Surface topographic study of chalcogenide thin films of GexSb(As)40-xS50Te10 glasses.

The surface topography and fractal properties of GexSb(As)40-xS50Te10 (x=10, 20, 27 at.%) films, evaporated onto glass substrates, have been studied by atomic force microscopic imaging at different scales. The surface of the chalcogenide films is smooth (<5 nm roughness), isotropic and having some particular differences in texture. All films are self-similar with Mean Fractal Dimension in the range of 2.25-2.63. The films with GexSb40-xS50Te10 composition are more uniform in terms of surface morphology (grains structure) than those with GexAs40-xS50Te10 composition for which the film surface exhibits a superimposed structure of large particles at x=10 and 20 at.%.

Rapid purification method for fumonisin B1 using centrifugal partition chromatography.

Fumonisin B1 (FB1) is a highly toxic mycotoxin produced by fungal strains belonging to the Fusarium genus, which can be found mainly in maize products, and is gaining interest in food safety. To produce large amounts of pure FB1, a novel purifying method was developed by using centrifugal partition chromatography, which is a prominent member of the liquid-liquid chromatographic techniques. Rice cultured with Fusarium verticillioides was extracted with a mixture of methanol/water and found to contain 0.87 mg of FB1 per gram. The crude extracts were purified on a strong anion-exchange column and then separated by using a biphasic solvent system consisting of methyl-tert-butyl-ether-acetonitrile-0.1% formic acid in water. The collected fractions were analysed by flow injection-mass spectrometry and high-performance liquid chromatography coupled with Corona-charged aerosol detector and identified by congruent retention time on high-performance liquid chromatography and mass spectrometric data. This method produced approximately 120 mg of FB1 with a purity of more than 98% from 200 g of the rice culture. The whole purification process is able to produce a large amount of pure FB1 for analytical applications or for toxicological studies.

Fumonisin contamination and fumonisin producing black Aspergilli in dried vine fruits of different origin.

Aspergillus niger isolates are able to produce fumonisins in high quantities on agar media with a low water activity. Several agricultural products fit this criterion, including dried vine fruits, dates and figs. Data on the occurrence and role of this species in fumonisin contamination of agricultural products with high sugar content are needed to clarify the importance of A. niger in human health. The mycobiota and fumonisin contamination of various dried vine fruit samples collected from different countries were examined to clarify the role of black Aspergilli in fumonisin contamination of such products. All except two of the examined samples were contaminated with black Aspergilli. Species assignment of the isolates was carried out using sequence analysis of part of the calmodulin gene. The range of fumonisin isomers present in the raisins samples, and produced by A. niger isolates collected from dried vine fruits was also examined using reversed-phase high-performance liquid chromatography/electrospray ionization-ion trap mass spectrometry (RP-HPLC/ESI-ITMS). Among the 30 A. niger/A. awamori isolates identified, 20 were found to be able to produce fumonisins (average contamination: 5.16 mg/kg; range: 0.017-19.6 mg/kg). The average fumonisin content of the 7 dried vine fruit samples which were found to be contaminated by potential fumonisin producing black Aspergilli was 7.22 mg/kg (range: 4.55-35.49 mg/kg). The isolates produced several fumonisin isomers also present in the dried vine fruit samples, including fumonisins B(1-4), 3-epi-FB(3), 3-epi-FB(4), iso-FB(1), and two iso-FB(2,3) forms. Fumonisin B(1) was detected for the first time in A. niger cultures. Most of these isomers have previously only been identified in Fusarium species. Our data indicate that A. niger and A. awamori are responsible for fumonisin contamination of dried vine fruits worldwide. The observed levels of contamination are alarming and pose a new threat for food safety.

Purification and preliminary characterization of a cold-adapted extracellular proteinase from Trichoderma atroviride.

Eleven cold-tolerant Trichoderma isolates were screened for the production of proteolytic activities at 10 degrees C. Based on the activity profiles determined with paranitroanilide substrates at 5 degrees C, strain T221 identified as Trichoderma atroviride was selected for further investigations. The culture broth of the strain grown at 10 degrees C in casein-containing culture medium was concentrated by lyophilization and subjected to gel filtration, which was followed by chromatofocusing of the fraction showing the highest activity on N-benzoyl-Phe-Val-Arg-paranitroanilide. The purified enzyme had a molecular weight of 24 kDa, an isoelectric point of 7.3 and a pH optimum of 6.2. The temperature optimum of 25 degrees C and the low thermal stability suggested that it is a true cold-adapted enzyme. Substrate specificity data indicate that the enzyme is a proteinase with a preference for Arg or Lys at the P1 position. The effect of proteinase inhibitors suggests that the enzyme has a binding pocket similar to the one present in trypsin.

Colony morphology mutants of Trichoderma harzianum with increased beta-1,4-N-acetyl-glucosaminidase production.

A total of 36 UV-induced mutants with altered colony morphology were isolated from strain Trichoderma harzianum T334, a potential biocontrol agent against plant pathogenic fungi with the ability to produce constitutively low levels of chitinases. The level of constitutive beta-1,4-N-acetyl-glucosaminidase production in standing and shaken cultures under non-inductive conditions was tested in mutants and compared to that of the parental strain. About 30% of the mutants showed significantly increased levels of enzyme production, with strain T334 col26a being the best producer. This mutant and the parental strain were subjected to in vitro confrontation assays with plant pathogenic Fusarium culmorum, Pythium debaryanum and Rhizoctonia solani strains. The mutant derivative could be characterized by significantly higher biocontrol index values than the parental strain in each experiment, suggesting, that mutants with improved constitutive extracellular chitinase secretion could be applied for biocontrol purposes against fungal plant pathogens.

Green Mold Diseases of Agaricus and Pleurotus spp. Are Caused by Related but Phylogenetically Different Trichoderma Species.

ABSTRACT Producers of champignon (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus) are facing recent incidents of green mold epidemics in Hungary. We examined 66 Trichoderma strains isolated from Agaricus compost and Pleurotus substrate samples from three Hungarian mushroom producing companies by a polymerase chain reaction-based diagnostic test for T. aggressivum, sequence analysis of the internal transcribed spacer region 1 (ITS1) and ITS2 and (selectively) of the fourth and fifth intron of translation elongation factor 1alpha (tef1alpha), and restriction fragment length polymorphism of mitochondrial DNA. Seven Trichoderma species were identified: T. aggressivum f. europaeum (17 isolates), T. harzianum (three isolates), T. longibrachiatum (four isolates), T. ghanense (one isolate), T. asperellum (four isolates), T. atroviride (nine isolates), and a still undescribed phylogenetic species, Trichoderma sp. DAOM 175924 (28 isolates). T. aggressivum f. europaeum was exclusively derived from A. bisporus compost, whereas Trichoderma sp. DAOM 175924 exclusively occurred in the substrate for Pleurotus cultivation. Sequences of the latter strains were co-specific with those for Trichoderma pathogens of P. ostreatus in Korea. The widespread occurrence of this new species raises questions as to why infections by it have just only recently been observed. Our data document that (i) green mold disease by T. aggressivum f. europaeum has geographically expanded to Central Europe; (ii) the green mold disease of P. ostreatus in Hungary is due to the same Trichoderma species as in Korea and the worldwide distribution of the new species indicates the possibility of spreading epidemics; and (iii) on mushroom farms, the two species are specialized on their different substrates.

Rapid identification of clinical Trichoderma longibrachiatum isolates by cellulose-acetate electrophoresis-mediated isoenzyme analysis.

Cellulose-acetate electrophoresis was used to investigate isoenzyme polymorphism among ten clinical and 11 non-clinical isolates of Trichoderma. Initial testing of 13 enzyme systems for activity and resolution of bands showed that seven were appropriate for identifying the different species. Each of the enzyme systems investigated (glucose-6-phosphate dehydrogenase, glucose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, peptidases A, B and D, and phosphoglucomutase) was diagnostic for at least one species. On the basis of the results of isoenzyme analysis, several isolates identified originally as Trichoderma pseudokoningii, T. koningii or T. citrinoviride were re-identified as T. longibrachiatum, in agreement with sequence analysis data for the internal transcribed spacer region of the isolates. The availability of a quick, inexpensive and reliable diagnostic tool for the identification of T. longibrachiatum isolates is important, as most clinical Trichoderma isolates belong to T. longibrachiatum. Furthermore, as many different enzyme systems are available, the method may also be suitable for the identification of other clinically relevant fungal species.

Peptaibols and related peptaibiotics of Trichoderma. A review.

Peptaibols and the related peptaibiotics are linear, amphipathic polypeptides. More than 300 of these secondary metabolites have been described to date. These compounds are composed of 5-20 amino acids and are generally produced in microheterogeneous mixtures. Peptaibols and peptaibiotics with unusual amino acid content are the result of non-ribosomal biosynthesis. Large multifunctional enzymes known as peptide synthetases assemble these molecules by the multiple carrier thiotemplate mechanism from a remarkable range of precursors, which can be N-methylated, acylated or reduced. Peptaibols and peptaibiotics show interesting physico-chemical and biological properties including the formation of pores in bilayer lipid membranes, as well as antibacterial, antifungal, occasionally antiviral activities, and may elicit plant resistance. The three-dimensional structure of peptaibols and peptaibiotics is characterized predominantly by one type of the helical motifs alpha-helix, 3(10)-helix and beta-bend ribbon spiral. The aim of this review is to summarize the data available about the biosynthesis, biological activity and conformational properties of peptaibols and peptaibiotics described from Trichoderma species.

Extracellular proteases of Trichoderma species. A review.

Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.

Comparative study of potential virulence factors in human pathogenic and saprophytic Trichoderma longibrachiatum strains.

Potential virulence factors of 9 saprophytic and 12 clinical Trichoderma longibrachiatum strains were examined in the present study, in order to compare their capacity to cause infection in humans. All of the strains were able to grow at temperatures up to 40 degrees C and at pH values ranging from 2.0 to 9.0. Carbon and nitrogen source utilization experiments revealed that all of the strains were able to utilize a series of basic amino acids both as sole carbon and nitrogen sources. The MIC values of the tested antifungal drugs were found to be 0.016-8 microg/ml for amphotericin B, 64-256 microg/ml for fluconazole, 0.5-32 microg/ml for itraconazole and 0.008-1 microg/ml for ketoconazole in the case of the examined isolates. Metabolites of the strains inhibited the growth of different bacteria, furthermore, compounds produced by three clinical isolates reduced the motility of boar spermatozoa, indicating their toxicity to mammalian cells as well. On the whole, there were no significant differences in the examined features between strains derived from clinical or soil samples. The question, however, whether all environmental Trichoderma longibrachiatum strains have the capacity to cause infections or not, remains still unanswered.

Production of extracellular proteases by human pathogenic Trichoderma longibrachiatum strains.

Species belonging to the filamentous fungal genus Trichoderma are well known as potential candidates for the biological control of plant pathogenic fungi and as cellulase producers of biotechnological importance. Several data were published in the last decade also about the clinical importance of this genus, indicating that Trichoderma strains may be potential opportunistic pathogens in immunocompromised patients. However, there is a lack of information about the potential virulence factors of clinical Trichoderma strains. This study was designed to examine the extracellular proteolytic enzymes of six clinical T. longibrachiatum isolates. Supernatants from induced liquid cultures of the examined strains were screened for proteolytic enzyme activities with 11 different chromogenic p-nitroaniline substrates. The production of trypsin-like, chymotrypsin-like and chymoelastase-like protease activities cleaving N-Benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide, N-Succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, and N-Succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, respectively, was common among the strains examined. Separation of trypsin- and chymotrypsin-like activities by column chromatography revealed, that both systems are complex consisting of several isoenzymes. The pH-dependence of these two protease systems was also studied. Based on the results, the different isoenzymes seem to have different optimal pH values. Extracellular proteolytic enzymes may be involved in the pathogenecity of Trichoderma strains as facultative human pathogens.

In vitro water activity and pH dependence of mycelial growth and extracellular enzyme activities of Trichoderma strains with biocontrol potential.

AIMS: Water activity (aw) and pH are probably the most important environmental parameters affecting the activities of mycoparasitic Trichoderma strains. Therefore it is important to collect information on the effects of these factors on mycelial growth and on the in vitro activities of extracellular enzymes involved in nutrient competition (e.g. beta-glucosidase, cellobiohydrolase and beta-xylosidase) and mycoparasitism (e.g. N-acetyl-beta-glucosaminidase, trypsin-like protease and chymotrypsin-like protease) of Trichoderma strains with biocontrol potential. METHODS AND RESULTS: Water activity and pH dependence of the linear mycelial growth of five examined Trichoderma strains belonging to three different species groups was examined on yeast extract and soil extract media. Maximal growth rates were observed at aw 0.997 and pH 4.0 in the case of all strains. The activities of the examined extracellular enzymes at different aw and pH values were determined spectrophotometrically after incubation with chromogenic p-nitrophenyl and p-nitroaniline substrates. Maximal enzyme activities were measured at aw 0.950 for beta-glucosidase, trypsin-like protease and chymotrypsin-like protease, at 0.910 for cellobiohydrolase and at 0.993 for beta-xylosidase and N-acetyl-beta-glucosaminidase enzymes. Optimal pH values are suggested to be at 5.0 for beta-glucosidase, cellobiohydrolase and N-acetyl-beta-glucosaminidase, at 3.0 for beta-xylosidase, at 6.0 for trypsin-like protease and between 6.0 and 7.0 for chymotrypsin-like protease activities, respectively. CONCLUSIONS: Extracellular enzymes of the examined mycoparasitic Trichoderma strains are able to display activities under a wider range of aw and pH values than those allowing mycelial growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Data about the effects of aw and pH on mycelial growth and extracellular enzyme activities of Trichoderma reveal useful information about the applicability of biocontrol strains in agricultural soils with specific water and pH relations.

Conformational analysis of endomorphin-1 by molecular dynamics methods.

Endomorphin-1 (EM1, H-Tyr-Pro-Trp-Phe-NH2) is a highly potent and selective agonist for the mu-opioid receptor. A conformational analysis of this tetrapeptide was carried out by simulated annealing and molecular dynamics methods. EM1 was modeled in the neutral (NH2-) and cationic (NH-) forms of the N-terminal amino group. The results of NMR measurements were utilized to perform simulations with restrained cis and trans Tyr1-Pro2 peptide bonds. Preferred conformational regions in the Phi 2-Psi 2, Phi 3-Psi 3 and Phi 4-Psi 4 Ramachandran plots were identified. The g(+), g(-) and trans rotamer populations of the side-chains of the Tyr1, Trp3 and Phe4 residues were determined in chi 1 space. The distances between the N-terminal N atom and the other backbone N and O atoms, and the distances between the centers of the aromatic side-chain rings and the Pro2 ring were measured. The preferred secondary structures were determined as different types of beta-turns and gamma-turns. In the conformers of trans-EM1, an inverse gamma-turn can be formed in the N-terminal region, but in the conformers of cis-EM1 the N-terminal inverse gamma-turn is absent. Regular and inverse gamma-turns were observed in the C-terminal region in both isomers. These beta- and gamma-turns were stabilized by intramolecular H-bonds and bifurcated H-bonds.

Characterization of CDw92 as a member of the choline transporter-like protein family regulated specifically on dendritic cells.

CDw92 is a 70-kDa surface protein broadly expressed on leukocytes and endothelial cells. In this manuscript, we present the molecular cloning of the CDw92 molecule by using a highly efficient retroviral expression cloning system. Sequence analysis of the CDw92 cDNA revealed a length of 2679 bp. The 1959-bp open reading frame encodes a protein of 652 amino acids. Computational analysis of the CDw92 protein sequence indicates 10 transmembrane domains, three potential N-linked glycosylation sites, and an amino acid stretch in the C-terminal region that is related to the immunoreceptor tyrosine-based inhibitory motif. Comparison of the sequence of the CDw92 clone presented in this study with various database entries show that it is a C-terminal variant of human choline transporter-like protein 1, a member of a recently identified family of multitransmembrane surface proteins. Furthermore, we found that CDw92 is stably expressed on monocytes, PBLs, and endothelial cells, as we did not yet find modulation of expression by various stimuli on these cells. In contrast to this factor-independent expression of CDw92, we detected a specific regulation of CDw92 on monocyte-derived dendritic cells (Mo-DCs). Maturation of Mo-DCs by ionomycin or calcium ionophore resulted in down-regulation of CDw92 and incubation of these cells with IL-10 in a specific re-expression. Moreover, targeting of CDw92 on LPS-treated Mo-DCs by CDw92 mAb VIM15b augmented the LPS-induced IL-10 production 2.8-fold. Together, these data suggest a crucial role of the CDw92 protein in the biology and regulation of the function of leukocytes in particular DCs.

Research training between 14 and 18 in Hungary.

The first five years of a new program to organize high-level scientific research training for gifted high school students in Hungary are described. Besides giving unique research opportunities for talented students in their most receptive age, the program already helped the establishment of almost 100 scientific research clubs in Hungarian high schools, provided a focal point for science training of high school teachers and helped regional cooperation in Central-Eastern Europe.

Comparison of porcine and semisynthetic human insulins using euglycemic clamp-derived glucose-insulin dose-response curves in insulin-dependent diabetes.

In order to compare the biologic effectiveness of porcine and semisynthetic human insulins, a euglycemic clamp method was used in eight insulin-dependent diabetic subjects. Each subject was tested for each insulin on separate days. In order to derive glucose-insulin dose-response curves for both insulins, sequential but constant infusion rates of 0.2, 0.5, 1.0, and 2.0 mU/kg/min were performed. Plasma glucose levels attained during the euglycemic clamp were 96 +/- 3 mg/dL. At each insulin infusion rate, the steady-state glucose infusion rate required to maintain euglycemia was measured. At each increment of insulin infused, steady-state glucose infusion rates for porcine insulin were 1.12 +/- 0.22, 1.90 +/- 0.59, 4.28 +/- 0.61, and 9.37 +/- 0.66 mg/kg/min compared with 1.27 +/- 0.42, 2.38 +/- 0.20, 4.25 +/- 0.43, and 8.87 +/- 0.67 mg/kg/min for semisynthetic human insulin. By ANOVA, no significant difference was noted between the two insulins. Because insulin infusion rates may not result in predictable circulating free insulin levels in subjects who have circulating insulin antibodies, free insulin levels were determined. When steady-state glucose infusion rates were compared with free insulin levels achieved at the four insulin infusion rates, dose-response curves for both porcine and semisynthetic human insulins were virtually identical. These data suggest that semisynthetic human insulin has equivalent biologic effects on overall glucose metabolism compared with porcine insulin in insulin-dependent diabetes.

Gaba-ergic and dopaminergic regulation of thyroid stimulating hormone. Effects of baclofen and metoclopramide.

The effects of the GABA analogue, baclofen, and the dopamine antagonist, metoclopramide, on basal and TRH-stimulated TSH release were studied in 6 normal male volunteers. Basal TSH secretion was not influenced by baclofen (10 mg orally three times daily for 3 days) or metoclopramide (10 mg i.v.), given alone or together. Baclofen produced a blunting of the TRH-stimulated TSH release (p less than 0.05), which persisted after metoclopramide administration. It is speculated that GABA and its analogues exert an inhibitory effect on TSH secretion, presumably at the level of the pituitary, and this effect is not mediated by dopamine.

GABA-ergic and dopaminergic mechanisms in gonadotrophin secretion in males--effects of baclofen and metoclopramide.

The GABA analogue, baclofen, and the dopamine antagonist, metoclopramide, were studied with respect to their effects on basal and LRH-induced LH and FSH release in 6 normal male volunteers. Basal gonadotrophin secretion was unchanged following the administration of baclofen or metoclopramide given alone or in combination. LRH-stimulated LH release was significantly blunted after metoclopramide administration in the baclofen pre-treated volunteers. Serum LH concentration (mean +/- SD) in the control phase was 30.1 +/- 17.2 mIU/ml and was 19.4 +/- 9.6 mIU/ml after baclofen plus metoclopramide (P less than 0.02). LRH-stimulated values, however, were unaffected by baclofen or metoclopramide when the drugs were given alone. LRH-stimulated FSH release was not significantly influenced by baclofen or metoclopramide given alone or in combination. Basal Prl secretion increased significantly when baclofen and metoclopramide were given separately and in combination. Basal Prl concentration (mean +/- SD) increased from 14 +/- 2 ng/ml to 18.7 +/- 4.8 ng/ml after baclofen and to 111.5 +/- 31.9 ng/ml after metoclopramide (P less than 0.01). The rise in serum Prl concentration, however, was not significantly different when measured after metoclopramide alone (111 +/- 31.9 ng/ml) or after metoclopramide and baclofen (112 +/- 33.3 ng/ml). It is proposed that GABA and dopamine exert opposing effects on LH secretion in normal men.

Comparison of the biologic activity of porcine and semisynthetic human insulins using the glucose-controlled insulin infusion system in insulin-dependent diabetes.

Semisynthetic human insulin is prepared from porcine pancreas by chemical methods involving the substitution of porcine B-30 alanine with threonine. To compare the effectiveness of porcine and semisynthetic human insulins, eight insulin-dependent diabetic patients were evaluated during two separate periods using a glucose-controlled insulin infusion system. During each 36-h period, patients received either porcine or semisynthetic human insulin. Patients ingested mixed meals. The mean daily insulin requirements for porcine and semisynthetic human insulins were 84 +/- 9 U and 85 +/- 6 U (+/- SEM), respectively (P = NS). Mean blood glucose values were similar at 95 +/- 1 mg/dl for porcine and 101 +/- 3 mg/dl with semisynthetic human insulin (P = NS). Prior metabolic control or insulin antibody levels did not correlate with intravenous insulin requirements. These studies indicate that semisynthetic human insulin is as effective as porcine insulin in maintaining near-normal blood glucose control in short-term intravenous studies using artificial pancreas techniques in insulin-dependent diabetes.

Comparison of porcine insulin and human insulin (Novo) using the glucose-controlled insulin infusion system, glucose-insulin dose-response curves, and the outpatient effectiveness of human insulin (Novo) in insulin-dependent diabetes.

The usefulness of human insulin (Novo) in the treatment of diabetes was studied in eight insulin-dependent diabetic patients. Evaluation was carried out through three methods: (1) glucose-controlled insulin infusion system on two separate days during the patients' ingestion of mixed meals; (2) euglycemic clamp method; and (3) evaluation of patients for 3 mo using short- and intermediate-acting insulins. Employing the glucose-controlled insulin infusion system resulted in mean daily insulin requirements of 84 +/- 9 U and 85 +/- 6 U SEM for porcine and human insulin, respectively (P = NS). Plasma glucose levels attained during the euglycemic clamp were 96 +/- 3 mg/dl. At each insulin infusion rate, the steady-state glucose infusion rates for porcine insulin were 1.12 +/- 0.22, 1.90 +/- 0.58, 4.28 +/- 0.61, and 9.37 +/- 0.66 mg/kg/min compared with 1.27 +/- 0.42, 2.38 +/- 0.20, 4.25 +/- 0.43, and 8.87 +/- 0.67 mg/kg/min for human insulin. No complications of either insulin preparation were observed in the 3-mo patient evaluation. Human insulin was associated with lower levels of circulating insulin antibody (P less than 0.03). In addition, hemoglobin A1c levels decreased from 8.4 to 6.9% (P less than 0.035), and normal hemoglobin A1c levels were maintained in patients using portable insulin infusion pumps.