Band 64, Heft 3-4

3-Substituted benzylidene-1,3-dihydro-indoline derivatives were tested for their in vitro antibacterial activity against the Gram-negative bacteria Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and the Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus, and for their their in vitro antifungal activity against Candida krusei and Candida albicans. The minimum inhibitory concentration (MIC) values were determined by the 2-fold serial dilution technique in Mueller Hinton broth and Sabouraud dextrose agar using antibacterial and antifungal assays, respectively. For comparison of the antimicrobial activity, rifampicin, ampicillin trihydrate, gentamicin sulfate, and ofloxacin were used as reference antibacterial agents, and fl uconazole and amphotericin B were employed as reference antifungal agents. The most active compound 10 showed notable inhibition against Bacillus subtilis, Staphylococcus aureus, and Candida krusei. Compounds 1 and 6 were found slightly effective against Klebsiella pneumoniae and Escherichia coli. In addition, compounds 13 and 14 showed inhibition against Bacillus subtilis and Staphylococcus aureus. Indole derivatives were also tested in vitro for replication of the HepAD38 cell line and compared with lamivudine (3TC, L-2′,3′-dideoxy-3′-thiacytidine). The IC 50 values of the compounds were found to be >1000 μM against HBV except for compound 13 which exhibited activity with an IC 50 value of 500 μM.

Some 5-(1,2,3-triazol-1-ylmethyl)uridine derivatives were synthesized via the 1,3-dipolar cycloaddition of a 5-azidomethyluridine derivative with substituted acetylenes. The antiviral activities of these compounds against hepatitis A virus (HAV, MBB cell culture-adapted strain) and Herpes simplex virus type-1 (HSV-1) were tested.

The ability of two calix[4]arene derivatives, namely 25,27-p-tert-butylcalix[4]dithiooxabenzocrown (1) and 25,27-p-tert-butylcalix[4]trithiooxabenzocrown (2), to produce chromosomal aberrations in root meristematic cells of Allium cepa and micronuclei (MN) in normochromatic erythrocytes (NCE) of Balb/c mice was investigated. NCE are normal mature red blood cells with a full complement of hemoglobin but lack ribosomes. In the first test, the root tips were treated with a series of concentrations of the two test chemicals ranging from 10 - 7 to 10 - 4 M for 24 or 48 h. Both compounds caused concentration-dependent increases in the percentage of aberrant cells and reductions in the mitotic index. These effects depended, to some extent, on the duration of the treatment. The most conspicuous chromosomal abnormalities were c-mitosis, chromosome bridges, chromosome breaks, chromosome lags as well as micronuclei and multinuclei. In the second test, acridine orange fluorescent staining was applied to evaluate the incidence of MN in NCE of mice intraperitoneally injected with varying contents of the two test chemicals (0.02 - 0.08 mg/mouse). The two chemicals induced dose-dependent MN formation as compared to the negative control. The second compound had more pronounced cytogenetic influence than the first one. Mitomycin C (MMC, 14 mg/kg body weight), employed as positive control, produced more obvious effects on the parameters investigated.

Three hydroxycinnamic acid derivatives conjugated with glycine-containing oxazole were synthesized. The prepared compounds were tested for their antioxidant activity using the 1,1-diphenyl-2-picrylhydrazyl (DPPH ・ ) test. Among the tested hydroxycinnamic acid amides the highest DPPH scavenging activity has been found for the sinapic acid amide.

The series of complexes: cis-[Pd(PMSA) 2 X 2 ], cis-[Pt(PMSA) 2 X 2 ], trans-[Pt(PMSA) 2 I 2 ] and [Pt(PMSA) 4 ]Cl 2 (PMSA = N-3-pyridinylmethanesulfonamide; X = Cl, Br, I), previously synthesized and characterized by us, as well as the free ligand PMSA, were tested for their cytotoxic activity without electroporation - against murine leukemia F4N and human SKW-3 and MDA-MB-231 tumour cell lines - and with electroporation - against the latter two cell lines. The majority of the complexes exhibited cytotoxic effects (IC 50 < 100 μmol/l) under the conditions of electroporation. Both cis- and trans-[Pt(PMSA) 2 I 2 ] had pronounced cytotoxic effects (29 - 61 μmol/l against MDA-MB-231 cells).

The fungus Trichoderma viride was isolated from the Caribbean sponge Agelas dispar, which was collected from waters around the island of Dominica. Its EtOAc extract, exhibiting mild radical scavenging properties, was mass cultivated and found to produce a new pyranone derivative, trichopyrone (1), and ten compounds, namely four sorbicillinoid polyketide derivatives, trichodermanone A-D (2 - 5), two hexaketide derivatives, rezishanone (6) and vertinolide (7), three known dodecaketides, trichodimerol (8), bislongiquinolide (trichotetronine, 9), and bisvertinol (10), as well as 2-furancarboxylic acid (11). The structures of all compounds were determined by interpretation of their spectroscopic data (1D and 2D NMR, MS, UV and IR). The biological activities of all isolates were evaluated in a series of bioassays (radical scavenging, antioxidant, antimicrobial, inhibition of HIV-1 RT). The majority had very weak or no effects in the applied test systems

A new thiophene, echinothiophenegenol (1), together with seven known thiophenes was isolated from the crude ethanol extract of roots of Echinops grijisii Hance. The structure of 1 was elucidated on the basis of spectroscopic data. Compounds 2 and 5, isolated from the plant for the first time, and compounds 1 - 7 were tested for their cytotoxicity against two human cancer cell lines, HL60 and K562. The thiophenes showed better activity than the bithiophenes

Antioxidant agents against reactive oxygen species can be used for several cosmetic and medicinal applications. This study’s objective was to evaluate the antioxidant activities of Polytrichastrum alpinum (Hedw.) G. L. Sm. (Polytrichaceae), an Antarctic moss species collected from King George Island (Antarctica). The identifi cation of the moss species was performed on the basis of morphological characteristics and molecular sequencing of the 18S rRNA gene. Two benzonaphthoxanthenones: ohioensins F and G, were isolated from the extract after several chromatographic procedures. The various in vitro antioxidant capacities of a methanolic extract of P. alpinum and the isolated compounds were evaluated by analyzing the scavenging capacities of free radicals of 2,2-azino-bis(3-ethylbenzthiazoline- 6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH), the total phenol assay with Folin-Ciocalteu reagent, the ferric ion (Fe 3+ ) reducing power and the nitric oxide (NO) scavenging activity and compared to those of commercial standards for each assay. The experimental data showed that even the crude extract of P. alpinum exhibited potent antioxidant activity. The antioxidant activity was increased two- to seven-fold for the purified compounds. The antioxidant activities of both purified compounds were found to be more or less the same in all experiments. However, the obtained data showed that the Fe3+ reducing power of the purified compounds and crude methanolic extract was almost the same suggesting the presence of other stronger reducing agents in the methanolic extract which could not be isolated in the present experiment. Therefore, further work on the isolation of these stronger antioxidant agents from this moss specimen of the extreme environment is warranted. Developments of laboratory mass culture techniques are anticipated to achieve bulk production of the active constituents for commercial application.

Six triterpene acids identified as betulinic, oleanolic, ursolic, 3-epimaslinic, alphitolic and euscaphic acids have been isolated from a dichloromethane extract of hairy root cultures of Ocimum basilicum L. (Lamiaceae). These cultures were obtained by genetic transformation using Agrobacterium rhizogenes. The extract as well as the isolated compounds were evaluated for their hepatoprotective activity by measuring their effect on the oxidative stress status of liver, induced by carbon tetrachloride, in albino rats and in liver homogenate in vitro. All tested compounds displayed hepatoprotective activity comparable to oleanolic and ursolic acids.

A phytochemical study of the stem bark of Vismia laurentii resulted in the isolation of a tetracyclic triterpene, tirucalla-7,24-dien-3-one (1), and seven other known compounds: 3-geranyloxyemodin (2), vismiaquinone A (3), vismiaquinone B (4), bivismiaquinone (5), epifriedelinol (6), betulinic acid (7) and stigmasta-7,22-dien-3-ol (8). The structure of all these compounds was elucidated by spectroscopic means. The stem bark extract and compounds 1 and 3 showed good antimalarial activity against the W2 strain of Plasmodium falciparum.

Tetrahydroquinoline (1), prepared by a traditional synthetic method, the imino Diels- Alder reaction, was biotransformed by Mortierella isabelina to afford a new compound, 2, characterized by spectroscopic methods.

The common effect of NH 4 + , NO 3 - , KH 2 PO 4 and sucrose on the biosynthesis of galanthamine by a Leucojum aestivum shoot culture was studied. Polynominal regression models were elaborated for the description of the galanthamine biosynthesis as a consequence of variation of the investigated variables (NH 4 + between 0.20 and 0.54 g/L; NO 3 - between 1.44 and 3.44 g/L; KH 2 PO 4 between 0.10 and 0.24 g/L, and sucrose between 30.00 and 60.00 g/L). Optimization procedures allowed us to establish the optimal concentrations of the investigated variables and to propose the modified MS nutrient medium, with 4.50 g/L KNO 3 , 0.89 g/L NH 4 NO 3 , 1.25 g/L (NH 4 ) 2 SO 4 , 0.10 g/L KH 2 PO 4 and 60 g/L sucrose, for the galanthamine production by a Leucojum aestivum shoot culture. The proposed modified MS medium provided considerable increase of both the production yield and the relative content of the target alkaloid in the alkaloid mixture.

Brassinosteroids (BRs) are involved in multiple plant growth and development processes, such as cell elongation, photomorphogenesis, flowering time control, and stress responses. The phytohormone abscisic acid (ABA) is crucial to plant development and adaptation to stressful environments. The receptors and pathways of BRs and ABA have been deeply studied. But the relationship between them remained largely unknown and there are only few reports about it. Our experiments showed that the BR-deficient and BR-insensitive Arabidopsis mutants det2, bri1 - 5 and bri1 - 9 were more sensitive to ABA than the wild type (Ws-2), especially the det2 and bri1 - 9 mutants. Germination, hypocotyl and root elongation, and stomatal apertures of the mutants were more severely inhibited by ABA. All the results suggest that BRs counteract ABA in regulating plant growth, and the interaction may be complicated. The possible mechanisms are discussed.

Previous studies showed that salicylic acid (SA)-deficient transgenic Arabidopsis expressing the salicylate hydroxylase gene NahG had a higher tolerance to moderate salt stress. SA may potentiate the stress response of germination and growth of Arabidopsis seedlings by inducing reactive oxygen species (ROS). However, the detailed mechanism for a better adaption of NahG plants to moderate salt stress is largely unknown. In the present study we found that a higher GSH/GSSG (glutathione/oxidized glutathione) ratio and ASA/DHA (ascorbic acid/dehydroascorbate) ratio in NahG plants during the stress may be the key reason for their stress-tolerance advantage. NahG plants actually could not produce more active antioxidant enzymes than the wild-type ones under natural conditions, but maintain higher activities of glutathione reductase (GR) and dehydroascorbate reductase (DHAR) during the stress. Hereby, the reduced glutathione and reduced ascorbic acid contents are higher in NahG plants under salt stress. However, NahG plants do not adapt better under severe salt stress. All antioxidant enzyme activities, GSH/GSSG ratio and ASA/DHA ratio declined substantively at 400 mM NaCl stress in both NahG and wild-type seedlings.

Pueraria candollei Wall. ex Benth. var. mirifica (Airy Shaw & Suvat.) Niyomdham was investigated for callus induction using Murashige and Skoog (MS) medium containing different plant growth regulators. After 8 weeks of culture, 66 - 100% of leaf or stem explants formed calli. Calli from stem explants cultured on MS medium supplemented with 0.5 mg/l thidiazuron (TDZ) gave the maximum of shoot induction (16%) and the highest level of total isoflavonoids [(50.39 ± 7.06) mg/g dry wt], which was 7-fold higher than that of the native tuber [(7.04 ± 0.29) mg/g dry wt]. These results suggest that addition of TDZ to the culture medium markedly enhances the production of isoflavonoids in calli induced from stem explants of P. candollei var. mirifica.

Fatty acids are essential components of almost all biological membranes. Additionally, they are important in energy storage, as second messengers during signal transduction, and in post-translational protein modification. De novo synthesis of fatty acids is essential for almost all organisms, and entails the iterative elongation of the growing fatty acid chain through a set of reactions conserved in all kingdoms. During our work on the biosynthesis of secondary metabolites, a 450-kDa protein was detected by SDS-PAGE of enriched fractions from mycelial lysates from the basidiomycete Omphalotus olearius. Protein sequencing of this protein band revealed the presence of peptides with homology to both α and β subunits of the ascomycete fatty acid synthase (FAS) family. The FAS encoding gene of O. olearius was sequenced. The positions of its predicted 21 introns were verified. The gene encodes a 3931 amino acids single protein, with an equivalent of the ascomycetous β subunit at the N-terminus and the α subunit at the C-terminus. This is the first report on an FAS protein from a homobasidiomycete and also the first fungal FAS which is comprised of a single polypeptide.

Essential oil isolated from lemongrass (Cymbopogon fl exuosus) mutant cv. GRL-1 leaves is mainly composed of geraniol (G) and geranyl acetate (GA). The proportion of G and GA markedly fluctuates during leaf development. The proportions of GA and G in the essential oil recorded at day 10 after leaf emergence were ~ 59% and ~ 33% respectively. However, the level of GA went down from ~ 59 to ~ 3% whereas the level of G rose from ~ 33 to ~ 91% during the leaf growth period from day 10 to day 50. However, the decline in the level of GA was most pronounced in the early (day 10 to day 30) stage of leaf growth. The trend of changes in the proportion of GA and G has clearly indicated the role of an esterase that must be involved in the conversion of GA to G during leaf development. We isolated an esterase from leaves of different ages that converts GA into G and has been given the name geranyl acetate esterase (GAE). The GAE activity markedly varied during the leaf development cycle; it was closely correlated with the monoterpene (GA and G) composition throughout leaf development. GAE appeared as several isoenzymes but only three (GAE-I, GAE-II, and GAE-III) of them had significant GA cleaving activity. The GAE isoenzymes pattern was greatly influenced by the leaf developmental stages and so their GA cleaving activities. Like the GAE activity, GAE isoenzyme patterns were also found to be consistent with the monoterpene (GA and G) composition. GAE had an optimum pH at 8.5 and temperature at 30 °C. Besides GAE, a compound with phosphatase activity capable of hydrolyzing geranyl diphosphate (GPP) to produce geraniol has also been isolated.

Microbiologically influenced corrosion (MIC) caused by sulfate-reducing bacteria (SRB) has been investigated in an oilfield injection water system. Strain CW-01 was isolated from an oilfield and strain CW-04 was isolated from biofilm dirt of pipeline walls. The strains were facultative anaerobes, non-motile, Gram-positive, pole flagellum, and spore-forming curved rods. The growth was observed over the temperature range 20-70 °C. Strain CW-01 grew optimally at 37 °C. The pH range for growth was 3.0-11, optimal at pH 6.0. Strain CW-04 grew optimally at 48 °C. The pH range for growth was 3.0-10, optimal at pH 7.2. The strains grew at a very broad range of salt concentrations. Optimal growth was observed with 1.5 g/L NaCl for strain CW-01 and 0.7 g/L NaCl for strain CW-04. The strains showed most similarity in physiological characteristics, except for acetone and saccharose. Analysis of the 16S rDNA sequences allowed strains CW-01 and CW-04 to be classified into the genus Desulfotomaculum. The corrosion speciality of the strains had been comparatively investigated. Especially SRB’s growth curve, bearable oxygen capability, drug fastness and corrosion rate had been analyzed. The results showed that it is difficult to prevent bacterial corrosion caused by these two strains.

A field test carried out in an industrial greenhouse in Lithuania revealed the attractiveness of synthetic methyl salicylate (MeSa) to two dipteran species: the vinegar fl y, Drosophila busckii (Drosophilidae), and the cluster fl y, Pollenia rudis (Calliphoridae). The attractant for the former fl y species was especially effective, as sticky traps containing 0.25 ml of MeSa captured (814 ± 55) D. busckii flies/trap on average compared to (12 ± 4) flies/trap in control traps. The mean capture of P. rudis [(42 ± 4) flies/trap] was significantly higher in MeSa-baited traps compared to the control traps [(13 ± 4) flies/trap]. The presence of MeSa in emissions of many fruits suitable for D. busckii feeding allows to attribute this attractant to kairomones. In case of P. rudis, MeSa should be attributed to synomones (compounds beneficial for both receiver and sender), because adult flies feeding on flowers act as pollinators. This is the first report on the field-active attractant for D. busckii and the second for P. rudis.

Three new pyridine alkaloids were detected in the pygidial glands of some Stenus species. The chemotaxonomic significance of the occurrence of these alkaloids and stenusine in different Stenus species is discussed. The antimicrobial properties of (Z)- and (E)-3-(2- methyl-1-butenyl)-pyridine and the deterrent activities of stenusine and norstenusine were investigated.

Total amounts and patterns of bromoisoxazoline alkaloids of Aplysina sponges from Croatia (Mediterranean Sea) were analyzed along an underwater slope ranging from 1.8 to 38.5 m. Total amounts of alkaloids varied from sample to sample and showed no correlation with depth. In contrast, striking differences of alkaloid patterns were found between sponges from shallow sites (1.8 - 11.8 m) and those collected from deeper sites (11.8 - 38.5 m). Sponges from shallow depths consistently exhibited alkaloid patterns typical for Aplysina aerophoba with aerophobin-2 (2) and isofistularin-3 (3) as main constituents. Sponges from deeper sites (below 11.8 m) resembled Aplysina cavernicola with aerothionin (4) and aplysinamisin- 1 (1) as major compounds. The typical A. cavernicola pigment 3,4-dihydroxyquinoline- 2-carboxylic acid (6), however, could not be detected in A. aerophoba sponges but was replaced by the A. aerophoba pigment uranidine (5) which appeared to be present in all sponge samples analyzed. During transplantation experiments sponges from sites below 30 m featuring the A. cavernicola chemotype of bromoisoxazoline alkaloids were translocated to shallower habitats (10 m). The alkaloid patterns in transplanted sponges were found to be stable over a period of 12 months and unaffected by this change in depth. In a further experiment, clones of Aplysina sponges from shallow depths of 5 - 6 m resembling the A. aerophoba chemotype were either kept in situ under natural light conditions or artificially shaded by excluding photosynthetically active radiation (PAR). Neither 4 nor 1 were detected in artificially shaded sponges over an observation period of 12 months. In summary, two chemically distinct types of Aplysina sponges were discovered in this study that proved to be remarkably stable with regard to the bromoisoxazoline patterns and unaffected either by changing the light conditions or depth. It is not clear presently whether the Aplysina sponges collected from depths < 11.8 m represent a new chemotype of A. cavernicola lacking the pigment 6 or whether we have incidentally come across a so far undescribed species of the genus Aplysina.

The aim of the present work was to investigate the Schistosoma mansoni and Fasciola gigantica cross-reactivity between adult worms and egg homogenates of the parasites. Immunoprophylactic effects of crude Schistosoma mansoni worms and egg antigens mixed with or without saponins extracted from Atriplex nummularia were studied followed by challenge with 80 cercariae of Schistosoma mansoni. Our results showed that post 1st immunization with schistosome egg antigens (SEA) there was a significant change (P ≈ 0.05) in the IgM levels against Fasciola egg homogenate (FgEH) without saponins. Post 2 nd immunization with SEA mixed with saponins the levels of IgM increased significantly (P ≈ 0.05) against Fasciola worm homogenate (FgWH) as compared with a non-immunized group. Post 2 nd immunization the level of IgG was significantly elevated (P ≈ 0.05) by SEA mixed with saponins against FgWH. Post 2 nd immunizations with SEA mixed with saponins showed a signifi cant change (P ≈ 0.05) in IgG levels against FgEH. These results clearly demonstrated that there is a cross-reactivity between Schistosoma mansoni eggs and Fasciola gigantica worms and eggs. Saponins were found to be immunostimulatory adjuvants in our study

FK506 binding proteins (FKBPs) in cells are known as immunophilins. We have identifi ed and characterized a cDNA encoding an endoplasmic reticulum (ER) immunophilin, FKBP23, from pig liver by nested PCR. The predicted amino acid sequence of pig FKBP23 shows high identity to those of human FKBP23 and mouse FKBP23. It possesses a conserved FKBP-type peptidylprolyl cis-trans isomerase (PPIase) domain and EF-hand domain. We constructed a plasmid to express pFKBP23. Furthermore, we proved that the recombinant pFKBP23 can specifi cally bind to natural BiP, the main protein of the molecular chaperone Hsp70 in ER lumen; the binding is interrelated with the Ca 2+ concentration just as the FKBP23 from mice.

Rab5 is an important small GTPase involved in endocytosis and membrane trafficking. Rab5-binding proteins can be identified using Rab5 affinity chromatography with nonhydrolyzable GTP analogues such as GTPγS or GppNHp. However, this method requires significant quantities of the GTP analogue and is thus time-consuming and expensive. In the present report we show a faster and more cost-effective method that does not use a GTP analogue but uses constitutively the active Rab5 mutant (Rab5Q79L) as a ligand. To validate this method, the binding of EEA-1 was confirmed and several novel Rab5-binding proteins were also identified by 2-dimensional electrophoresis and liquid chromatographymass spectrometry/mass spectrometry (LC-MS/MS).

7-Ketocholesterol (7-Kchol, oxidized cholesterol) is an important mediator of cell death in atherosclerosis mediated by up-regulated Nox 4 gene expression. In the current study using the human colon cancer HT-29 cell line, we have demonstrated that 7-Kchol promotes endoplasmic reticulum (ER) stress via gene up-regulation of ER chaperone and membrane kinases.