Band 55, Heft 11-12

From Pseudomonas sp. CFML 95-275 a pyoverdin was isolated with a cyclopeptidic substructure. It could be shown that this pyoverdin is identical with one obtained from Pseudomonas fluorescens BTP 7 for which a lactone structure had been deduced from the interpretation of a FAB spectrum. The elucidation of the correct structure of the pyoverdin is described.

From Pilotrichella flexilis various flavonoids have been isolated. This species contains in contrast to P. cuspidata not only 3′,3‴-binaringenin and related compounds, but also some hitherto unknown biflavonoids based on eriodictyol and aureusidin. The structures of two new compounds are elucidated spectroscopically. Possible causes for the differences of the flavonoid patterns between the two species of the genus Pilotrichella are discussed.

From gametophytes of Rhizogonium distichum have been isolated the biflavone dicranolomin and five triluteolins. Two of the triluteolins, which were named rhizogoniumtriluteolin and distichumtriluteolin, were new compounds; their structures have been elucidated spectroscopically.

A new quinolone alkaloid, megistolactone (1) was isolated from the bark of Sarcomelicope megistophylla. Its structure has been elucidated on the basis of MS and NMR data. From a biogenetic point of view, this compound should be considered as an oxidation product of 1,2,3,4-tetra-O-subsituted acridone alkaloids, which are also present in the bark.

In the present study we report the isolation of a phenylpropanoid glucoside, plantamajoside, together with rosmarinic acid, chlorogenic acid and luteolin-7-O-monoglucoside. This is the first report of these compounds from Plantago lagopus.

These studies were undertaken to characterise resorcinolic lipids (5-n-alk(en)ylresorcinols) composition and to determine their seasonal fluctuations in fruit pulp and leaves of Ginkgo biloba L. Resorcinolic lipid concentrations were consistently higher in fruit pulp than in leaves. In pulp, several mono- and di-unsaturated homologs of alkylresorcinols were the predominant group of analysed lipids. Contrary to the fruit pulp, only 5-n-pentadecylresorcinol was demonstrated in leaves. Initially, the alkylresorcinol’s content both in pulp and leaves increased until June - July and decreased following seeds ripening. This trend continued until senescence of leaves in late September and October.

Essential oils from Ferulago asparagifolia Boiss., F. galbanifera (Miller) W. Koch, F. humilis Boiss. (Endemic), F. trachycarpa Boiss. growing in Turkey were evaluated against 15 microorganisms for their antifungal and antibacterial activity using an agar tube dilution and microdilution broth susceptibility assay, respectively. The essential oil compositions were investigated by GC/MS. Inhibitory effects against Escherichia coli, Enterobacter aerogenes, Candida albicans, Gaeumannomyces graminis var. tritici, Sclerotium rolfsii and Fusarium moniliforme were remarkable. Results are discussed in comparison with the chemical composition of the essential oils.

Four bacteria, identified as Pseudomonas aeruginosa, Alcaligenes eutrophus, Bacillus subtilis and Micrococcus'luteus were isolated from crude oil polluted soils using anthracene as the sole carbon and energy source. All the organisms utilized n-hexadecane, n-tetradecane, diesel oil, engine oil and naphthalene as sole carbon sources. None could utilize hexane, cycloheptane, xylene, benzene, toluene, phenol, fluoranthene,and kerosene as carbon sources. Highest cell density obtained with 0.1% (w/v) anthracene were 4.5 × 10 7 (cfu/ml), 8.6 × 10 6 (cfu/ml), 5.4 × 10 6 and 2.4 × 10 6 (cfu/ml) respectively, for P. aeruginosa, A. eutrophus, B. subtilis and M. luteus after 30 days incubation. Growth of the organism s on a Nigerian crude oil resulted in a residual oil concentration of 22.2% , 33.3% , 39.3% ,44% and 91.7% respectively, for P. aeruginosa, A. eutrophus, B. subtilis, M. luteus and the noninoculated control on the 14 th day. Ring fission enzymes of the meta pathway were detected in induced cells of P. aeruginosa and A. eutrophus while ortho pathway enzymes were detected in B. subtilis and M. luteus. P. aeruginosa and A. eutrophus had specific catechol-2,3-dioxygenase activities of 3.8 ± 0.183 and 0.64 ± 0.032 μmol / min × mg protein respectively while catechol-1,2-dioxygenase activities of 1.95 ± 0.029 and 1.89 ± 0.026 μmol / min x mg protein were detected in B. subtilis and M. luteus respectively. This work , highlights the capability of these unreported tropical strains of A. eutrophus, B. subtilis and M. luteus as anthracene degraders.

Flavonoids are secondary plant metabolites whose anticancer properties are actually being studied from an epidemiological and pharmacological point of view. They are believed to be implicated in the lower risk of some forms of cancer observed in Asian countries, due to their capacity to control cell proliferation, to act on certain regulatory enzymes as protein kinases or topoisom erases. Based on these precedents, three flavonols isolated from a cytotoxic butanol extract from Retama sphaerocarpa Boissier have been assessed to study their topoisom erase I and II activity. Two new rhamnazin glycosides were found to have the ability to stabilize the cleavage com plex hum an D N A topoisom erase I at concentrations in the 100- 250 μm range, acting as topoisom ersase I poisons.

A crude enzyme solution prepared from fronds of Porphyra sp. showed rem arkable oxygen uptake activity when linoleic acid was added as a substrate. Fatty acid oxidizing activity was mainly present in the soluble fraction of the crude homogenate. The activity was purified 769- fold from mature fronds by ammonium sulfate fractionation, ion-exchange and hydrophobic chromatography. SDS-PAGE analysis of the purified proteins indicated that its subunit size was about 13 kDa. Gel filtration chromatography equipped with a photodiode array detector revealed that the activity was associated with a protein having a molecular weight of 12,500- 13,000. It eluted with a chromophore having the maximum absorbance at 417 nm, thus, the a protein was suggested to be a heme protein. The spectrophotometric property of the protein was highly similar to that of cytochrome c suggesting that it has heme c as a prosthetic group. The protein showed highest oxygenation activity against linoleic acid, and α-linolenic acid and arachidonic acid followed, but oleic acid could not be oxidized. From linoleic acid the protein form ed 9- and 13-hydroperoxides to the same extent, and both were shown to be racemic. These results showed that the oxidizing activity is accountable to a cytochrome, but not to a typical lipoxygenase.

From the cultures of the spore-derived mycobionts of the lichen Pyrenula japonica, two new xanthones, 1,8-dihydroxy-3-hydroxym ethyl-5-m ethoxyxanthone and 1,2,8-trihydroxy-5-methoxy-3-methylxanthone, were isolated along with 1,7-dihydroxy-3-methylxanthone, 1,5,8- trihydroxy-3-methylxanthone, 1,8-dihydroxy-5-methoxy-3-methylxanthone, emodin and sclerotiorin. Their structures were determined by spectroscopic methods. Sclerotiorin was isolated for the first time from lichen mycobionts. Radical scavenging activities of the isolated xanthones were also studied.

A highly sensitive fluorometric assay for the determination of monooxygenase activity in liver microsomes is described. The assay is based on the use of 3-chloro-7-methoxy-4-methylcoumarin which is demethylated to 3-chloro-7-hydroxy-4-methylcoumarin. The rate of formation of 3-chloro-7-hydroxy-4-methylcoumarin was recorded as an increase of fluorescence (λ A = 380 nm, λ F = 480 nm ) with time. When 3-chloro-7-methoxy-4-methylcoumarin was incubated in the presence of MgCl 2 and NADPH with rat liver microsomes, a continuous increase of the fluorescence could be measured. The reaction proceeded linearly for about 10 min and at least up to a concentration of 0.1 mg/ml of microsomal protein. Besides 3-chloro-7-hydroxy-4-methylcoumarin a hydroxylated derivative of the substrate was formed as a second metabolite during the incubation. Using an excitation wavelength of 480 nm and a fluorescence/em ission w avelength of 480 nm, the fluorescence of this substance (λ A = 338 nm, λ F = 422 nm ) amounted only to about 1% of the fluorescence of the main product. The use of 3-chloro-7-methoxy-4-methylcoumarin as substrate enables the fluorometric determination of the O-dealkylation activity of a cytochrom e P450-dependent monooxygenase system in rat liver which is inducible by phenobarbital but not by 3-methylcholanthrene.

Autumnal tints are one of the most manifest and fascinating natural phenomena, but the mechanism of chlorophyll (Chl)-breakdown in deciduous trees has not been fully elucidated. In this study, we analyzed the composition of Chl-related compounds and determined the activities of initial Chl-degrading enzymes in Ginkgo leaves at various stages in the process of autumnal coloring. Only pheophytin a (Pheo a, Mg-free Chl a) was detected in yellow leaves by HPLC analysis, and the activity of Mg-dechelatase in yellow leaves was found to be higher than in green leaves. These findings showed that the removal of magnesium from Chl a occurred in advance of dephytylation in the Ginkgo.

ʟ Ultrastructural and imm unocytochemical investigations gave evidence that cyanophycin (multi--arginyl-poly-ʟ-aspartate) granules accumulate in the cyanobacterium Synechocystis sp. strain PCC 6803 under nutrient deficient growth conditions, especially under phosphate limitation. Besides nutrient deficiency, growth of Synechocystis PCC 6803 on ʟ-arginine or ʟ-asparagine as sole N-source also led to high increase of cyanophycin synthesis, while growth on the combination of ʟ-arginine or ʟ-asparagine with nitrate only caused minor cyanophycin accum ulation. Growth of Synechocystis PCC 6803 on ʟ-arginine as sole N-source caused substantial morphological and physiological changes, such as severe thylakoid membrane degradation with partial loss of pigments and photosynthetic activity leading to a phenotype almost like that seen under nutrient deficiency. In contrast to the wild type, the PsbO-free Synechocystis PCC 6803 mutant could grow on ʟ-arginine as sole N-source with only minor morphological and physiological changes. Due to its fairly balanced growth, the mutant accumulated only few cyanophycin granules. ʟ-arginine degrading activity (measured as ornithine and ammonium formation) was high in the PsbO-free mutant but not in the wild type when cells were grown on ʟ-arginine as sole N-source. In both cells types the ʟ-arginine degrading activity was high (although in the PsbO-free mutant about twice as high as in wild type), when cells were grown on ʟ-arginine in combination with nitrate, and as expected very low when cells were grown on nitrate as sole N-source. Thus, net cyanophycin accumulation in Synechocystis PCC 6803 is regulated by the relative concentration of ʟ-arginine to the total nitrogen pool, and the intracellular ʟ-arginine concentration is greatly influenced by the activity of the ʟ-arginine degrading enzyme system which in part is regulated by the activity status of photosystem II. These results suggest a complex interrelation between cyanophycin synthesis, ʟ-arginine catabolism , and in addition photosynthesis in Synechocystis PCC 6803.

The light-harvesting complex II of the purple bacteria has two strong near infrared electronic absorption bands, around 800 (B800) and 850 (B850) nm, arising from the Q y transitions of bacteriochlorophyll a. It was previously reported that under some specific acid/chaotropic conditions the B850 bacteriochlorophylls of the light-harvesting complex II of Ectothiorhodospira sp. are strongly reorganised. Part of these pigments absorbs at 843 nm while another set absorbs around 858 nm. The current work should investigate whether a mix of two different complexes could generate the 843- and 858-nm bands. Acid/chaotropic conditions inducing the reorganisation of B850 were reproduced on a sample bound to an ionic-exchange column. The chromatographic pattern was found strongly homogeneous. The findings indicate that the heterogeneity of the reorganised B850 results from two forms of differently structured bacteriochlorophylls bound to the same polypeptide backbone.

After root application of [7α- 3 H]-7β-hydroxysitosterol and [3α,6β- 3 H 2 ]-6α-hydroxylathosterol these sterols could be detected in the leaves and phloem sap feeding aphids. These results imply that the phloem sap is a sterol transport system in barley plants.

A seven fold increase in the rate of respiratory O 2 uptake was observed 24 h after slicing of potato tuber disks. The maximum activity of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was 5 -7 times greater than that of ATP-dependent phosphofructokinase (PFK) in fresh or aged potato slices. Thus, PFP may participate in glycolysis which supplies respiratory substrate in potato tubers. The PFP activity of desalted extracts determined in the absence of fructose-2,6-bisphosphate (F2,6BP) increased by 4.5 fold 24h after slicing. However, maximal PFP activity determined with saturating (1 μm) F2.6BP was not changed. The K a values of PFP for F2,6BP was lowered from 33 to 7 nᴍ after 24 h of aging treatment. This increased susceptibility of the PFP activity to its allosteric activator, F2,6BP, may be involved in the increased respiration in wounded disks of potato tubers. Imunoblotting experiments indicated that both the α (66 kDa) and the β (60kDa) subunits of PFP were present in fresh or 24h aged tuber slices.

The primary reactions leading to Al toxicity in plant cells have not yet been elucidated. We used soybean ( Glycine max [L.] Merr.) cell suspension cultures to address the question whether lipid peroxidation plays an important role in Al toxicity. Upon transfer to an Alcontaining culture medium with a calculated Al 3+ activity of 15 μᴍ soybean cells showed a distinct and longtime increase in lipid peroxidation within 4h. At the same time a drastic loss of cell viability was observed. Butylated hydroxyanisole (BHA) and N,N′-diphenyl-pphenylenediamine (DPPD), two lipophilic antioxidants, were able to almost completely suppress lipid peroxidation in Al-treated cells at a concentration of 20 μᴍ. This effect was dosedependent for DPPD and was observed at minimum concentrations of 1 -2 μᴍ. When lipid peroxidation was suppressed by DPPD or BHA cell viability remained high even in the presence of toxic Al concentrations. These results suggest that Al-induced enhancement of lipid peroxidation is a decisive factor for Al toxicity in suspension cultured soybean cells.

10-Hydroxystearic acid seems to be widely distributed in nature: Bacteria generate it by hydroxylation of oleic acid, but it was found also as constituent of plants, in cancer cell cultures and in mammalian tissue homogenates. Investigation of 10-hydroxystearic acid, obtained from mammalian tissue homogenates, revealed its identity with that of bacteria. Thus not 10-hydroxystearic acid is widely distributed in nature but its producers: bacteria. When biological material is processed in aqueous media, lipases are activated, these cleave membrane phospholipids. Thus liberated oleic acid is the substrate for widespread bacteria which are introduced into the media when the work up procedure is done in not sterile surrounding. The bacteria transform then oleic acid to 10R-hydroxystearic acid.

Analyses of red pepper extracts which had been pretreated with lipase type VII (EC 3.1.1.3.) from Candida rugosa showed for the first time pepper carotenoid esters to be substrates of this enzyme. However, the extent of enzymatic hydrolysis depends on the respective carotenoid and was not quantitative compared to chemical saponification. After enzymatic cleavage, 67-89% of total capsanthin, 61-65% of total zeaxanthin, 70-81% of total β-cryptoxanthin and 70-86% of total violaxanthin were detected in free form. Nevertheless, the method described here offers the possibility to cleave in part several carotenoid esters originating from red pepper quickly and under comparatively mild reaction conditions. Replacement of the generally performed alkaline hydrolysis by enzymatic cleavage allows the resulting product to be used in food industry as “natural” coloring agent e.g. to colour cheese and jellies.

The antioxidative activity of two series of amphiphilic compounds from a group of quaternary ammonium salts has been investigated. They were so-called bifunctional surfactants synthesized to be used as common pesticides or as antioxidants. The latter application was to be ensured by providing the compounds studied with an antioxidant group. Studies on antioxidative possibilities of those compounds were performed on pig erythrocytes. Due to their hydrophobic parts, they anchor in the erythrocyte membrane and influence the degree of lipid oxidation in the erythrocyte membrane subjected to UV radiation. It was found that compounds of both series decreased the oxidation of the membrane lipids. The inhibition of this oxidation increased with the length of their hydrophobic chains up to fourteen carbon atoms. The compounds of the longest hydrophobic chains showed a somewhat weaker antioxidative activity. Of the two series studied compounds were more effective having bromide ions as counterions. The corresponding compounds of a second series (chlorides) protected erythrocyte significantly weaker against oxidation. The effect of the compounds on fluidity of the erythrocyte membrane has been studied in order to explain the oxidation results. Change in fluidity of the erythrocyte ghost membranes was found also dependent on length of the hydrophobic part of the compounds and was more pronounced in the case of bromide surfactants. The final conclusion is that the compounds studied can be succesfully used as antioxidant agents of good efficacy.

Transformation of 12,13-epoxy-11-hydroxy-9-octadecenoic acid and 4,5-epoxy-N-acetylsphingosine by addition of porcine liver homogenate and human liver microsomes, respectively was investigated. Both epoxides were converted to corresponding dioles by porcine liver homogenate, but not by human liver microsomes, suggesting location of the hydrolyzing enzymes not in the microsomes, but within the cell wall.

Six isolates of Bacillus thuringiensis isolated from soil samples confirmed to be toxic to mosquito larvae were differentiated using a PCR-Based technique. Three of these isolates initially identified using a serological technique were further differentiated with the PCR amplification of the δ-endotoxin target sequences. Using the total DNA of isolates as tem plate, at least four isolates yielded amplicons one or all the crystal protein genes, cryl a, b, c, or II with sizes ranging from 238-1070 bp. None of these isolates yielded an amplicon for any of Cry IV A, B and D tested. Of the four isolates identified by PCR technique one isolate remained unidentified by serology.

Immunoscreening of a C. paradoxa expression library against water soluble muroplast (“cyanelle”) proteins resulted in isolation of a clone encoding the nucleus-encoded muroplast class-II fructose-1,6-bisphosphate aldolase (class-II FBA § ). Its nucleotide sequence was determined. The 1432 bp insert, derived from a single-copy gene transcript, bears a reading frame of 1206 bp in length, representing 402 amino acids with 346 amino acids of mature protein. The leading amino acids match structural features necessary for precursor import across chloroplast envelope membranes. In phylogenetic tree topology, the investigated mature FBA clusters within type B FBAs with Synechocystis sp. as nearest neighbor. This is the first report of a Type B class-II FBA sequence of plastids.

In the membrane of mouse macrophages two gangliosides were detected which inhibit the division of murine mastocytoma P815 tumor cells. The two gangliosides were incorporated into the cytoplasmatic membrane of mastocytoma cells. The concentration necessary to achieve a complete inhibition of P815 tum or cell division is about 1 [_im for both effective gangliosides. Macrophage ganglioside-induced inhibition of cell division is accompanied by morphological changes of the mastocytoma cells. While the cells are rounding, their diameter increases and serotonin and granules appear in the cytoplasm of the enlarged cells. Our findings suggest that macrophage gangliosides may differentiate mastocytoma cells into mast cells.

In a previous study we have reported that both (E,Z )-8,10-dodecadienol (E,Z ) and (Z,Z )- 8,10-dodecadienol (Z,Z ) isomers inhibit the attraction of male codling moth, Cydia pomonella L. when added to (E,E)-8,10-dodecadienol (E,E ) while the (E,E)-8,10-dodecadienol (Z,E ) isomer induces slight increase in the number of males attracted to the pheromone source. In the p resent study, we have tested the behavioral activity of the individual geometrical isomers E,Z; Z,E and Z,Z . A few number of codling moth males flew to the Z,E -isomer while the other two isomers (i.e. E,Z and Z,Z ) did not elicit any upwind orientation. Analysis of the flight behavior to the E,E- and Z,E-isomer showed significant differences in most of the flight parameters evaluated. Based on the biological observations and molecular modeling, we suggest that the behavioral activity of the Z,E-isomer is due to presence of specific receptors for this isomer on male antennae and not to its structural resemblance to the E,E-isomer. These results underline the importance of the Z,E-isomer in sex attraction of male codling moth.

Lichen, Parmotrema stuppeum (P. stuppeum ) was successively extracted with benzene and acetone. Both the extracts were fractionated on 1 % oxalic acid impregnated silica gel column to obtain four phenolic com pounds. The structures of com pounds were identified by 1 H and 13 C NMR spectra as methyl orsenillate, orsenillic acid, atranorin and lecanoric acid respectively. Antioxidant activity of benzene extract, acetone extract and isolated com pounds w ere evaluated in a β-carotene-linoleate model system. The pure com pounds showed moderate antioxidant activity. This is the first report on the isolation and characterisation of com pounds from the lichen P. stuppeum as well as on their antioxidant activity.

A novel isoquinolinone alkaloid, iseluxine (1), has been isolated from the epigeal parts of Iseia luxurians (Moric.) O’Donell (Convolvulaceae), a climber indigenous to the tropical Americas. Structural elucidation was achieved by HRM S, 1 H NMR, 13 C NMR , and HMBC spectroscopy. N- and / or O-m ethyl derivatives of 1 are already known from certain Magnoliidae families, e.g., the Fumariaceae, the Lauraceae, or the Papaveraceae. Iseluxine, the “missing link” in the biosynthesis of these methyl derivatives from dopamine, is the first isoquinolinone alkaloid characterized by a catechol substructure.

Six sesquiterpene lactones, two new (3 and 6) and four know n (1, 2, 4 and 5) w ere isolated from the leaves of the Mexican Neurolaena macrocephala (A steraceae). The furanoheliangolide 6, containing 4β,5-dihydro-9α-hydroxy- atripliciolide as basic structure, was found for the first time in nature. The chem otaxonom ic im portance of this phytochem ical work is discussed.

Citrus peels were successively extracted with hexane, chloroform and acetone using a soxhlet extractor. The hexane and chloroform extracts were fractionated into alcohol-soluble and alcohol-insoluble fractions. These fractions were tested against different gram positive and gram negative bacteria. The EtOH-soluble fraction was found to be most effective. Fractionation of EtOH -soluble fraction on silica gel column yielded three polymethoxylated flavones, namely desmethylnobiletin, nobiletin and tangeretin. Their structures were confirm ed by UV, 1 H, 13 C NMR and mass spectral studies. The findings indicated a potential of these natural compounds as bio preservatives in food applications.

Seasonal variations on the content of harpagoside in Scrophularia scorodonia L. (Scrophulariaceae) were investigated using plants collected monthly from January to December in 1995. During growth of this species the percentage of harpagoside was the highest during the maxim um development of the plant, specially in July. H arpagoside levels differed among leaves, stems and flowers of S. scorodonia. Leaves were distinguished from other plant parts by higher levels of harpagoside. Drying at ambient temperature influenced the yield of harpagoside com pared with the results of plant drying by microwave.

When adult males of the smaller brown planthopper, Laodelphax striatellus were fed on 2% crude rice (leaf and stem ) extract containing 15% sucrose there were characteristic stylet sheaths deposited on parafilm membrane. Further bioassays with the butanol-soluble fr. of the extract revealed that it is highly effective for the insects. When the butanol fr. was charged on an ODS open column and eluted in sequence with 20, 40 and 100% m ethanol in w ater, the ODS-40% m ethanol fr. was shown as the most effective one. F urther separation of the ODS-40% methanol fr. resulted in six effective components. These components acted to stimulate very high probing response on L. striatellus only when they are combined. This activity was found to be similar to those of the ODS-40% methanol fr. and the original crude rice plant extract. Two of the active components were identified as tricin 5-O-glucoside and tricin 7-O-glucoside, respectively, through spectroscopic analyses.