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Resistance of cancer cells to cytostatics is caused by a number of mechanisms that are often combined. These include reduced cell entry or increased efflux, increased DNA repair, defects of, apoptotic pathways, increased cytostatic degradation as well as elevated levels of intracellular thiols of glutathione and metallothioneins (MT). It has been reported that high concentrations of thiol groups in the cytoplasm bind platinum alkylation derivatives and chemorezistence is due to the transfer of platinum from the cytostatic to MT, which inactivates them. Because we have shown an increase in MT levels in resistant neuroblastoma (NB) lines, but not in sensitive lines after incubation with platinum cytostatics, we have considered MT-3 for NB cells in our previous studies.SiMa NB cell lines transfected with vector containing human MT-3 and GFP or GFP only (control). Expression Microarray Human Cancer 3711 ElectraSense medium density 4 × 2k array slides with 1,609 DNA probes (Custom Array, Bothell, WA, USA), MT-3 expression and most expressed genes validated by real-time polymerase chain reaction. Sensitivity to CDDP (cisplatin) - MTT assay, clonogenicity test, Western blott caspase cleavage and free oxygen radicals fluorescence microscopy after CellROX Deep Red Reagent staining. Levels of MT-3 mRNA in 23 samples of high-risk NB, normal human cortex and bovine adrenal glands were investigated by reverse transcription polymerase chain reaction.Expression microarray showed downregulation 3 and overexpression of 19 genes in MT-3 transfected NB cells. Using gene ontology, over-expressed genes have been shown to drive senescence-induced oncogenes (CDKN2B and ANAPC5), and the genes of glutathione S-transferase M3, caspase 4 and DNAJB6 (chaperone neuronal proteins) were also expressed. We have demonstrated a reduced sensitivity of MT-3 transfected cells to CDDP (24h IC50 of 7.48 ± 0.97 and 19.81 ± 1.2 μg/ml), a higher number of colonies after incubation with CDDP, reduced caspase 3 after incubation with CDDP and lower free oxygen radicals after induction of CDDP. High-grade NB cells expressed MT-3 significantly more than non-tumoral adrenal cells but failed to show a clear relationship to disease course.We have demonstrated the relationship between MT-3 and senescence-induced oncogene genes and some other genes relevant to cell fate (glutathione S-transferase M3, caspase 4 and DNAJB6) and a significant proportion of MT-3 on CDDP resistance. High levels of MT-3 in high-risk NB could be one of the causes of frequent relapses in this tumor.Key words: neuroblastoma - metallothionein 3 - chemoresistance The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.This work was supported by AZV CR grant 15- 28334A. Submitted: 17. 2. 2018Accepted: 16. 4. 2018.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) represent a small family of enzymes called cholinesterases. These enzymes are in the organisms either soluble or anchored through anchoring proteins collagen Q (ColQ) and proline-rich membrane anchor (PRiMA). Knowledge of molecular biology and genetics of cholinesterase and their anchoring proteins resulted in the preparation of mutant mice with the absence of different molecular forms of cholinesterases. So far a number of mutant mice were prepared with a genetic modification on the genes encoding cholinesterases or anchoring proteins. The mice with mutation in the genes encoding the cholinesterases are: the mice with the absence of AChE, mice with the absence of BChE, mice with a deletion of exon 5 and 6 in the AChE gene and mice with the absence of AChE in muscles. The mice with a mutation in the genes encoding anchoring proteins include the mice with the absence of AChE and BChE anchored by ColQ and mice with the absence of AChE and BChE anchored by PRiMA. The study of adaptation changes results from the absence of cholinesterases led to the enrichment of existing knowledge about cholinesterases and the cholinergic nervous system.absence of cholinesterases mutant mice acetylcholinesterase butyrylcholinesterase.

The aim of our work was to confirm an immunohistochemical profile of routine markers of epithelial and neuroendocrine differentiation in eleven cases of Merkel cell carcinoma, as well as to study the expression of two markers of early phases of neuronal differentiation, namely reelin and class III beta-tubulin, markers which have not yet been studied in Merkel cell carcinomas. In all the investigated tumours the characteristic "dot-like" pattern of cytokeratin 20 immunoexpression, as well as negative immunostaining for cytokeratin 7 and thyroid transcription factor 1 (TTF-1) were disclosed; all the tumours showed neuroendocrine differentiation, expressing either neuron specific enolase (NSE) or chromogranin A(CgA), or both. An interesting finding was observed when the anti-cytokeratin monoclonal antibody MNF 116 was used. The characteristic "dot-like" pattern was detected in high proportion of tumours, including two samples of local recurrence of one of the carcinomas, where neoplastic cells have lost the expression of cytokeratin 20. The majority (91%) of Merkel cell carcinomas included in our group showed positive immunodetection of class III beta-tubulin when TU-20 antibody was used, while TuJ-1 immunostaining was surprisingly negative in all the investigated tumours. Detection of reelin was negative in almost all the studied Merkel cell carcinomas except for cases, where neoplastic cells revealed weak focal immunostaining in a minor portion of neoplastic cells.

Nestin is one of intermedial filaments exprimed in proliferating progenitor cells of the CNS and PNS (central and peripheral nervous system). Postnatal reexpression of the protein occures mainly in CNS tumors and correlates with a high grade of malignancy. The aim of our study is assessment of the nestin expression in benign and malignant skin melanocytic lesions with respect to presume a prognostic role of this protein. We examined 127 bioptic specimens, including 42 nodular melanomas (NM), 32 superficial spreading melanomas (SSM), 10 dysplastic nevi and 43 common intradermal or dermoepidermal nevi. We proved significant increase in nestin expression in melanoma groups, especially in nodular melanomas, where nestin was localized mainly in the peripheral, invasive areas of the tumor mass.Detection of nestin expression might be used as an additional melanocytic tumour marker.

In order to assess an impact of acute changes in blood volume on levels of B-type natriuretic peptides (BNP and NT-proBNP) 30 patients with a heart disease and chronic renal insufficiency on chronic dialysis program were assessed. An acute fluid restriction of 3750 ml on an average lead to decreased filling of the left heart ventricle (echocardiography revealed a reduced size of the left atrium, change in E/A parameters and deceleration time, and decreased end-diastolic volume of the left ventricle). Prior to dialysis normal levels of BNP (2.58 +/- 1.21 pg/ml) and elevated levels of NT-proBNP (193.2 +/- 117.7 pg/ml) had been indicated. Following dialysis a statistically significant decrease of BNP concentration in venous blood was proved, however it was "masked" by hemoconcentration. NT-proBNP level in venous blood remained unchanged. Correlation between BNP and NT-proBNP was not proved before dialysis nor after it. Correlation between BNP levels and echocardiographic parameters was not confirmed and a weak negative correlation with ejection fraction was proved in NT-proBNP. A BNP assessment could play an important role in the evaluation of acute changes in heart compensation. On the other way, NT-proBNP concentration is stable and is a long term marker of synchronisation of fluid circulation and function of the left ventricle. An importance of its assessment is probably rather prognostic.

The paper deals with a group of 157 patients followed for the period of four years in view of the relation of concentrations of BNP or NT-proBNP to overall mortality. In the introduction the authors describe their experience with natriuretic peptides; their own data follow. The Kaplan-Meier analysis of probability of survival proved to possess a strong predictive efficiency of the concentrations of BNP or NT-proBNP for overall mortality of the observed cohort.

Recently in the literature information is found on estimation of natriuretic peptides in the differential diagnosis of dyspnoea. Because in the Czech Republic since the beginning of 2002 routine estimation of NT-proBNP is available (analyzer Elecsys 2010), the objective of our work was to find out whether it is possible to use in the everyday practice of a district hospital estimation of NT-proBNP to differentiate dyspnoea with affection of the heart muscle from other types of dyspnoea.A group of 33 patients from the medical department of the Sternberk hospital was examined who attended on account of dyspnoea and lacked signs of acute coronary syndrome. All probands were diagnosed on the basis of defined criteria; according to the final diagnosis the patients were divided into three groups: group "LV" was formed by dyspnoic patients with organic affection of the left ventricle and signs of congestion in the lesser circulation, group "non-LV" was formed by patients where no organic affection of the left ventricle was found but other heart disease was present. Group "non-C" was formed by patients where a cardiac cause of dyspnoea was ruled out. In all patients on admission NT-proBNP was assessed.33 probands were examined, 18 men and 15 women, mean age 74.5 years. 25 probands the dyspnoea was classified as dyspnoea with affection of one of the cardiac compartments [19 of them had signs of organic affection of the left ventricle (group "LV")]; in 6 probands no signs of organic left ventricular affection were found (group "Non-LV"). The remaining 8 patients had no signs of any disease of the heart muscle, valves, septa, endocardium and pericardium (group "Non-C). The baseline values of NTpro-BNP were closely associated with the NYHA classification (grade II--median 55.3 pmol/l (469 ng/l, grade III--median 399.3 pmol/l (3384 ng/l), grade IV--median 724.7 pmol/l (6294 ng/l), the differences were statistically significant, p0.05). The dyspnoic probands with concurrent affection of some cardiac compartment (groups "LV" and "Non-LV") had a NT-pro BNP concentration significantly higher than probands without affection of the heart (group "Non-C") (median 589.5 pmol (4996 ng/l as compared with 62.9 pmol/l (533 ng/l, p0.01). In the group of probands with heart disease probands with affections of the left ventricle (group "LV") had significantly higher NT-proBNP values than subjects without affection of the LV and without any heart disease (groups "Non-LV" and "Non-C") (median 670.6 pmol/l (5683 ng/l) as compared with 187.5 pmol/l (1589 ng/l), p0.01). In hospitalized probands after treatment along with improved cardiopulmonary compensation also a significant drop of NT pro-BNP occurred (median 303 pmol/l (3967.7 ng/l to 211 pmol/l (2561 ng/l), p0.05). When looking for associations between anamnestic, laboratory and clinical data we found that the value of NT-proBNP is associated with dyspnoea with cardiac affection (groups "LV" + "Non-LV", correlation coefficient 0.48), with the left ventricular ejection fraction (correlation coefficient 0.52) and the baseline NYHA classification (correlation coefficient 0.36). In the examined group we did not find an association between NT-proBNP and age, sex, diabetes mellitus, hypertension, the presence of atrial arrhythmias, aortal stenosis, or the width of the left atrium. When using as cut-off for NT-proBNP 59 pmol/l (500 ng/l), the sensitivity of NT-proBNP for dyspnoea with affection of the cardiac compartments was 92% and the specificity 67%.Assessment of NT-proBNP is an important diagnostic acid in the differential diagnosis of dyspnoea.

Expression of cyclin-dependent kinase 5 (Cdk5) gradually increases during embryonal development until it reaches maximal levels that persist in the adult brain. Cdk5 represents a key component in the reelin signalling cascade. Activity of p35/Cdk5 is critical for the development of normal, six-layered structure of the mammalian brain cortex. Although it is not required for splitting of the cortical pre-plate into the marginal zone and sub-plate, a process that is affected in absence of the reelin and/or Dab1, it is substantial for the later-born neurons to bypass sub-plate neurons and to enter into the embryonal cortical plate. The defect of p35/Cdk5 disrupts the classical inside-out histogenetic pattern of neocortex. Neurons that fail to migrate past older layers of cells accumulate beneath the sub-plate thus forming an abnormal, inverted outside-in configuration in p35/Cdk5 defective animals. Exploration of neuronal migration disturbancies can shed new light on some diseases of human brain development including different heterotopias and lissencephaly. From an evolutionary point of view, the involvement of Cdk5 in the reelin signalling cascade could define a new step in the brain gray matter development.

Many observations indicate that genetic factors play an important role in the aetiology of autism. Up to now, however, no genetic markers have been convincingly identified which influence the predisposition to this disorder. Complex genetic analysis of autistic patients and their families may therefore lead to the identification of features which could help to direct further search for the predisposing genes.We have analysed a sample of 20 patients with autism spectrum disorders. The patients have been subjected to clinical genetic examination, cytogenetic analysis and DNA analysis of the FMR1 gene. In the sample studied we have observed more boys (15/20), various degree of mental retardation (18/20), high frequency of complications during pregnancy (10/20) and delivery (10/20), increased incidence of psychiatric disorders, behavioural abnormalities and suicides among the relatives, and increased head circumference and unusually formed ears in the probands. Three patients had different chromosomal aberrations or variants (t(21;22), inv(9) and inv(10)). One patient harboured expansion of the trinucleotide repeat sequence in the FMR1 gene on the full mutation level which is characteristic for the fragile X syndrome, and one patient is suspected to suffer from the Rett syndrome.Our observations confirm and extend the results reported in the literature. Most interesting are mainly the macrocephaly which may be associated with the recently described increased neonatal levels of neural growth factors in autistic individuals, ear malformations which may indicate aberrations in the HOXA1 gene pathway, the occurrence of chromosomal inversions recurrent in autism, and peculiarities in the pedigrees of the patients.

Twenty tumors of the peripheral sympathetic nervous system were investigated using a spectrum of antibodies against vimentin, neurofilament triplet, S-100 protein, neuronal specific enolase (NSE), chromogranin, synaptophysin, and vasoactive intestinal polypeptide. There were two ganglioneuromas, seven stroma rich neuroblastomas (composite ganglioneuroblastomas), five undifferentiated and six differentiating stroma poor neuroblastomas (NB) included in the series. Formalin-fixed, paraffin embedded material was used. The results showed that reactivity of the antibodies was relatively high, except the reactivity against synaptophysin. The tumor cell population showed a heterogeneous positivity in all cases. Only some undifferentiated NB were positive with the employed antibodies, which reduces the diagnostic benefit in a group of NB in which diagnostic demands of the immunohistochemistry are most important. The best results in undifferentiated NB were obtained with polyclonal antibody against NSE. This antibody is, however, not specific. Positive results of the immunohistochemistry in this group of tumors should be evaluated with caution.