Application of water framework directive in Hungary: development of biological classification systems.

The classification according to the Water Framework Directive (WFD) includes numerous challenges in contrast with the previously applied water qualification standards. The most important element of the ecological status, the biological one, is based on five groups of living organisms: phytoplankton, phytobenthon, macrophytes, macro-invertebrates and fish. The results of a three-year research project financed by the Ministry of Environment and Water (MoEW) and the Hungarian Academy of Sciences (HAS) are reported in this work. The objective of the project was the elaboration of a proposal for biological classification according to the WFD for the related groups of living organisms. In the course of the project the biological characteristics to be measured were selected for each of the above listed groups which served as the basic data for Biological Quality Elements (BQEs). In the BQEs we estimated the type-specific reference values for most of the Hungarian surface water types. Then we created the structure of the qualification system for these groups, including specification of class boundaries between the five classes for the Environmental Quality Ratio (EQR) values on the basis of expert estimation. A Non-Taxonomic Periphyton Index (NTPI, not included in the WFD) was also developed and tested for qualification. The elaborated classification systems were tested on the basis of existing scarce data for numerous Hungarian water types.

Influence of 3-methylcholanthrene on liver nucleolar and nucleoplasmic activities of protein kinases and RNA polymerases.

The experiments were designed to investigate some details of the action of 3-methylcholanthrene (3-MC) on the regulation of transcription. After a single intraperitoneal dose of 3-MC a significant increase in the activities of both nucleolar and nucleoplasmic protein kinases in hepatic cells of young rats was found. The maximal stimulation took place 24 hr after the administration of 3-MC and the extent of activation was much greater in the nucleolar fraction. There is a significant elevation of the activities of both functional forms, free and template-engaged, of RNA polymerase A 24 hr after a single injection of 3-MC. Free and engaged forms of extranucleolar RNA polymerase B show a different behaviour: after 24 hr of 3-MC administration the engaged form is markedly enhanced while the activity of the free enzyme shows a significant decrease. The more moderate increase in total RNA polymerase B activity is obviously preceded by a transfer of the enzyme from 'free' to 'engaged' form. Since the enhancement of protein kinase activities was accompanied by the stimulation of nuclear RNA polymerases we suggest that both kinds of enzymes are involved in an epigenetic mechanism of the inducing action of 3-MC on cytochrome P1-450.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of underivatized and permethylated gangliosides.

Underivatized and permethylated gangliosides have been studied by the matrix-assisted laser desorption (MALO) ionization technique. The samples investigated included commercially available and highly purified gangliosides from the human brain containing up to five sialic acid residues. Several permethylated gangliosides have also been studied, and MALD has proven successful in analyzing multicomponent mixtures of glycolipids with different fatty acyl residues. During the studies a variety of matrix and wavelength combinations have been tested in both the positive and negative ion modes. The best results have been obtained with the matrices 2,5-dihyd roxybenzoic acid, 4-hydrazinobenzoic acid, 1,5-diaminonaphthalene, and 6-aza-2-thiothymine. Negative ion mass spectra of the underivatized gangliosides have always been of better quality than the positive ion mass spectra; exhibiting better signal-to-noise ratio, better resolution, less fragmentation, and less adduct formation with Na(+) and K(+). With increasing number of sialic acid substituents the molecular ion region became less and less resolvable leading to broadened peaks even in the negative ion mode. Fragmentation could frequently be observed in the negative ion mode, and it was pronounced in the positive ion mode. The major fragmentation pathways corresponded to loss of sialyl groupts) and to decarboxylation of one of the sialyl residues. For underivatized gangliosides the typical sample amount used was 10-20 pmol, Permethylation led to a significant improvement in sensitivity (two orders of magnitude); the detection limit of permethylated gangliosides was about 10 fmol. The higher stability of the permethylated compounds was indicated by the fact that positive ion mass spectra exhibited only a marginal extent of fragmentation.

Matrix-assisted laser desorption ionization mass spectrometry with 2-(4-hydroxyphenylazo)benzoic acid matrix.

A novel matrix substance, 2-(4-hydroxyphenylazo) benzoic acid, or HABA, has been found to be very advantageous for matrix-assisted ultraviolet laser desorption ionization mass spectrometry. This compound has been successfully used for the desorption of peptides, proteins, and glycoproteins up to approximately 250 kDa. For these materials, the most abundant analyte-related peaks correspond to [M + H](+) ions and multiply protonated molecules. Comparisons with sinapic acid, 2,5-dihydroxybenzoic acid, and α-cyano-4-hydroxycinnamic acid indicate that the new matrix provides comparable sensitivity for peptides and smaller proteins but results in better sensitivity for larger proteins and glycoproteins in protein mixtures. Other matrices discriminate against the higher mass components in these cases. Somewhat reduced mass resolution has been found for smaller proteins, but for larger proteins and glycoproteins the best mass resolution can often be obtained with the new matrix. For other classes of compounds that form ions predominantly via cation attachment, at least as good sensitivity and even better resolution have been obtained. Derivatized glycolipids and synthetic polymers have been studied in detail. For the analysis of many synthetic polymers, the best performance in terms of sensitivity and mass resolution has been observed with HABA matrix. Mass resolution was higher for cation adducts than for the protonated peptide molecules in the same mass range. The new matrix exhibits greatly extended (in time) analyte ion production and reproducibility. Owing to the uniform sample surface with this matrix, barely any spatial variation of the ion signal could be observed. In addition, many hundreds of single-shot mass spectra could be accumulated from the same spot, even for larger proteins.

Novel neuropeptide Y processing in human cerebrospinal fluid from depressed patients.

In vitro processing of neuropeptide Y (NPY) in cerebrospinal fluid (CSF) of patients with depression was monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Single peptide bonds in NPY were cleaved to yield N- or C-terminal fragments. Multiple cleavage to form internal peptides was unimportant. Degradation rates varied between individuals, whereas the product distributions were fairly constant. Other peptides did not evidence such proteolysis. MALDI-TOF MS will facilitate extensive investigations of NPY processing that could provide the basis for clinical assays and illuminate the pathophysiology related to depression.

Utility of non-covalent complexes in the matrix-assisted laser desorption ionization mass spectrometry of heparin-derived oligosaccharides.

Molecular weights of heparin-derived oligosaccharides ranging from disaccharides to hexadecasaccharides have been determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. While these compounds ionize poorly or not at all when used as such, a strong signal can be obtained of their ionic complexes formed with a basic peptide or protein. The molecular weight of the sulfated oligosaccharide is determined by subtracting the mass of the basic component from that of the complex. Optimization of the experimental conditions resulted in sub-picomole sensitivity, in the elimination of sulfate loss and of the interference from attachment of inorganic cations. Synthetic peptides (Arg-Gly)10 and (Arg-Gly)15 were specifically designed as complexing agents for synthetic and natural heparin fragments up to decasaccharides. Accurate molecular weight determination on chemically homogeneous oligosaccharides (+/- 0.05%) unambiguously identified the number of saccharide units, and the number of O,N-sulfate and N-acetyl groups. For oligosaccharides larger than decasaccharides, a small basic protein, angiogenin (M(r) = 14,120), was used to form the complex (an inhomogeneous hexadecasaccharide fraction was the largest available for this study). For inhomogeneous samples larger than decasaccharides, the mass accuracy is lower (+/- 0.2-0.3%) but still suffices to determine the number of saccharide units present and to estimate the number of sulfate groups, except it is no longer possible to differentiate one sulfate from two N-acetyl groups (delta = 4 Da). However, taking into account known regularities of sulfation and acetylation, the specificity of heparin lyases and chemical degradation steps, the method promises to contribute significantly to the determination of the primary structure of heparin and other sulfated glycosaminoglycans.

[Clinical significance of serological markers of Helicobacter pylori]. / A Helicobacter pylori szerológiai markereinek klinikai jelentösége.

Helicobacter pylori (H. pylori) is a Gram-negative, curved bacillus known since 1983. It is supposed to play role in the pathogenesis of certain gastroduodenic diseases, e.g. non-ulcer dyspepsia (NUD), chronic inflammation, ulcers, etc. Serum samples of 70 patients who were examined for stomach complaints with gastroscopy and those of 22 healthy persons were analysed. The purpose of the study was to evaluate anti-H. pylori IgG, IgM and IgA antibodies using ELISA method. Whether the antibodies can be detected or not, 8 possible variations exist, each of them denoting certain state of infection. These states are not always going parallel with the macroscopic pictures revealed by gastroscopy, but there are some obvious congruences. Results show that serologic examination cannot replace gastroscopy but on the other hand in follow-up tests and examinations as well as in understanding the aetiology of different gastroduodenal diseases it can play an important role.

[Diagnostic value of alkaline phosphatase isoenzymes]. / Az alkalikus foszfatáz izoenzimek diagnosztikai jelentösége.

Among the serum enzymes used for diagnostic purposes the alkaline phosphatase (AP) is one of the oldest and the most frequently applied laboratory tests. Increased activity found in the serum is a consequence of certain hepato-biliary disorders, different bone diseases, endocrine syndromes, kidney illnesses and malignant tumors as well. From the study of the pattern of the isoenzymes of the serum-AP information of practical importance may be gained by which many differential-diagnostic problems can be solved. For the clinical-chemical laboratories methods that use electrophoretic separation on different membranes or gels are recommended. Patterns characteristic for different diseases may well be differentiated from normal constellation. AP-isoenzymes help to reveal cholestases at early stages, some disorders of bone metabolism and malignant processes as well.

The art and practice of systems biology in medicine: mapping patterns of relationships.

Systems biology has developed in recent years from a technology-driven enterprise to a new strategic tool in Life Sciences, particularly for innovative drug discovery and drug development. Combining the ultimate in systems phenotyping with in-depth investigations of biomolecular mechanisms will enable a revolution in our understanding of disease pathology and will advance translational medicine, combination therapies, integrative medicine, and personalized medicine. A prerequisite for deriving the benefits of such a systems approach is a reliable and well-validated bioanalytical platform across complementary measurement modalities, especially transcriptomics, proteomics, and metabolomics, that operates in concert with a megavariate integrative biostatistical/bioinformatics platform. The applicable bioanalytical methodologies must undergo an intense development trajectory to reach an optimal level of reliable performance and quantitative reproducibility in daily practice. Moreover, to generate such enabling systems information, it is essential to design experiments based on an understanding of the complexity and statistical characteristics of the large data sets created. Novel insights into biology and system science can be obtained by evaluating the molecular connectivity within a system through correlation networks, by monitoring the dynamics of a system, or by measuring the system responses to perturbations such as drug administration or challenge tests. In addition, cross-compartment communication and control/feed-back mechanisms can be studied via correlation network analyses. All these data analyses depend critically upon the generation of high-quality bioanalytical platform data sets. The emphasis of this paper is on the characteristics of a bioanalytical platform that we have developed to generate such data sets. The broad applicability of Systems Biology in pharmaceutical research and development is discussed with examples in disease biomarker research, in pharmacology using system response monitoring, and in cross-compartment system toxicology assessment.