. Infused i.v. MDL 74,405 induced a dose-related reduction of myocardial infarct size in pentobarbitone-anaesthetised rats subjected to 60 min coronary artery ligation followed by 30 min reperfusion. Similarly i.v. infusion of MDL 74,405 beginning 10 min before coronary artery occlusion (60 min) until 30 min after the onset of reperfusion (8 days) caused a decrease of infarct size associated with an increase in aortic flow. Plasma levels of creatine phosphokinase were significantly reduced. In isolated infarcted hearts, obtained from MDL 74,405-treated rats after 8 days of reperfusion and perfused according to the Langendorff technique, an increase in the contractility index (+) and (-) dP/dtmax was apparent. In isolated non-infarcted rat hearts subjected to 30 min no-flow global ischaemia, perfusion with MDL 74,405 resulted in an increase in heart rate and the contractility indices (+) dP/dtmax, and left ventricular systolic pressure during reperfusion. In conclusion MDL 74,405, is a cardioselective free radical scavenger, that reduces myocardial infarct size and attenuates post-ischaemic dysfunction.

Effect of incomplete ischemia and reperfusion of the rat brain on the density and affinity of alpha-adrenergic binding sites in the cerebral cortex. Prevention of changes by stobadine and vitamin E.

The effect of incomplete 4 hr ischemia and subsequent 1 hr reperfusion of the rat brain on the density and affinity of alpha-adrenergic binding sites was investigated. To assess the involvement of oxygen-derived free radicals in the development of ischemic injury, we tested the effect of stobadine and vitamin E on putative changes of the binding parameters of alpha-adrenergic binding sites in ischemic and reperfused rat brains. Compared to the group of sham operated animals decreased density and increased affinity of [3H]dihydroergocryptine binding sites was found in cerebrocortical membranes of rats subjected to 4 hr incomplete ischemia and 1 hr reperfusion. The reduction of Bmax and Kd induced by ischemia and reperfusion of the brain was prevented by stobadine and vitamin E administration. Neither incomplete ischemia nor incomplete ischemia and subsequent reperfusion of the rat brain exerted significant effect on [3H]rauwolscine binding to alpha 2-adrenergic binding sites. Our results suggest that brain ischemia and reperfusion may affect the density and affinity of alpha 1- rather than of alpha 2-adrenergic binding sites. The beneficial effect of stobadine and vitamin E indicates that increased generation of oxygen free radicals might play a role in the development of these changes.

Protection of infarcted, chronically reperfused hearts by an alpha-tocopherol analogue.

Free radicals may cause part of the irreversible injury which occurs during myocardial infarction and reperfusion. In the present study MDL 73404, a hydrophilic, cardioselective, free radical scavenger analogue of alpha-tocopherol, was evaluated for its effects on infarct size as well as on indicators of reperfusion injury. A pentobarbitone-anaesthetised rat model of coronary artery ligation (60 min; followed by 8 days of reperfusion) was used. Intravenous infusion of MDL 73404 (3 mg/kg per h) began 10 min before occlusion until 30 min after the onset of reperfusion. MDL 73404 reduced (P < 0.02) the elevated serum levels of thiobarbituric acid reactive substances and plasma levels of creatine phosphokinase (P < 0.01). An increase in cardiac output and heart rate together with a decrease (P < 0.01) in infarct size was evident in rats that had received MDL 73404, 8 days previously. Isolated infarcted hearts obtained from rats after 8 days of reperfusion had greater (P < 0.01) + dP/dt max, -dP/dt max, left ventricular systolic pressure and coronary flow after MDL 73404 compared to saline-treated controls. Infusion of [14C]MDL 73404, during the time of occlusion resulted in a concentration of 14.5 +/- 2.2 mg eq/g in the non-ischaemic ventricular tissue and a concentration of 3.0 +/- 0.4 mg eq/g in the area at risk. After infusion for the 30 min of reperfusion, 6.4 +/- 0.2 mg eq/g was detected in the non-ischaemic ventricular tissue but only 3.1 +/- 0.5 mg eq/g in the area at risk.(ABSTRACT TRUNCATED AT 250 WORDS)

Short cerebral ischemia and subsequent reperfusion and treatment with stobadine.

Lipid peroxidation and activities of antioxidative enzymes were studied in the brain cortex after short (15 min) cerebral ischemia and reperfusion (10 min) in rats. Conjugated dienes (CD) and thiobarbituric acid-reactive substances (TBARS) were significantly elevated in the group of rats with ischemia followed by reperfusion in comparison to the ischemic animals. Superoxide dismutase (SOD) activity significantly increased in the group of animals with ischemia and reperfusion. No significant changes in the activities of glutathione peroxidase (GP) were observed. Stobadine administered before ischemia or before reperfusion decreased the level of TBARS. Stobadine probably prevents malondialdehyde (MDA) formation from hydroperoxide or might elevate the activity of aldehyde dehydrogenase. In contradiction to the findings after long-lasting (4 h) ischemia and subsequent reperfusion, no decrease in the concentration of CD or in the activity of SOD or GP was found.

Cytogenetic analysis of an adenoid cystic carcinoma of the Bartholin's gland. A rare, semimalignant tumor of the female genitourinary tract.

Cytogenetic analysis has been performed on short-term cultures from a 56-year-old woman suffering from an adenoid cystic carcinoma of Bartholin's gland. Beside a normal female karyotype, the tumor revealed an abnormal cell line with complex chromosome changes involving the chromosomes 1, 4, 6, 11, 22, and 14. The mainly structural and nonbalanced rearrangements led to the loss of the chromosome segments 1p31----qter, 4q22----q28, 6p12----qter, 11p11.2----pter, 14q24----qter, and 22q13----qter. Clonal numerical aberrations were not observed. To our knowledge, such a tumor has to-date not been cytogenetically investigated.

Sister chromatid exchanges in patients with carcinoma in situ of cervix uteri.

Sister chromatid exchange (SCE) was analyzed in lymphocytes of 21 patients with carcinoma in situ of cervix uteri and 19 control subjects. The mean SCE frequencies were 8.92 +/- 0.31 (n = 417) and 6.94 +/- 0.23, (n = 375) per metaphase in patients and controls, respectively. The increase of SCE levels in cancer patients was highly significant in respect to controls (p less than 0.001). Together with data of other authors in patients with precancerous and cancerous lesions of the cervix, our results suggest that there is no correlation between SCE rate and severity of cancerous lesions.

Effect of stobadine on brain lipid peroxidation induced by incomplete ischemia and subsequent reperfusion.

The ability of stobadine (ST) to prevent lipid peroxidation was tested in incomplete rat cerebral ischemia induced by 4 hour ligation of the common carotid arteries with a subsequent 10 min reperfusion. The extent of lipid peroxidation was determined by the measurement of the level of conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS). The levels of CD and TBARS were significantly elevated in brain cortex samples from animals subjected to ischemia followed by reoxygenation in comparison with ischemic samples without reperfusion, samples from sham operated or control animals. The concentration of CD and TBARS significantly decreased in animals treated with therapeutic doses of ST (2 mg/kg) administered i.v. immediately before reperfusion or 10 min after the onset of reperfusion. Stobadine was more effective than the known lipid antioxidant vitamin E, given in a dose of 30 mg/kg.day i.m. over 3 consecutive days prior to ischemia. The beneficial effect of ST on survival of rats was more effective in comparison with vitamin E. Significant changes were found in the activities of the antioxidative enzymes, i.e. increase in superoxide dismutase (SOD) and decrease in glutathione peroxidase (GP) in brain cortex samples from animals subjected to ischemia followed by reoxygenation. Stobadine prevented these changes. Catalase (CAT) activity was not detectable. It may be concluded from the increased SOD activity that oxygen radicals play a significant role in cerebral ischemia followed reperfusion. In addition to its antioxidant effect, stobadine probably prevents superoxide radical generation. The mechanism of xanthine oxidase inhibition is not involved in preventing superoxide radical generation by stobadine. Stobadine maintained high GP activity, probably by preventing glutathione oxidation.

Effect of the steroidal alkaloid buxaminol-E on blood pressure, acetylcholinesterase activity and (3H)quinuclidinyl benzilate binding in cerebral cortex.

Pharmacological properties of the steroidal alkaloid buxaminol-E isolated from Buxus sempervirens varietas bullata were studied in relation to the cholinergic nervous system. In anaesthetised cats buxaminol-E (1.25 mumol/kg) initially induced a small short-lasting increase in blood pressure followed by marked hypotension. Atropine (1.47 mumol/kg) inhibited the hypotensive effect almost completely and methylatropine (1.47 mumol/kg) partially. Buxaminol-E and the muscarinic agonists McN-A-343 and bethanechol inhibited the binding of the muscarinic antagonist (3H)quinuclidinyl benzilate in cat and rabbit cerebral cortex membranes in decreasing order of potency. In about 10-fold higher concentrations they also inhibited acetylcholinesterase activity in cat cerebral cortex in the same order of potency. The results suggest that the hypotensive effect of buxaminol-E could be due to both central and peripheral activation of muscarinic receptors, and to a lesser degree to inhibition of acetylcholinesterase activity.

[The central effects of a steroid alkaloid, Buxaminol-E, in conscious cats]. / Centrálne úcinky stereoidného alkaloidu Buxaminolu-E u bdelých maciek.

Buxaminol-E injected i.v. to conscious cats evoked hypothermia, tachypnoe, anorexia, salivation, defecation, decrease of spontaneous activity and sensitivity to painful stimulus and agitation during its administration. The above mentioned effects of B--E, with the exception of the antinociceptive action which was not examined and of the initial excitation, were observed also after intracerebroventricular (i.c.v.) administration of B--E, and they were depressed by atropine administered i.c.v. Our findings suggest a central cholinergic action of B--E in conscious cats. Paroxysmal tonic-clonic convulsions and circling observed only after i.c.v. administration of B--E and piloerection, ataxia and urination were not inhibited by atropine administered i.c.v.

Analysis of the cardiovascular changes evoked by microinjection of NaCl into the nucleus tractus solitarii of rats: evidence for a Na+/Ca2+ relationship.

The effect of various ionic solutions microinjected into the nucleus tractus solitarii (NTS) on blood pressure, heart rate and respiration rate was investigated in urethane-anesthetized rats. Unilateral microinjection of solutions containing NaCl (154 mM), NaCl (154 mM) and KCl (2.8 mM) or NaNO3 (154 mM) into a restricted area of the NTS evoked acute decreases in blood pressure, heart rate and respiration rate. However, calcium chloride (0.36-3.3 mM), present in the microinjection solutions, reduced the decrease in all 3 recorded parameters in a concentration-dependent manner. A disturbance in the Na+/Ca2+ ionic ratio may account for the changes evoked by the administered solutions. These results indicate the presence of a restricted area in the NTS which is sensitive to changes in some vegetative functions.

Time course of lipid peroxidation during incomplete ischaemia followed by reperfusion in rat brain.

The time course of lipid peroxidation was studied in the rat brain cortex after ischaemia and reperfusion. The ischaemia was induced by 4-hour occlusion of both common carotid arteries and was followed by reperfusion of different duration (10, 30 or 60 min). The extent of lipid peroxidation was determined by measurement of conjugated dienes (CD) and TBA reactive products. Maximal values of CD and TBA reactive products were found after 10- and 30-minute reperfusion. This indicated the most suitable time interval for studying the effect of antioxidants and oxygen radical scavengers in this model of brain ischaemia.

Cardiovascular effects of increasing sodium chloride concentrations microinjected into the nucleus tractus solitarii of the rat.

Unilateral microinjection of increasing sodium chloride concentrations (154.0-256.6 mM) into the nucleus tractus solitarii (NTS) of urethane-anesthetized rats evoked hypotension and bradycardia. The effect was elicited only in a restricted area of the mediocaudal NTS. Microinjection of demineralized water, an isotonic sucrose solution and artificial cerebrospinal fluid were without effect. The possible existence of sodium-sensitive neurons in the NTS is discussed.

Antihistaminic activity of stobadine in the peripheral and central nervous system.

Stobadine in low doses induced hyperpolarization, and in high doses depolarization and inhibition of transmission in the cat superior cervical ganglion. The ganglionic depolarizing action of histamine was inhibited by stobadine in low doses. Stobadine partially depressed ganglionic responses to isoprenaline, serotonin, M1 muscarinic agonist McN-A-343 and clonidine only in high doses. Stobadine administered into the third cerebral ventricle of anaesthetized cats evoked dose-dependent hypotension without changes in heart rate. Intracerebroventricular (i.c.v.) administration of histamine evoked hypertension followed by hypotension. Both effects were inhibited by i.c.v. pretreatment with stobadine. These results indicate that in nervous tissue stobadine antagonises histamine responses presumably due to H1-receptor stimulation.