Introduction: The aim of the study was to review surgically treated patients with a pineal cyst with respect to an indication, therapy and outcome, and to provide an A to Z summary of pineal cysts spanning embryology to therapeutic possibilities. Material and methods: We retrospectively evaluated patients treated at the Department of Neurosurgery, Masaryk's Hospital in Usti nad Labem between 2002 and 2012. All demographic data, symptoms, investigations and management data were recorded and evaluated. Results: Eight patients underwent a surgery (all female, median age 15.5 years), infratentorial-supracerebellar approach was used in all cases and the mean size of the cyst was 21 x 16 mm. Six patients presented with "typical" symptoms (headache, nausea, faintness, visual impairment), one with precocious puberty and one with progressive comatose condition. Mean follow-up time was 2.7 years. After the surgery, symptoms resolved completely in five patients (including the patient with precocious puberty) and partially in three. Conclusion: If correctly indicated, surgical resection is the optimal therapeutic approach to symptomatic pineal cysts. Based on our experience and published literature, we continue to consider the infratentorial-supracerebellar approach as a gold standard for surgical treatment of pineal cysts.

The myocardial extracellular matrix plays an important role in maintaining the structural and functional integrity of the heart and is centrally involved in post-myocardial infarction repair processes. We analysed some genetic and proteomic aspects that could play an important role in the development of myocardial infarction. Matrix metalloproteinases are enzymes that contribute strongly to the degradation of extracellular matrix components. In this study the serological levels of MMP-2 and MMP-9 were investigated using immunological testing in 34 patients with myocardial infarction and 34 matched control subjects. The serum levels of MMPs were determined by ELISA. Changes in serum levels were characterized within 24 h and after 6 months post myocardial infarction. Significantly higher levels of MMP-2 (299.47 ± 117.61 ng/ml) and MMP-9 (93.56 ± 53.74 ng/ml) were determined in patients with myocardial infarction compared to the controls, in both cases P < 0.001. MMP-9 levels decreased significantly in the 6 months after cardiac event, whereas the levels of MMP-2 were almost equal to the post-infarction ones. While comparing the results from four patients that died of cardiovascular cause within 6 months we found significantly higher MMP-2 (435.00 ± 55.83 ng/ml, P = 0.003) and MMP-9 (166.25 ± 41.07 ng/ml, P = 0.018) values. Microarray analysis was used to determine the gene expression of selected genes for MMPs and their regulators from peripheral blood. The selected genes did not show satisfactory results that could have a potential implication for diagnostics of tissue degeneration.

Development of ascending aortic dilatation (AAD) in about 10 % of patients operated for aortic valve disease (AVD) is probably based on intrinsic pathology of the aortic wall. This may involve an abnormality in the process of extracellular matrix remodelling. The present study evaluated the serum levels of specific metalloproteinases (MMP-2 and MMP-9) and investigated the gene for transforming growth factor receptor 2 (TGFBR2) in 28 patients with AVD associated with AAD (mean age 60.6 years), in 29 patients (68.9 years) with AVD without AAD, and in 30 healthy controls (45.3 years). The serum levels of MMPs were determined by ELISA. Further, we focused on genetic screening of the received February 10, 2013. accepted March 13, 2013. The research was partially supported by the projects Ln00B107 and 1M06014 of the Ministry of education, Youth and sports of the Czech republic and by the long-term strategic development financing of the Institute of Computer Science (RVO:67985807). Corresponding author: Jana Šímová, Department of anthropology and Human genetics, Faculty of science, Charles university in Prague, Viničná 7, 128 43, Prague 2, Czech Republic. e-mail:

Development of ascending aortic dilatation (AAD) in about 10 % of patients operated for aortic valve disease (AVD) is probably based on intrinsic pathology of the aortic wall. This may involve an abnormality in the process of extracellular matrix remodelling. The present study evaluated the serum levels of specific metalloproteinases (MMP-2 and MMP-9) and investigated the gene for transforming growth factor receptor 2 (TGFBR2) in 28 patients with AVD associated with AAD (mean age 60.6 years), in 29 patients (68.9 years) with AVD without AAD, and in 30 healthy controls (45.3 years). The serum levels of MMPs were determined by ELISA. Further, we focused on genetic screening of the TGFBR2 gene. Plasma MMP-2 concentrations were significantly higher in the groups of patients compared to the controls: median 1315.0 (mean 1265.2 ± SD 391.3) in AVD with AAD, 1240.0 (1327.8 ± 352.5) in AVD without AAD versus 902.5 (872.3 ± 166.2) ng/ml in the healthy controls, in both cases P < 0.001. The serum levels of MMP-9 were significantly higher in AVD with AAD patients [107.0 (202.3 ± 313.0)] and in AVD without AAD patients [107.0 (185.8 ± 264.3)] compared to the healthy controls [14.5 (21.2 ± 24.8) ng/ml], in both cases P < 0.001. No significant correlation was observed between plasma MMP-2 and MMP-9 and ascending aorta diameter. Genetic screening did not reveal any variation in the TGFBR2 gene in the patients. Measurement of MMP levels is a simple and relatively rapid laboratory test that could be used as a biochemical indicator when evaluated in combination with imaging techniques.

Surface plasmon resonance (SPR) biosensors capable of in real time detection of Cronobacter at concentrations down to 106 cells mL(-1) in samples of consumer fresh-whole fat milk, powder whole-fat milk preparation, and powder infant formulation were developed for the first time. Antibodies against Cronobacter were covalently attached onto polymer brushes of poly(2-hydroxyethyl methacrylate) (poly(HEMA)) grafted from the SPR chip surface. The lowest detection limit, 10(4) cells mL(-1), was achieved in phosphate buffered saline (pH 7.4) with sensors prepared by covalent immobilization of the same antibodies onto a self assembled monolayer (SAM) of hexa(ethylene glycol) undecanethiol (EG(6)). However, when the EG(6) based sensors were challenged with milk samples the non-specific response due to the deposition of non-targeted compounds from the milk samples was much higher than the specific response to Cronobacter hampering the detection in milk. Similar interfering fouling was observed on antifouling polymer brushes of hydroxy-capped oligoethylene glycol methacrylate and even a 10 times higher fouling was observed on the widely used SAM of mixed hydroxy- and carboxy-terminated alkanethiols. Only poly(HEMA) brushes totally suppressed the fouling from milk samples.The robust well-controlled surface initiated atom transfer radical polymerization of HEMA allowed the preparation of highly dense brushes with a minimal thickness so that the capture of antigens by the antibodies immobilized on the brush layer could take place close to the gold SPR surface to provide a stronger optical response while the fouling was still suppressed. A minimum thickness of 19nm of poly(HEMA) brush layer was necessary to suppress completely non-specific sensor response to fouling from milk. (C) 2011 Elsevier B.V. All rights reserved.

Nuclease from tomato (TBN1) was produced by in planta biotechnology purified and tested for its anticarcinogenic properties. The nuclease was cytostatic after its intratumoral administration to nude mice bearing human melanoma or prostate carcinoma or after tumor targeting by TBN1 administration intravenously as conjugate with polyethylene glycol (PEG). Inhibitory effects of TBN1 on tumor growth were comparable to effects of bovine seminal RNase (BS-RNase), but the inhibition was reached at about ten times lower protein concentration. Simultaneously, TBN1 exhibited a lower degree of embryotoxicity compared to BS-RNAse and other nucleases. TBN1 showed significant stability in vivo, because it was readily detected after its administration intratumorally or intravenously by the fluorescence methods. Intravenous administration of TBN1-PEG caused significant inhibition of tumor proliferation without obvious degenerative changes, while direct administration of TBN1 into melanoma tumors led to rapid tumor tissue degeneration. The fact can be essential for the mode of TBN1 biological action that mature nuclease is a small (36 kDa) thermostable glycoprotein that has ability to destroy human 28S, 18S, 7S and 5.8S RNA, circular RNAs, double-stranded RNA in vitro and shows DNase and 3'nucleotidase activities.

An indirect competitive enzyme immunoassay of Enterobacter sakazakii has been developed. The rabbit polyclonal antibodies to heat-labile or heat-stable antigen of the type strain E. sakazakii CNCTC 5739(T) were prepared for these purposes. The detection limits of enzyme immunoassays were within the range 0.6-14.4 x 10(5) cells/ml. Antibodies raised to heat-labile antigen were serotype-specific. Although they contain non-specific IgG fractions binding periplasmatic and cytosol proteins, the interactions of these immunoglobulins are not manifested under conditions of ELISA developed.

The competitive enzyme immunoassays for detection of Campylobacter jejuni, C. coli and C. fetus subsp. fetus have been developed. Rabbit and hen immunoglobulins were prepared for these purposes. The working conditions of ELISAs, such as the concentrations of immunoreactants, incubation temperatures and time, and the composition of the substrate have been established. The detection limits were in the range 5.0 10(4)-3.2 10(6) cfu/ml. The application of chemiluminescent substrates did not result in any significant improvement of the assay's detectability and sensitivity. Prepared antibodies showed rather high specificity and cross-reactivity profiles, and both rabbit and hen immunoglobulins were similar. Only IgY to C. jejuni cross-reacted with seven strains of C. jejuni and two other Campylobacter spp.A limited number of naturally and artificially contaminated food samples were tested. The results obtained by means of an enzyme immunoassay were compared with those obtained from PCR or commercially available Singlepath (R) Campylobacter GLISA-Rapid Test. Poultry products were naturally contaminated with Campylobacters. The wild species were identified as C. jejuni and C. coli.

Rabbit IgG raised against whole cells ofYersinia enterocolitica O:3, O:9 and against a group of pathogenicY. enterocolitica strains (serotypes O:3, O:5,27, O:8. and O:9) were prepared. The antibody limiting titers were within the range of 1:9.5 × 104−1:7.5 × 105. The immunoblotting analysis ofYersinia lipopolysacchides separated by SDS-PAGE showed that IgG against the single serotype O:3 interacted with high-molar-mass LPS of O:3 whereas other antibodies were bound to low-molar-mass LPS of serotypes O:3, O:5,27, O:9 and strainY. enterocolitica (CNCTC Y 2/68). IgG against the group of pathogenic serotypes also weakly interacted with low-molar-mass LPS of serotypes O:5, O:6,30, and O:10. The cross-reactivity of the antibodies withY. pseudotuberculosis Ia and/orY. rohdei b, d, e, f, i, which was observed by means of dot-blotting procedure using the whole bacterial cells as an antigen, was shown not to be caused by LPS of these bacteria. The prepared antibodies were used in the development of indirect competitive ELISA. At the optimum concentration of the immunoreactants the detection limits were within the range of 3–7 × 106 colony-forming units per mL.

Objectives: Our research is a pilot study that specializes in the molecular genetic investigation of the TNNT2 gene in Czech patients with HCM/FHC disease. This study was initiated with exons 9 and 11 of TNNT2 because of their crucial role in the binding ability of cardiac troponin T to alpha-tropomyosin, and continued with analyses in other regions of the gone.Methods: Hundred and eighty-one Czech probands with HCM/FHC were enrolled in this study. The study group consisted of 24 families with FHC and probands without FHC history but with HCM diagnosis. The clinical diagnosis was based on echocardiography. DNA was isolated from peripheral blood lymphocytes and subsequently analyzed by the polymerase chain reaction (PCR), followed by DNA sequencing analyses, which were cross-sequenced.Results. The Delta Glul 60 mutation was observed in a sequence of the TNNT2 gene in a patient with the severe form of hypertrophic cardiomyopathy. No sequence alteration was found in exons 9 and 11 of the TNNT2 gene found in the rest of the DNA samples.Conclusion: The AGlul 60 mutation was observed in patients with severe forms of hypertrophic cardiomyopothy. This region is responsible for binding troponin T to alpha-tropomyosin. This mutation may lead to functional and structural effects on the troponin T protein. Mutations in this region are reported relatively rarely and therefore it was unique to observe the AGlul 60 mutation in our study.

Summary Objectives: Our research is a pilot study that specializes in the molecular genetic investigation of the TNNT2 gene in Czech patients with HCM/FHC disease. This study was initiated with exons 9 and 11 of TNNT2 because of their crucial role in the binding ability of cardiac troponin T to α-tropomyosin, and continued with analyses in other regions of the gene. Methods: Hundred and eighty-one Czech probands with HCM/FHC were enrolled in this study. The study group consisted of 24 families with FHC and probands without FHC history but with HCM diagnosis. The clinical diagnosis was based on echocardiography. DNA was isolated from peripheral blood lymphocytes and subsequently analyzed by the polymerase chain reaction (PCR), followed by DNA sequencing analyses, which were cross-sequenced. Results: The ΔGlu160 mutation was observed in a sequence of the TNNT2 gene in a patient with the severe form of hypertrophic cardiomyopathy. No sequence alteration was found in exons 9 and 11 of the TNNT2 gene found in the rest of the DNA samples. Conclusion: The ΔGlu160 mutation was observed in patients with severe forms of hypertrophic cardiomyopathy. This region is responsible for binding troponin T to α-tropomyosin. This mutation may lead to functional and structural effects on the troponin T protein. Mutations in this region are reported relatively rarely and therefore it was unique to observe the ΔGlu160 mutation in our study.

Bovine seminal ribonuclease (BS RNase), a dimeric homolog of bovine pancreatic ribonuclease (RNase A), is known to display special biological activities namely cytotoxicity for human tumor cells. Because some plant ribonucleases have a similar mass weight and structure as the animal ribonuclease, effects of a commercial product of Mung bean (Phaseolus aureus) nuclease (PhA) were studied on proliferation of ML-2 human tumor cells, as well as it's aspermatogenic, embryotoxic, immunogenic, and immunosuppressive activity, and therapeutic efficiency in athymic mice bearing human melanoma tumor. Concerning the antiproliferative activity, PhA nuclease was almost non-effective in vitro on ML-2 cells and also immunosuppressive activity on human lymphocyte in mixed culture was very low compared to that of BS RNase. However, significant antitumor activity was detected on human melanoma tumor after intratumoral or intraperitoneal administration into the mice. Furthermore conjugate of PhA nuclease with polyethylene glycol (PEG) injected seven times at the dose of 10 microg intraperitoneally showed identical antitumor activity as that of bovine seminal ribonuclease (BS RNase) injected by the same way at ten times higher dose. Both PhA and BS RNases exerted strong aspermatogenic effect on the width of spermatogenic layers while RNase A administration at ten times higher concentration was ineffective. PhA nuclease when compared by means of antibody cross reaction with RNase A, BS RNase and wheat leaf neutral RNase (WLN-RNase) was found to be immunologically similar to RNase A and WLN-RNase, meanwhile BS RNase showed much higher antigenicity in comparison with them.

Previously we have shown that monomeric RNase A has no significant biological activity, whereas its oligomers (dimer to tetramer) prepared by lyophilizing from 50% acetic acid solutions, show remarkable aspermatogenic and antitumor activities. Furthermore, conjugates prepared by chemical binding of native RNase A to polyethylene glycol (PEG) have shown a significant aspermatogenic and antitumor activities. In this work we show that the chemical conjugation of PEG to the RNase A C-dimer, and to the two RNase A trimers (NC-trimer and C- trimer) decreases the aspermatogenic activity of the oligomers while increasing their inhibitory activity on the growth of the human UB900518 amelanotic melanoma transplanted in athymic nude mice. Moreover, the PEG-conjugated RNaseA oligomers are devoid, like the free oligomers, of any embryotoxic activity.

Summary Objectives: Our research is a pilot study that specializes in the molecular genetic investigation of the TNNT2 gene in Czech patients with HCM/FHC disease. This study was initiated with exons 9 and 11 of TNNT2 because of their crucial role in the binding ability of cardiac troponin T to α-tropomyosin, and continued with analyses in other regions of the gene. Methods: Hundred and eighty-one Czech probands with HCM/FHC were enrolled in this study. The study group consisted of 24 families with FHC and probands without FHC history but with HCM diagnosis. The clinical diagnosis was based on echocardiography. DNA was isolated from peripheral blood lymphocytes and subsequently analyzed by the polymerase chain reaction (PCR), followed by DNA sequencing analyses, which were cross-sequenced. Results: The ΔGlu160 mutation was observed in a sequence of the TNNT2 gene in a patient with the severe form of hypertrophic cardiomyopathy. No sequence alteration was found in exons 9 and 11 of the TNNT2 gene found in the rest of the DNA samples. Conclusion: The ΔGlu160 mutation was observed in patients with severe forms of hypertrophic cardiomyopathy. This region is responsible for binding troponin T to α-tropomyosin. This mutation may lead to functional and structural effects on the troponin T protein. Mutations in this region are reported relatively rarely and therefore it was unique to observe the ΔGlu160 mutation in our study.

Subcutaneous application of bovine RNase A conjugated to HYase (bovine hyaluronidase), polyethylene glycol (PEG) and HYase+PEG resulted in a marked reduction of the width of the spermatogenic layers of the mouse testes. The number of sperms in caput epididymidis was significantly decreased in mice injected with conjugated RNase A. There was not any significant embryotoxic effect of free RNase A even conjugated with HYse, PEG and HYse+PEG. The immunogenicity, expressed in production of antibodies against free RNase A or conjugates with PEG, was very low. However, the immunogenic action of this enzyme conjugated only to HYase was much higher and produced the same immunogenicity as HYase itself. The immunogenic effect of RNase A+HYase conjugate decreased when PEG was joined to this conjugate. The inhibitory effect of RNase A conjugated to HYase, PEG and HYase+PEG on human ML-2 cells studied in vitro, was practically ineffective. On the other side, when RNase A conjugated to HYase or PEG was administered intraperitoneally into the mice bearing human melanoma, the antitumor effect was pronounced.

An indirect enzyme immunoassay for rapid detection ofCampylobacter jejuni subsp.jejuni O:23 has been developed. Optimum concentrations of immobilized cells, polyclonal chicken IgY, and rabbit anti-IgY antibody-horseradish peroxidase conjugate were 3.1 CFU/nL, 10 µg/mL, and 8 µg/mL, respectively. Under such conditions, the detection limit reached 50 CFU/µL, limit of quantification being 480 CFU/µL. By testing 5 chromogens.viz. 1,2-benzenediamine, 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), 3,3′,5,5′-tetramethylbenzidine, bi(4,4′-anisidine) and 3-methyl-2-benzothiazolinone hydrazone, in horseradish peroxidase substrate, 1,2-benzenediamine or 3,3′,5,5′-tetramethylbenzidine as H-donors in the enzyme substrate provided the highest ELISA sensitivity. The applied polyclonal antibody was specific for homogeneous antigen. The cross-reactions were observed only with one strain ofC. sputorum subsp.sputorum (21.5 %) and with G+ bacteriumMicrococcus luteus (6.1 %). Preliminary tests have been performed with a limited number of artificially contaminated food samples. No matrix effects on the ELISA sensitivity were observed. The results (by means of ELISA) were comparable with those given by both a standard cultivation method performed according toČSN ISO 10272 and commercially available Singlepath® Campylobacter GLISA-Rapid Test.

RNase A (bovine pancreatic ribonuclease) and BS-RNase (bovine seminal ribonuclease) are monomeric and dimeric enzymes, respectively, with aspermatogenic and antitumor activities. While the aspermatogenic and, in some experimental situations, the antitumor effects of the RNase A are only minor, the activity of BS-RNase in these phenomena is very significant. These differences can be annulled by means of conjugation of the enzymes with PEG (polyethylene glycol) chains. Aspermatogenic activity was studied histologically following subcutaneous injections of RNase A and BS-RNase conjugates in ICR mice, and the antitumor activity in athymic nude mice with growing human melanoma with i.p. injection of these conjugated ribonucleases. The experiments proved that RNase A, when conjugated to PEG, produced identical aspermatogenic and antitumour effects as BS-RNase conjugated to this polymer. Immunogenicity of RNase A and BS-RNase did not change substantially after the conjugation with PEG polymers. Binding of produced antibodies to both ribonucleases attached to PEG, however, was substantially reduced.

Salmonella enteritidis and Listeria monocytogenes were detected in real time using a surface plasmon resonance (SPR) sensor based on prism excitation of surface plasmons and spectral interrogation. The respective antibodies against the pathogens were immobilized on the gold sensor surface as a covalently crosslinked double-layer or covalently bound on a crosslinked albumin layer. Salmonella and Listeria could be detected by the sensor at concentrations down to 106cell/ml. The sensor sensitivity was comparable with that of standard ELISA in which the same antibodies were used.