Band 42, Heft 9-10

In the Bryozoan Conopeum seuratum six esterified monoterpene alcohols, three free monoter-pene alcohols, three monoterpene ketones, two monoterpene aldehydes, four esters of diterpene acids, two diterpene acids and one triterpene acid were identified, most of them new for marine organisms. Some non-terpenoid compounds were identified too. Preliminary investigation on the terpenoids of the Black Sea sponge Halichondria panicea was performed and its terpenoid composition was compared with those of C. seuratum.

The phosphonolipids from whole Cicada oni have been isolated by preparative thin-layer chromatography (TLC) in methanol-water (2:1, v/v) and have been identified by TLC, infra-red spectroscopy, phosphono phosphorus determinations and elemental microanalyses. The presence has been confirmed of the phosphono analogues of phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin and of ceramide aminoethyl phosphonate.

Anabasine is the major volatile product in the poison gland exudate of Messor ebeninus, acting as a defensive compound. Exudates of the poison gland also contain minor, yet unidentified, components that are possibly responsible for the alarm behavior that is also elicited by the venom. Dufour’s gland secretion is characterized by aliphatic hydrocarbons of which 1-pentadecene predominates. Upon exposure to Dufour’s gland secretion the ants recruited to the emitting source, but did not exhibit any aggressive behavior. The possible concordant effects of both adnexal glands secretions is discussed.

The bud exudates of Populus nigra, P. trichocarpa, and P. grandidentata × tremuloides were analyzed for the presence of phenolics and flavonoids. A novel natural product, caffeic acid y.y-dimethylallyl ester, was identified from P. nigra, along with further phenolics such as ρ-hydroxy-acetophenone, dimethyl caffeic acid, cinnamoyl cinnamata and vanillin. The flavonoid aglycones correspond to those reported earlier. Propolis samples from the Sonoran Desert were shown to exhibit the flavonoid pattern that is typical for poplar bud exudates, thus confirming this material as the major source for bee-glue. Only a single flavonoid might originate from the leaf exudate of a desert shrub.

Chain elongation of the methyl esters of linoleic and linolenic acids by one, two, or three methylene groups followed by elimination of the functional groups leads to (Z,Z)-6,9-alkadienes and (Z,Z,Z)-3,6,9-alkatrienes that function as pheromones in various insects. All of the simple reactions involved proceed in excellent yield, they do not require expensive reagents, high pressure or high temperature. The route of synthesis is applicable on a large scale. Rigorous analyses prove the purity of the hydrocarbons prepared.

1 H and 13 C NMR spectra are documented for the novel flavone-6,8-di-C-glycosides isolated from the liverwort, Apometzgeria pubescens. The existence of rotational isomerism evident from these spectra is shown to be general for 8-C-monohexosylflavones and of possible diagnostic value.

Chemical analysis of the culture filtrates of Fusarium culmorum CMI 14764 has demonstrated the presence of seven trichothecene mycotoxins. Major metabolites are 3-acetyldeoxynivalenol and 7α,8α-dihydroxycalonectrin. with 3,15-diacetyldeoxynivalenol, deoxynivalenol. calonectrin, isotrichodermin and 12,13-epoxytrichothec-9-ene (EPT) as minor products. The occurrence of the rarely encountered unsubstituted trichothecene EPT is significant in that this compound may function as a common intermediate in the biosynthetic pathways to all natural trichothecenes. The structures of the known trichothecenes isolated from F. culmorum suggest a route in which EPT is sequentially oxygenated to the more complex deoxynivalenol derivatives.

When incubated in liquid culture with Aspergillus flavus, the prenylated pterocarpan (-)-edunol [2-(3,3-dimethylallyl)-3-hydroxy-8,9-methylenedioxypterocarpan (1)] was converted into a dihydrofurano-pterocarpan (2), a dihydropyrano-pterocarpan (3). and a 2,3-dihydro- dihydroxyprenyl-substituted pterocarpan (4).

The prenylated isoflavone, 2,3-dehydrokievitone [5,7,2′,4′-tetrahydroxy-8-(3,3-dimethylallyl)- isoflavone, 1], was metabolized by both Aspergillus flavus and Botrytis cinerea to yield the same three compounds, a dihydrofurano-isoflavone (DK-M1, 2), a dihydropyrano-isoflavone (DK- M2, 3), and a glycol (DK-M3, 4). Although structure 3 has previously been assigned to lunatone, an isoflavone from CuCl 2 -stressed lima bean (Phaseolus lunatus) seedlings, a comparison of spectroscopic (MS, 1 H NMR) data indicated that the Phaseolus compound was not identical with metabolite DK-M2 found in Aspergillus and Botrytis cultures. Evidence is presented to suggest that lunatone is probably identical with the furano-substituted metabolite DK-M 1 (2).

The major constituent of the yellow frond exudate of the fern Notholaena sulphured was identified by spectroscopic methods as 3,5,2′-trihydroxy-7-methoxy-8-acetoxy flavone and its structure was confirmed by synthesis. This novel natural flavonoid was also detected in the frond exudate of five other Notholaena species. In the yellow form of N. sulphurea, the rare 5,2′- dihydroxy-7,8-dimethoxy flavone was also found, along with some trivial flavonoids. The white form of N. sulphurea produces three dihydrochalcones that are accompanied by some kaempferol methyl ethers and apigenin-7-methyl ether. The 3-acetoxy as well as the 3-butyroxy and the 4′- butyroxy derivatives of 7-methyl aromadendrin were also identified in this material. One of them was shown to belong to the extremely rare group of 3-cis-dihydroflavonols.

Piperoyl-CoA, a potential intermediate in the biosynthesis of piperine. the pungent principle of pepper, was synthesized via N-succinimidyl piperate and subsequent transesterification with CoA- SH. UV-spectrophotometry of the HPLC-purified compound revealed, besides a maximum at 260 nm, a pronounced second absorption maximum at 368 nm due to the thioester linkage for which, as determined via the Fe 3+ -complex of piperoyl hydroxamate and with [ 14 C]piperoyl-CoA, a molar extinction coefficient of 30.8 × 10 6 [cn 2 mol -1 ] was calculated.

The activity of geraniol- 10-hydroxylase was greatly increased when Catharanthus roseus cells were transferred to a production medium for monoterpene indole alkaloids. Its activity pattern showed a closer relationship than tryptophan decarboxylase to the pattern of indole alkaloid accumulation. The intermediate accumulation of tryptamine and its later incorporation into ajmalicine indicated that the coordination of the two precursor pathways for the monoterpene indole alkaloid formation was not synchronized under the chosen culture conditions. A better tuning of these two pathways may shorten the time period for induction of indole alkaloid biosynthesis.

Not less than 1 nmol of methionine and phenylalanine were detected as fluorescent spots on agar plates with ophthalaldehyde reagent under UV light. Microorganisms were grown on thin- agar-layer coated filter papers placed on nutrient agar plates, and then transfered onto new plates lacking nutrients by moving the papers. After the background amino compounds were removed by the diffusion to the bottom plates, the paper cultures were moved and incubated on assay plates containing N-acetylmethionine or N-acetylphenylalanine. The amino acids formed around the colonies were visualized by the ophthalaldehyde procedure to indicate aminoacylase activity of microorganisms. The substrate- and stereospecificity of the enzyme was shown for some strains on the agar plates by this procedure.

Small angle X-ray scattering studies on ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) from spinach reveal a configurational change in its quaternary structure upon the transition of the molecule from the activated form occurring in the presence of CO 2 and Mg 2+ to the deactivated form obtained when CO 2 and Mg 2+ are removed by extensive dialysis under nitrogen. Present structural models are comparable to models which were postulated previously for the same enzyme but isolated from the hydrogen bacterium Alcaligenes eutrophus [O. Meisen- berger, I. Pilz, B. Bowien, G. P. Pal, and W. Saenger, J. Biol. Chem. 259, 4463-4465 (1984)]. The radius of gyration is R = 47.5 ±0.2 nm for the active spinach Rubisco. Upon deactivation, R changes to 49.2 ± 0.2 nm, suggesting a more elongated quaternary structure. The observed differ­ence in deactivation behaviour in ambient and in nitrogen atmosphere indicates a higher affinity of this spinach enzyme to CO 2 with respect to the same enzyme from Alcaligenes eutrophus.

The content of starch, malate, potassium and the activity of phospho-enolpyruvate carboxylase (PEPC) were analyzed by quantitative histochemistry in mesophyll cells of different zones along the axis of lyophylized primary elongating leaves of barley. The concentrations of potassium and malate were low in the region of the intercalary meristem (close to the point of grain attachment), but concentrations of solutes increased abruptly and stoichiometrically (equivalent basis) in the elongation zone (2 to 10 mm from the leaf base), where they contributed approximately -0.17 megapascal to the solute potential. Although this solute concentration represents only a minor portion of the reported solute potential, the specific nature of the change, its correlation with a decrease of starch content, and the distribution of the activity of PEPC indicate cell expansion in barley could be augmented by a potassium malate osmoregulatory system that utilizes starch as a source of carbon skeletons.

Biotransformation of citral (1) was studied with four strains of Botrytis cinerea using grape must (A), a synthetic medium (B) and mixtures of A and B. Whereas in A complete metabolization of 1 without evidence of any volatile product was observed, in B nerol (2) and geraniol (3) were found as predominant volatile bioconversion products; in minor amounts (E,Z)-2,6-dimethylocta- 2,6-dien-1,8-diol (4) and (E,E)-2,6-dimethylocta-2,6-dien-1,8-diol (5). 2-methyl-2-hepten-6-one (6), 2-methyl-2-hepten-6-ol (7), 2-methyl-2-hepten-6-one-1-ol (8) and 2-methyl-y-butyrolactone (9) were identified. Using small amounts of A in B (1:700 to 5:700) low yields of 2 and 3 were obtained, whereas the quantities of 4, 5. 6 and 8 increased. Quantitatively, the results were strongly dependent on the strains used. The bioconversion products were all identified by capil­lary gas chromatography (HRGC) and coupled HRGC techniques, i.e. -mass spectrometry (HRGC-MS) and -Fourier transform infrared spectroscopy (HRGC-FTIR).

The cell line CP-3 of Catharanthus roseus produces low levels of indole alkaloids during station­ary phase of growth, but increased levels when transferred to growth-limiting production media. Highest specific yields of alkaloids were usually achieved 8-15 days after transfer of the cells to the production medium. We attempted to reduce the time required for alkaloid production by altering preculture conditions, and by adding an elicitor preparation known to stimulate alkaloid accumulation in other cells. Reduction of 2,4-D in the last growth medium before transfer to production medium and a high initial inoculum load into the production medium resulted in rapid and greatest alkaloid accumulation (up to 2 mg ajmalicine/g dry mass or 40 mg/1 within 7 days). The addition of elicitor to the cell suspension stimulated high levels of tryptamine biosynthesis and accumulation under all culture conditions. Additionally, two cell lines derived from CP-3 cells and maintained on NAA/kinetin containing medium were selected for rapid growth in the absence of 2,4-D and were characterized with respect to their ajmalicine production. At a high inoculum load these cells produced up to 1.3 mg/g or 20 mg ajmalicine/1 when cultivated on the growth medium. The biotechnological utility of such cell lines is discussed.

Determination of chloride contents in aqueously isolated chloroplasts from spinach by amperometric titration indicated chloride concentrations of 60 to 100 mmol 1 -1 (based on a chloroplast volume of 25 μl mg -1 chlorophyll). However, when analyzed by anion chromatography, chloride contents in chloroplasts were much lower (1-8 mmol 1 -1 ). In leaf extracts chloride concentrations obtained with both methods were rather similar. Boiling of chloroplast extracts prior to chloride titration reduced indicated chloride contents by a factor of three or four, but did not change results obtained with anion chromatography. It is concluded that chloroplasts contain large amounts of silver complexing agents giving rise to a drastic overestima­tion of chloroplast chloride concentrations when measured by amperometric titration. Boiling and centrifugation of extracts apparently precipitates these compounds only insufficiently.

Activities of acetyl-CoA synthetase (EC 6.2.1.1), adenylate kinase (EC 2.7.4.3) and glycerate kinase (EC 2.7.1.31) were found in leaf extracts of iojap mutant maize (Zea mays) which lacks the protein-synthesizing apparatus in plastids. These activities represented at least one-half of the rates observed for normal maize plants. Since the proteins studied are localized either preferen­tially (adenylate kinase) or exclusively (acetyl-CoA synthetase, glycerate kinase) in chloroplasts, the high activities seen for iojap mutant can not be attributed to some outerchloroplastic isoforms of these enzymes. The data indicate that in maize the three proteins are synthesized by an outerchloroplastic translation system.

Triethyl lead (Et 3 Pb + ), a highly toxic oxidation product of the anti-knock agent tetraethyl lead (Et 4 Pb) was shown to act as anion (Cl - /OH - ) antiporter in plant membranes, dissipating energy- dependent ion gradients, membrane potentials, and consequently turgor. This mechanism was demonstrated with tonoplast-type vesicles isolated from coleoptiles of Zea mays L. The ATP- driven H + accumulation within those vesicles was abolished already at nano-molar levels of Et 3 Pb + , but only in the presence of Cl - . In intact cells the turgor dependent indole-3-acetic acid induced elongation growth of coleoptile segments of Avena sativa L. was inhibited by Et 3 Pb + at micro-molar levels and after a lag of 15-20 min. This lag might be due to a slow penetration of the agent through the waxy cuticle and the cell wall.

The pH dependence of two, flash-induced slowly decaying delayed luminescence components - resulting from charge recombination of Q A - and Q B - with the S 2 and S 3 states (S 2 Q A - + S 3 Q A - and S 2 Q B - + S 3 Q B - recombinations) - in untreated and herbicide treated chloroplasts was investigated within a pH range of 5.5 to 8.5. The component associated with Q A - showed the same pH-dependence in untreated and in DCMU-treated chloroplasts: its half-decay time increased with increasing pH reflecting an appr. +5 meV/pH change in the free energy of activation. Contrary to this the component associated with Ob exhibited an opposite behaviour: its half-decay time decreased with increasing pH and the free energy of activation changed with about -5 meV/pH. It is concluded that both acceptor and donor side effects are involved in the pH-dependence of slow phase delayed luminescence components.

The directed and non-directed locomotion of human polymorphonuclear leukocytes on a glass surface was compared to Brownian and drift motion. The average track velocity was measured under different conditions. The track velocity of colchicine treated cells was the same as control cells. However, cytochalasin B treated cells and cytokineplasts had a reduced track velocity compared with the control cells. The non-directed locomotion was investigated by measuring the mean square displacement as a function of time. The diffusion constant. D. which quantitates the random walk process, and the characteristic time, x, which governs the migration of the cell, was calculated. The value of the diffusion constant depended on the cell treatment: For control cells 261 μm 2 /min, for colchicine treated cells 145 μm 2 /min. for cytochalasin B treated cells 55 μm 2 /min, and for cytokineplasts 47 μm 2 /min. The characteristic time was about 40 s. The measurement showed that the non- directed locomotion can be described by the Brownian motion. The directed locomotion was investigated by a necrotactic assay and quantitated by the McCutcheon index. This index was for control cells 0.85 ±0.07, for colchicine treated cells 0.8 ± 0.07, and for cytokineplasts 0.75 ± 0.1. The measurement showed that the directed locomo­tion can be described by a process which is called drift mode. From this method of analysis it was determined that the important organelles of the cell for the directed and the non-directed locomotion are: (i) A part of the plasma membrane, (ii) the microfilaments, and (iii) an unstructurated part of the cytoplasme. The microtubules of the cell are only of minor importance for the directed and the non-directed locomotion.

A comparison between blood and thrombuses is made on behalf of T 1 and T 2 relaxation data. Thereby, the time interval between the withdrawal and the measurement of blood forms a parameter of investigation. Among the thrombuses the surgically removed ones are compared with those aggregated in blood during course of time. As the most outstanding result one can see that the formation of thrombuses changes significantly the T 1 and T 2 relaxation times of 1 H und 23 Na. The values of the relaxation times obtained for those thrombuses which have aggregated within the blood are thereby comparable with those relaxation times found for the surgically removed ones.

In an earlier study it was shown that an isotonic salt solution within a measuring chamber of a cytopherometer is rotating caused by Lorentz forces, if the magnetic earth field crosses the electric field in the chamber. This may be a model of the ability of certain higher organisms to recognize the direction of the magnetic earth field. The topic of this study was the possible effect of the magnetic earth field in humans. It is shown that the duration of REM latency is influenced by the position of sleepers in N-S or E-W direction: it is shortened in E-W direction (p = 0.02).

An automatically-regulated pressure probe is described, which facilitates the direct and continuous measurement of turgor pressure and water relation parameters in plant cells. An optoelectronic sensor drives a feed back system so that the position of the oil/cell sap interface in the measur­ing capillary remains constant. The sensor is easily applied to most measurement set-ups by mounting it on a focusing screen connected to the microscope used for observation. The position of the oil/cell sap boundary is sensed by its movement along the image of the capillary on the screen and can be set to any appropriate position. The optical coupling of the regulation signal is advantageous because it leaves the experimenter free to measure the electrical or other parameters of the cell. Experiments on cells of Eremosphaera viridis have shown that the device allows much faster and more exact measurements of water rela­tion parameters, and over longer periods, than the earlier method.

Dihydroflavonol 4-reductase from Petunia flowers cata­lyzes the reduction of dihydroquercetin to leucocyanidin and, in particular, of dihydromyricetin to leucodelphinidin, whereas reduction of the simple dihydroflavonol dihydro- kaempferol to leucopelargonidin could not be observed. This special substrate specificity of dihydroflavonol 4-re­ductase is most probably the reason for the observations that delphinidin derivatives are the main end products of anthocyanin biosynthesis in Petunia flowers, whereas an- thocyanins based on pelargonidin are rarely found and, if present, are only formed in very small amounts.