Band 46, Heft 5-6

A series of new flavonoid aglycones have been identified in the frond exudates of the fern Cheilanthes argentea, in five species of Notholaena, in Pityrogramma triangularis, and in Platyzoma microphylla. These aglycones comprise several rare flavonoids and five novel natural products: 5,7,8-trihydroxy-3-methoxy-6-methyl flavone, 3,5,2′-trihydroxy-7,8,4′-trimethoxy flavone, 5,2′-dihydroxy-3,7,8-trimethoxy flavone, 5,7,4′-trihydroxy-2′-methoxy flavanone, and 3,5,4′-trihydroxy-6,7,8-trimethoxy flavone. The novel flavonoids were characterized by their N M R spectral data

A further investigation of the isoflavonoid components of Piscidia erythrina, Lupinus alhus and L. luteus has revealed the presence of 3-hydroxy- and 3-methoxy-coumaranochroman- 4-ones. These compounds are the first known representatives of a new type of naturally occurring isoflavonoid. Using a combination of chemical and spectroscopic methods, the com pounds were identified as 3,5,7,4′-tetrahydroxy-5′-methoxy-3′-(3,3-dimethylallyl)coumaranochroman- 4-one (piscerythrol, 1), 3,5,7,4′-tetrahydroxy-6,3′-di(3,3-dimethylallyl)coumaranochroman- 4-one (lupinol A, 3), 5,7,4′-trihydroxy-3-methoxy-6,3′-di(3,3-dimethylallyl)- coumaranochroman-4-one (lupinol B, 6) and 3,5,7,4′-tetrahydroxy-6-(3,3-dimethylallyl)- coumaranochroman-4-one (lupinol C, 7). All four isoflavonoids were found to have the cis relative configuration at C-2/C-3. Possible biogenetic relationships between the coumaranochroman- 4-ones and other isoflavonoids (isoflavones and coumaronochrom ones) found in Piscidia and Lupinus are briefly discussed

The prenylated flavonoids, topazolin [5,7,4′-trihydroxy-3-methoxy-6-(3,3-dimethylaIlyl)- flavone], piscerythrone [5,7,2′,4′-tetrahydroxy-5′-methoxy-3′-(3,3-dimethylallyl)isoflavone] and piscidone [5,7,4′,5′-tetrahydroxy-2′-methoxy-6′-(3,3-dimethylallyl)isoflavone] were metabolized by Aspergillus flavus and Botrytis cinerea to give a variety of products. Topazolin and piscerythrone were converted by both fungi to compounds similar to those previously obtained from luteone (6-prenyl) and licoisoflavone A (3′-prenyl) respectively. The 6′-prenylated isoflavone piscidone was metabolized only by B. cinerea to give the corresponding dihydropyrano- isoflavone as a major product. Neither fungus was found to metabolize the 8-prenylated pyranoflavanonol lupinifolinol

Bisaborosaol A (1), a novel sesquiterpene belonging to the bisabolane class, was isolated from Rosa rugosa leaves. The absolute configuration of 1 at C-4 was shown to be inverted in comparison with bisabolanoids from Compositae. Bisaborosaol A (1) and its corresponding carboxylic acid (2) may be biogenetically related to the carotanoids which constitute the major sesquiterpenes of R. rugosa leaves

Five novel bisabolane sesquiterpenes possessing a tetrahydrofuran ring or a hydroperoxy group were isolated from Rosa rugosa leaves and their structures elucidated by chemical and spectroscopic methods. These highly oxidized sesquiterpenes were structurally related to bisaborosaol A which is the major bisabolanoid of the plant

Cell free protein extracts from heterotrophic anthraquinone (“Rubiatype”) producing cell suspension cultures of three different Galium species and from a photoautotrophic phylloquinone (vitamin K ,) producing cell suspension culture of Morinda lucida Benth. (Rubiaceae) catalyzed the synthesis of o-succinylbenzoic acid (OSB) from isochorismic acid and a-oxoglutaric acid in the presence of thiamine diphosphate and Mn 2+ . At least two intermediates in the conversion of isochorismic to o-succinylbenzoic acid were detectable. One of these intermediates is likely to be identical to 2-succinyl-6-hydroxy-2,4- cyclohexadiene-l-carboxylate (SHCHC), a metabolite known to occur in bacterial mutants (menD + ) blocked in o-succinylbenzoic acid synthesis. The structure of the second intermediate is as yet unknown. A third intermediate may also occur

Cell suspension cultures of Coleus blumei Benth. producing high amounts of rosmarinic acid were used to study the biosynthetic pathway of this caffeic acid ester. One of the involved enzymes, the hydroxyphenylpyruvate reductase (HPPR), is characterized in this paper. HPPR catalyzes the NAD (P)H dependent reduction of p-hydroxyphenylpyruvate to p-hydroxyphenyllactate. The enzyme developed maximal activity at an incubation temperature of 37 °C and at a pH of 6.5 to 7.0. The reaction proceeded linearly for an incubation time of 60 min and up to a protein concentration of 0.2 mg per assay. As electron donor HPPR accepted NADH and NADPH with K m -values of 190 µm and 95 µm respectively. The enzyme reduced differently substituted hydroxyphenylpyruvates but not β-phenylpyruvate. The apparent K m -values for the various substrates were at 10 µ M for p-hydroxyphenylpyruvate, at 130 µm for 3,4-dihydroxyphenylpyruvate and at 250 µ M for 3-methoxy-4-hydroxyphenylpyruvate. HPPR was competitively inhibited by rosmarinic acid and pyruvate with K i -values of 210 µ M and 200 µ M respectively. Caffeic acid, p-coumaric acid and cinnamic acid did not affect the enzyme activity but p-coumaroyl-CoA inhibited HPPR

The uptake and sequestration of alkaloids by latex vacuoles of P. somniferum sedimenting at 900 x g and 1100 x g were studied; both populations take up morphine effectively. Morphine uptake by the 1100 x g vacuoles was stimulated by exogenous ATP and, after uptake of large amounts of alkaloid, both populations were stimulated by exogenous ATP to take up further morphine; this effect was no longer present in 900 x g vacuoles isolated from the latex of more mature capsules. Uptake by 1100 x g vacuoles was more sensitive to temperature than that of 900 x g vacuoles. Determination of the proton gradient across the vacuolar membrane (△pH), controlled dissipation of △pH through the use of NH 4 Cl, and correlation with morphine uptake demonstrated that morphine uptake was strongly dependant on the maintenance of △pH. Inhibitors designed to produce loss of protons from the vacuole had, however, little effect on the system. Nitrate, as an inhibitor of tonoplast ATPase, had an inhibitory effect on morphine uptake by the 1100 x g vacuoles. Uptake of meconate, [ 35 S]sulphate and L -[U 14 C]malate, important acid constituents of the 900 x g vacuoles was investigated. Uptake of sulphate and malate was continuous and saturation was not reached; the rate of uptake of sulphate and malate was much lower than that of morphine and other alkaloids, and uptake of meconate by these vacuoles was not detected. Studies on specificity of alkaloid uptake with the 900 x g vacuoles indicated no absolute preference for either the ( + )- or the (-)-isomer of codeine. Noscapine uptake was stimulated by ATP under conditions where ATP had no effect on morphine uptake. Nicotine was not taken up, but low levels of caffeine and l-m ethoxycanthin-6-one were taken up, but less effectively than morphine or noscapine. Uptake of alkaloids by P. somniferum latex vacuoles is dependant on the maintenance of tonoplast △pH and an ATPase generates this △pH. Sequestration appears to involve protonation and anion-cation stabilization involving meconate and sulphate. The specificity of alkaloid uptake suggested no clear correlation with pK or lipophilicity, and some sort of channel mechanism, more related to alkaloid shape is suggested

An enzyme from leaves of sumach (Rhus typhina) was partially purified that catalyzes the β-glucogallin (l-O-galloylglucose)-dependent galloylation of 1,6-digalloylglucose, thus forming 1,2,6-trigalloylglucose and free glucose. This acyltransferase had a molecular weight of ca. 750,000 and a pH optimum at 5.0-5 .5. Besides β-glucogallin (K m = 3.9 m M ) , also related 1-O-phenylcarboxylglucoses acted as acyl donors. On the other hand, the acceptor substrate, 1,6-digalloylglucose (K m = 0.9 m M ) , could only be replaced by 1,6-diprotocatechuoylglucose (relative activity 46%); however, also tri-, tetra-, and pentagalloylglucoses were galloylated. A pronounced stimulation of the enzymatic reaction was observed upon addition of penta- or hexagalloylglucose into the assay mixtures. The systematic name “ β-glucogallin: 1,6-di-O-galloylglucose 2-O-galloyltransferase” (EC 2.3.1. - ) is proposed for the enzyme

Biotransformation of β-damascone (1) was studied with four strains of Botrytis cinerea using grape must (A) and a mixture of A with synthetic medium (B). Whereas in A 4-hydroxy-β-damascone (2), 3-hydroxy-β-damascone (3), and 2-hydroxy-β-damascone (4) were found as volatile bioconversion products, in B additionally 4-oxo-β-damascone (5) was detected. Furthermore, 9-hydroxy-8,9-dihydro-β-damascone (6) was identified as chemically formed by-product. Quantitatively, the results were strongly dependent on the strain and medium used. Incubation of 1 with two B. c. strains in medium Bled to an almost selective formation (93 and 95%) of 2 and the highest yield of products (30 and 66%). The bioconversion products were identified by capillary gas chromatography (H RG C) and coupled HRGC techniques, i.e. - mass spectrometry (HRGC-MS) and - Fourier transform infrared spectroscopy (HRGC-FTIR) after extractive sample preparation

Pseudomonas strains of the so-called fluorescent group usually produce several pyoverdins which differ only in the nature of a dicarboxylic acid bound amidically to the chromophor. For the pyoverdins isolated from the culture medium of Pseudomonas fluorescens 12 it is shown that succinic acid is an artefact formed by hydrolysis of succinic amide, and that a-ketoglutaric acid is transformed enzymatically to glutamic acid. This process is reversed after the phase of exponential growth of the bacteria. The ratio C 4 - vs. C 5 -acids changes with the culture time and with increasing Fe 3+ content of the medium in favor of the latter

The heterocyst-forming cyanobacterium Anabaena variabilis (ATCC 29413) is deficient in catalase activity, but especially heterocysts are rich in superoxide dismutase and peroxidases. It exhibits a light-dependent methylviologen-catalyzed oxygen uptake which is accompanied by a massive release of hydrogen peroxide. Methylviologen caused a transient concentrationdependent inhibition of nitrogenase ending in a complete loss of activity. Suppression of hydrogen peroxide accumulation by the photosynthesis inhibitor D C M U (diuron, 3-[3,4-dichlorophenyl]- 1, 1-dimethylurea), or by addition of catalase prevented nitrogenase inhibition indicating that methylviologen affects nitrogenase activity via hydrogen peroxide produced by photosynthetic electron transport. Immunospecific Western blotting of cell extracts separated on SDS polyacrylamide gel electrophoresis showed that the transient inhibition of nitrogenase is accompanied by rapid modification of its Fe-protein followed by enzyme destruction. In a cell-free system the enzyme exhibited only moderate sensitivity towards hydrogen peroxide but was found susceptible against activated oxygen species generated by the xanthine/xanthine oxidase system and free iron. The results indicate that H 2 O 2 : itself has little direct effect on nitrogenase, but endogenous peroxide produced by methylviologen in the light may initiate oxygen radical formation leading to nitrogenase modification and subsequently its destruction

Fast protein liquid chromatography on Superose 6 of crude extracts from the chlorophyllfree mutant no. 20 of the unicellular green alga Chlorella kessleri reveals two possibly oligomeric forms of pyruvate kinase (2.7.1.40). Their occurrence is markedly altered in the course of heterotrophic growth with changing levels of exogenous glucose as carbon source with only one enzyme species with a MW of 400 kDa existing in growing cells, two forms of 400 and 580 kDa in resting cells. Substrate affinity towards PEP of the 400 kDa form is better than that of the 580 kDa species; responses to the effector AMP are different as well. In vitro, addition of PEP or of AMP leads to the formation of higher MW enzyme species with MW of 730, 1050 and 1400 kDA without affecting the total activity. In vivo alterations in the levels of several metabolites including PEP upon addition of glucose have been shown to occur. Therefore, it is discussed, whether changes in the concentration of intermediates and effectors may provide the mechanism for the increased rate of carbohydrate degradation by affecting the occurrence and/or ratio of the various PK forms with different kinetic and regulatory properties. Upon blue light irradiation, which also stimulates carbohydrate breakdown of the Chlorella mutant cells, the distribution of PK is shifted towards the species with higher substrate affinity, a result being in accordance with the above conception

The effect of two 5-n-alk(en)ylresorcinol* (17:1 and 17:0) homologs at the concentrations of 5 - 20% on the thermotropic properties of dipalmitoylphosphatidylcholine in hydrated bilayers has been studied. The effect is different, depending on unsaturation of the aliphatic chain of resorcinol derivative. Saturated homologue ( 5 - 2 0 m ol% ) induces disappearance of the pretransition , increase of the main transition temperature and a half-width of the sition peak. Unsaturated homologue shifts pretransition towards higher temperatures, and similarly to the saturated one increases transition half-width but decreases the transition temperature, decreases the transition heat content and induces some phase separation

The interaction of the bipyridylium herbicides diquat and paraquat with the proteins gliadin and human serum album in (HSA) as well as with cellulose and the effect of various ions on the strength of interaction were studied by charge-transfer chromatography. Both compound showed very similar behaviour. The ions decreased in each case the strength of interaction, the effect depended nonlinearly on the ion concentration and was of saturation character. Gliadin showed the weakest interaction. HSA binds the bipyridylium com pounds in an ion-dependent manner, the strength of interaction is higher than that of cellulose at higher ion concentration. This finding indicates an ion-mediated interaction of unknown character between the bipyridylium herbicides and HSA

The recently developed methods of non radioactive in situ hybridization of chromosomes offer new aspects for chromosome analysis. Fluorescent labelling of hybridized chromosomes or chromosomal subregions allows to facilitate considerably the detection of specific chromosomal abnormalities. Formany biomedical applications (e.g. biological dosimetry in the low dose range), a fast scoring for aberrations (e.g. dicentrics or translocations) in required. Here, we present an approach depending on fluorescence in situ hybridization of isolated suspension chromosomes that indicates the feasibility of a rapid screening for specific chromosomes or translocations by slit scan flow cytometry. Chromosomes of a Chinese hamster x human hybrid cell line were hybridized in suspension with biotinylated human genomic DNA . This DNA was decorated with FITC by a double antibody system against biotin. For flow cytometry the chromosomes were stabilized with ethanol and counterstained with DAPI or propidium iodide (PI). An experimental data set of several hundred double profiles was obtained by two parameter slit scan flow cytometry and evaluated automatically. The evaluation algorithm developed allowed a classification of chromosomes according to the number of centromeres and their chromosomal positions in less than 1 msec per individual profile. Approximately 20% of the measured DAPI profiles showed a bimodal distribution with a significant centromeric dip indicating a “ normal” chromosomal morphology and a correct alignment in the flow system. In many cases, profiles of a “normal” bimodal fluorescence distribution of the DNA stain (DAPI, PI) were correlated with a “ normal” FITC profile. Due to their centromeric indices these profiles agreed well to the expected human chromosomes of the cell line. In some cases of “ normal” DAPI (PI) profiles, “aberrant” FITC profiles were observed. These were interpreted as interspecies translocation chromosomes. For all results, there was a good agreement between flow cytometry and microscopic observations (digital image analysis)

A novel enzyme inhibitor of RNA -directed DNA -polym erases of avian myeloblastosis and murine leukemia virus was isolated from fermentations of an tasmanian Podoscypha species. Its structure was elucidated by spectroscopic methods and oxidative degradation as (E)-4,5- dioxo-2-hexadecenoic acid (1). The enzyme inhibitor, which was named podoscyphic acid, did not inhibit DNA and RNA synthesis in permeabilized L 1210 cells nor did it affect RNA synthesis in isolated nuclei of L 1210 cells. 1 inhibits protein synthesis in whole L 1210 cells and rabbit reticulocyte lysate and shows very weak antimicrobial and cytotoxic properties. The testing of ethyl (E) 4,5-dioxo-2-hexadecenoate (2) and (E)-4-oxo-2-tetradecenoic acid (11) revealed the importance of the free y-oxoacrylic acid unit for the biological activities of 1

The response of fly photoreceptors to light stimuli of high intensity consists of a fast transient depolarization caused by the activation of light-dependent channels and a delayed after depolarization. The after depolarization was reversibly reduced or abolished when the extracellular Na + had been replaced by Li + , tris or choline. Na + specificity, the dependence on the concentrations of Na + and Ca 2+ as well as the polarity of the effect suggest that electrogenic Na + - Ca 2+ exchange contributes to the after depolarization. Prolonged incubation in Na + -free media also led to the reversible suppression of the fast response component, which is most probably due to an increase in the intracellular Ca 2+ concentration caused by the inhibition of Na + -Ca 2+ exchange. Evidence is presented that the Na + - Ca 2+ exchange system in fly photo receptors also transports Sr 2+

Bumps, the elementary excitatory events of the Limulus ventral nerve photo receptor following a weak flash of light were recorded under voltage clamp conditions. The statistical distribution of various bump parameters and their changes caused by weak conditioning pre-illumination are described, and the influence of lowered external Ca 2+ -concentration together with normal or raised Mg 2+ -concentration (15 °C). 1) Weak conditioning pre-illumination causes desensitization: the bump current amplitude, bump duration , bump area (current-integral), and the bump latency are diminished, the more, the stronger the conditioning flash, i.e. the light adaptation. Very weak conditioning pre-illumination causes facilitation, expressed by an increase in number and size of the observed bumps. The average bump latency, however, is already shortened under these conditions. 2) Lowering the external Ca 2+ -concentration from 10 mmol/l to 250 (µmol/1 has its primary effect on the dark -adapted photoreceptor (without substantially reducing the ability for light adaptation ). It causes the following average changes: the amplitudes, durations, current-integrals, and the latencies of current bumps are greatly enlarged and the number of bumps is raised. 3) Raised magnesium concentration from 50 to 100 mmol/l can partially compensate for the lack of calcium ; however, it enhances the effect of calcium deficiency on the latency, i.e. it further enlarges the average latencies. The results can be explained on the basis of our model of bump generation by two assumptions. 1) Lowering the external calcium concentration causes a decrease in the cytosolic Ca 2+ -level without substantially reducing the intracellular calcium stores from which the light-adapting calcium release is fed. The lowered cytosolic Ca 2+ -concentration induces an “extra” dark adaptation resulting in greater bumps and more bumps exceeding the threshold of recognition. The bump latency, however, which behaves differently from all other bump parameters, is determined by a separate calcium -dependent reaction where magnesium competes with calcium antagonistically. 2) Facilitation is due to cooperativity of transmitter binding in order to open the ion channels

Shape deformation of the thin, area non-compressible spherical shell separating two different media, subjected to an external, alternating, homogeneous electric field is considered. The function relating shape deformation of the shell to the shear stress in its plane is presented. Shear stress develops as a result of Maxwell stress acting on both shell surfaces. All media are considered as lossy. The theoretical model predicts that the shear stress reaches its maximal value at the shell “equator” in relation to the field direction. The magnitude of the shear stress depends on the strength and frequency of the electric field, on the geometric parameters of the shell, and on the electric parameters of the system studied. The foregoing was applied to an analysis of cell deformation, e.g., an erythrocyte in an electric field. The analysis predicts that the maximal shear stress in the membrane develops at field frequencies close tof = 10 7 Hz. This is comparable to the electrocompression stress of the same membrane, maximal at the cell “pole”. At lower frequencies, compression predominates over shear stress

Bud Exudates of three Betulaceae were analyzed for minor flavonoid aglycones. Three flavonoids not previously reported were found in Alnus koehnei, four in Betula nigra. A novel flavonol, morin-3,2′,4′-trimethyl ether, is described for Ostrya carpinifolia

A new method for the covalent modification of polymers in a two-step process is described. Squaric acid diethylester reacts with primary or secondary aliphatic amines selectively to ester amides. It is shown for the first time that this class of compounds react with polymeric systems containing amine functionalities (e.g. ε-amino groups of proteins) in aqueous solution, forming stable squaric acid diamide linkages. Three different monoamides were synthesized and the reaction rates of diamide formation versus hydrolysis were determined. Detailed kinetic studies revealed that unwanted hydrolytic side-reactions are more than ten times slower than the formation of squaric acid diamides

The effect of mixtures of air pollutants (SO 2 , NO 2 , O 3 ) on phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) activities from needles of four-year-old Norway spruce (Picea abies [L.] Karst.) in closed fumigation chambers were analyzed. Three months exposure to high concentrations of O 3 (1030 µg/m 3 ) together with SO 2 (1030 µg/m 3 ) or NO 2 (615 µg/m 3 ) resulted in a dramatic increase in PEPCase activities