Volume 61, Issue 11-12

In this study the enhancement effect of Sophora pachy- carpa roots’ acetone extract on the antibacterial activity of gentamycin was evaluated against Staphylococcus au­reus. Disc diffusion and broth dilution methods were used to determine the antibacterial activity of gentamy­cin in the absence and presence of plant extract and its various fractions separated by TLC. A clinical isolate of S. aureus was used as test strain. The active component of the plant extract involved in enhancement of genta- mycin’s activity had R f = 0.72 on a TLC plate. The spec­tral data ( 1 H NMR. 13 CNMR) of this compound re­vealed that this compound was 5,7,2′,4′-tetrahydroxy-8- lavandulylflavanone (sophoraflavanone G), previously isolated from Sophora exigua. In the presence of 0.03 μg/ mL of sophoraflavanone G the MIC value of gentamy­cin for S. aureus decreased from 32 to 8 μg/mL (a four­fold decrease). These results signify that the ultra-low concentration of sophoraflavanone G potentiates the an­timicrobial action of gentamycin suggesting a possible utilization of this compound in combination therapy against Staphylococcus aureus.

Six species of lichens, such as Usnea florida, Usnea barbata, Usnea longissima, Usnea rigida, Usnea hirta and Usnea subflorida, were collected from different areas of Anatolia (district of Antalya, Karabük, Çankırı, Giresun and Trabzon) in Turkey. Their usnic acid amounts in acetone extracts were determined by HPLC. In addition, antimicrobial activities of these extracts were determined against Escherichia coli (ATCC 35218), Enterococcus faecalis (RSKK 508), Proteus mirabilis (Pasteur Ens. 235), Staphylococcus aureus, Bacillus subtilis and Bacillus megaterium. It was shown that with increasing amount of usnic acid, the antimicrobial activity increased. Usnic acid contents of Usnea species varied between 0.22-6.49% of dry weight.

Proteases play a regulatory role in a variety of pathologies including cancer, pancreatitis, thromboembolic disorders, viral infections and many others. One of the possible strategies how to combat with these pathologies seems to be the use of low molecular inhibitors. Natu­ral products were evaluated in the in vitro antiprotease assay on serine proteases (trypsin, thrombin and urokinase) and on the cysteine protease cathepsin B. We found interesting results for β-ursolic acid isolated from Salvia officinalis, which significantly inhibited all tested proteases in vitro in the micromolar range. β-Ursolic acid showed the strongest inhibition activity to urokinase (IC 50 = 12 μᴍ) and cathepsin B (IC 50 = 10 /μᴍ) as proteases included in tumour invasion and metastasis indicated possible anticancer effectivity. Therefore, we tested the ability of β-ursolic acid at doses of 50, 75 and 100 mg/kg given i.p. to inhibit lung coloniza­tion of B16 mouse melanoma cells in vivo. We found, that β-ursolic acid significantly de­creased the number of B16 colonies in the lungs of mice at the dose 50 mg/kg (p < 0.05).

A soluble viscous protein was purified from yam (Dioscorea opposita Thunb.) tuber mucilage tororo by chromatographic steps, and its functional properties were estimated. The purified dioscorin having the molecular weight of about 200 kDa exhibited high scavenging activities against hydroxyl radicals (IC 50 = 195.1 μg/ml) and superoxide anion radicals (IC 50 = 92.7 μg/ml). Moreover, it showed extremely high angiotensin I-converting enzyme inhibitory activity (IC 50 = 41.1 μg/ml). The results suggested that yam D. opposita tuber has a wide spectrum of strong antioxidative and antihypertensive activities and it could be utilized as a source of natural antioxidant.

Solanum asterophorum Mart. (Solanaceae) is a shrub popularly known as “jurubeba-defogo” in the northeast of Brazil. In the present work, the methanol extract (SA-MeOH, 3-750 μg/mL) and isojuripidine (10 -7 - 3 x 10 -4 ᴍ), a steroidal alkaloid obtained from S. asterophorum Mart. leaves, inhibited phasic contractions induced by both 1 μᴍ histamine [IC 50 = (225.8 ± 47.4) μg/mL and (3.5 ± 0.8) x 10 -5 ᴍ] or 1μᴍ acetylcholine [IC 50 = (112.5 ± 20.6) μg/mL and (2.3 ± 0.4) \ 10 -5 ᴍ] in guinea-pig ileum, respectively. The extract and isojuripidine also relaxed the ileum (SA-MeOH, 1-750 μg/mL, and isojuripidine, 10 -9 - 3 x 10 -4 ᴍ) pre-contracted with 1 μᴍ histamine [EC 50 = (101.1 ± 17.4) μg/mL and (1.2 ± 0.3) x 10 -6 ᴍ] or 1 μm acetylcholine [EC 50 = (136.8 ± 21.1) μg/mL and (1.9 ± 0.4) x 10 -6 ᴍ] or 40 mᴍ KCl [EC 50 = (149.4 d 19.5) μg/mL and (1.8 ± 0.7) x 10 -6 ᴍ], respectively, in an equipotent and concentration-dependent manner. This effect is probably due to inhibition of calcium influx through voltage-operated calcium (Ca v ) channels. To confirm this hypothesis, we evaluated their effect on cumulative CaCl 2 curves in depolarizing medium nominally without Ca 2+ . SA-MeOH (27, 243, 500, and 750 μg/mL) and isojuripidine (3 x 10 -8 , 10 -6 , 3 x 10 -5 , and 3 x 10 -4 m) inhibited the contractions induced by CaCl 2 , in a concentrationdependent manner. The concentration-response curves to CaCl 2 , in the presence of SAMeOH and isojuripidine, were shifted downward in relation to a control curve in a nonparallel manner resulting in reduction of the maximum effect [E max = (71.2 ± 9.2); (57.4 ± 9.2); (43.8 ± 3.4); (41.5 ± 2.4) and (90.6 ± 4.8); (74.7 ± 8.7); (66.4 ± 3.9); (31.3 ± 4.1)%, respectively]. SA-MeOH and isojuripidine present spasmolytic action in guinea-pig ileum due to a partially blockade of calcium influx through Ca v channels.

Chemical reactions performed by fungi have been used as a modern tool in chemistry. In this work, we show the tryptophan biotransformation with Psilocybe coprophila on liquid culture medium. The results prove once more the versatility of fungi in performing a wide range of industrially attractive chemical reactions.

Keywords The course of transformations of the pharmacological steroids: testosterone propionate, 4-chlorotestosterone acetate, 17β-estradiol diacetate and their parent alcohols in Fusarium culmorum AM282 culture was compared. The results show that this microorganism is capable of regioselective hydrolysis of ester bonds. Only 4-ene-3-oxo steroid esters were hydrolyzed at C-17. 17β-Estradiol diacetate underwent regioselective hydrolysis at C-3 and as a result, estrone - the main metabolite of estradiol - was absent in the reaction mixture. The alcohols resulting from the hydrolysis underwent oxidation at C-17 and hydroxylation. The same products (6β- and 15α-hydroxy derivatives) as from testosterone were formed by transformation of testosterone propionate, but the quantitative composition of the mixtures obtained after transformations of both substrates showed differences. The 15α-hydroxy deriv­atives were obtained from the ester in considerably higher yield than from the parent alcohol. The presence of the chlorine atom at C-4 markedly reduced 17β-saponification in 4-chloro- testosterone acetate. Only 3β,15α-dihydroxy-4α-chloro-5α-androstan-17-one (the main prod­uct of transformation of 4-chlorotestosterone) was identified in the reaction mixture. 6β- Hydroxy-4-chloroandrostenedione, which was formed from 4-chlorotestosterone, was |not de­tected in the extract obtained after conversion of its ester.

From Pseudomonas sp. PS 6.10, when grown in a casamino acid medium, a pyoverdin was isolated whose primary structure could be deduced from its mass spectrometric fragmentation pattern and amino acid analysis. It belongs to the smallest representatives of this group of siderophores comprising only six amino acids in its peptide chain. When grown in a succinate minimal medium the corresponding ferribactin considered to be the biogenetic precursor of the pyoverdin was obtained as the major component.

The primary aim of this work was to determine the interactions of an oxindole alkaloid (mitraphylline) isolated from Uncaria tomentosa with β-amyloid 1-40 (Aβ1-40 protein) apply­ing the capillary electrophoresis (CE) method. Specifically the Hummel-Dreyer method and Scatchard analysis were performed to study the binding of oxindole alkaloids with Aβ1-40 protein. Prior to these studies extraction of the alkaloid of interest was carried out. Identifica­tion of the isolated alkaloid was performed by the use of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) combined with electrospray ionization mass spectrometry (ESI-MS). The proposed approach was proved to be an efficient and accurate method for specific compound isolation and identification purposes. Moreover, ana­lytical information from the CE approach can be considered as the valuable tool for binding constant determination. The binding constant of mitraphylline with Aβ1-40 protein deter­mined by the Hummel-Dreyer method and Scatchard analysis equals K = 9.95 x 10 5 m -1 . The results obtained showed the significant binding of the tested compound with Aβ1-40 protein. These results are discussed and interpreted in the view of developing a strategy for identifica­tion of novel compounds of great importance in Alzheimer disease therapy.

A novel analytical approach involving the addition of an ionic liquid into the mobile phase of the thin-layer chromatography (TLC) system during the optimization of chromatographic separation of peptides was demonstrated. Different behavior of peptides in the TLC sytem was observed after the addition of 1,3-dimethylimidazolium methyl sulfate to the eluent in comparison to the system without the ionic liquid. The objective of the work was to study the effect of the addition of different contents of ionic liquid to the mobile phase comprising mostly water and to observe the behavior of peptides’ retention. The potential usefulness of environmentally friendly ionic liquids for the optimization of separation of peptides was demonstrated. An increase of R f values was observed with increasing the ionic liquid content in the mobile phase. The benefits of the used approach were related to the separation achieved. Finally, quantitative structure-retention relationships (QSRR) were used for the studies on the predictions of peptides’ retention in the TLC systems with the addition of ionic liquid in terms of the predictions performed recently in HPLC systems.

The effect of drought stress at supraoptimal temperature on free proline and polyamine levels was compared in wild type and transgenic soybean (Glycine max cv. Ibis) plants having increased proline levels. Since glutamate and arginine are precursors of both proline and polyamines, it was assumed that the genetic manipulation of proline levels would also affect the polyamine levels. The proline and spermine concentrations increased, while the putrescine concentration generally decreased or did not change after the treatments in both genotypes. Following drought higher proline and lower spermine levels were detected in the transgenic plants compared to the wild type ones, which could be explained by the increased use of their common precursors for proline biosynthesis in the transgenic plants.

MANB36, a secrete endo-β-1,4-D-mannanase produced by Bacillus subtilis B36, was puri­fied to homogeneity from a culture supernatant and characterized. The optimum pH value for the mannanase activity of MANB36 is 6.4 and the optimum temperature is 50 °C. The enzyme activity of MANB36 is remarkably thermostable at 60 °C and the specific activity of MANB36 is 927.84 U/mg. Metal cations (except Hg 2+ and Ag + ), EDTA and 2-mercaptoetha- nol (2-ME) have no effects on enzyme activity. This enzyme exhibits high specificity with the substituted galactomannan locust bean gum (LBG). The gene encoding for MANB36, manB36, was cloned by PCR and sequenced. manB36 contains a single open reading frame (ORF) consisting of 1104 bp that encodes a protein of 367 amino acids. The predicted mo­lecular weight of 38.13 kDa, calculated by the deduced protein of the gene manB36 without signal peptide, coincides with the apparent molecular weight of 38.0 kDa of the purified MANB36 estimated by SDS-PAGE. The mature protein of MANB36 has been expressed in Escherichia coli BL21 and the expressed mannanase has normal bioactivity.

In addition to the importance of many Dioscorea species (yams) as starchy staple food, some representatives are known and still used as a source for the steroidal sapogenin diosgenin, which, besides phytosterols derived from tall-oil, is an important precursor for partial synthesis of steroids for pharmaceutical research and applications. While in edible yams the diosgenin content should be as low as possible, a high yield of the compound is preferable for cultivars which are grown for the extraction of sterols. In the past, miscalculations and insufficiently precise techniques for quantification of diosgenin prevailed. Therefore we set out to re-evaluate the steroid content of a world collection of Dioscorea species, using leaves as sample material. We optimized diosgenin quantification techniques and fingerprinted the whole collection with the DNA amplification fingerprinting (DAF) technique. Total diosgenin contents ranged from 0.04 to 0.93% of dry weight within the collection. Several Dioscorea cultivars can be characterized via their DAF fingerprint patterns.

O 3 is the major component of photochemical oxidants and gives rise to visible injuries on plant leaves. In O 3 -exposed plants, O - 2 is produced before the formation of the injury, but the role that O - 2 plays in plant response to O 3 exposure is still unknown. To clarify its role, we observed the behavior of plants during O 3 exposure after pretreatment with tiron, which is an O - 2 scavenger. When tiron-pretreated tobacco cv. Bel W3 was exposed to O 3 , leaf damage was attenuated. In O 3 -exposed tobacco, tiron inhibited increases in the levels of ethylene and salicylic acid, which promote leaf injury. Tiron pretreatment also suppressed increases in the expression of O 3 -induced genes. These results suggest that O - 2 is involved in many plant responses induced by O 3 exposure. Bel B, a tobacco cultivar that is genetically related to Bel W3, is reported to be more resistant to O 3 than Bel W3, but the reason for this difference is unclear. We investigated the differences between the responses of Bel B and tiron-pretreated Bel W3 to O 3 exposure, and we discuss the reasons for the resistance to O 3 by comparing the phenotype of Bel B with that of tiron-pretreated Bel W3.

The hybrid sensory histidine kinase Slr1759 of the cyanobacterium Synechocystis sp. strain PCC 6803 contains multiple sensory domains and a multi-step phosphorelay system. Immuno blot analysis provided evidence that the histidine kinase Slr1759 is associated with the cytoplasmic membrane. The gene slr1759 is part of an operon together with slr1760, encoding a response regulator. A comparative investigation was performed on Synechocystis sp. strain PCC 6803 wild type (WT) and an insertionally inactivated slr1759-mutant (Hik14) which also lacks the transcript for the response regulator Slr1760. The mutant Hik14 grew significantly slower than WT in the early growth phase, when both were inoculated with a low cell density into BG11 medium without additional buffer and when aerated with air enriched with 2% CO 2 . Since the aeration with CO 2 -enriched air results in a decrease of the pH value in the medium, the growth experiments indicated that Hik14 is not able to adjust its metabolic activities as rapidly as WT to compensate for a larger decrease of the pH value in the medium. No significant differences in growth between Hik14 and WT were observed when cells were inoculated with a higher cell density in BG11 medium or when the BG11 medium contained 50 mm Epps-NaOH, pH 7.5, to prevent the pH drop. This Hik14 phenotype has so far only been seen under the above defined growth condition. Results of photosynthetic activity measurements as well as Northern blot-, immuno blot-, and metabolite analyses suggest that the two-component system Slr1759/Slr1760 has a function in the coordination of several metabolic activities which is in good agreement with the complex domain structure of Slr1759. The direct targets of this two-component system have so far not been identified.

During our earlier investigations we have observed a prominent preference of the linker histone H1 for binding to a cis-platinated DNA (a synthetic fragment with global type of platination in respect to targets for cisplatin) comparing with unmodified and trans-Pt-modified DNA. In the present work we report our recent experimental results on the binding of the linker histones H1 and H5 to a cisplatin-modified synthetic DNA fragment containing a single nucleotide target d(GC/CG) for inter-platination. Surprisingly, no preferential binding of linker histones to cis-inter-platinated DNA was observed by means of the electromobilityshift assay. The same negative results were obtained with a part of the linker histone molecule suggested to be responsible for DNA-binding - its globular domain. Contrary, the data with another nuclear protein with similar DNA-binding properties as linker histones - HMGB1 - showed a strong afinity for interaction with DNA containing interstrand cross-links.

A soluble fructose-1,6-bisphosphate aldolase enzyme has been purified 50.2-fold (2.36%) at the homogeneity from the electric organ of Electrophorus electricus by one step of DEAE- 52 anion exchange chromatography followed by Superose-12 gel filtration-FPLC. Like other aldolase enzymes the E. electricus protein is a dimer with two identical subunits of 45 kDa. The N-terminal (20 residues) revealed a high homology with S. aurata (75%, goldfish), R. ratus and M. musculus (mouse, 80%) enzymes.

The geno- and cytotoxicity of chromate, an important environmental pollutant, is partly attributed to the flavoenzyme-catalyzed reduction with the concomitant formation of reactive oxygen species. The aim of this work was to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) and glutathione reductase (GR, EC 1.6.4.2) in the mammalian cell cytotoxicity of chromate, which was evidenced controversially so far. The chromate reductase activity of NQO1 was higher than that of GR, but lower than that of lipoamide dehydrogenase (EC 1.6.4.3), ferredoxin:NADP + reductase (EC 1.18.1.2), and NADPH: cytochrome P-450 reductase (EC 1.6.2.4). The reduction of chromate by NQO1 was accompanied by the formation of reactive oxygen species. The concentration of chromate for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (22 ± 4) μᴍ. The cytotoxicity was partly prevented by desferri- oxamine, the antioxidant N(N′-diphenyl-p-phenylene diamine and by an inhibitor of NQO1, dicumarol, and potentiated by 1,3-bis-(2-chloroethyl)-l-nitrosourea (BCNU), which inacti­vates GR. The NADPH-dependent chromate reduction by digitonin-permeabilized FLK cells was partly inhibited by dicumarol and not affected by BCNU. Taken together, these data indicate that the oxidative stress-type cytotoxicity of chromate in FLK cells may be partly attributed to its reduction by NQO1, but not by GR. The effect of BCNU on the chromate cytotoxicity may indicate that the general antioxidant action of reduced glutathione is more important than its prooxidant activities arising from the reactions with chromate.

BCR/ABL oncogene, as a result of chromosome aberration t(9;22), is the pathogenic principle of almost 95% of human chronic myeloid leukemia (CML). Imatinib (STI571) is a highly selective inhibitor of BCR/ABL oncogenic tyrosine kinase used in leukemia treatment. It has been suggested that BCR/ABL may contribute to the resistance of leukemic cells to drug and radiation through stimulation of DNA repair in these cells. To evaluate further the influence of STI571 on DNA repair we studied the efficacy of this process in BCR/ABLpositive and -negative cells using single cell electrophoresis (comet assay). In our experiments, K562 human chronic myeloid leukemia cells expressing BCR/ABL and CCRF-CEM human acute lymphoblastic leukemia cells without BCR/ABL expression were employed. The cells were exposed for 1 h at 37 ∞C to N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) at 5 μᴍ, mitomycin C (MMC) at 50 μᴍ or to γ-radiation at 15 Gy with or without a 24 h preincubation at 1 μᴍ of STI571. The MTT cells survival after 4 days of culture showed that STI571 enhanced the cytotoxity of the examined compounds in the K562 line. Further it was found, that the inhibitor decreased the efficacy of DNA repair challenged by each agent, but only in the K562 expressing BCR/ABL. Due to the variety of DNA damage induced by the employed agents in this study we can speculate, that BCR/ABL may stimulate multiple pathways of DNA repair. These results extend our previous studies performed on BCR/ABLtransformed mouse cells onto human cells. It is shown that BCR/ABL stimulated DNA repair in human leukemia cells. In conclusion we report that STI571 was found to inhibit DNA repair and abrogate BCR/ABL-positive human leukemia cells therapeutic resistance.

The aim of this study was to investigate the in vitro genotoxic effects of the anticancer drugs fotemustine and vinorelbine on human lymphocytes and to determine individual and sex-related responses to these drugs. Fotemustine is a DNA-alkylating drug while vinorelbine is a semi-synthetic Vinca alkaloid. The study was carried out with twenty independent healthy donors for each drug. We have tested the ability of these drugs to induce chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) as well as effect on the mitotic index (MI) in cultured human lymphocytes. Fotemustine was shown to induce CAs and SCEs at all concentrations tested (2, 4 and 8 μg/ml) in a dose-dependent manner. Additionally it also decreased the mitotic index in a similar dose-dependent manner. Vinorelbine had no effect on structural CAs, but it significantly increased the numerical CAs at all doses tested (0.5, 1 and 2 μg/ml). Vinorelbine also induced SCE events and increased the MI values. Two-way analyses of variance were used to compare the individual and gender-related susceptibilities to fotemustine and vinorelbine with respect to the CA, SCE and MI values. The results indicated that individuals in fotemustine treatment groups showed different genotoxic responses with respect to CA and SCE induction and additional findings indicated a gender-specific response in this group. Individuals in the vinorelbine test group also exhibited statistically significant numerical CA, SCE and MI responses to vinorelbine. A statistically significant gender-related SCE response to this drug was also evident. This study indicates that these drugs have potentially harmful effects on human health.