Enzyme Production of Pleurotus ostreatus Evaluated by the Three‐Way Principal Component Analysis

The effect of the composition of culture media and fermentation time on the production of β‐glucosidase, xylanase, laccase, manganese‐dependent and independent peroxidases by the edible fungus Pleurotus ostreatus was determined. Culture medium separately containing potato, pepper, and tomato extracts and the enzyme activities were assessed to 31.5 days of fermentation. Three‐dimensional principal component analysis (3D‐PCA) followed by the plots of the first two elements of component matrices was employed for the elucidation of the similarities and dissimilarities between the parameters. It was established that the dimensionality of the original 3D matrix (9, 5, 4) could be reduced to 2, 1, 2 with 4.54 % loss of information. The plots demonstrated that the culture media displayed large differences in their capacity to promote enzyme production and that the presence of pepper and tomato extracts exerted the greatest influence on the enzyme activities. The effect of the fermentation time was manifested only after 14 d of fermentation and the highest differences were observed after 28 and 31.5 d of fermentation. Except laccase and manganese‐dependent peroxidase, the enzyme activities also differed considerably dependent on the composition of the fermentation broth. 3D‐PCA followed by the plots of component matrices is a valuable tool for the simultaneous assessment of three dimensional data matrices in biotechnological processes.     

Hungarian experience with non-Hodgkin's lymphoma in childhood

Between 1990 and 2004, 230 children with non-Hodgkin's lymphoma (NHL) were treated according to the Berlin-Frankfurt-Münster (BFM) protocols (NHL-BFM-90 and -95) in Hungary. The aim of the present study was to summarize our experience with these protocols, to assess the survival rates and to compare the Hungarian data with the international results. The male-to-female ratio was 2.59:1, the mean age at the time of diagnosis was 10 years and 1 month. Ninety-one children had lymphoblastic/T-NHL (LB/T-NHL), 108 B-NHL and 31 anaplastic large cell lymphoma (ALCL). Twenty-eight patients had relapse after a mean time of 13 months from the time of the initial diagnosis. In the above mentioned period, 16 children underwent autologous stem-cell transplantation. Nine patients with B-NHL got anti-CD20 immunotherapy. The five-year overall survival (OS) of our patients is 77.8%+/-3%, the event-free survival (EFS) is 75.1%+/-3%. The 5-year OS and EFS rates were not statistically different in the three histology groups (OS: 71.6%+/-5%, 82.7%+/-4% and 80.3%+/-7%; EFS: 68.7%+/-5%, 81.1%+/-4% and 73.9%+/-8% in LB/T-NHL, B-NHL and ALCL, respectively). We can conclude that non-Hodgkin's lymphoma has a quite good prognosis among the malignant pediatric diseases. The cure rate is over 75%. The Hungarian results are comparable with other international data. In the last five years the mortality during induction was reduced from 10% to 2% and the OS is about 10% better than it was before. In case of relapse or residual disease, therapeutic results can be improved with stem-cell transplantation with or without immunotherapy.     

Biomarker indicators of chemoprevention among subjects occupationally exposed to metals

Chemoprevention with chelating agent Humetta for three months was performed, due to anaemia and other haematologic disorders, immunotoxicological alterations and/or increased chromosome aberration rate among galvanisers and goldsmiths occupationally exposed to precious and heavy metals. Twenty-two of altogether 47 subjects took part voluntarily in the chemoprevention, and the rest of the subjects served as untreated controls. Complex clinical laboratory testing including detailed anamneses; genotoxicological and immunotoxicological monitoring were performed before and after administration of chemopreventive agent. After chemoprevention a significant improvement was observed in anaemia and serum glucose levels, while a less marked improvement was found in serum cholesterol levels and liver functions. Altered chromosome aberration and apoptotic cell fraction also tended to normalise after treatment. Immunological parameters were not affected by the treatment. The obtained results may suggest that chemoprevention with chelating agents as Humetta can help in the prevention of harmful effects of occupational exposures to metals.     

The somatostatin analogue peptide TT-232 induces apoptosis and chromosome breakage in cultured human lymphocytes

Somatostatin receptors are supposed to be important in the regulation of apoptosis. In this study, we measured apoptosis occurring spontaneously, or induced by the synthetic somatostatin analogue, the peptide TT-232. We examined isolated human peripheral blood lymphocytes (PBL) from 32 nurses exposed bedside to cytostatic drugs, 12 chronic lymphoid leukaemia (CLL) patients prior to treatment, and 19 unexposed, healthy donors without anamnestic occupational exposure to genotoxic agents. Cells were stimulated by phytohaemagglutinin-P (PHA) and cultured for 69 h with or without 15 microg/ml TT-232, respectively. Cell kinetic parameters and apoptosis were determined by flow cytometry after staining with FITC-labeled anti-BrdU and propidium iodide (PI) and the results on spontaneous and peptide-induced apoptosis were compared with the obtained chromosome aberration frequencies (CA). The peptide TT-232 unexpectedly induced chromosome breakage in addition to apoptosis. The mean spontaneous apoptotic fractions were 6.65+/-0.89%, 6.46+/-0. 53%, and 3.07+/-0.57%, and the mean CA yields in the samples without TT-232 were 1.74+/-0.46%, 2.44+/-0.40%, and 4.50+/-1.05%, for healthy subjects, nurses, and CLL patients, respectively. A total of 15 microg/ml TT-232 treatment in healthy subjects increased the mean CA frequency (10.38+/-1.57%), as well as the apoptotic cell fraction (2.63+/-0.45 times higher than the corresponding untreated sample). In TT-232-treated PBLs of nurses, CA remained unchanged and the mean apoptotic cell fraction showed only a slight increase (1.24+/-0.11 times higher than the untreated). Among CLL patients, TT-232 treatment significantly increased both CA (up to 17.83+/-4.04%) and the ratio of apoptotic cells (21.78+/-11.00 times higher than the untreated). These results demonstrated significant differences in apoptosis sensitivity in controls, nurses and CLL donors, after 15 microg/ml TT-232 treatment. Data also indicate that the induced CA yields in CLL donors with high CA are in correlation with TT-232-induced apoptosis.     

Voxel-Wise Motion Artifacts in Population-Level Whole-Brain Connectivity Analysis of Resting-State fMRI

Functional Magnetic Resonance Imaging (fMRI) based brain connectivity analysis maps the functional networks of the brain by estimating the degree of synchronous neuronal activity between brain regions. Recent studies have demonstrated that “resting-state” fMRI-based brain connectivity conclusions may be erroneous when motion artifacts have a differential effect on fMRI BOLD signals for between group comparisons. A potential explanation could be that in-scanner displacement, due to rotational components, is not spatially constant in the whole brain. However, this localized nature of motion artifacts is poorly understood and is rarely considered in brain connectivity studies. In this study, we initially demonstrate the local correspondence between head displacement and the changes in the resting-state fMRI BOLD signal. Than, we investigate how connectivity strength is affected by the population-level variation in the spatial pattern of regional displacement. We introduce Regional Displacement Interaction (RDI), a new covariate parameter set for second-level connectivity analysis and demonstrate its effectiveness in reducing motion related confounds in comparisons of groups with different voxel-vise displacement pattern and preprocessed using various nuisance regression methods. The effect of using RDI as second-level covariate is than demonstrated in autism-related group comparisons. The relationship between the proposed method and some of the prevailing subject-level nuisance regression techniques is evaluated. Our results show that, depending on experimental design, treating in-scanner head motion as a global confound may not be appropriate. The degree of displacement is highly variable among various brain regions, both within and between subjects. These regional differences bias correlation-based measures of brain connectivity. The inclusion of the proposed second-level covariate into the analysis successfully reduces artifactual motion-related group differences and preserves real neuronal differences, as demonstrated by the autism-related comparisons.     

Distribution of PACAP-38 in the central nervous system of various species determined by a novel radioimmunoassay

Pituitary adenylate cyclase activating polypeptide (PACAP) occurs in two molecular forms: PACAP-38 and PACAP-27. Soon after the isolation and chemical characterization of PACAP, the first radioimmunoassay (RIA) methods have been developed, but it is a still rarely used laboratory technique in the field of PACAP research. The aim of the present study was to develop a novel, highly specific PACAP-38 assay to investigate the quantitative distribution of PACAP-38 in the central nervous system of various vertebrate species under the same technical and experimental conditions. Different areas of the brain and the spinal cord were removed from rats, chickens and fishes and the tissue samples were processed for PACAP-38 RIA. Our results indicate that the antiserum used in the RIA is C-terminal specific, without affinity for other members of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon peptide family. The average ID50 value was 48.6+/-3.4 fmol/ml determined in 10 consecutive assays. Detection limit for PACAP-38 proved to be 2 fmol/ml. PACAP-38 immunoreactivity was present in the examined brain areas of each species studied, with highest concentration in the rat diencephalons. High levels of PACAP-38 were also detected in the rat telencephalon, followed by spinal cord and brainstem. The central nervous system of the fish also contained considerable concentrations of PACAP-38, whereas lowest concentrations were measured in the central nervous system of the chicken.     

Comparison of Human Hepatoma HepaRG Cells with Human and Rat Hepatocytes in Uptake Transport Assays in Order to Predict a Risk of Drug Induced Hepatotoxicity

Human hepatocytes are the gold standard for toxicological studies but they have several drawbacks, like scarce availability, high inter-individual variability, a short lifetime, which limits their applicability. The aim of our investigations was to determine, whether HepaRG cells could replace human hepatocytes in uptake experiments for toxicity studies. HepaRG is a hepatoma cell line with most hepatic functions, including a considerable expression of uptake transporters in contrast to other hepatic immortalized cell lines. We compared the effect of cholestatic drugs (bosentan, cyclosporinA, troglitazone,) and bromosulfophthalein on the uptake of taurocholate and estrone-3-sulfate in human and rat hepatocytes and HepaRG cells. The substrate uptake was significantly slower in HepaRG cells than in human hepatocytes, still, in the presence of drugs we observed a concentration dependent decrease in uptake. In all cell types, the culture time had a significant impact not only on the uptake process but on the inhibitory effect of drugs too. The most significant drug effect was measured at 4 h after seeding. Our report is among the first concerning interactions of the uptake transporters in the HepaRG, at the functional level. Results of the present study clearly show that concerning the inhibition of taurocholate uptake by cholestatic drugs, HepaRG cells are closer to human hepatocytes than rat hepatocytes. In conclusion, we demonstrated that HepaRG cells may provide a suitable tool for hepatic uptake studies.