Prostaglandin F2α Causes Fast Degenerative Changes in Ovulated Mouse Oocytes.

The effects of prostaglandin F2α on the cytoskeleton and membrane organelles of oocytes was investigated by culturing ovulated mouse oocytes in its presence (50 or 100 ng/ml) for 3 h. Tubulin, fibrillar actin, membranes and chromatin were visualized by specific antibodies, phalloidin, lipophilic dye DiOC6 and Hoechst 33342, respectively. Control oocytes were characterized by a meiotic spindle with chromosomes aligned at its equator, and a cortical layer of microfilaments with an actin cap. Intracellular membranes were localized mostly in the central region in metaphase I and in a broader volume, but still excluding the cell periphery, in metaphase II, and were slightly concentrated around the chromosomes. In oocytes treated with 50 ng/ml prostaglandin, cortical actin staining was diminished, the membrane distribution was clustered, and chromosomes showed signs of misalignment despite the apparently preserved spindle. In cells treated with 100 ng/ml prostaglandin, both the spindle and the actin cortex had degenerated or disappeared as microscopic objects. Metaphase plates were on average broader and more disorganized than in the 50 ng/ml group, and the distribution of membrane organelles had become uniform. These effects, to our knowledge observed for the first time, did not require presence of the cumulus during the incubation. They could be regarded as acceleration of the oocyte postovulatory aging, in which cytoskeletal deterioration seemed to have a leading role.

The use of unfixed bone marrow trephines for multicolour flow cytometry.

An adequate bone marrow aspirate is essential for a rapid diagnosis of acute leukaemia by multicolour flow cytometry enabling the simultaneous assessment of multiple antigens on the cell surface as well as intracellular or nuclear ones. In the context of acute leukaemia, it is important to have a diagnosis of the blasts lineage as soon as possible to decide the appropriate treatment. This is sometimes delayed due to difficulties in obtaining a bone marrow aspirate due to a "dry tap". In this study we evaluated retrospectively cell markers results by flow cytometry of unfixed bone marrow trephines of 65 patients with leukaemia at diagnosis and including a few after treatment. Our aims were: 1) To compare cell markers results between bone marrow trephine (BMT) and bone marrow aspirate (BMA) 24 cases and BMT with peripheral blood (PB) 14 cases in paired samples to establish if they were reproducible with results of the unfixed bone marrow trephine biopsies. 2) To ascertain a precise diagnosis in 27 (42%) of the cases in which only a bone marrow trephine was available. We demonstrated that unfixed bone marrow trephine provides an adequate and representative cell suspension for flow cytometry and it is a powerful tool when no other material (bone marrow aspirate or peripheral blood) is available to make a rapid diagnosis. Furthermore when marrow aspirate or peripheral blood paired samples were available, flow cytometry results obtained were identical across all the sample types. Applicability to the clinical laboratory: We described a method to obtain a cell suspension from core biopsies that can easily be implemented routinely in a laboratory that performs diagnostic flow cytometry immunophenotyping. This method is simple, inexpensive and it doesn't require extra equipment.

Determinants of the host-guest interactions between α-, ß- and γ-cyclodextrins and group IA, IIA and IIIA metal cations: a DFT/PCM study.

The most widely used native cyclodextrins are α-, ß- and γ-cyclodextrins containing six, seven or eight α-d-glucopyranoside units in the ring, respectively. Although the ligation properties of these host molecules have been extensively studied, a number of questions regarding their metal binding and selectivity remain unaddressed: to what extent do the size and flexibility of the host α-, ß- and γ-cyclodextrins influence their metal affinity/selectivity? Which metal is the most preferred binding partner of α-, ß- and γ-cyclodextrins? How do the charge, size and preferred coordination number of the metal cation shape its interactions with the host cyclodextrin? Can the guest metal cation inflict structural alterations in the host molecule and, if so, how do these changes correlate with the metal's properties? In the present study, by employing density functional theory (DFT) calculations combined with polarizable continuum model (PCM) computations, we try to answer these questions by evaluating the thermodynamic parameters of the IA, IIA and IIIA group metal binding to α, ß- and γ-cyclodextrins. We assess how the interaction between the two binding partners depends on (1) the size, valence state and preferred coordination number of the guest metal cations, (2) the size and flexibility of the host molecule, and (3) the dielectric properties of the environment. The series of group IA (Na+ and Rb+), IIA (Mg2+ and Sr2+) and IIIA (Al3+ and In3+) metal cations have been chosen for the task as they allow us to study the effect of various metal parameters (variable charge, ionic radius and coordination number) on the strength and form of the interactions with the host cyclodextrins.

Dynamics of Lamins B and A/C and Nucleoporin Nup160 during Meiotic Maturation in Mouse Oocytes.

This study was aimed at elucidating the fate of three important nuclear envelope components - lamins B and A/C and nucleoporin Nup160, during meiotic maturation of mouse oocytes. These proteins were localized by epifluorescence and confocal microscopy using specific antibodies in oocytes at different stages from prophase I (germinal vesicle) to metaphase II. In immature germinal vesicle oocytes, all three proteins were detected at the nuclear periphery. In metaphase I and metaphase II, lamin B co-localized with the meiotic spindle, lamin A/C was found in a diffuse halo surrounding the spindle and to a lesser degree throughout the cytoplasm, and Nup160 was concentrated to the spindle poles. To our knowledge, this is the first report on nucleoporin localization in mammalian oocytes and the first successful detection of lamins in mature oocytes. While the distribution patterns of both lamins closely paralleled the respective stages of mitosis, Nup160 localization in metaphase oocytes corresponded to that in mitotic prometaphase rather than metaphase. The peculiar distribution of this nucleoporin in oocytes may reflect its role in meiosis-specific mechanisms of spindle assembly and its regulation.

Nitrous oxide speeds the reduction of distressing intrusive memories in an experimental model of psychological trauma.

BACKGROUND: Post-traumatic stress disorder (PTSD) involves maladaptive long-term memory formation which underlies involuntary intrusive thoughts about the trauma. Preventing the development of such maladaptive memory is a key aim in preventing the development of PTSD. We examined whether the N-methyl d-aspartate receptor (NMDAR) antagonist gas nitrous oxide (N2O) could reduce the frequency of intrusive memories by inhibiting NMDAR-dependent memory consolidation in a laboratory analogue of psychological trauma. METHOD: Participants were randomized to inhale N2O (N = 25) or medical air (N = 25) after viewing a negatively valenced emotional film clip ('trauma film'). Participants subsequently completed a daily diary assessing frequency of intrusive thoughts relating to the film clip. A week later, participants completed an explicit memory recall task related to the film. RESULTS: Post-encoding N2O sped the reduction in intrusive memory frequency, with a significant reduction by the next day in the N2O group compared to 4 days later in the air group. N2O also interacted with post-film dissociation, producing increased intrusion frequency in those who were highly dissociated at baseline. Sleep length and quality the night after viewing the film did not differ between the groups. CONCLUSION: N2O speeds the reduction of intrusive analogue trauma memory in a time-dependent manner, consistent with sleep-dependent long-term consolidation disruption. Further research with this drug is warranted to determine its potential to inoculate against enduring effects of psychological trauma; however, caution is also urged in dissociated individuals where N2O may aggravate PTSD-like symptomatology.

DOES THE PATTERN OF CLONAL EVOLUTION IN THE KARYOTYPE OF PATIENTS WITH ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES DEPEND ON THE TYPE OF THE PRIMARY CHROMOSOMAL ABERRATIONS?

The aim of our study was to define if the type of primary chromosomal aberrations (CA) of the karyotype of patients with Acute myeloid leukemia (AML) and Myelodysplastic syndromes (MDS) determines the way and the rate of karyotype development. Conventional cytogenetic analysis was carried out on 248 AML and 105 MDS patients at diagnosis. Clonal evolution (CE) was found in 40% (51 of 128) of AML patients and in 47.5% (19 of 40) of MDS patients having CA in their karyotype. The first pattern we established was for the most frequent CA which initiate CE in 28 patients with a complex karyotype. These CA were non-balansed rearrangements in the following regions: 5q, 7q, 11q, 3q, monosomy 5, monosomy 7. The second pattern of CE was regarding the most frequent aneuploidias (+8, +11, +21, -Y, and the third pattern concerned balanced CA. We found significant difference in the distribution of karyotypes in different stages of progression between the first and the other two groups (p < 0.001). No statistical difference was found between the patterns in the second and the third group CA (p > 0.5).

IS THE AMPLIFICATION OF c-MYC, MLL AND RUNX1 GENES IN AML AND MDS PATIENTS WITH TRISOMY 8, 11 AND 21 A FACTOR FOR A CLONAL EVOLUTION IN THEIR KARYOTYPE?

The aim of our study was 1) to define if the amplification of c-MYC, MLL and RUNX1 genes is related to the progressive changes of the karyotype in patients with AML and MDS with trisomy 8, 11 and 21 (+8, +11 and +21) in bone marrow and 2) can that amplification be accepted as part of the clonal evolution (CE). Karyotype analysis was performed in 179 patients with AML or MDS with the different chromosomal aberrations (CA) aged 16-81. The findings were distributed as follow: initiating balanced CA (n = 60), aneuploidia (n = 55), unbalanced CA (n = 64). Amplification of c-MYC, MLL and RUNX1 genes by means of fluorescence in situ hybridization (FISH) was found in 35% (7 out of 20) of AML and MDS patients with +8, +11 u +21 as single CA in their karyotype; in 63.6% of pts (7 out of 11)--with additional numerical or structural CA and in 75% (9 out of 12)--with complex karyotype. We assume that the amplification of the respective chromosomal regions in patients with +8, +11 and +21 is related to CE. Considering the amplification as a factor of CE, we established 3 patterns of karyotype development depending on the type of the initiating CA in it. Significant statistical differences were found between the three patterns regarding the karyotype distribution in the different stages of progression (p < 0.001).

Numerical chromosome disorders in the common marmoset (Callithrix jacchus)--comparison between two captive colonies.

BACKGROUND: Chromosomal analyses were performed for marmosets from two colonies - Deutsches Primatenzentrum (DPZ) and Biomedical Primate Research Centre (BPRC). Chlorine-based disinfectants are used in DPZ; no chemical disinfection is applied in BPRC. METHODS: The rates of chromosomal non-disjunction, polyploidy and endoreduplication were investigated after G-banding. RESULTS: For DPZ monkeys, the mean rates of non-disjunction were 7.6% for bone marrow and 11.3% for lymphocytes. The polyploidy level was 2.5% in bone marrow and 0.8% in blood. Frequency of endoreduplication in bone marrow and in leucocytes was 0.5% and 0.8%, respectively. For BPRC, the rate of non-disjunction in leucocytes (1.3%) was significantly lower than that for DPZ; the polyploidy rate (0.2%) in blood was lower than that in DPZ; endoreduplication was not observed. CONCLUSION: The levels of chromosomal disorders are elevated for DPZ colony. We suggest that the increased rate of chromosomal disorders in DPZ marmosets can be related to the chemical disinfection of their environment.

[Investigation of cytokeratin and vimentin intermediate filaments in polycystic ovaries (PCOS) -- presence and specific structure of Balbiani body in primordial follicles].

Despite of the importance of cytoskeletal proteins for ovarian function and pathology, very few studies have addressed the presence and distribution of these proteins in polycystic ovaries. We investigated sections of human polycystic ovarian tissue for vimentin and a set of cytokeratins by epifluorescence. The studied proteins showed strong colocalization. Positive reaction was detected in two main ovarian compartments: with weak intensity in follicular cells and very strong in perinuclear position in oocytes of primordial follicles. Epifluorescent study of the oocytes from primordial follicles allowed us to identify the immunopositive structure in oocytes as Balbiani body, a transient organelle responsible for establishing oocyte polarity and ooplasm gradients in nonmammalian vertebrates. Our results suggest functional interaction of different types of cytoplasmic intermediate filament proteins in polycystic ovaries and a possible importance of the Balbiani body for human oogenesis in norm and pathology.

Amplification of c-MYC and MLL Genes as a Marker of Clonal Cell Progression in Patients with Myeloid Malignancy and Trisomy of Chromosomes 8 or 11.

Gene amplification (amp) is one of the basic mechanisms connected with overexpression of oncogenes. The c-MYC (located in 8q24) and MLL (located in 11q23) are the most often over represented genes that lead to a rapid proliferation of the affected cell clone in patients with myeloid neoplasms. Assessment of the level of amp c-MYC or amp MLL in the cases with trisomy 8 (+8) or trisomy 11 (+11) and myeloid malignances is necessary for a more precise estimation of the disease progression. A total of 26 patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) were included in the study: 18 with +8, six with +11 and two with complex karyotypes suspected of the partial trisomy. Routine cytogenetic analysis and fluorescent in situ hybridization (FISH) were applied to indicate the chromosome alterations and genes amp in the bone marrow cells. Amp c-MYC was observed in 12 from 18 (66.7%) patients with +8. All the patients with +11 demonstrated a different level of amp MLL. In most of the cases with MDS (9/10), the coincidence of the +8 or +11 with amp c-MYC or amp MLL, respectively, leads to transformation to AML and/or short overall survival. Our data suggest that amp c-MYC and amp MLL develop in conformity with +8 and +11, especially in cases with progressive deviations in the karyotype as an aggressive expansion of an aberrant cell clone and appearance of additional chromosome anomalies.

Improvement of seminal parameters with Prelox: a randomized, double-blind, placebo-controlled, cross-over trial.

In a randomly allocated, double-blind, placebo-controlled, cross-over design, 50 infertile patients were treated for 1 month with placebo or a combination of l-arginine aspartate and Pycnogenol (Prelox). Semen samples were examined at 4 week intervals according to WHO criteria. Treatment with Prelox increased significantly the semen volume, concentration of spermatozoa, percentage of motile spermatozoa and percentage of spermatozoa with normal morphology compared with placebo. The placebo had no influence on the parameters of seminological analysis. Intake of Pycnogenol for 1 month improved the fertility index to normal values. After treatment, the fertility index decreased again to infertile status. No unwanted effects were reported. Prelox seems to be a promising alternative to treat patients with mild infertility.

Improvement of erectile function with Prelox: a randomized, double-blind, placebo-controlled, crossover trial.

In a randomly allocated, double-blind, placebo-controlled, crossover design, 50 patients with mild to moderate erectile dysfunction (ED) were treated for 1 month with placebo or a combination of L-arginine aspartate and Pycnogenol (Prelox). Patients reported sexual function from diaries. Testosterone levels and endothelial NO synthase (e-NOS) were monitored along with routine clinical chemistry. Intake of Pycnogenol for 1 month restored erectile function to normal. Intercourse frequency doubled. e-NOS in spermatozoa and testosterone levels in blood increased significantly. Cholesterol levels and blood pressure were lowered. No unwanted effects were reported. Prelox is a promising alternative to treat mild to moderate ED.

[Sperm parameters in male idiopathic infertility after treatment with prelox].

UNLABELLED: The diagnosis of male infertility is determinate after assessment of sperm quality and clinical study. In nearly 30% of the cases nevertheless detailed clinical and laboratory study it can't be discovered the cause and on the bases of exclusion criteria set the diagnosis idiopathic infertility. The object of our study was investigation of the group patients (n=50) with idiopathic infertility treated with Prelox and to be studied its effects on spermatozoa parameters. MATERIAL AND METHODS: The study design was double-blind, placebo-controlled, cross-over, randomized study, including introduction period (1 month), two therapeutic periods (each one of 1 month) separated with 1 month wash out period and concluding period of 1 month. There was applied a new method for treatment with mechanism of action stimulation the production cGMP of spermatozoa endothelial nitric oxide synthase (eNOS). This is not surprising achieving results show improvement of sperm quality. The methods of the study were: 1. Assessment of the conventional semen analysis (according the criteria of WHO, 1999). 2. Spermatozoa function tests. 3. Spermatozoa-cervical mucus penetration tests. RESULTS: The obtained results showed improvement of sperm quality, in the middle-aged men the therapeutic answers was better than in younger. In conclusion the therapy with Prelox improve sperm parameters in men with idiopathic infertility. Pycnogenol (one of the constituents of Prelox) has powerful antioxidative influence ameliorating spermatozoa function.

[Assessment of sperm quality as a male factor in couple's infertility].

The purpose of the present review is to present laboratory assessment of some studies of human seminal fluid and spermatozoa - cervical-mucus interactions according the last (the fourth edition) of WHO guideline, 1999. There are discussed additional tests for spermatozoa function in connection with male factor in couple's infertility. It is outlined the signification of calculation of some spermatozoa indices for clinical practice. The assessment built on the entire population spermatozoa defines the fertility ability of the small portion spermatozoa reaching the oocyte and leading to fertilization.

New real-time analytical applications of electrochemical quartz crystal microbalance Stoichiometry and phase composition monitoring of electrodeposited thin chalcogenide films.

The electrochemical quartz crystal microbalance (EQCM) has recently gained increasing popularity as a powerful tool for electrochemical interface examinations. This paper reports on its great potential for developing new real-time analytical protocols. Potentiostatic coulometry is used in conjunction with quartz crystal microgravimetry (QCMg) with controlled hydrodynamics to develop two new analytical procedures: real-time stoichiometry monitoring of electrodeposited binary chalcogenide films and phase composition quantification of electrodeposited ternary chalcogenide compounds. The newly developed EQCM methods are illustrated on the examples of Ag2+deltaSe-electrodeposited films and the electroformation of ternary CuAgSe-films. Both compounds have been successfully used as ion-selective membranes for flow-injection detectors. The experimental set-up including hydrodynamic control, the main strategy of the approach used and its scope and limitations are broadly discussed. The obtained data allow for real time profile monitoring of either stoichiometry (Ag2+deltaSe-case) or phase composition (CuAgSe-case).

[Selecting function of female genital tract in relation with spermatozoa interaction].

The present study includes 50 infertile couples followed sperm parameters: morphology and others indices for ability of penetration in ovulatory cervical mucus during 12 months. It is discussed the possibility of which spermatozoa can participate in the fertilization and the impotence and place of the female genital tract for the selection of morphologically abnormal spermatozoa before reaching the place of fertilization.

Treatment of erectile dysfunction with pycnogenol and L-arginine.

Penile erection requires the relaxation of the cavernous smooth muscle, which is triggered by nitric oxide (NO). We investigated the possibility of overcoming erectile dysfunction (ED) by increasing the amounts of endogenous NO. For this purpose, we orally administered Pycnogenol, because it is known to increase production of NO by nitric oxide syntase together with L-arginine as substrate for this enzyme. The study included 40 men, aged 25-45 years, without confirmed organic erectile dysfunction. Throughout the 3-month trial period, patients received 3 ampoules Sargenor a day, a drinkable solution of the dipeptide arginyl aspartate (equivalent to 1.7 g L-arginine per day). During the second month, patients were additionally supplemented with 40 mg Pycnogenol two times per day; during the third month, the daily dosage was increased to three 40-mg Pycnogenol tablets. We obtained a sexual function questionnaire and a sexual activity diary from each patient. After 1 month of treatment with L-arginine, a statistically nonsignificant number of 2 patients (5%) experienced a normal erection. Treatment with a combination of L-arginine and Pycnogenol for the following month increased the number of men with restored sexual ability to 80%. Finally, after the third month of treatment, 92.5% of the men experienced a normal erection. We conclude that oral administration of L-arginine in combination with Pycnogenol causes a significant improvement in sexual function in men with ED without any side effects.

Health risk for children from lead and cadmium near a non-ferrous smelter in Bulgaria.

In an interdisciplinary approach the exposure to lead and cadmium of the population living in the vicinity of a Bulgarian non-ferrous metallurgical plant was studied to determine the routes of exposure. Two exposed villages situated 4 and 6 km SW and W, respectively,--in the plume of the plant's emissions--and a village for comparison situated 14 km SE of the plant were selected for the study. The blood of children aged 3-13 years from the exposed villages (n = 111) and the village for comparison (n = 18) was analyzed for lead (PbB) and cadmium (CdB) as well as for free erythrocyte protoporphyrin (FEP) and haematological parameters. A personal questionnaire concerning nutrition habits and life-style was filled in by interviewers of the children's parents. The cadmium levels in blood were within the variation range stated for Bulgaria. The exposed children had a mean CdB of 0.38 +/- 0.18 microgram/l (range 0.10-0.90 microgram/l) and the comparison group 0.31 +/- 0.35 microgram/l (0.06-1.42 micrograms/l). The mean PbB value measured in the exposed children was 240 +/- 96 micrograms/l (77-631 micrograms/l), while the mean value of PbB for the reference group was 149 +/- 57 micrograms/l (63-285 micrograms/l). Individual PbB levels > 150 and > 200 micrograms/l were found in 85% and 62%, respectively, of the exposed children and FEP > 80 micrograms/dl erythrocytes was detected in 21%. There was a concentration-response and a concentration-effect relationship (r = 0.51, D > 0.001) between PbB and FEP. According to the questionnaire only 10.3% of the interviewed families in the two exposed villages buy all food from the market, the others produce a significant part of their food themselves. The PbB levels of children who consume certain foods of local production (cabbage, cereals, poultry, and beef) were significantly higher compared to the PbB values of children whose families purchase these foods from the market. No significant relationship between lead in children's blood and the use of tin and ceramic utensils, drinking and irrigation water, hygiene habits, family tobacco smoking habits, playgrounds and traffic intensity were revealed. There was a significantly higher incidence of prematurely born babies in the exposed population. The proven relationship between lead intake via the food chain and existing significant health risks is the basis for risk communication. Measures aimed at healthier nutrition and life-style intend to contribute to decreasing the risk of the population living in the smelter region.

Bone marrow micrometastases as a prognostic factor in early breast cancer patients.

PURPOSE: To assess the prognostic significance of bone marrow micrometastases (BMMM) in patients with early breast cancer. PATIENTS AND METHODS: Cytological and cytochemical assessments of bone marrow aspirates were carried out on 100 patients with early breast cancer. The bone marrow was obtained by trephine biopsy or aspiration from both upper iliac crests during general anesthesia. The median follow-up period after surgery was 72 months. The statistical analysis of the data was carried out using the x(2) test and Kaplan-Meier univariate methodology. RESULTS: BMMM were found in 28 (28%) of all studied women. Bone metastases were proved in 13 (46.4%) of the 28 cases with BMMM within a period of 21 to 50 months after surgery (median 41.3 months). In the patients without BMMM, disease dissemination was diagnosed in 6 (8.3%) of them. In 3 (4.1%) cases bone metastases were proved, while in the other 3 (4.1%) pulmonary metastases were found. A significant relative risk (RR=11.43) of skeletal metastases occurrence with 95% confidence interval (95% CI) from 3.48 to 36.16 was established in patients with BMMM. Survival analysis of all 100 women showed an average survival period of 6.34 years for 95% of the patients (95% CI 6.20- 6.48). The difference in survival between the 2 groups with or without BMMM was not statistically significant. CONCLUSIONS: 1) The information of bone marrow status could be used as an additional prognostic criterion to identify a subgroup of early breast cancer patients with a high risk for development of bone metastases. 2) The detection of BMMM is an additional adverse prognostic factor, irrespective of tumour size and axillary node status and might be used to define the need of preventive therapy to reduce the incidence of bone metastases.