Volume 49, Issue 11-12

Atrichum undulatum as well as Polytrichum formosum are containing each six different coumarin glycosides of which three are common to both species. From the nine different glycosides eight are new natural compounds. The structures have been elucidated spectroscopically.

The volatile components of the essential oils of four Chinese medicinal Plants (Micromeria biflora, Elsholtzia souliei, Eupatorium adenophorum, Asarum petelotii) were investigated by GC and GC-MS. Large amounts of terpenes (geranial, naginataketone, p-cymene, β-himachalene), or phenol ethers (apiole, elemicin, 1,2,3,4-tetramethoxy-5-allyl-benzene) were identified in the oils.

A new piperidine amino acid, 2,4,5-piperidinetricarboxylic acid (11) was isolated from the poisonous mushroom, Clitocybe acromelalga. The structure determination and its biogenetic potential are discussed.

Flavonoids and other phenolic compounds were determined in 15 samples of propolis originating from China, from Brazil, and from Uruguay. A total of 24 compounds were identified using mainly HPLC and a few other analytical methods. The most abundant compounds found were benzoic acid and benzaldehyde derivatives, flavones, flavonols. and flavanones. 80% of the samples contained at least 22 g/100 g of flavonoids, primarily acacetin, isorhamnetin, apigenin, and pinocembrin. The flavonoid patterns were sufficiently distinctive to permit discrimination between propolis from China, from Uruguay, and from Brazil.

The roots of 13-day-old soybean ( Glycine max L.) and 7-day-old wheat ( Triticum aestivum L.) hydroponic plants were exposed to [ 14 C]-3,4-dichloroaniline (1.0 and 0.4 mg/1 (6.2 and 2.5 μᴍ) , respectively) and harvested after 48/120 h (soybean) and 72 h (wheat). Both species metabolized the xenobiotic almost quantitatively to N-(β-D-glucopyranosyl)-3,4-dichloroaniline, N-malonyl-3,4-dichloroaniline, 6′-O-malonyl-N-(β-D-glucopyranosyl)-3,4-dichloroaniline and non-extractable residues. In the soybean experiments 58.8 (48 h) and 54.6% (120 h) of the applied radioactivity were found in the nutrient; this fraction consisted primarily of N-malonyl-3,4-dichloroaniline. 37.3/24.1% (48/120 h) were detected in the plants. In the soybeans, 36.2 and 52.7% (48/120 h) of the absorbed 14 C were translocated, mainly into hypocotyls, primary and secondary leaves. After 120 h, the main metabolite was N-malonyl- 3,4-dichloroaniline (38.5% ); considerable levels of this metabolite accumulated in the primary and secondary leaves (10.4 and 10.4%). The glucosides were mainly found in the roots of the soybean plants. Totals of 23.5 and 35.1% (48/120 h) were transformed to non-extractable residues. In wheat, 78.3% of the applied 3,4-dichloroaniline was absorbed after 72 h. This fraction was partially translocated to the leaves, but most of the residues remained in the roots (90.3% of absorbed 14 C). In wheat, a total of 45.6% was transformed to nonextractable residues. The soluble radioactivity in the roots consisted of nearly equal amounts of the glucosides and the N-malonyl conjugate. The processes observed in soybean and wheat roots resembled those of the respective cell suspension cultures published previously.

The deletion mutant Bradyrhizobium japonicum ΔP22 produces a structurally altered exopolysaccharide. The nodulation of two cultivars each of Glycine max and Glycine soja, and cultivars of Macroptilium atropurpureum and Vigna radiata, infected with this mutant was examined in order to analyze the role of the exopolysaccharide in the infection process of plants with a determinate nodule type. All host plants analyzed exhibited delayed nodulation and formed fewer nodules per plant. The extent of the impairments depended on the genotype of the host plant. Morphological studies confirmed these results. In V radiata later steps in nodule development proceeded without further disturbances, whereas with G. soja PI 407287 minor changes were detected. In contrast, the inoculation of G. soja PI 468397 and M. atropurpureum lead to the formation of nodules most of which were not infected by Bradyrhizobium japonicum ΔP22 (Inf - ). However, on M. atropurpureum at least some effective nitrogen-fixing nodules developed. Such nodules did not emerge from G. soja PI 468397. Inf - nodules were arrested in an early stage of nodule development, and symptoms of plant defense responses were observed.

Yellow colouration of Dahlia variabilis is mainly provided by isoliquiritigenin 4′-glucoside and butein 4′-glucoside. Incubation of petal extracts with uridin 5′-diphosphoglucose and isoliquiritigenin or butein led to the formation of one product which was identified as the respective 6′-deoxychalcone 4′-glucoside. Glucosylation of hydroxyl groups in other positions was not observed. Naringenin chalcone and eriodictyol chalcone were not accepted as substrates. The 4′-glucosylation of isoliquiritigenin and butein showed a broad pH optimum ranging from pH 7 to 8 and was stimulated by Mg 2+ , Ca 2+ and Mn 2+ . N-Ethylmaleimide, p-hydroxymercuribenzoate, Cu 2+ , Zn 2+ , and Fe 2+ clearly reduced the activity of the enzyme. The apparent K m values for UDP-glucose, isoliquiritigenin and butein were 90, 26 and 276 [μᴍ respectively.

Pathways involved in the conversion of phosphoenolpyruvate (PEP) to pyruvate, the final step in glycolysis, were investigated after transfer of stationary-phase cells from suspension cultures of Catharanthus roseus to fresh complete or phosphate (Pi)-deficient Linsmaier and Skoog medium. In addition to pyruvate kinase (PK). enzymes that can function in an alternative pathway, namely, PEP carboxylase (PEPC), NAD-malate dehydrogenase and NAD-malic enzyme, were present in significant amounts in C. roseus cells. The activity of PEPC in Pi-starved cells was more than twice that in cells in the complete medium (Pi-fed cells), while that of PK in Pi-starved cells was lower than that in Pi-fed cells. No significant differences were observed in the levels of NAD-malate dehydrogenase and NAD-malic enzyme between these two types of cell. At cytosolic pH, the K m value of PEP (45 μᴍ ) for PEPC was lower than that for PK (100 (μᴍ). The activity of PEPC was inhibited by malate, citrate, aspartate and ATP. The activity of PK was also inhibited by ATP, but to a lesser extent. The cellular levels of PEP, adenylates and malate, which are substrates and effectors of PK and PEPC, in Pi-fed and Pi-starved cells suggest that the contribution of PEPC to the metabolism of PEP increased in Pi-starved cells in vivo. Evidence for operation of a bypass from malate to pyruvate in vivo was supported by the rapid release of 14 CO 2 from organic compounds derived from fixed NaH 14 CO 3 and from [4- 14 C]malate.

Although yeasts have been intensively investigated in photobiology, directional response of yeast growth to light has never been observed. The present data demonstrate for the first time phototropism in yeast, the basidiomycetous yeast Sporobolomyces salmonicolor. The effective spectral band is blue light - suggesting that a blue-light receptor similar to that in other plants is involved in yeast photophysiology. Further studies on yeast phototropism could help identification of the photoreceptor and throw new light on the mechanisms of signal transduction and response.

Glutamine synthetase (EC 6.3.1.2) activity of a non-photosynthetic mutant of Chlorella kessleri is markedly enhanced under blue and slightly increased under red light. In both cases, the effect is largest after 6 h of irradiation. In blue light, saturation is reached at about 10 μEm -2 s -1 ; in red light, it is not even indicated at 62 μE m -2 s -1 . Semilogarithmic plots of both intensity dependencies reveal different slopes, indicating envolvement of two separate photoreceptors. This feature is supported by different effects of pulse irradiation: The response to 15 min of red light irradiation (λ max 650 nm) increases in subsequent darkness. It reaches the same value as in continuous light after 6 h. The response to 15 min of blue light irradiation (λ max 441 nm) increases also in subsequent darkness. However, after 6 h it reaches only 30% of the value obtained by continuous irradiation. It is concluded that, glutamine synthetase of Chlorella is controlled by two different photoreceptors both independent of photosynthesis. There is evidence of two forms of glutamine synthetase, the intracellular distribution and specific light regulations of these are discussed.

The relative increase of the 7-ethoxycoumarin-O-deethylase and the 6,7-dimethoxycoumarin- O-demethylase activities were found 93 and 236% using a reconstituted cytochrome P 4502B 1 :NADPH-P450 reductase system by adding to the reaction mixtures their own products. The assays were irradiated during the reactions with the excitation wavelength maximum of their products umbelliferone (λ E = 365 nm) or scopoletin (λ E = 398 nm), respectively. Addition of the products to the reaction mixtures without irradiation (dark reaction) had no activating effect on the specific activities of the 7-ethoxycoumarin-O-deethylase or the 6,7-dimethoxycoumarin-O-demethylase. The relative increase of the specific activities is dependent on the excitation light intensities and was at maximum when the light intensity at the sample cuvette was 0.4 mW/cm 2 . The activation energies of the P 4502B 1-dependent 7-ethoxycoumarin-O-deethylation reaction obtained from Arrhenius plots with and without added umbelliferone and irradiation with λ E = 365 nm are 14.7 kJ/mol and 33.5 kJ/mol, respectively, in the temperature range of 27-37 °C. The irradiation energy of the fluorescent product umbelliferone change the catalytic mechanism, which has a two times lower activation energy in the presence of the irradiated product umbelliferone. Umbelliferone and scopoletin have highest fluorescence intensities in the wavelength range of the blue light (440-480 nm). The photochemical action spectrum of the 7-ethoxycoumarin-O-deethylase of the P4502B 1:reductase system is also found to be in the wavelength range of 420-470 nm. No activation effect was seen with irradiating light lower than 400 nm. Obviously the fluorescence light of the products are due for the extremely high activation effect. This is the first report that the products umbelliferone and scopoletin are photoreceptors and phototransducers.

Pinicoloform, a novel unbranched acyclic compound containing a trichloromethyl group, has been isolated from extracts of the mycelia of the Basidiomycete Resinicium pinicola. It was isolated because of its ability to induce morphological and physiological differentiation of mammalian cells, although it also exhibits antibiotic and cytotoxic activities. The structure of pinicoloform was determined by spectroscopic methods.

A number of safeners like naphthalene-1,8-dicarboxylic acid anhydride (naphthalic anhydride) or dichloroacetyl-hexahydro-3,3,8-α-trimethylpyrrolo-[1 ,2 α]-pyrimidine-6-(2H̲)-one (BAS 145138) drastically decreased the accumulation of protoporphyrin IX induced by a peroxidative cyclic imide (chlorophthalim, N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide), or p-nitrodiphenyl ether (acifluorfen methyl, 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoicmethylester). This effect was observed with etiolated maize and cress seedlings during a 16-h illumination period with these herbicides and 0.1 to 1 mᴍ safener present. The safeners did not affect the inhibition of protoporphyrinogen oxidase, the target enzyme of peroxidative herbicides. Mixed function oxidase inhibitors did not influence this safening effect. A microsome preparation from safener-treated maize seedlings did neither degrade protoporphyrin IX nor protoporphyrinogen IX.

(1-Amino-2-phenylethyl)phosphonic acid (APEP), (1-amino-2-phenylethyl)phosphonous acid (APEPi), α-aminooxy-β-phenylpropionic acid (AOPP) and several other phenylalanine analogues are potent inhibitors of (S)-phenylalanine ammonia-lyase (PAL) in vitro and in vivo. The ability of these compounds to inhibit (S)-phenylalanine-tRNA synthetases (PRSs) from wheat germ, soybean, and baker’s yeast has been investigated and compared to the inhibition of PAL. APEP and APEPi were found to inhibit the tRNA phe -aminoacylation reactions catalyzed by the three PRSs studied in vitro in a competitive manner with respect to (5)-phenylalanine. (R)-APEP inhibits the PRSs with apparent K i values of 144 μᴍ for wheat germ (app. K m for (S)-phe 5.2 μᴍ) , 130 μᴍ for soybean (app. K m for (S)-phe 0.9 μᴍ) , and 1096 μᴍ for baker’s yeast (app. K m for (S)-phe 5.5 μᴍ ) . The apparent K i values for (R)-APEPi are 315 μᴍ , 160 μᴍ , and 117 μᴍ , respectively. APEP and APEPi inhibit the ATPpyrophosphate exchange reactions catalyzed by the PRSs from wheat germ and baker’s yeast, but they are not activated and do not serve as substrates in these reactions. AOPP has no affinity to any of the three PRSs, whereas it is a potent inhibitor of PAL. In light of our in vitro results with PRSs from different sources it appears unlikely that the PAL inhibitors we have studied have any significant inhibitory effect on this essential step in protein synthesis in vivo.

Eight photoautotrophic cell cultures of Chenopodium rubrum, which are resistant against the photosystem II inhibitor metribuzin, were characterized for their growth parameters, chlorophyll content and photosynthetic capacity. Herbicide resistance of the eight lines results from different mutations in the D 1 protein of photosystem II, which is the target for different photosystem II inhibitors. In the presence of 10 -5 ᴍ metribuzin the eight lines showed substantial growth reduction depending on the degree of resistance, and this effect is explained by a reduced electron transport in photosystem II. The impaired photosynthetic capacity of the green cells in the presence of high metribuzin concentrations, leads to compensation effects similar to shade accommodation of plants. Adaptation includes an increase of the chlorophyll content, a decrease of the chlorophyll a/b ratios as well as an increase of thylakoid stacking and cell number per unit fresh weight. In the absence of the herbicide photosynthetic electron transport is not impaired, as indicated by measurements of electron transfer rates in photosystem II and flash-induced reduction kinetics of P-700 + . In summary the alterations of the D 1 protein of the eight cell lines do not result in a reduced electron transport in photosystem II.

The polymerase chain reaction was used to prepare gene-specific probes for several Lhc genes coding for chlorophyll a/b-binding proteins of cress (Lepidium sativum L.). Due to the presence of about 150 basepairs of the coding region, the isolated clones could be attributed to Lhc a3 (1 clone), Lhc b1 (5 clones), Lhc b2 (1 clone) and Lhc b3 (1 clone) genes. Probes prepared from the 3′ non-coding regions of the clones did not cross-hybridize; they were specific for 3 different Lhc b1 transcripts and one each of Lhc b2, Lhc b3 and Lhc a 3 transcripts. The transcript levels were higher in leaves than in cotyledons of light-grown seedlings; they decreased significantly in cotyledons from week 1 to week 4. The levels of 2 Lhc b1 transcripts (detected with probes cd 1 and cd 2) changed from 1 week old cotyledons (30% c d l, 28% cd 2) to 3 months old leaves (14% c d l), 44% cd 2), stems (11% c d l, 56% cd 2) and fruits (15% cd 1, 62% cd 2, all values percent of total transcripts), whereas transcript levels of another Lhc b1 gene (detected with probe cd 3) and of a Lhc a 3 gene remained nearly constant. The level of Lhc b2 and Lhc b3 transcripts were 1 - 2 orders of magnitude smaller than those of the other Lhc transcripts. The data obtained with cress plants are compared with published data from other plants.

Pisatin elicitors including fragments of chitosan and partially N-deacetylated chitins (DACs) strongly induced (+)-α,2′,4,4′-tetrahydroxydihydrochalcone, (-)-4′,7-dihydroxyflavanone, and 4,4′-dihydroxy-2′-methoxychalcone in Pisum sativum. These three flavonoids with moderate or weak antimicrobial activities were effectively induced by the treatment of pea with either the biotic elicitors or an abiotic elicitor CuCl 2 . The examination of chitosan derivatives with different molecular size and different N-acetylation for the flavonoidinducing activity in pea epicotyl assay revealed that the structural features required for the flavonoids induction were different from those required for (+)-pisatin induction. (+)-2- Hydroxypisatin, a possible intermediate in pisatin degradation pathway, which has been isolated from pea seedlings treated with the abiotic elicitor or infected by fungi was not induced by the chitosaccharides in pea epicotyl assay. These findings suggest that pea plants could distinguish invading pathogens by the difference of structural features of chitosaccharides derived from chitin, a major constituent of fungal cell walls. Thereby, the plant could accumulate antimicrobial compounds responsible for the chemical defense against invading pathogens.

A method was developed for carrying out gas-exchange and chlorophyll-fluorescence measurements simultaneously during fumigation of spruce twigs with peroxidic photooxidants. It is thus now possible to investigate how a pollutant affects distinct sectors of the photosynthetic apparatus of the plant: whereas fluorescence reveals any changes in the primary light reaction, CO 2 gas-exchange measurements supply information about the biochemical reactions of the Calvin cycle. Results of short-time fumigation with 750 ppb ozone are presented here. Gas-exchange and fluorescence data are affected strongly in early summer, but not in autumn. The assimilation rate decreases significantly: primarily as a result of Rubisco activity and possibly because of direct inhibition of the electron-transport chain as well. Closure of the stomata leads to further reduction in the assimilation rate. Though no damage becomes visible on the needles, the perturbance of the photosynthetic apparatus caused by ozone fumigation is not reversible within 24 h.

The hypermodified ureidonucleoside N6[((9-β-ᴅ-ribofuranosyl-9H-purine-6-yl)amino)carbonyl]- ʟ-threonine (5) is a constituent of transfer ribonucleic acid (tRNA) and is secreted as a tRNA catabolite in body fluids such as blood, milk and urine. Compound 5 and the isomeric ureidonucleosides bearing ᴅ-threonine (9), ʟ-allo- (7) and ᴅ-allo-threonine (11) as side chain moieties were synthesized on a preparative scale. The amido protons of 5 and 9 cause two separate 1 H NMR signals whereas 7 and 11 cause multiplets. The 13 C NMR signals of all carbon atoms of the allo-amino acid side chains (7, 11) are shifted downfield of the corresponding signals in compounds 5 and 9. The chemically protected intermediate compound adenosine urethane (3) is potentially of interest in the analysis of amino acids because it may be converted to nucleosides of the ureido type by reaction with amino acids in biological matrices.

One of the key obstacles in lectin research is the lack of specific techniques for their detection. Novel markers, biotin-labeled carbohydrates, could contribute to overcome this problem. Being at least 10 times more sensitive than neoglycoproteins in the membranescreening assays, they also enable direct detection of lectins in complex mixtures. The markers were synthesized by linking biotin to one, and a carbohydrate (galactose or glucose) to the other amino group of (the amino acid) lysine. After synthesis the markers were chromatographically purified on lectin (RCA for galactose marker, ConA for glucose marker) and avidin affinity columns. The applicability of the markers to detect lectins was demonstrated e.g. with sponge extracts (from Geodia cydonium). Following incubation with biotin-labeled carbohydrates covalent cross-linking between lectins and markers was induced by UV radiation. After transfer to the blotting membrane, lectins were detected with deglycosylated antibiotin antibody labeled with alkaline phosphatase. Besides for the cross-linking technique, the biotinylated carbohydrate markers were also used for detection of lectins on the nitrocellulose membrane in gene library screening and slot blotting.

Using the two-dimensional gel electrophoresis and two systems for computer analysis of the resulting maps, we obtained a good quantitative reproducibility of individual spots of silver-stained and Coomassie Blue-stained gels from mouse fetus liver samples. The GelScan- XL system was established for the evaluation of silver-stained gels. The Gel-Image software has shown to be capable of the analysis of Coomassie Blue-stained gels and autoradiographs. For these systems the linear relationship between defined concentrations of protein loaded on the gel and the resulting integrated intensities of spots were examined. The results show that both systems are efficient to detect quantitative changes in protein expression correlated with malformations in mouse fetuses.

In whole body extracts of the canthariphilous pyrochroid Schizotus pectinicornis and the cantharidin-producing oedemerid Oedemera femorata cantharidin contents were determined by means of quantitative gas chromatography. Adults and all developmental stages of both species contain this terpenoid, but in considerably different amounts. Cantharidin contents differ significantly between the sexes of adult O. femorata: 8.4 μ,g/female, 3.5 μg/male. Values for Sch. pectinicornis are more than 30-fold lower than those of O. femorata and no intersexual difference in the cantharidin content could be detected. Eggs of O. femorata contained cantharidin and larvae of this species are capable of synthesizing this terpenoid. The total amount of cantharidin increases in successive instars. In Sch. pectinicornis, the content of cantharidin decreases from egg to first instar. Feeding and copulation experiments with [ 2 H 2 ]cantharidin indicate that in O. femorata no or only very small amounts of cantharidin are transferred from males to females at copulation. Thus, males do not invest in the protection of their offspring by cantharidin gifts to the mate. For Sch. pectinicornis an intersexual transfer of labelled cantharidin during copulation was detected. About 45% of the entire cantharidin content in mated females derived from fed males. Analyses of eggs and first instar larvae show that a paternal allocation of 2 [H 2 ]cantharidin to developmental stages exists for this canthariphilous pyrochroid.

Electroantennogram (EAG) studies of the formicine genera Lasius and Formica (Hymenoptera, Formicidae) were carried out with 3,4-dihydroisocoumarins, recently identified as trail pheromones within this ant subfamily. The here investigated formicine species Lasius niger, Formica rufa, F. fusca and F. sanguinea were all responsive towards stimuli of synthetic 3,4-dihydroisocoumarins. Synthesized samples of natural 3,4-dihydroisocoumarins, found in the hindgut of these species evoke the highest electrophysiological responses while structure analogues were less efficient. Number and position of the methyl groups of test substances were important factors for structure activity in EAG. F. fusca and L. niger showed a stereoisomeric differentiation in EAG responses between (R)- and (S)-isomers of 8-hydroxy-3,5,7- trimethyldihydroisocoumarin, a component in the hindgut of both species. Higher electrophysiological activity for (R)-8-hydroxy-3,5,7-trimethyl-3,4-dihydroisocoumarin in comparison to the (S)-isomer agrees with better behavior responses of previous trail following tests in the case of Lasius niger.

Extracts of whole adult males of Gryllus bimaculatus show distinct maxima of free ecdysteroids on the 8th, 12th and 14th day after imaginal moult at a breeding temperature of 25 °C. Isolated OEFC of 11 days old males secrete in vitro free as well as conjugated ecdysteroids, although the latter predominate. The bulk of free ecdysteroids seem s to consist of E and 20 E according to their retention times in two HPLC systems. Apart from the polar ones, apolar m etabolites appear during incubation. By esterase the apolar metabolites are mostly split into polar ones, partly into 20 E and E. Further treatment of the polar metabolites with helicase leads again to E and 20 E. OEFC transform [ 3 H]E preferably into apolar and polar conjugates, with dominance of the apolar ones at the beginning and of polar ones in the further course of incubation. Only a small quantity of free 20 E is found during this experiment, but it can be produced in significant quantities by splitting the polar conjugates. N o conversion of [ 3 H]cholesterol into [ 3 H]ecdysteroids could be detected.

Extracts from Erodium cicutarium L. (EC) and some standard substances present in EC extracts were tested for their antioxidative properties during Fe 2+ -induced triglyceride oxidation. Hydrophobic fractions such as petroleum ether (PEE), benzene (BE) and chloroform I (ChE I) extracts as well as hydrophilic fractions i.e. water (WE) and ethyl acetate (EAE) showed an antioxidative effect. Among the tested substances vitamin C and some polyphenolic compounds; tannin, (+)-catechin and gallic acid exhibited antioxidative activity, stronger than the one of the mentioned extracts.

[1- 13 C]o-Succinylbenzoic acid is incorporated to approximately 50% into each of the keto groups of the quinone ring of juglone in JugIans regia. The same labelled precursor is transformed into the keto group of position 1 in lawsone, the pigment of Impatiens balsamina, suggesting two different intermediates in the biosynthesis of these simple 2- or 5-hydroxy-1,4-naphthoquinones.

None of six published preparation procedures for circular bacterial plasmids was satisfactory to purify the endogenous plasmid of a nitrate-reducing strain (AD2) of the phototrophic bacterium Rhodobacter capsulatus. By modifying the method of N. T. Hu and B. L. Marrs (1979), Arch. Microbiol. 121, 61-69 , the 115 kb plasmid of the latter strain was prepared to a highly pure state. Digestion of the plasmid with restriction endonuclease Eco RI yielded 21 fragments with sizes ranging from 0.3 to 18.4 kb. Except for the three largest ones (18.4 kb, 16.9 kb and 11.2 kb), all fragments were cloned into the vector plasmid pU C 8, amplified in E. coli JM83 and characterized by H ind III restriction analysis.

An antibody of a tumor marker (α-fetoprotein) conjugated with peroxidase was dispersed in organic solvents including synthetic polymers, and the interaction of the antibody with the polymers was investigated. It was found that the antibody-conjugated enzyme is associated with the polymers dissolved in hydrophobic organic solvents. The association correlated with the polymerization degree, concentration, and nature of the polymers.