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.

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.

The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer.

A new matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometer is described for sequencing peptides. This instrument combines the advantages of high sensitivity for peptide analysis associated with MALDI and comprehensive fragmentation information provided by high-energy collision-induced dissociation (CID). Unlike the postsource decay technique that is widely used with MALDI-TOF instruments and typically combines as many as 10 separate spectra of different mass regions, this instrument allows complete fragment ion spectra to be obtained in a single acquisition at a fixed reflectron voltage. To achieve optimum resolution and focusing over the whole mass range, it may be desirable to acquire and combine three separate sections. Different combinations of MALDI matrix and collision gas determine the amount of internal energy deposited by the MALDI process and the CID process, which provide control over the extent and nature of the fragment ions observed. Examples of peptide sequencing are presented that identify sequence-dependent features and demonstrate the value of modifying the ionization and collision conditions to optimize the spectral information.

Accurate mass measurements using MALDI-TOF with delayed extraction.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is now an essential tool in biopolymer analysis. Sensitivity and mass range are unsurpassed, but mass measurement accuracy and resolution have been limited. With delayed extraction and a reflecting analyzer, mass measurements using MALDI-TOF can be made with an accuracy of a few parts per million (ppm). It is possible to distinguish Lys from Gln in peptides, and to determine the elemental composition of smaller molecules (mass 100-500). In database searching strategies, a smaller mass window, resulting from an increase in mass accuracy, greatly decreases the number of possible candidates. Mass measurement accuracy with errors less than 5 ppm is demonstrated on a mixture of 12 peptides ranging in mass from ca. 900 to 3700 Da. Mass measurements on 13 peaks in an unseparated tryptic digest of myoglobin gave results with an overall average error less than 3.5 ppm, with a maximum error of 7 ppm.

Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: implications for proteome mapping.

Matrix-assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of alpha- and beta-chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one-dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time-lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa alpha-subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)-polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.

Sequencing oligonucleotides by exonuclease digestion and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A protocol was developed for sequencing oligonucleotides by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Oligonucleotides were partially hydrolyzed in separate time-course digestions with 3' --> 5' and 5' --> 3' acting phosphodiesterases in a MALDI-compatible buffer or in the MALDI matrix itself. The partial digests were analyzed by MALDI-TOF mass spectrometry employing an instrument equipped with delayed ion extraction and the sequence was inferred from the mass differences between adjacent peaks. Resolution, mass accuracy, and sensitivity were considerably enhanced with delayed extraction, in comparison with the standard MALDI technique. Much longer lengths of DNA can be unambiguously sequenced with delayed extraction MALDI compared with standard MALDI-MS.

DNA sequencing by delayed extraction-matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry was used to detect and order DNA fragments generated by Sanger dideoxy cycle sequencing. This was accomplished by improving the sensitivity and resolution of the MALDI method using a delayed ion extraction technique (DE-MALDI). The cycle sequencing chemistry was optimized to produce as much as 100 fmol of each specific dideoxy terminated fragment, generated from extension of a 13-base primer annealed on 40- and 50-base templates. Analysis of the resultant sequencing mixture by DE-MALDI identified the appropriate termination products. The technique provides a new non-gel-based method to sequence DNA which may ultimately have considerable speed advantages over traditional methodologies.

Applications of delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry to oligonucleotide analysis.

A delayed ion extraction technique is shown to dramatically improve mass resolution and the overall quality of matrix-assisted laser desorption ionization (MALDI) mass spectra of oligonucleotides. Isotope limited mass resolution was obtained on samples up to 10-kDa molecular mass in linear mode, and as high as 7500 mass resolution (defined at half peak height) was observed in reflector mode. This performance is as good as that achieved to date for peptides and proteins. Applications included the detection of oxidized byproducts of phosphorothioate DNA and separation of components differing only by 15 Da at 9.5-kDa molecular mass. In addition to single components, complex mixtures could also be analyzed at greatly improved performance over conventional MALDI. An example is shown for sequence verification of an oligonucleotide of 31 bases in length by analyzing the failure products. Mass accuracy was adequate to verify sequences of oligodeoxyribonucleotides up to 9500-Da molecular mass. Fast fragmentation taking place between the ionizing pulse and the extraction pulse is demonstrated to be a sequencing tool for small oligonucleotides. By proper selection of matrix material, wavelength, and irradiance, fast fragmentation can be promoted efficiently. Fragment ions tend to form from cleavage of phosphodiester bonds, as previously observed in infrared MALDI.

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.

Mass spectrometric molecular-weight determination of highly acidic compounds of biological significance via their complexes with basic polypeptides.

Highly acidic compounds that are difficult to ionize by matrix-assisted laser desorption ionization give excellent spectra when mixed with a basic peptide or protein to form a noncovalent complex. This phenomenon makes it possible to determine the molecular weights of polysulfated, -sulfonated, and -phosphorylated biomolecules such as cysteic acid-containing peptides, oligonucleotides, heparin-derived oligosaccharides, and suramin (a drug containing two trisulfonated naphthalene moieties). Peptides and small proteins rich in arginine were used as the basic components. The extent of complex formation correlates with the number of phosphate and sulfate groups in the acidic component and with the number of arginines in the basic component. Neither the acidic amino acid residue aspartic and glutamic acid nor the basic lysine and histidine contribute to complex formation. For oligonucleotides, histone H4 was found to be the best complexing agent investigated. The analytical utility of the complex formation is demonstrated by the molecular-mass determination of acidic compounds from 500 to 6000 Da at the picomole or sub-picomole level with an accuracy of +/- 0.1% or better and by the absence of alkali cation adducts.

Generation of large radical ions from oligometallocenes by matrix-assisted laser desorption ionization.

Non-polar polymers containing ferrocene, ferrocenylnaphthalene, and ruthenocenylnaphthalene groups in their repeating units were studied by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry. Sample preparation for these polymers utilized tetrahydrofuran as solvent and several new matrices such as dithranol, 9-nitroanthracene, and quinizarin (all of these are anthracene derivatives). By comparing the mass spectra of oligometallocenes recorded with different matrices at different wavelengths (337 nm and 2.94 microns) and laser desorption ionization mass spectra recorded without matrix, it could be verified that the major analyte-related peaks in the MALDI mass spectra corresponded to the radical molecular ions. Radical ions have not been seen in the MALDI mass spectra of biopolymers such as proteins, peptides, and carbohydrates. Radical formation was demonstrated for samples in the mass range (1 kDa-13 kDa). Even in the presence of potential cationization sites such as methyl ester groups in the repeating units of some polyferrocene samples, MALDI mass spectra were dominated by radical ions of the analyte. Two possible mechanisms for radical formation in MALDI are discussed. Comparison of results with different matrices suggested that the distribution of masses observed in the mass spectra and characterized by the polydispersity index was independent of the matrix, but significant differences (5%) in the average molecular weights of the mass distributions were found.

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.

[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.

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.

[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.

Results and prognosis following surgery for malignant tumors of the skull base.

A series of 155 malignant craniofacial tumors referred to a specialized craniofacial center for surgical treatment are presented. One quarter of these were primary and three quarters recurrent. The statistics of long-term follow-up are presented, and it is concluded that this type of surgery for these difficult problems is worthwhile. The increasing experience gained from operating on and following up these patients is presented.

Acute monosymptomatic aseptic meningitis caused by Toxoplasma gondii.

Acute monosymptomatic aseptic meningitis was observed in a 4 year old male patient. Toxoplasma gondii tachyzoites were detected in Giemsa-stained smears prepared from the CSF. Inoculation of mice gave the same result. The patient was cured after the application of pyrimethamine and sulpha drugs. On basis of the smears, the serological results and data in the literature, a direct infection through the nasal cavity has been assumed.