Volume 50, Issue 11-12

Two polybrominated C 15 -acetogenins (1,2) isolated from a Mediterranean sponge previously and a new polybrominated bicyclic ether with a bromoallenic side chain (3) were isolated from Laurencia paniculata and Laurencia obtusa respectively. The structures of these compounds were elucidated by spectroscopic methods.

The fungus Lentinus crinitus DR-5 (Basidiomycotina) produces in submerse culture the new natural products 2,2-dimethyl-6-methoxy-4-chromanone 1, its alcohol 3 and (S)-2,2-di-methyl-3-hydroxy-6-methoxy-4-chromanone 2. Beside these merohemiterpenes the antibioti-cally active hirsutane sesquiterpenes 5 -8 were isolated. The formation of these metabolites displayed strong dependence from the carbon source in the medium. While lactose favoured the formation of the antibiotic sesquiterpene ketones 5 and 6, they were suppressed with mannose where the hemiterpenes 1 and 4 and the hirsutane-diol 8 were the dominating metabolites.

Four novel anthraquinone derivatives, named Dendryol A, B, C, and D, were isolated from the culture filtrate of Dendryphiella sp. Their structures were elucidated from spectral data and single-crystal X-ray diffraction analysis. The four compounds showed phytotoxic activity against barnyardgrass at more than 1 μl application of 5000 μg/ml.

In order to elucidate chemical structure-odor correlation in the all isomers of n-nonen-1- ols, an entire series of these alcohols were synthesized stereo-selectively in high purity. For unequivocal syntheses of them, geometrically selective hydrogenation of the respective acetylenic compound was adopted. The synthesized alcohols were converted to their 3,5-dinitrobenzoate derivatives with 3,5-dinitrobenzoyl chloride, and then purified by repeated recrystallization. Chemical structure-odor correlations in all the isomers of n-nonen-1-ols were elucidated by introducing a novel method to evaluate odor characteristics and by treating the obtained data statistically with the principal component analysis method (Cramer et al., 1988). The odor profiles of the tested compounds were attributable largely to the positions of the carbon- double bond. The geometries of compounds had only a little effect. With the principal component analysis, the odor profiles of the series of compounds were successfully integrated into the first and the second principal components. The first component (PC-1) consisted of combined characteristics of fruity, fresh, sweet, herbal and oily-fatty, in which herbal and oily-fatty were conversely correlated each other to the position of double-bond of the tested compounds. Of these, only (6Z)-nonen-1-ol deviated markedly from the correlation, indicative of some special interaction between the spatial structure of this compound and the sensory machinery of human.

Contrary to normal fruit, plums infected with Taphrina pruni show various disease symptoms. A comparative study of chemical constituents has proved, by means of GC and GC/MS. that only infected plums contain 4-penta-decylpyridine. Its structure was confirmed by synthesis: a phosphinenickel complex catalyzed the reaction between 4-bromopyridine and penta-decylmagnesium bromide. Extraction and chromatography of the cultivated fungus have demonstrated that the com­pound is a fungal metabolite.

Anaerobic sulphide oxidation in the light was studied with seven cyanobacterial strains isolated from the southern Baltic Sea. All strains oxidized sulphide (1.2-3.5 mM) stoichio-metrically to thiosulphate. No elemental sulphur could be detected as end product of anoxy-genic photosynthesis. Sulphite as intermediate sulphur compound was released into the growth medium during anaerobic sulphide oxidation by these cyanobacteria. The formation of sulphite from sulphide was inhibited by the photosynthesis inhibitor DCMU. The cyanobacterium Oscillatoria sp. strain BO 32 produced another so far not identified intermediate sulphur compound which was probably stored within the cells.

The NADPH-protochlorophyllide oxidoreductase, an enzyme catalysing the light-driven conversion of protochlorophyllide to chlorophyllide, was studied in the greening mutant C- 2A′ of the unicellular green alga Scenedesmus obliquus. Studies of the enzyme activity in vitro showed strong dependence on the presence of glycerol and the detergent Triton X-100. Prerequisite for the formation of a photoactive enzyme complex is a sufficient preincubation time with the substrates PChlide and NADPH. A continuous assay system, reading the absorbance increase at the wavelength of chlorophyllide, was used to determine the kinetic constants. The K m value for NADPH is 4.2 μᴍ, the V max is 5.9 pmol · s -1 . The K m and V max for protochlorophyllide are 0.19 μᴍ and 6.5 pmol · s -1 , respectively. The pH-dependence of the reaction exhibits a broad maximum between pH 7-8.5 typically for an enzyme active during chloroplast development, when pH-changes might be expected. The obtained kinetic data outline that the light dependent formation of chlorophyll in vivo is not limited by the substrates PChlide and NADPH, indicating that only light is the triggering factor in the very early greening process.

Wild type tobacco N. tabacum var. John William’s Broadleaf and the tobacco aurea mutant Su/su were permanently grown under 700 ppm CO 2 in air. In comparison to plants grown under 350 ppm CO 2 in air but under otherwise identical conditions growth was substantially enhanced. Gas exchange measurements carried out by mass spectrometry show that the rate of photosynthesis in the wild type and in the mutant is increased by more than 100%. The photorespiratory rate in the wild type measured as 18 O 2 -uptake in the light in the “700 ppm CO 2 -plants” is not reduced to the extent expected or deduced from experiments in which the 350 ppm system responds under in vitro conditions to 700 ppm CO 2 . An analysis of the induction kinetics of room temperature fluorescence kinetics of the adapted (700 ppm CO 2 ) system and the control system (350 ppm CO 2 ) under various CO 2 -partial pressures shows that permanent growth under the elevated CO 2 -partial pressure leads to a structural modification of the photosynthetic apparatus.

, 1995 C5-Pathway, Glutamyl-tRNAglu-Synthetase (E.C.6.1.1.17), Misacylation of tR N A glu, Amidotransferase, Scenedesmus obliquus In a previous paper we described the purification of a glutamyl-tRNA synthetase from the unicellular green alga Scenedesmus obliquus, m utant C-2A'. We now dem onstrate that, firstly, this enzyme is capable of mischarging plastidal tR N A gln from barley with glutamate, as well as it regularly charges the plastidal tR N A glu from Scenedesmus. Secondly, we show that the mischarged glutamyl-tRNAgln is subsequently am idated by a glutamyl-tRNA am idotransfer­ ase to form the glutaminyl-tRNAglri required for plastidal protein biosynthesis. This phenom ­ enon could already be dem onstrated for higher plant chloroplasts, mitochondria, cyanobact­ eria and gram-positive bacteria, as far as investigated. As recently shown the applied glutamyl-tRNA synthetase from Scenedesmus is a plastidal enzyme. In this paper we prove by treatm ent with m onobromobim ane and cyanogen bromide that the "regular" substrate of the enzyme, tR N A glu from Scenedesmus, is a plastidal tRNA with the plastid-specific sulfur modification in the anticodon. In the case of cyanogen bromide treatm ent, a total inactivation of the tRNA was achieved, revealing the presence of a sulfur modification in the plastid-tRNA glu anticodon.

A purification scheme for the enzyme phosphinothricin-N-acetyltransferase (PAT) originating from Streptomyces viridochromogenes {pat-gene product from Streptomyces virido­chromogenes) and mediating herbicide resistance to transgenic plants was developed. The enzyme was isolated from a transformed and overproducing Escherichia coli strain. With a combination of ammonium sulfate fractionation, chromatography on DEAE-Sephadex A50-, Phenylsepharose-, Hydroxylapatite-and FPLC-Superose 12-columns it was possible to obtain PAT which was at least 90 % homogeneous on the basis of SDS-PAGE. The properties of the isolated PAT were compared with the properties of PAT from S. hygroscopicus (bar-gene product from S. hygroscopicus) previously isolated and characterisized by Botterman, J., Gossele, V., Thoen, C., Lauwereys, M. (1991), Gene 102, 33-37. Differences were observed in the molecular masses of the two native enzymes (PAT from S. viridochrogenes being a dimer of 40 kD and PAT from S. hygroscopicus being a monomer of 21 kD), and in the temperature sensitivity of the two enzymes (the PAT from S. viridochromogenes being slightly more temperature stable than PAT from S. hygroscopicus). However, since the pat and the bar-gene are to 85 % homologous, substantial similarities exist between the two enzymes especially in the kinetic values and the substrate specificity. The isolated S. virido­chromogenes PAT did not acetylate putative substrates present in the plant cell. Antibodies were raised against the isolated protein. This antiserum was able to detect PAT in transgenic plants and therefore is suitable to analyse the fate of the protein in such plants under various stress conditions.

In submerged cultures, when grown in the presence of pyrene. the saprophytic and plant inhabiting basidiomycete Crinipellis stipitaria was found to produce 6,8-dihydroxy-3-methyl-isocoumarin. The amount of 6,8-dihydroxy-3-methylisocoumarin formed depended on the concentration of pyrene in the medium. In cultures incubated without pyrene or with less than 2.5 mg/l no formation of 6,8-dihydroxy-3-methylisocoumarin was observed. Besides pyrene, chrysene, another four ring polycyclic aromatic hydrocarbon, induced isocoumarin formation whereas anthracene, phenanthrene or fluoranthene were not effective. During metabolization experiments with [4,5,9,10- 14 C]pyrene (0.4 mCi (14.8 kBq)/mmol), labelled 6.8-dihydroxy-3-methylisocoumarin (specific activity 15 μCi (0.6 kBq)/mmol) was isolated, in addition labelled acetic acid (specific activity 0.1 μCi (4 Bq)/mmol) was identified. When C. stipitaria JK 364 was incubated for 14 days with 5 μCi (0.2 kBq) sodium [2- 14 C]acetate and 10 mg/l pyrene. labelled 6.8-dihydroxy-3-methylisocoumarin was isolated from the culture. The results are in agreement with the polyketide origin of the isocoumarin and proof the breakdown of pyrene to acetate which is a central intermediate in the pathway from pyrene to 6,8-dihydroxy-3-methylisocoumarin.

In a screening for inhibitors of fungal melanin biosynthesis a novel test system was success­ fully employed. With this test system, based on the production of dihydroxynaphthalene melanin (DHN melanin) by a Lachnellula species in agar cultures, extracts of cultures of basidiomycetes, ascomycetes and deuteromycetes were tested. From fermentations of Col-lybia dryophila, 6-methylpurine (1) and 6-methyl-9-β-ᴅ-ribofuranosylpurine (2) were isolated as the active principles and 3,5-dichloro-4-methoxybenzoic acid (3) was obtained from cultures of Stropharia squamosa. The corresponding alcohol (4) and aldehyde (5) previously isolated from several basidiomycetes, were also active in this test system. In a screening of fungal metabolites, 1-methoxy-8-hydroxynaphthalene and 1,8-dimethoxynaphthalene inhibited biosynthesis of DHN melanin. Among the inhibitors identified, compounds 3 -5 were the most selective ones. They were the only metabolites without cytotoxic activities.

The interaction of an organophosphorus insecticide methylparathion (O.O-dimethyl 0-4-nitrophenyl phosphorothioate) with double-stranded DNA was characterized by UV and circular dichroism (CD) spectroscopy. Two kinds of DNA were employed: calf thymus DNA (CT DNA) and a synthetic two-stranded oligomer of sequence 5′-d(TTGGATCCGAATT-CAAGCTT)-3′ Melting curves and CD spectra were taken for the DNAs in the presence of the insecticide at methylparathion/DNA base pair molar ratio of 0.5. The insecticide evoked a decrease of the melting temperature and a broadening of the transition range for CT DNA. Similar effects were observed for the synthetic oligomer but they were less pronounced than in the case of CT DNA. Methylparathion evoked a slight shift and an increase in the amplitude of the negative band in the CD spectra of both DNAs. Obtained results indicate that methylparathion may perturb the thermal stability and conformation of DNA, which is an evidence that the insecticide has an ability to interact directly with DNA.

Hydrogenperoxide exogenously administerd to the suspension-cultured cells of Pueraria lobata stimulated a rapid disappearance of the constitutive isoflavonoids (malonylglucosides of daidzein and genistein) from the methanol soluble fraction of the cells, in a similar manner as observed in the cells treated with yeast extract or glycoprotein preparation of Phyto­phthora megasperma f. sp. glycinea. Rapid generation of H 2 O 2 was detected within 10 min after elicitation at the cell surface, presumably preceded by the activation of plasma membrane NAD(P)H-oxidases. Pretreatment of some representative NAD(P)H-oxidase inhibitors such as SHAM, DEDTC and KCN (1 mᴍ each) to the cells 1 h before elicitation resulted in almost complete inhibition of the elicitor-stimulated metabolism of isoflavonoids for 4 h, whereas treatment of the inhibitors at 1 h after elicitation showed no significant effect on the rapid isoflavonoid metabolism. These findings suggest that this type of response to elicitation could be regarded as a plausible initial defense strategy prior to the appearance of de novo synthesized defensive barriers such as phytoalexins and lignin in plant-pathogen interaction.

Black lipid membranes were prepared on a Teflon septum separating electrically the two chambers of a Teflon cuvette, using the technique of Mueller et al., (Nature 194. 979 (1962)). An external, static magnetic field was applied, whose intensity varied from 0 G to 100 G at the membrane location. Field applications higher than 10 G are effecting higher leakage currents, increased capacity and faster breakdown of the bilayer state, as compared to the absence of a magnetic field. If bilayers were doped with chlorophyll a, these effects were increased. Quantum mechanical and thermodynamical phenomena on membranes will be discussed as possible origins of these effects.

The inhibition effect of selected amphiphilic quaternary alkyl-substituted ammonium salts on oxidation of the lecithin liposome membrane was studied, induced by ultraviolet light and Fe 2+ ions with ascorbic acid added. The salts differed in alkyl chain length Ri having the following alkyl substitutents: R1 = C 9 H 19 ; R2 = C 10 H 21 ; R3 = C 12 H 25 ; R4 = CH 2 OC 14 H 29 ; R5 = CH 2 OC 16 H 33 . It was found that all the salts used induced inhibition in oxidation of the liposome membrane, both that induced by UV light and ferrous ions. The antioxidant activity of the salts studied, dependent on the chain length and concentration, can be represented by the relation R1 < R2< R3 < R4 < R5. A similar dependence was obtained when the hemolytic activity of compounds was studied. Activity of salts increase with alkyl chain length.

In this report it is shown that glycerol (0.5-10% v/v) stimulate the C 12 -E 9 -solubilized renal (Ca 2+ +Mg 2+ )ATPase in the presence of low concentrations of free Ca 2+ (< 10 -6 ᴍ). At 4% (v/v), the polyol decreases the K 0.5 for Ca 2+ from 1.15 to 0.22 μᴍ at the high-affinity site, and a very-high-affinity Ca 2+ component appears. This component has a K 0.5 ≤10 -9 ᴍ and its maximal velocity is about one-third that of the fully activated enzyme (at 10-20 μᴍ Ca 2+ ), which is not affected by glycerol (21.1 and 20.2 nmol·mg -1 ·min -1 in the absence and presence of the polyol, respectively). The low-affinity, inhibitory component of the Ca 2+ curve (50-1000 μᴍ ) is also unaffected by glycerol. With 0.07 μᴍ free Ca 2+ and soluble enzyme, the stimulatory effect of glycerol saturates at ≈10% (v/v). In contrast, with 17 μᴍ free Ca 2+ , glycerol has little effect up to 10% (v/v), and then progressively inhibits ATPase activity. These data indicate that the effect of the polyol is modulated by the occupancy of the high-affinity Ca 2+ sites. In native vesicles, the stimulation promoted by low concentrations of glycerol at low concentrations of Ca 2+ is accompanied by inhibition of active Ca 2+ transport, indicating that, in these conditions, the polyol uncouples ATPase activity and ATP-driven Ca 2+ influx.

The aerial parts of Bidens aurea (Aiton) Sherff have been used in folk medicine as an antiulcer agent. The efficacy of an ether extract to prevent gastric mucosal damage produced by several necrotic agents was studied in rats. The best efficiency was against lesions induced by 30% NaCl with a significant decrease of damage at all doses tested (250, 125 and 62.5 mg kg -1 ). In contrast, only the highest dose (250 mg kg -1 ) was effective on gastric erosions induced by 0.6 ɴ HCl. This effect could not be related with the mucus gel production only since the groups treated with of absolute ethanol or 30% NaCl before and then received 250 mg kg -1 of the ether extract increased significantly the amount of gastric mucus and its glycoprotein content. However, the ether extract of Bidens aurea enhanced the production of mucosal non-protein SH groups at all doses assayed after treatment with absolute ethanol and 0.6 ɴ HCl. With NaCl-induced gastric injury, also the highest dose (250 mg kg -1 ) produced an increase of this parameter. The results suggest that a slight augmentation of the mucus gel concentration and the increase of non-protein sulfhydryl compounds, could con­ tribute to the functional protection mechanism mediated by a flavonoid fraction of B.aurea in these experimental models.

The performance of enzyme immunoassay (EIA) and radioimmunoassay (RIA) in the quantitative analysis of ecdysteroids was compared. The EIA was found to be at least equivalent to the RIA with respect to analytical range and sensitivity and to be more comfortable with respect to safety and time saving. When biological samples were analyzed by both assays a good correlation (r = 0.83) was found. Since the EIA has certain advantages over the RIA, we now recommend the use of the former assay for the quantification of ecdysteroids.

The molecular phylogeny of 11 Old World and 5 New World vultures was inferred from nucleotide sequences of the mitochondrial cytochrome b (cyt b) gene. According to this analysis carrion-feeding has evolved independently at least three times during evolution: 1.) In the New World vultures, which are clearly separated from vultures of the family Accipitridae; 2.) in the Neophron-Gypaetus clade which is positioned at the base of the Accipitrid tree and 3.) in the Gyps-Aegypius-complex which encloses the largest group of Old World vultures. Thus the genetic data clearly show that the carrion-feeding lifestyles and associated morphologies shared by New and Old World vultures are rather based on convergence than on close genetic relatedness. Employing the cyt b sequences of 12 other members of the Falconiformes and 10 members of the Ciconiiformes (sensu Sibley and Monroe, 1990) the phylogenetic relationship between the three clades of vultures and these other taxa was assessed. New World Vultures appear to share distant ancestry with storks but a close relationship is unlikely.

There is no doubt that evolution can only operate in accordance with the laws of thermodynamics. To transform this simple perception into a statement that can be quantified it is necessary to examine how the entropy (or the in formation content) changes by means of the two main factors of evolution: selection and mutation . In the present paper this aim was reached by using the methods of information theory. That evolution becomes possible the change of information by mutation and selection must not be negativ in the long run. Consequently an upper limit for the information content of the genom must be assumed. If the evolution as a self-optimizing process gains this upper limit it reaches a dynamic equilibrium at which the increase of entropy by mutation balances the decrease of entropy by selection. In particular it was examined under which circumstances of dynamic equilibrium mastergenotypes (genotypes that can preserve their specific sequence over a long time) can exist.

From Bartramia mossmanniana the following five biflavonoids were isolated: bartramiaflavone, anhydrobartramiaflavone, philonotisflavone, 2,3-dihydrophilonotisflavone and 5′,3‴-dihydroxyamentoflavone.

Ten flavonoids and chlorogenic acid were isolated from the leaves and stems of Cryptocarya alba (Lauraceae). The flavonoids were identified as isorhamnetin. kaempferol, quercetin and their glycosides isorhamnetin-3-O-rhamnoside, isorhamnetin-3-O-galactoside, isorhamnetin-3-O-glucoside, kaempferol-3-O-galactoside, quercetin-3-O-rhamnoside (quercitrin), quercetin-3-O- galactoside (hyperoside), and quercetin-3-O-glucoside (isoquercitrin).