Volume 44, Issue 1-2

9 β-Hydroxymarasmic acid, a new sesquiterpenoid, marasmic acid and isovelleral were isolated from cultures of a basidiomycete. Comparison of the antimicrobial activity of the three natural compounds together with two n-butyl ether derivatives of marasmic acid revealed MICs against bacteria in the range of 0 .2 -20 ng/ml. The antifungal, cytotoxic and phytotoxic activities of isovelleral were similar to those exhibited by marasmic acid, whereas the marasmic acid deriva­ tives were considerably less active than the parent compound. Isovelleral was the only compound to show hemolytic activity.

Roots of three species of the genus Coreopsis (Compositae), C. tinctoria, C. lanceolata and C. grandiflora were investigated for the occurrence of phenylpropanoids. In addition to phenylpropanoids already known from other Coreopsis species some new derivatives of eugenol- and Z-coniferyl alcohol were found. In this report we present their structures together with their 1 H NMR. 13 C NMR and MS data. Two aromatic acetylenes - one of them not previously described for Coreopsis species - were identified as well.

Phytochrome from etiolated oat seedlings (Avena sativa L.) was investigated by “native” gel electrophoresis and by isoelectric focusing. At pH 8 . 8 the Pfr form migrated faster than the Pr form in electrophoresis. We assume a difference in the surface charge rather than a difference in shape for the phytochrome forms. This assumption was confirmed by isoelectric focusing which clearly showed relatively more negative charge in the Pfr form than in the Pr form. The role of the peptide region from residue 323 to 360 is discussed in this connection. It carries 9 negatively charged residues, it is exposed only in the Pfr form and it has already been described as a signal region for rapid protein degradation (PEST sequence, see Rogers et al., Science 234, 364-368, 1986). The experiments on electrofocusing revealed a microheterogeneity of phytochrome which was present in the native state as well as in the completely unfolded state. The most probable reason could be either posttranslational modification or genetic polymorphism of phytochrome in oat.

Incorporation of L-[2- 2 H]phenyl-[2- 2 H]alanine and L-phenyl-[2- 13 C, 15 N]alanine into cytochalasin D by Zygosporium masonii involved the complete loss of both the α- 2 H- and the α- 15 N-atom. Incorporation of a mixture of L-phenyl-[ 15 N]alanine and L-[U- 14 C]phenylalanine into cytochalasin D and protein amino acids (phenylalanine, leucine, isoleucine) was accompanied by a substantial loss of 15 N with respect to 14 C. These effects are attributed to rapid exchange reactions taking place while L-phenylalanine is part of the intracellular pool of amino acids. In addition, the medium- and concentration-dependent incorporation of the carbon skeleton of exogeneous D-phenylalanine into cytochalasin D is reported. In a peptone-based complex medium, D-phenylalanine is poorly incorporated. Throughout the whole concentration range (0-250 mg/l), the incorporation rates are less than 10% of those of L-phenylalanine. In a minimal medium containing NH 4 NO 3 as nitrogen source however, D-phenylalanine is preferred over the natural enantiomer by a factor of 1.28 up to 6.78, depending on the concentrations of exogeneous D - and L-phenylalanine. These effects are attributed to the medium-dependent activities of different amino acid transport systems responsible for the uptake of D - and L-phenylalanine in Z. masonii.

The duckweed Lemna gibba G 1 was used as a model to study inhibitory sites with the herbicide and glutamate analogue glufosinate (PPT). Growth and chlorophyll formation were partly inhibited by 25 n-M, completely suppressed by 250 (im PPT. Photosynthesis showed partial inhibition within few hours, dark respiration ( 0 2 consumption) increased already within one hour. In the presence of 1 mM PPT in the light, the ammonium pool of Lemna increased to 600% within few hours, later to 1000%. The overall amino acid pool exhibited a slower increase to 300%, the nitrate pool only a slight increase, while total phosphate remained almost unchanged. In the dark all these effects were less pronounced than in the light. Nitrate, nitrite and phosphate uptake were partially inhibited by PPT, especially after 19 h PPT pretreatment. Nitrate reductase activity in vitro, after PPT treatment in vivo, showed an inhibition similar to that of nitrate uptake. Ammonium was not taken up but released under the same conditions. The data are explained by a combined effect of PPT, by inhibition of glutamine synthetase leading to accumulation of ammonium from photorespiration and proteolysis, by membrane depolarization and inhibition of anion/proton cotransport, by secondary uncoupling of phosphorylation, and by secondary inhibition of nitrate reductase activity.

Participation of singlet molecular oxygen ( 1 O 2 ) in peroxidation of dopa and dopamine was studied by measurements of chemiluminescence spectra, the influence of solvents with various lifetime of O 2 ( 1 Δ g ) and O 2 ( 1 Δ g )-quenchers on quantum yield of chemiluminescence. A decrease of absorption and fluorescence intensities of 1,3-diphenylizobenzofuran (DPBF) was also studied in the presence of dopa and dopamine oxidized with alkaline H 2 O 2 as a criterion for the involvement of 1 O 2 .

The polyphosphate component in MP NMR spectra of the Gram-positive Propionibacterium acnes increased after hyperthermia treatment. The cells were exposed to temperatures in the interval from 15 °C to 45 °C. The amount of polyphosphate increased with increasing temperature. There were no temperature induced changes in the other phosphorous components seen in the spectra with exception of a decrease in ATP for higher temperatures. The increase in polyphosphates was less than that obtained from cells irradiated by near ultra-violet light.

Absract Several enzymes involved in polyamine biosynthesis namely ornithine, arginine and S-adenosylmethionine decarboxylase as well as spermidine synthase, were analyzed in partially purified wheat extracts. For all enzymes effective inhibitors were found. Among them the most interesting was l-aminooxy-3-aminopropane, which inhibited all three decarboxylases. Classical polyamine biosynthesis inhibitors like difluoromethylornithine, difluoromethylarginine. methyl glyoxal bis- (guanylhydrazone) and cyclohexylamine were also inhibitory on plant enzymes. A remarkable difference in the amount of arginine and ornithine decarboxylase existed in wheat. Arginine decarboxylase seems to be more important at least during the early stage of development. Influence of polyamine synthesis inhibitors on polyamine levels is more likely to come from arginine decarboxylase inhibitors. As inhibitors of all essential enzymes involved in plant polyamine biosynthesis were found, the study of the importance of polyamines in plant physiology will be considerably facilitated.

Inhibitors of enzymes involved in polyamine biosynthesis which stop the growth of bacteria, fungi and animal cell systems were analyzed for their potential to interfere with plant cell systems. Several compounds were found to be potent inhibitors of plant enzymes, namely the decarboxylases of ornithine, arginine and S-adenosylmethionine as well as the aminopropyltransferase. Application of enzyme inhibitors (α-difluoromethylornithine. l-aminooxy-3-aminopropane, a-difluoromethylarginine, methylglyoxal bis(guanyl-hydrazone), cyclohexylamine) or combinations of these on whole plant systems resulted neither in a large decrease in polyamine content nor in a significant growth reduction. The lack of response cannot be explained by hindered inhibitor uptake into plants. Inhibition of single enzymes may induce alternative biosynthetic pathways.

The high levels of polyamines generally found in cotyledons of seedlings might be exported to hypocotyls and radicles. 14 C-Labelled putrescine applied to a sugar beet seedling through a cotyledon was found in small amounts as putrescine in the hypocotyl or radicle within a few minutes. Significant amounts of the labelled putrescine, however, were metabolized rapidly to compounds which could not be extracted into the organic phase on dansylation.

Substrate specificity of soybean lipoxygenase-1 (EC 1.13.11.12) was studied using as synthetic substrate analogs an entire series of (ω-6 Z,ω-9 Z)-C 13 ~ C 20 (rdienoic acids and (ω-6 Z,ω-9 Z)- C 12 ~ C 20 (i-dienols. The relative activity of lipoxygenase-1 against linoleic acid (C 18:2 COOH) was the highest among the dienoic acids and that of (6Z.9Z)-pentadecadienol (C 15:2 OH) was the highest among the dienols. Soybean lipoxygenase-1 oxygenated almost at to- 6 position of the dienoic acids independently of the chain length. With the dienols as substrate, the position of oxygenation was much influenced by the chain length. Above all, C 15:2 OH was selectively oxygenated at ω-10 rather than at ω-6 . Kinetic analyses revealed that longer and more hydrophobic dienols had higher affinity but lower velocity of oxygenation reaction. But higher velocity was obtained when smaller K m value was observed for the dienoic acids. To clucidate stereospecificity of the oxygenation to C 15:2 OH, optically active authentic samples were prepared via biological asymmetric reduction with baker’s yeast. The ω- 6 oxygenation to C 15:2 OH was found to be (S)-specific (R/S = 26/74) as is that to C 18:2 COOH. whereas the ω-10 oxygenation showed low enantioselectivity (R/S - 60/40).

Chlorobium phaeobacteroides contains two soluble basic c-type cytochromes, a flavocytochrome c-552 and a small cytochrome c-555. Both electron transfer proteins were highly purified by ion exchange chromatography and gel filtration. The flavocytochrome c-552 exhibits maxima at 552 nm, 523 nm and 416 nm in the reduced state and at 409.5 nm with two shoulders at 440 nm and 480 nm in the oxidized form. The best purity index (A 280 /A 416 )obtained was 0.65. The molecular properties of this flavocytochrome are as follows: isoelectric point, pH 9.5 - 10; redox potential, +63 mV; molecular weight, 56,000. Cytochrome c-555 is a small basic hemoprotein with an isoelectric point of pH 9.5 - 10, a molecular weight of 9,500 and a midpoint redox potential of +105 mV. The best purity index {A 280 /A 418 ) obtained was 0.176. The oxidized form of this cytochrome has a maximum at 411.5 nm, while the reduced state shows three maxima (α-band at 554.5 nm; β-band at 523 nm, and γ-band at 418 nm). The a-band is asymmetrical with a typical shoulder at 551 nm.

Biosynthesis of bacteriochlorophyll c and a in the thermophilic phototrophic prokaryote. Chloroflexus aurantiacus Ok-70-fl, was strongly inhibited by the antibiotic gabaculin (3-amino 2,3- dihydrobenzoic acid), an inhibitor of the glutamate-C 5 -pathway of ö-aminolevulinate (ALA) synthesis. The key enzyme of the Shemin-pathway of ALA formation, ALA synthase (EC 2.3.1.37), was not detected in cell extracts of Chi. aurantiacus. However, the extracts catalyzed ALA formation from glutamate 1-semialdehyde, a reaction being highly sensitive to gabaculin.

Lysed cells as well as different fractions of isolated, disrupted chloroplasts from light grown Euglena gracilis were tested for their capability of porphyrin biosynthesis. It is shown that the formation of the soluble porphobilinogenase (PBG-ase) fraction is inhibited by cycloheximide indicating its biosyntheses on 80S ribosomes, whereas the formation of the membrane bound chloroplast PBG-ase is chloramphenicol sensitive. Different pH-optima are demonstrated for the two enzymes. From the presented results it is deduced from those isolated from dark grown cells.

The preparation of the four stereoisomers of propiconazole (TILT ® ) is described. Their inhibition of the 14α-C-demethylation of the sterol nucleus is examined and compared with the inhibition by the four stereoisomers of etaconazole (SONAX ® ). The quantitative structure-activity relationships (QSAR) of substituted 1,3-dioxolane-2-yl-methyltriazoles and 1,3-dioxane-2-ylmethyltriazoles on in vivo fungicidal activity are investigated.

In this investigation, the effect of bialaphos (phosphinothricyl-alanyl-alanine) on the enzymes involved in NH 4 + -assimilation - glutamine synthetase, glutamine-2-oxoglutarate aminotransferase, glutamate dehydrogenase - is examined and compared to the effect of phosphinothricin (glufosinate) on the same enzymes. Bialaphos was given to whole plants (in vivo) and to leaf homogenate (in vitro). The investigation showed that bialaphos has an inhibiting effect on glutamine synthetase in vivo, but not in vitro. In contrast to this, phosphinothricin inhibits glutamine synthetase in vitro as well as in vivo. It was found that bialaphos, similar to phosphinothricin, does not inhibit glutamine-2-oxoglutarate aminotransferase and glutamate dehydrogenase in vivo or in vitro. Only at bialaphos concentrations exceeding 10 mM, there is an inhibition of glutamate dehydrogenase in vitro. Using radioactive [ 3 H]bialaphos (phosphinothricyl- 3 H-alanyl-alanine) it could be demonstrated that in the plant, bialaphos is split into phosphinothricin and alanine. The phosphinothricin released is probably the active herbicide component. Consequently, the herbicidal effects of phosphinothricin and bialaphos are the same.

The application of bialaphos (phosphinothricyl-alanyl-alanine) effects a quick photosynthesis inhibition under atmospheric conditions (400 ppm CO 2 , 21% O 2 ). However, under conditions (1000 ppm CO 2 , 2% O 2 ) under which photorespiration cannot occur there is no photosynthesis inhibition. In the previous investigation it could be shown that bialaphos splits in plants into phosphinothricin and alanine. The inhibition of glutamine synthetase through freed phosphinothricin results in an NH 4 + -accumulation and a decrease in glutamine. With the addition of glutamine, photosynthesis inhibition by bialaphos can be reduced. An NH 4 + -accumulation takes place under atmospheric conditions as well as under non-photorespiratory conditions; though in the latter case, in less amounts. After adding glutamine and other amino acids the NH 4 + -accumulation increases especially. This indicates that NH 4 + -accumulation cannot be the primary cause for photosynthesis inhibition by bialaphos. The investigations indicate that for the effectiveness of either bialaphos or phosphinothricin, a process in connexion with photorespiration plays a considerable role. The glyoxylate transamination in photorespiration could be inhibited, which results probably on a glyoxylate accumulation. Corresponding investigations showed inhibition of photosynthesis as well as a direct inhibition of RubP-carboxylase with glyoxylate.

Cells of the cyanobacterium Synechococcus 6301 were grown under illumination whose spectral composition favoured absorption either by the phycobilisome (PBS) light-harvesting antenna of photosystem II (PS II) or by the chlorophyll (Chi) a light-harvesting antenna of photosystem I (PS I). Cells grown under PS I-light developed relatively high PS II/PS I and PBS/Chl ratios. Cells grown under PS II-light developed relatively low PS II/PS I and PBS/Chl ratios. Thus, the primary difference between cells in the two acclimation states appeared to be the relative concentration of PBS-PS II and PS I complexes in the thylakoid membrane. Measurements of the quantum yield of oxygen evolution suggested a higher efficiency of cellular photosynthesis upon the adjustment of photosystem stoichiometry to a specific light condition. The quantum yield of oxygen evolution was nevertheless lower under PBS than Chi excitation, suggesting quenching of excitation energy in the photochemical apparatus of PS II in Synechococcus 6301. This phenomenon was more pronounced in the PS II-light than in the PS I-light grown cells. Room temperature and 77 K fluorescence emission spectroscopy indicated that excess excitation energy in the PBS was not transferred to PS I, suggesting the operation of a non-radiative and non-photochemical decay of excitation energy at the PBS-PS II complex. This non-photochemical quenching was specific to conditions where excitation of PS II occurred in excess of its capacity for useful photochemistry.

Biotypes of Amaranthus retroflexus L ., A. hybridus L., A. bouchonii Thell. and Chenopodium album L. insensitive to atrazine were collected from maize monoculture where atrazine had been applied extensively. Atrazine-resistant biotypes of A. retroflexus and A. hybridus showed phenmedipham and lenacil co-resistance and atrazine-resistant biotype of C. album showed fenuron co-resistance. An atrazin-resistant biotype of A. bouchonii with co-resistance to diuron was not resistant to fenuron, lenacil and phenmedipham.

Plasma membrane Mg 2+ , Ca 2+ ATPases were isolated from Dunaliella parva by differential centrifugation and subsequent sucrose gradient centrifugation and analyzed for their properties with special emphasis on ecophysiological requirements of this extremely salt-tolerant alga. Most properties (V max - and K M -values, substrate specificity, vanadate and DES sensitivity, resistance against ouabain) indicate that the ATPases of the plasma membrane of D. parva are basically of the same type as that found in the plasma membrane of other algae or higher plants. However, some interesting deviations from the normal characteristics of plasma membrane ATPases of plants were observed for the Dunaliella ATPases. These modifications partially may reflect adaptations of the ATPase and/or the microenvironment of the ATPase to the highly saline environment of this alga; 1) The plasma membrane ATPase of D. parva requires unusually high concentrations of divalent cations (up to 100 mM Mg 2+ or Ca 2+ ) for maximal activity. Both cations can substitute for each other. 2) The plasma membrane ATPase of D. parva is extremely resistant against salt. It was stimulated by NaCl or KC1 at concentrations up to 800 mM , whereas at higher salt concentrations the enzyme was inhibited. However, about 2.5 M NaCl was required for halfmaximal inhibition of ATPase activity. 3) The ATPase was inhibited by inhibitors of anion transport such as SITS and D ID S . which suggests direct or indirect involvement of ATPase in anion transport. The possible functions of the plasma membrane ATPases are discussed with special emphasis on problems related to the hypersaline environment of this alga.

In this investigation low, non-solubilizing concentrations of the strong anionic detergent SDS were used to perturbate the interaction of Ca 2+ and P i with their respective binding domains on the sarcoplasmic reticulum Ca-transport ATPase. Rising SDS concentrations produce a two-step decline of Ca 2+ -dependent ATP hydrolysis. At pH 6.15, SDS differently affects high affinity Ca 2+ binding and phosphorylation by inorganic phosphate and releases the “mutual exclusion” of these two ligand binding steps. The degree of uncoupling is considerably more pronounced in the presence of 20% Me 2 SO. The reduction of Ca 2+ binding by SDS is demonstrated to be a result of decreased affinity of one of the two specific high affinity binding sites and of perturbation of their cooperative interaction. Higher SDS partially restores the original high Ca 2+ affinity but not the cooperativity of binding. Phosphorylation exhibits a higher SDS sensitivity than Ca 2+ binding: Increasing SDS competitively inhibits and then completely abolishes phosphoenzyme formation. Thus. SDS binds to the phosphorylation domain, evidently involving the Lys 352 residue of the ATPase molecule; this is accompanied by a more unspecific concentration-dependent SDS effect, probably mediated by hydrophobic force, which, finally, suppresses phosphorylation. Me 2 SO does neither qualitatively affect the SDS-dependent chemical properties of the vesicular material nor the SDS-dependent perturbation of the investigated reaction steps.

A new polyclonal antiserum against spinach CF 1 subunit delta was produced in rabbits. It decorates only one band at 21 kDa in Western immunoblots of thylakoid proteins and does not react in ELISA with δ-free four subunit CF 1 (-δ); therefore it is regarded monospecific. The polypeptide used as immunogen had been purified by HPLC. Earlier antisera against CF 1 δ crossreact with CF 1 subunit β. The new antiserum 306 contains different antibodies; some can be absorbed with thylakoids, i.e. by δ within the assembled CF 0 CF 1 complex on the membrane, others still react in ELISA with isolated CF 1 . The former antibodies agglutinate thylakoids and inhibit PMS cyclic photophosphorylation. Therefore we conclude that part of the surface of CF 1 subunit δ is exposed within the quaternary structure of the ATP-synthase complex of photosynthetically active thylakoids, but part of the surface of δ is shielded. Trypsination of isolated δ occurs at several sites, but this protease does not attack δ in situ, nor does aminopeptidase. Staphylococcus aureus protease V8 digests CF 1 δ after isolation at residues Asp 53 , Glu 61 , Glu 95 and Glu 106 , but has no access to these residues of δ in situ. Thus CF 1 subunit δ seems to be shielded within the CF 0 CF 1 complex to a large degree. Direct agglutination of thylakoids by anti δ serum 306 was weak and could be improved tenfold by a Coombs serum (goat anti rabbit gammaglobulin), whereas an anti β serum agglutinated directly. From this we conclude that δ is not accessible at the top of the enzyme; the exposed part is extending below the large subunits a and β and oriented towards the membrane.

Incubation of synthetic 12-hydroxy-(3 Z)-dodecenal (3 ZHD) with tea chloroplasts revealed that 12-hydroxy-(2 E)-dodecenal (2 EHD) was the product of isomerization reaction of 3 ZHD during the incubation. The isomerization was caused also by incubation of 3 ZHD with the heat-inactivated chloroplasts. The isomerization activity was extracted with hot ethanol. These results indicate that the isomerization was caused by heat-stable factor(s) but not by enzyme(s) in tea chloroplasts.

Cell walls of the unicellular cyanobacterium Synechocystis sp. PCC 6714, isolated from cell homogenates, were found to be unusually resistant against extraction with various detergents, organic solvents, chaotropic agents, and proteases. The major outer membrane proteins (M r 67,000; 61,000; 94,000) were solubilized by differential SDS-extraction and purified by preparative SDS-PAGE. The extracted proteins, reconstituted into lipid bilayer membranes, formed two types of pores with single-channel conductances of 2.2 nS (pore diameter of 1.4 nm) and 0.3 nS (pore diameter not determined), respectively. Carotenoids and lipopolysaccharide were found to be associated with the extracted major proteins.

Propolis is a natural composite balsam, manufactured by honey bees, and active biochemically. Some of its biological effects are attributed to its ability to stimulate enzymatic systems, which in many instances involve trace elements. In the present study we assayed propolis and its ethanolic extract (EEP) for trace elements, and quantified them with the aid of neutron activation analysis. Four elements that have been detected in propolis and EEP for the first time are Na. Cr, Co (ppm) and Ag (ppb). The wide concentration range of the inorganic components in various batches of propolis and EEP is due to the difference in their extraction capacities, and to the variability in the environmental pollutants and the flora at the beehive sites.

The tetrahydroisoquinoline alkaloids salsolidine (1-methyl- 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline) and 1- carboxysalsoline (1-carboxy-1-methyl-6-hydroxy-7-methoxy- 1,2,3,4-tetrahydroisoquinoline), which are themselves not substrates for the enzyme catechol-O-methyltransferase, are - contrary to a hitherto existing view - able to inhibit competitively the methylation of the catecholamine metabolite 3,4-dihydroxybenzoic acid by the enzyme enriched from rat liver. The inhibitor constants were determined to be K i = 0.19 mM for salsolidine and K i = 0.44 mM for 1-carboxysalsoline.