Showing papers by "Jožef Stefan Institute published in 1997"

Structural and functional aspects of papain-like cysteine proteinases and their protein inhibitors.

Abstract: Cysteine proteinases are widely distributed among living organisms According to the most recent classifications (Rawlings and Barrett, 1993, 1994), they can be subdivided on the basis of sequence homology into 14 or even 20 different families, the most important being the papain and the calpain families The papain-like cysteine proteinases are the most abundant among the cysteine proteinases The family consists of papain and related plant proteinases such as chymopapain, caricain, bromelain, actinidin, ficin, and aleurain, and the lysosomal cathepsins B, H, L, S, C and K Most of these enzymes are relatively small proteins with Mr values in the range 20000-35000 (reviewed in Brocklehurst et al, 1987; Polgar, 1989; Rawlings and Barrett, 1994; Berti and Storer, 1995), with the exception of cathepsin C, which is an oligomeric enzyme with Mr approximately 200000 (Metrione et al, 1970; Dolenc et al, 1995) A number of cysteine proteinases are located within lysosomes Four of them, cathepsins B, C, H and L, are ubiquitous in lysosomes of animals, whereas cathepsin S has a more restricted localisation (Barrett and Kirschke, 1981; Kirschke and Wiederanders, 1994) The enzymes, except cathepsin C, are endopeptidases (reviewed in Kirschke et al, 1995), although cathepsin B was found also to be a dipeptidyl carboxypeptidase (Aronson and Barrett, 1978) and cathepsin H also an aminopeptidase (Koga et al, 1992) Cathepsin C is a dipeptidyl aminopeptidase, but at higher pH it exhibits also dipeptidyl transferase activity (reviewed in Kirschke et al, 1995) Among the lysosomal cysteine proteinases, cathepsin L was found to be the most active in degradation of protein substrates, such as collagen, elastin and azocasein (Barrett and Kirschke, 1981; Maciewicz et al, 1987; Mason et al, 1989), arid cathepsin B the most abundant (Kirschke and Barrett, 1981) All the enzymes are optimally active at slightly acidic pH, although their pH optima for degradation of synthetic substrates vary from 55 for cathepsin L to 68 for cathepsin H (reviewed in Kirschke et al, 1995) Several other lysosomal cysteine proteinases, such as cathepsins N, T and K, are known, although their properties are less well characterised (reviewed in Kirschke et al, 1995) In particular cathepsin K has attracted recent interest (Bromme et al, 1996; Shi et al, 1995; Bossard et al, 1996; Drake et al, 1996) and was found to be expressed specifically in osteoclasts (Drake et al, 1996) with properties similar to cathepsin L (Bossard et al, 1996)

Industrial applications of CrN (PVD) coatings, deposited at high and low temperatures

Abstract: Industrial applications of CrN (PVD) coatings are entering an expanding but selective range of mass manufactured goods. They may be prepared as single, low and high temperature CrN coatings and double TiN+CrN coatings. In this work, depositions of CrN at high temperatures were performed by a low voltage thermionic arc in a BAI 730M apparatus, while at low temperatures (below 250 °C), the plasma-beam sputtering process in a SPUTRON apparatus was used. We studied the following critical parameters that influence the quality of the coatings and applied the performance tests used in industrial practice: adhesion and scratching coefficient, microhardness, surface topography, oxidation and corrosion resistance. The performance tests were made with the assistance of technicians as well as in 12 Slovenian factories. CrN coatings were deposited at 480 °C for wear and corrosion protection in cold forming and cutting of copper in commutator manufacturing, in forming of aluminium components in automotive production and for surface improvement of moulds (made of H11 steel) in Al-Si die casting under pressure. Deposition temperatures of 180–220 °C, obtained in the SPUTRON apparatus, were required to improve cold forming tools made of alloyed tool steels (e.g. D2 and D3). The lowest obtainable temperature of 140 °C in the SPUTRON gave a CrN coating of high quality for practical use. These coatings were used to protect electrodeposited and electropolished nickel moulds (models) in artificial teeth production. Double TiN+CrN coatings were used as a highly abrasive resistant coating in the production of rotors (in the electromotor industry), and in cold forming and forging in mass manufacturing of screws. The results and performance tests were compared with the available data, mostly published on forming, milling, deep drawing of copper, nickel and titanium and their alloys, and on die casting of aluminium and Al-alloys.

Cathepsins B, H, and L and their inhibitors stefin A and cystatin C in sera of melanoma patients.

Abstract: The levels of cathepsins (Cats) B, H, and L and their inhibitors stefin A and cystatin C were determined in the sera of 43 patients with metastatic melanoma, in 54 patients with treated cutaneous melanoma with no evidence of metastatic disease, and in 30 healthy blood donors, using quantitative ELISAs. The levels of Cats B and H and cystatin C were significantly higher within the group of metastatic melanoma patients compared with the healthy controls. The median Cat B was 4.8 versus 3.6 ng/ml (P < 0.013), the median Cat H was 13.7 versus 4.9 ng/ml (P < 0.0001), and the median cystatin C was 470 versus 320 ng/ml (P < 0.02). Cat H was also significantly increased within the group of melanoma patients with no metastasis, with a median of 9.6 ng/ml. Cat B was found to correlate with Cat L (r = 0.36; P < 0.02) and cystatin C (r = 0.41; P < 0.008). The serum level of Cat H was significantly increased in patients showing no response to the chemoimmunotherapy as compared to the level in responders. Metastatic melanoma patients with high contents of Cat B and Cat H experienced significantly shorter overall survival rates than the patients with low levels of each enzyme (Cat B: P < 0.003 and relative risk, 2.5; Cat H: P < 0.006 and relative risk, 2.4, using medians as cutoff values). The other potential factors for prognosis for this group of patients revealed moderate (histological type and age) or no (tumor thickness, sex, and lymph node metastasis) prognostic significance. Similarly, no difference in survival was found for stefin A, cystatin C, and Cat L. These results suggest that the serum levels of Cats B and H could serve as prognostic factors for patients with advanced melanoma.

First Order Regression

Abstract: We present a new approach, called First Order Regression (FOR), to handling numerical information in Inductive Logic Programming (ILP). FOR is a combination of ILP and numerical regression. First-order logic descriptions are induced to carve out those subspaces that are amenable to numerical regression among real-valued variables. The program FORS is an implementation of this idea, where numerical regression is focused on a distinguished continuous argument of the target predicate. We show that this can be viewed as a generalisation of the usual ILP problem. Applications of FORS on several real-world data sets are described: the prediction of mutagenicity of chemicals, the modelling of liquid dynamics in a surge tank, predicting the roughness in steel grinding, finite element mesh design, and operator‘s skill reconstruction in electric discharge machining. A comparison of FORS‘ performance with previous results in these domains indicates that FORS is an effective tool for ILP applications that involve numerical data.

A MRI spatial mapping technique for microvascular permeability and tissue blood volume based on macromolecular contrast agent distribution

Abstract: A rapid and automated method for two-dimensional spatial depiction (mapping) of quantitative physiological tissue characteristics derived from contrast enhanced MR imaging was developed and tested in disease models of cancer, inflammation, and myocardial reperfusion injury. Specifically, an established two-compartment kinetic model of unidirectional mass transport was implemented on a pixel-by-pixel basis to generate maps of tissue permeability surface area product (PS) and fractional blood volume (BV) based on dynamic MRI intensity data after administration of albumin-(Gd-DTPA)30, a prototype macromolecular contrast medium (MMCM) designed for blood pool enhancement. Maps of PS and BV in disease models of adenocarcinoma, intramuscular abscess inflammation, and myocardial reperfusion injury clearly depicted zones of increased permeability (up to approximately 500 microl/cc/h--compared to inflammation > tumors. A rapid, automated mapping technique derived from dynamic contrast-enhanced MRI data can be used to facilitate the identification and characterization of pathophysiologic abnormalities, specifically relative increases in blood volume and/or microvascular permeability.

Arsenic Compounds in Higher Fungi

Abstract: In 50 mushroom species (56 samples) from Slovenia, Switzerland, Brazil, Sweden, The Netherlands and USA, total arsenic was determined by radiochemical neutron activation analysis (RNAA). Arsenic concentrations ranged from 0.1 to 30 μg g−1 (dry mass). Arsenic compounds were determined in methanol extracts from the mushrooms by HPLC–ICP–MS. The aim of the study was not only to quantify arsenic compounds in mushrooms but also to uncover trends relating the methylating ability of a mushroom to its taxonomic or evolutionary status. The main arsenic compound found in many mushrooms (various puffballs, Agaricales and Aphyllophorales) was arsenobetaine. Arsenate [As(V)] was the main arsenic species in Laccaria fraterna and Entoloma rhodopolium and arsenite [As(III)] in Tricholoma sulphureum. A mixture of arsenite and arsenate was present in Amanita caesarea. Dimethylarsinic acid (DMA) and methylarsonic acid were present in many mushrooms, but generally as minor components. In Laccaria laccata, Leucocoprinus badhamii and Volvariella volvacea, DMA was the major metabolite. Arsenocholine (AC) and the tetramethylarsonium ion were present in a few species, generally at low concentrations, except for Sparassis crispa, in which AC was the main compound. Tri- methylarsine oxide was not found in any of the mushrooms. In some species small amounts of unknown compounds were also present. The possible taxonomic significance of the metabolite patterns and the predominance of arsenobetaine in more advanced fungal types are discussed. © 1997 John Wiley & Sons, Ltd.

Bov‐B Long Interspersed Repeated DNA (LINE) Sequences are Present in Vipera Ammodytes Phospholipase A2 Genes and in Genomes of Viperidae Snakes

Abstract: Ammodytin L is a myotoxic Ser49 phospholipase A2 (PLA2) homologue, which is tissue-specifically expressed in the venom glands of Vipera ammodytes. The complete DNA sequence of the gene and its 5′ and 3′ flanking regions has been determined. The gene consists of five exons separated by four introns. Comparative analysis of the ammodytin L and ammodytoxin C genes shows that all intron and flanking sequences are considerably more conserved (93–97%) than the mature protein-coding exons. The pattern of nucleotide substitutions in protein-coding exons is not random but occurs preferentially on the first and the second positions of codons, which suggests positive Darwinian evolution for a new function. An Ruminantia specific ART-2 retroposon, recently recognised as a S′-truncated Bov-B long interspersed repeated DNA (LINE) sequence, was identified in the fourth intron of both genes. This result suggests that ammodytin L and ammodytoxin C genes are derived by duplication of a common ancestral gene. The phylogenetic distribution of Bov-B LINE among vertebrate classes shows that, besides the Ruminantia, it is limited to Viperidae snakes (Vipera ammodytes, Vipera palaestinae, Echis coloratus, Bothrops alternatus, Trimeresurus flavoviridis and Trimeresurus gramineus). The copy number of the 3′ end of Bov-B LINE in the Vipera ammodytes genome is between 62000 and 75000. The absence of Bov-B LINE at orthologous positions in other snake PLA2 genes indicates that its retrotransposition in the V. ammodytes PLA2 gene locus has occurred quite recently, about 5 My ago. The amplification of Bov-B LINEs in snakes may have occurred before the divergence of the Viperinae and Crotalinae subfamilies. Due to its wide distribution in Viperidae snakes it may be a valuable phylogenetic marker. The neighbor-joining phylogenetic tree shows two clusters of truncated Bov-B LINE, a Bovidae and a snake cluster, indicating an early horizontal transfer of this transposable element.

Potato cysteine proteinase inhibitor gene family: molecular cloning, characterisation and immunocytochemical localisation studies

Abstract: Potato cysteine proteinase inhibitors (PCPIs) represent a distinct group of proteins as they show no homology to any other known cysteine proteinase inhibitor superfamilies, but they all belong to the Kunitz-type soybean trypsin inhibitor family. cDNA clones for five PCPIs have been isolated and sequenced. Amino acid substitutions occurring in the limited regions forming loops on the surface of these proteins suggest a further classification of PCPIs into three subgroups. Accumulation of PCPI was observed in vacuoles of stems after treatment with jasmonic acid (JA) using immunocytochemical localisation, implying that these inhibitors are part of a potato defence mechanism against insects and pathogens. Genomic DNA analysis show that PCPIs form a multigene family and suggest that their genes do not possess any introns.

Induction of Decision Trees and Bayesian Classification Applied to Diagnosis of Sport Injuries

Abstract: Machine learning techniques can be used to extract knowledge from data stored in medical databases. In our application, various machine learning algorithms were used to extract diagnostic knowledge which may be used to support the diagnosis of sport injuries. The applied methods include variants of the Assistant algorithm for top-down induction of decision trees, and variants of the Bayesian classifier. The available dataset was insufficient for reliable diagnosis of all sport injuries considered by the system. Consequently, expert-defined diagnostic rules were added and used as pre-classifiers or as generators of additional training instances for diagnoses for which only few training examples were available. Experimental results show that the classification accuracy and the explanation capability of the naive Bayesian classifier with the fuzzy discretization of numerical attributes were superior to other methods and estimated as the most appropriate for practical use.

Theoretical analysis of the formation of membrane microtubes on axially strained vesicles

Abstract: The formation of membrane microtubes (tethers) was analyzed by a theoretical study of the shape changes of an axisymmetrical phospholipid vesicle caused by a pulling axial force applied at the vesicle poles. Equilibrium vesicle shapes were obtained by variationally seeking the minimum of the sum of membrane local and nonlocal bending energies at constant vesicle volume, membrane area, and the distance between the vesicle poles. The effect of axial force on vesicle shapes was studied by examining the shape behavior of prolate axisymmetrical vesicles with equatorial mirror symmetry. For a vesicle with a given relative volume, the resulting shapes reside within a given region of the phase diagram for this vesicle as a function of the distance between vesicle poles and the relative difference between the areas of the membrane layers. The upper limit of this region was obtained by a variational procedure for the determination of vesicle shapes that correspond, at given vesicle volume, membrane area, and difference between the areas of membrane layers, to the maximum distance between the vesicle poles. It was shown that for finite values of the ratio between the nonlocal and local bending moduli, at high enough axial force the vesicle shape exhibits an elongated tubular ending at each pole. The equation for the radius of such a tubular ending obtained by the rigorous treatment presented matches the equation that has previously been used as an approximation in analyses of tether formation methods for the determination of membrane bending moduli. Furthermore, it is predicted that below a certain critical value of the ratio between the two bending moduli that depends on the vesicle volume, the shape characterized by the tubular endings is attained, with continuously increasing the axial force, by a discontinuous shape transformation.

The Expression of Lysosomal Proteinases and Their Inhibitors in Breast Cancer: Possible Relationship to Prognosis of the Disease.

Abstract: Proteolytic enzymes have been proposed as new biological prognostic indicators to facilitate decisions about treatment of breast cancer patients following surgery. We reported earlier that the activities of cysteine proteinases (CP), cathepsin (Cat) B and cathepsin (Cat) L and the expression of stefin A might be associated with breast tumor progression and prognosis. Here, the protein concentrations of Cats D, B and L and stefin A have been measured in a series of 60 matched pairs of breast tumours and control adjacent tissues, using ELIS As developed in our laboratory. Median tumor concentrations of Cat D (47 pm/mg), Cat B (222 ng/mg) and Cat L (88 ng/mg) were significantly (p<0.0005) increased by 7 fold, 27 fold and 6 fold, respectively. Much greater increases in the activities of Cat B (63 fold) and of Cat L (274 fold) were found, indicating enhanced activation of cysteine proteinases in tumors, due either to proteolytic activation of proCat B and proCat L and/or to a decrease in specific endogenous cystatins. However, the 1.6-fold decreased (p<0.0001) levels of inhibition by cystatins could not be entirely responsible for more than 100-fold increased ratio of CP:cystatins activity. Moreover, stefin A was either increased or decreased in tumor samples, resulting in a 1.4-fold median increase in tumors. Comparing the biological parameters with the established histo-pathological prognosticators, we found that the increased protein concentration of Cat B was associated with lymph node involvement (p<0.009) and higher stage (p<0.003), and both Cat B and Cat L activities were more increased in high grade tumours (p<0.05). Survival analysis revealed that stefin A was the most significant prognostic factor for disease-free (p<0.008) and overall survival (p<0.02), followed by increased Cat B activity and protein concentration. Cat L was of borderline significance while Cat D was not significant for prognosis. We conclude that enhanced activation of CP, due partially to an imbalance between cysteine proteinases and inhibitors is linked to the progression of breast cancer. Larger sample size is needed to confirm the prognostic significance of stefin A, Cat B and Cat L.

Expression of Cysteine Protease Inhibitors Stefin A, Stefin B, and Cystatin C in Human Lung Tumor Tissue

Abstract: In human lung tumor tissue specimen (n=73) concentrations of stefins A and B were found to be increased 2.0-fold (p<0.01) and 1.3-fold (p<0.01), respectively, as compared to matched normal tissue. Stefn A and B concentrations were higher in primary tumors than in secondary tumors, i.e. metastases from other organs to the lung (p<0.01; p<0.05, respectively). Cystatin C concentrations were rather low and did not differ between tumor and normal tissue. Both concentrations of stefins did not correlate with TNM stages. Stefin A was higher in squamous cell carcinoma than in adenocarcinoma (p<0.01), while stefin B did not show such a difference. At investigation of a relationship between survival probability of patients with primary tumors it was found that increased stefin B concentrations and total cysteine-protease-inhibitory activities but not stefin A concentrations were positively correlated with survival probability.

Conditions for Occam's Razor Applicability and Noise Elimination

Abstract: The Occam's razor principle suggests that among all the correct hypotheses, the simplest hypothesis is the one which best captures the structure of the problem domain and has the highest prediction accuracy when classifying new instances. This principle is implicitly used also for dealing with noise, in order to avoid overfitting a noisy training set by rule truncation or by pruning of decision trees. This work gives a theoretical framework for the applicability of Occam's razor, developed into a procedure for eliminating noise from a training set. The results of empirical evaluation show the usefulness of the presented approach to noise elimination.

On Multi-class Problems and Discretization in Inductive Logic Programming

Abstract: In practical applications of machine learning and knowledge discovery, handling multi-class problems and real numbers are important issues. While attribute-value learners address these problems as a rule, very few ILP systems do so. The few ILP systems that handle real numbers mostly do so by trying out all real values applicable, thus running into efficiency or overfitting problems.

The role of dopants in tailoring the microwave properties of Ba6-xR8+2/3x Ti18O54 R = (La–Gd) Ceramics

Abstract: Microwave dielectric ceramics with a high dielectric constant need to satisfy very high technical demands. They should possess extremely low losses to achieve high Q-values (Quality factor) a small temperature coefficient of resonant frequency (τf), and a relative permittivity (er) higher than 80. Industrial applications require very stringent electrical and dimensional tolerances, typically ± 0.5–1.0 ppm K-1 for a specified τf and ± 0.25% for a specified er. To meet such requirements ceramics based on BaO–R2O3 – TiO2 (R = La–Gd) are used. The investigation of this type of ceramic was stimulated by the observation that ceramics based on compositions in the TiO2-rich region of the system exhibit highly temperature stable electrical properties. Especially interesting are compositions within the solid solubility region with the general formula Ba6-xR8+2/3x Ti18O54. As the ionic radius of the rare earth decreases the extent of the solid solubility region becomes narrower, i.e., 0

Characterization of the Equilibrium Intermediates in Acid Denaturation of Human Stefin B

Abstract: Acid-induced denaturation of recombinant human stefin B was followed using circular dichroism (CD) and fluorimetry. By comparing different spectroscopic probes, a number of equilibrium intermediates were detected. In pH denaturation at very low salt concentration (0.03 M NaCl) four states can be distinguished: N – IN– I1– U, where N is the native state, IN is a native-like intermediate, I1 is an acid intermediate state with properties of a molten globule and U is the unfolded state. State I1 exhibits no near-ultraviolet CD but has some residual far-ultraviolet CD. It differs from U in its ability to increase fluorescence of 1-anilino-naphthalene 8-sulfonate (ANS). In 0.42 M salt, the pH denaturation is three-state between the dimeric native state N2 and intermediates IN2 and I2, which are also dimeric according to size-exclusion chromatography. The acid intermediate I2 is more structured than I1: it binds ANS to a lower extent an I1, its Tyr residues are protected from the solvent, it shows some near-ultraviolet CD and its far-ultraviolet CD is even more intense than that for the native state. 1H-NMR spectra confirmed the overall structural features of the acid intermediates. To obtain the enthalpies of unfolding, microcalori-metric measurements were performed under conditions where the acid intermediates are maximally populated (18°C): state IN from pH 5.0 to 4.6, 0.03 M salt; state I2 below pH 3.8, 0.42 M salt; and state I1 in equilibrium with IN at pH 4.05, 0.03 M salt. Enthalpies of unfolding for states IN and I2 were comparable to those of the native state. The enthalpy of unfolding for state I1 could not be determined.

Emergent Spatial Structures in Critical Sandpiles

Abstract: We introduce and study a new directed sandpile model with threshold dynamics and stochastic toppling rules. We show that particle conservation law and the directed percolation-like local evolution of avalanches lead to the formation of a spatial structure in the steady state, with the density developing a power law tail away from the top. We determine the scaling exponents characterizing the avalanche distributions in terms of the critical exponents of directed percolation in all dimensions.

Dynamic light scattering in polymer-dispersed liquid crystals

Abstract: A polymer dispersed liquid crystal was studied by dynamic light scattering. In the experiments, where no electric field is applied, the system exhibits additional slow dynamics to the ones observed in bulk nematic liquid crystals. This slow dynamics gradually disappears with an increasing electric field. While the measured time intensity correlation function does not depend on the scattering vector in the system where no field is applied due to the multiple scattering, its dependence on the electric field shows expected size effects. In confined systems, with typical size $d$, there are no eigenmodes of the orientational fluctuations in droplets with the wave vectors less than the minimal wave vector ${q}_{\mathrm{min}}\ensuremath{\sim}\ensuremath{\pi}/d$. We observed a quadratic dependence of the inverse relaxation time on the scattering vector down to a certain scattering vector, below which the relaxation time remains constant. The size calculated from this minimal scattering vector is in agreement with the average droplet size obtained from scanning electron microscope photographs and the atomic force microscope images of our sample. The electric field changes the temperature behavior of the inverse relaxation time near the nematic-isotropic phase transition, which increases with the temperature when the electric field is applied, but decreases when the field is absent.