Volume 43, Issue 7-8

From cell suspension cultures of Rauwolfia serpentina grown in an optimized production medium for the glucoalkaloid raucaffricine, a novel glucoalkaloid was isolated and identified as 17-O-acetyl-21-O-β-ᴅ-glucopyranosyl-ajmaline (acetylrauglucine). This alkaloid is formed in very small amounts (< 5 × 10 -4 %). The biogenetically related N α -demethylated base (acetyl-nor-rauglucine) and the deacetyl product rauglucine have also been detected in culture extracts. In addition 21(.R)-(β-D-glucopyranosyl)-hydroxy-sarpagan-17-al has been isolated and identified as an artifact which originates from raucaffricine.

Plant cell cultures effectively convert terpenoid and aromatic alcohols into glycosides. A quantitative comparison showed a culture of peppermint (Mentha piperita) to be the most active among four species examined. The synthesis of glycosides was affected by the concentration of substrate, incubation period, cell age and aggregation, and by light. Conversion rates of exogenous alcohols were greater than 70% under optimized conditions.

1. A highly purified sporopollenin fraction from Corylus avellana pollen was obtained using a gentle method employing hydrolyzing enzymes (pronase, lipase, cellulase, amylase, cellulysin) and an exhaustive extraction using different solvents. 2. The sporopollenin fractions were degraded by potash-fusion and nitrobenzene oxidation and the low molecular decomposition products were analyzed by TLC and HPLC. The investigation centered solely on the proof of phenolic compounds. 3. The degradation by potash-fusion yielded p-hydroxybenzoic acid as a main component, while the degradation by nitrobenzene oxidation (NBO) resulted in the formation of p-hydroxybenzaldehyde as the main component. In addition phenolic components such as p-coumaric acid, ferulic acid, vanillin and vanillic acid were formed to different degrees by using NBO as degradation procedure. 4. A comparison of the products formed following degradation of Corylus sporopollenin and “synthetic sporopollenin” shows, that phenolic compounds, if they indeed occurred as degradation products of “synthetic sporopollenin”, are generated only in extremely small quantities. It appears that, in contrast to several reports in the literature, phenols are integral constituents of natural sporopollenin. This view is supported by unpublished tracer experiments.

Rosmarinic acid biosynthesis can be stimulated in cell cultures of Coleus blumei by culturing the cells in medium with 4% sucrose. In enzyme extracts of these cells two new enzymes of rosmarinic acid biosynthesis were discovered. Hydroxyphenylpyruvate reductase reduces 4-hydroxyphenylpyruvate and 3,4-dihydroxyphenylpyruvate to 4-hydroxyphenyllactate and 3,4-dihydroxyphenyllactate, respectively, using NADH. Rosmarinic acid synthase transfers the caffeoyl moiety of caffeoyl-CoA to the non-phenolic OH-group of 3,4-dihydroxyphenyllactic acid, in course of which rosmarinic acid is formed.

Seeds of two Wrightia species, i.e. W. tinctoria and W. coccinea, synthesize triacylglycerols containing isoricinoleoyl (9-hydroxy-cis-12-octadecenoyl, 9-OH-Δ 12 18:1) moieties as the major lipid constituents. Seed maturation is accompanied by lipid accumulation and steep increase in the level of 9-OH-Δ 12 18:1 in the total lipids, while relative proportions of palmitic (16:0), oleic (18:1), and linoleic (18:2) acids decrease. 9-Hydroxy stearic acid (9-OH-18:0) is also detected in these seed lipids, and both 9-OH-Δ 12 18:1 and 9-OH-18:0 occur exclusively in the neutral lipids of mature and developing seeds. Developing seeds of both Wrightia species synthesize upon incubation with sodium [1- 14 C]acetate, besides common long-chain fatty acids, considerable proportions of 9-OH-Δ 12 18:1 and 9-OH-18:0, which are predominantly esterified in triacylglycerols. Substantial proportions of radiolabeled 9-OH-Δ 12 18:1 and 9-OH-18:0 are also formed upon anaerobic incubation of the developing seeds with the ammonium salts of [1- 14 C]18:2 and [1- 14 C]18:1, respectively. The data presented suggest that hydration of the Ay olefinic bond of 18:2 is a possible pathway of synthesis of 9-OH-Δ 12 18:1, however, hydration of the Δ 9 olefinic bond of 18:1 followed by Δ 12 desaturation of 9-OH-18:0 cannot be ruled out.

The content of tryptamine derivatives in Psilocybe semilanceata, a popular hallucinogenic mushroom, was measured by high-performance liquid chromatography. Most of the 52 samples have been collected at several localities in Switzerland during a 1-5 year period. The content of psilocybin and baeocystin varied in the range of 0.21-2.02% and 0.05-0.77%, respectively, whereas only traces of psilocin were present. The variability of the alkaloid level depending on origin, year of collection, size and part of mushrooms is discussed.

Chlorophyll a is hydroxylated quantitatively in the 13 2 -position, when chromatographed on silica gel thin-layer plates. This was shown by using HPLC as a non-destructive method für analyzing photosynthetic pigments. The hydroxy-group of Chl RC I, a 13 2 -hydroxy-20-chloro-chl a. described by Dörnemann and Senger (1986) and Scheer et al. (1986), is shown to be artificially introduced during the purification procedure by TLC.

The synthesis of 2-(E)-O-(4-hydroxy-cinnamoyl)-ᴅ,ʟ-galactaric acid (1a) is described. Starting with free galactaric acid (2) and 4-acetoxy-cinnamoyl-chloride (3), these compounds were esterified under regioselective conditions to racemic 2-(E)-O-(4-acetoxy-cinnamoyl)-galactaric acid (4). After deprotection of the p-coumaroyl moiety 1a was obtained as a photo-unstable product. Optical resolution of racemate l a on bovine serum albumin covalently bound to silica resulted in the isolation of the pure optical isomers. The (+)-dextrorotatory synthetic enantiomer l a proved to be identical with the natural factor.

Cell suspension cultures of two chickpea (Cicer arietinum L.) cultivars, resistant and susceptible towards the chickpea pathogen Ascochyta rabiei, were compared with regard to elicitor-induced changes in phytoalexin and isoflavone accumulation. The elicitor was isolated from fermentergrown mycelium of A. rabiei and it mainly consisted of glucose, mannose and N-acetylgalactosamin. Time course and dose response studies on elicitor action demonstrated that the cell culture of the resistant cultivar ILC 3279 accumulated large amounts of the pterocarpan phytoalexins medicarpin and maackiain within 8 h. The cell culture of the susceptible cultivar ILC 1929 accumulated only small amounts of the phytoalexins some 12 h after elicitor treatment. Growth of the cell cultures and the accumulation of isoflavones and isoflavone conjugates were not altered by elicitor treatment except for a higher accumulation of formononetin 7-O-glucoside-6″-O-malonate in cell culture ILC 3279 subsequent to the maximum of phytoalexin accumulation.

The extractable activities of thirteen enzymes of primary and secondary metabolism have been measured in chickpea (Cicer arietinum L.) cell suspension cultures after treatment with an elicitor from the fungus Ascochyta rabiei (Pass.) Lab. The cell culture, derived from the A. rabiei resistant cultivar ILC 3279, constitutively accumulated the isoflavones biochanin A and formononetin together with their 7-O-glucosides and the 7-O-glucoside-6″-malonates. After elicitor application the cells rapidly form the pterocarpan phytoalexins medicarpin and maackiain. Among the enzymes of primary metabolism only the glucose 6-phosphate dehydrogenase exhibited a significant increase in activity with a maximum four hours after application of the elicitor. In phenylpropane metabolism the activities of phenylalanine ammonia lyase and chalcone synthase were enhanced by the elicitor and exhibited highest levels after four hours. In contrast the chalcone isomerase activity was not influenced by the elicitor. A substantial enhancement occurred with the isoflavone 7-O-glucosyltransferase activity eight hours after elicitor application. The results suggest that in this cell culture the elicitor-induced biosynthesis of pterocarpan phytoalexins was accompanied with a rapid and transient increase of those enzyme activities which are located at branching points of related pathways, i.e. pentose phosphate cycle, general phenylpropane metabolism, flavonoid formation and isoflavone conjugation.

The absorption spectrum of the previously purified ʟ-amino acid oxidase from the cyanobacterium Anacystis nidulans has shown considerable variation with each preparation and the spectrum in several preparations was quite different from the absorption spectrum of other simple flavoproteins (E. K. Pistorius and A. E. Gau, Biochim. Biophys. Acta 849, 203, 1986). Here we show that the spectral complexity and variability of the ʟ-amino acid oxidase can be largely explained by the presence of a modified flavin derivative of yet unknown structure besides oxidized FAD and FAD semiquinone. After removal from the enzyme this modified chromophore has absorption maxima at 260, 396 and in the 600 nm region. This derivative of FAD seems to be formed in variable amounts during the purification of the enzyme. On the other hand, extraction of Anacystis photosystem II complexes which contain the flavoprotein, almost exclusively yields modified flavin derivatives and practically no authentic oxidized FAD. The spectrum of the chromophores which have been extracted from photosystem II complexes at different purification stages, is either similar (although not identical) to the spectrum of the chromophore extracted from the isolated ʟ-amino acid oxidase or similar to the spectrum of reduced flavin. All extracted chromophores show a fluorescence emission in the 420 to 560 nm region when excited with light of 390 nm. These results indicate that the flavin present in the ʟ-amino acid oxidase protein as well as in photosystem II complexes from A. nidulans rapidly undergoes modification reactions of yet unknown nature to yield several closely related FAD derivatives. This might possibly be the reason why so far no flavin has been detected in photosystem II. The presence of such modified flavin derivatives in photosystem II complexes of A. nidulans as shown here is an additional support of our hypothesis that an unusual flavin is functional on the donor side of photosystem II.

By means of the immunochemical methods of double immuno diffusion and tandem crossed immuno electrophoresis we have compared the bifunctional enzyme RuBP carboxylase/oxygenase from tobacco mutants which differ with respect to their rates of photosynthesis and photorespiration. The comparative studies were carried out with a monospecific antiserum to the enzyme of the wild type Nicotiana tabacum var. John William’s Broadleaf. RuBP carboxylase/oxygenase from the green, yellow-green and yellow phenotype of the mutants namely N. tabacum Su/su, N. tabacum Su/su var. Aurea, N. tabacum var. Consolation, N. tabacum var. NC 95 and N. tabacum Xanthi (D 523) are immunochemical^ identical to the enzyme of the wild type N. tabacum var. John William’s Broadleaf. Furthermore, immunochemical identity of the RuBP carboxylase/oxygenase exists between Nicotiana tabacum and other representatives of the Solanaceae such as Solanum tuberosum, S. lycopersicum and Datura suaveolens. In contrast to this only partial identity to the enzymes of the C 3 -plants Antirrhinum majus, Spinacia oleracea, Sinapsis alba, Petroselinum crispum, Allium porrum, Hordeum vulgare, Avena sativa to the enzymes of the green alga Chlorella vulgaris and to the blue-green alga Oscillatoria chalybea as well as to the enzyme of the C 4 -plant Zea mays is observed.

In the present paper the purification of a specific 4,5-dioxovalerate transaminase from pigment mutant C-2 A′ of the unicellular green alga Scenedesmus obliquus to apparent homogeneity is described. The newly isolated enzyme ʟ-glutamate: 4,5-dioxovalerate aminotransferase is not identical with ʟ-alanine: 4,5-dioxovalerate aminotransferase (EC 2.6.1.43) and ʟ-alanine: glyoxylate aminotransferase (EC 2.6.1.44). A procedure for the purification is described and the resulting homogeneous protein is characterized by its K ᴍ -values for oxo-substrates and amino donors, its pyridoxal phosphate requirement, reversability of the catalysis, pH-optimum, isoelectric point and its molecular weight.

The polynucleotides poly(U), poly(C), poly(A) and poly(G) have been y-irradiated in N 2 O- and N 2 O/O 2 (4:1)-saturated aqueous solutions. Hydroxyl radicals from the radiolysis of water react with the polynucleotides thereby producing among other lesions strand breaks. Strand breakage is connected with the formation of phosphomonoester end groups. Such end groups have been determined by measuring inorganic phosphate after a three hour incubation at 37 °C with acid or alkaline phosphatase. In the absence of oxygen G(phosphomonoester end groups) (in units of μmol J -1 ) are 0.47 (poly(U)), 0.17 (poly(C)) and ≤ 0.04 (poly(A) and poly(G)). In the case of poly(U) and poly(C) on heating the sample for one hour at 95 °C prior to incubation with phosphatases the above values increased by 0.14 and 0.07 μmol J -1 , resp., whereas such treatment of the purine polynucleotides still did not produce a measurable yield of phosphomonoester end groups. Comparing these values with G values for strand breakage taken from the literature, about two phosphomonoester end groups are formed per strand break in poly(U) while for poly(C) this ratio is about unity. The purine polynucleotides show very low yields of strand breakage in agreement with the negligible phosphomonoester yields. In the presence of oxygen G(phosphomonoester end groups) are 0.46 (poly(U)), 0.21 (poly(C)), and ≤ 0.04 (poly(A) and poly(G)). On heating, these values increase, most markedly for poly(U) and poly(C). This is possibly linked to the decomposition of unstable hydroperoxides which are formed in high yields in poly(U) and poly(C) (G = 0.7 and 0.19 μmol J -1 resp.). It is known that at least in the case of poly(U), base radicals attack a sugar moiety and are the main precursors of these lesions. G(phosphomonoester end groups) are considerably lower in the case of the purine polynucleotides. Whether this is due to an inability of the base radicals to attack a sugar moiety or has other reasons must remain an open question.

The distribution of isomers of β-carotene is reported in the CP-47 pigment-protein complex of photosystem II and photosystem I (PS I) from barley. The distribution of carotenoids from light harvester complex I I is reported. CP-47 lacks 9,9′-rá-carotene, but contains 9,13′-cis-carotene; PS I lacks 9,13′-cis but contains 9,9′-eis. More 9-cis, 9,13-cis, 9,15-cis, and epoxide is observed in CP-47 than in PS I.

The investigations described here were carried out in the context of our research project on the physiological, biochemical, and cytomorphological characterization of spruce trees growing in natural habitats and showing damage of varying intensity. Here we report on specific aspects of the photosynthetic apparatus. The aim of the measurements was to analyze whether or not the activity of the photosynthetic electron transport pathway is affected in damaged trees. The investigations were carried out on a 20 to 25-year-old spruce plantation in the Hunsrück mountains and on an 80-year-old spruce plantation in the Westerwald mountains. The photosynthetic electron transport rate was determined by photoreduction of 2,6-dichlorophenolindophenol. A decrease of the electron transport rate was shown in the damaged spruce trees in comparison to the apparently healthy trees. The investigation of the water splitting enzyme system - determined in the Hillreaction by feeding in electrons by means of diphenylcarbazide - indicates that the electron transport on the oxidizing side of photosystem II is impaired. The results imply that the photosynthetic electron transport chains in the thylakoid membranes of the spruce chloroplasts are sites of early injurious effects. This is in agreement with the electron microscopic analyses which show consistently that early damage occurs especially at the cellular membranes. This membrane damage is apparent even in the green needles of damaged spruce trees.

In order to obtain an insight into the damage of thylakoid membranes of spruce (Picea abies) trees with damage of varying intensity, investigations were performed on the content of Q в -protein, cytochrome f, and P-700 in chloroplasts of spruce needles from apparently healthy and from damaged trees. Needles from the second and third needle year and the seventh whorl were chosen. The investigations were carried out in 1986 on a 20 to 25-year-old spruce plantation in the Hunsriick mountains and on an 80-year-old spruce plantation in the Westerwald mountains. In damaged trees an unequivocal decrease in the content of Q в -protein, cytochrome f, and P-700 was found, even in needle groups that appear visibly green and healthy. The amount of cytochrome f decreased by 25% per dry weight (approximately to the same extent as chlorophyll); the content of Q в -protein and P-700, however, were more drastically reduced compared to the control trees (about 40% and 50%, respectively). These results of measuring the photosynthetic electron transport components imply that the thylakoid membranes are sites of early injurious effects.

The thermoluminescence and delayed luminescence characteristics of PS II α and PS II β centers were investigated in BBY particles and stroma thylakoids, respectively. The BBY particles exhibited a thermoluminescence band at 25 °C (B band) which was associated with the charge recombination of the S 2 Q в - redox couple and underwent period-2 oscillation in a sequence of flashes. In the flash-induced decay of delayed luminescence of BBY particles a component with a half-time of 34 s corresponded to the B thermoluminescence band and was also assigned to S 2 O в - charge recombination. No corresponding thermoluminescence or delayed luminescence components associated with the secondary acceptor Q в could be observed in the glow curve or delayed luminescence decay of stroma thylakoids. These observations indicate that unlike PS II α the PS II β centers are not associated with the two-electron gate, Q в .

In order to study the structure-activity relationships of bis(guanylhydrazone) type polyamine antimetabolites, trifluoromethylglyoxal bis(guanylhydrazone) (CF 3 -GBG), a close analog of the antileukemic drug methylglyoxal bis(guanylhydrazone) (mitoguazone. MGBG) was synthesized according to a novel modification of previous methods, yielding single crystals. Single-crystal X-ray crystallography revealed the presence of an isomer different from the one detected in the case of MGBG and all other bis(guanylhydrazones) so far studied. In contrast to MGBG, CF 3 -GBG was shown to be a very weak inhibitor of yeast adenosylmethionine decarboxylase, being thus devoid of value as a polyamine antimetabolite. In addition, the compound did not have antiproliferative activity against mouse L1210 leukemia cells in vitro. As long as analogous isomers of the two compounds are not available, no conclusions can be drawn about the reasons lying behind the drastical differences between their biological properties.

A procedure for silver staining is described which leads to the selective and reliable impregnation of nerve fibers in bleached skin of vertebrates and invertebrates. In combination with osmium, the protocol enhances the staining of secondary sensory cells of mechanosensory and electrosensory organs so that the innervation pattern of each organ and the number of sensory cells per organ can easily be evaluated. The technique can be also used for staining nerve fibers in whole embryos.

In this study, partially purified K + -H + ATPase from frog gastric mucosa were obtained by using differential and density gradient centrifugation. Optimum activity of K + -H + ATPase (V max ), Michealis-Menten constant (K m ) and Hill coefficient (h) were found as 83.3 μmol Pi·mg prot -1 h -1 , 95.2 μᴍ and 0.91, respectively. Enzyme preparations were more stable in glycerol solutions stored at -40 °C. Minimum activity lost was determined for samples stored in 40% (v/v) glycerol solution at -40 °C for two months.

A vital chemical communication can be observed in the Tunesian desert isopod Hemilepistus reaumuri. These crustaceans live in social units which are strictly closed to alien conspecifics. It was attempted to analyze the highly evolved family-specific recognition system by identifying their pheromonal compounds both from the extracts of surface washings of intact animals and from the exuvia.

A recent model of celestial e-vector analysis by the bee assumes that polarization information is transformed into modulation of perceived brightness while the bee scans the sky by rotating its field of view. It is shown that the suggested simple strategy to read compass information from the polarization pattern of the sky in natural conditions can work only in a part of the sky close to the zenith. The bee would need a different strategy for other regions of the sky.