Volume 51, Issue 3-4

Lipoxygenases catalyze the hydroperoxidation of polyunsaturated fatty acids and thus the first step in the synthesis of fatty acid metabolites in plants. Products of the LOX pathway have multiple functions as growth regulators, antimicrobial compounds, flavours and odours as well as signal molecules. Based on the effects of LOX products or on the correlation of increases in LOX protein and the onset of specific processes, a physiological function for LOXs has been proposed for growth and development and for the plant response to patho­gen infection and wound stress.

Chromopeptide siderophores (azotobactin 87-1 and -II) were isolated from an iron deficient culture medium of Azotobacter vinelandii ATCC 12837 (=DSM 87). Their structures were elu­ cidated by chemical degradation studies and spectroscopic methods, especially 2D-NMR-tech-niques. Total assignments of 1 H-, 13 C-, and 15 N-resonances based on 2D-HOHAHA-, 1 H/ 13 C-HMQC-, 1 H / 13 C-HMBC-, 1 H / 15 N-HMQC/TOCSY-, and 1 H/ 15 N-HMBC-experiments are given as well as sequential information derived from 1 H/ 1 H-NOESY-, 1 H / 13 C-HMBC-and 1 H/ 13 N-HMBC-experiments. Both Az 87-1 and Az 87-11 consist of a tetracyclic chromophore -(1S)8,9-dihydroxy-4-oxo-2,3,4,5-tetrahydro-1H,10cH-3a,5,10b-triazaacephenantrylene-1-carb-oxylic acid -and a decapeptide chain linked with the N-terminus to the carboxy group of the chromophore containing also modified, non-proteinogenic amino acids. The sequence L-Ser-D-Ser-L-Hse-Gly-D-threo-OHAsp-Hse-Hse-Hse-D-N 5 OH-N 5 -R-Hbu-Om-L-Hse was determined for Az 87-1, while Az 87-11 contains a C-terminal L-Hse-lactone instead. Iron is chelated by the catecholic group of the chromophore, the β-hydroxy aspartic acid, and the hydroxamate function formed by N 5 -hydroxyornithine and R-β-hydroxybutyric acid.

The fatty acid composition of seed oils of eight Ranunculaceae was determinated in order to characterize new sources of gamma-linolenic acid. Fatty acids were identified as fatty acid methyl esters (FAME) by capillary gas-liquid chromatography (GC) and capillary GC-Fourier transform infrared spectroscopy (FTIR). For trienic FAME the use of a cyanopropyl-polysiloxane stationary phase (CP-Sil 88) allowed the separation with high resolution of methyl ester of columbinic acid (trans-5,cis-9,cis-12 18:3) and gamma-linolenic acid (cis-6,cis-9,cis-12 18:3). The results confirmed the presence of columbinic acid in Thalictrum seed oils, and that of gamma-linolenic acid in Anemone and related species seed oils. The taxonomic subdivision of Ranunculaceae into sub-families and tribes, which resulted from morphological considerations, did not account for the above results.

The flagellar autofluorescent substance of the brown alga Scytosiphon lomentaria , which is probably involved in the photoreception of the phototaxis of flagellate cells, was investi­ gated. 4′,5′-Cyclic FMN (1) was isolated from the extract of whole mature plants for the first time as a natural product. Since the concentration of 4′,5′-cyclic FMN (1) was considerably low in vegetative plants, which do not contain fluorescent flagella, this substance is consid­ered to correspond to the flagellar fluorescent substance.

Trehalose is accumulated at very high concentrations in yeasts when this organism is sub­ mitted to a stress condition. This report approaches the question on the protective effect of trehalose and its degradation product, glucose, against structural and functional damage promoted by guanidinium on yeast cytosolic pyrophosphatase. Here it is shown that both, 1 ᴍ trehalose or 2 ᴍ glucose, are able to attenuate at almost the same extent the conforma­ tional changes promoted by guanidinium chloride on the pyrophosphatase structure. On the other hand, while 1 m trehalose increases 3.8 times the K i (from 0.15 to 0.57 ᴍ) for guanidi­ nium chloride inhibition of pyrophosphatase activity, 2 m glucose did not even duplicate this parameter (from 0.15 to 0.25 ᴍ). These data support evidences for a functional reason for the accumulation by yeasts of trehalose, and not other compound, during stress conditions.

In order to obtain a model which allows to directly study Mg 2+ influx into the cell, Mg 2+ deficiency was induced in the unicellular photoautotrophic flagellate Euglena gracilis. Lack of Mg 2+ in the culture medium leads to a number of morphological, biochemical, and physio­ logical changes in E uglena gracilis. The rate of cell division was reduced under Mg 2+ -free conditions. Subsequently an enlargement of the cells was observed and they changed from spindle-like to oval shape. The Mg 2+ -starved cells were well filled with paramylon granules, while their motility and vitality was not impaired. Concurrently with the larger cell size the protein-, carbohydrate-, and chlorophyll content of the cells increased. Further changes were observed in the surface carbohydrates. The proportion of cells with galactose, N-acetyl-galac-tosamine and mannose on the cell surface rose in the Mg 2+ -starved cultures, shown in a lectin-binding assay. Fucose was found on the pellicle of Mg 2+ -starved cells only. Cultivation of E uglena gracilis in Mg 2+ -free medium induced a drastic reduction of the intracellular Mg 2+ concentration already after 24 h (from 233 nmol/10 6 cells to 82 nmol/10 6 cells). When Mg 2+ was made available again, the Mg 2+ -starved cells took them up rapidly and the intracellular concentration of free Mg 2+ rose. As Mg 2+ depletion could be induced in Euglena gracilis easily by manipulating the culture conditions and as the cells remained viable, it was con­ cluded that this flagellate can be used as a model organism for studying the Mg 2+ uptake of eukaryotic cells.

Confocal fluorescence microscopic images were used to investigate the accumulation site of protoporphyrin IX (PPIX) within liverwort cells (Marchantia polymorpha L.) treated with peroxidizing herbicides as acifluorfen methyl (AFM). A high level of PPIX accumulation was observed in the cells during 12 h to 24 h after the addition of AFM . The results obtained from confocal fluorescence microscopic images gave clear evidence that the accumulation of PPIX occurred only in the chloroplasts, but was not observed in the cytosol or at the plasma membrane. The presence of PPIX in the chloroplasts strongly suggests that protoporphyrino­ gen (Protogen) accumulates by inhibition of protoporphyrinogen oxidase (Protox) which is the target enzyme for peroxidizing herbicides. The plastidic occurrence of PPIX provides evidence of either the presence of an additional herbicide-resistant Protox or of a non-enzy-matic Protogen-oxidation system in the Marchantia chloroplast.

The inhibitory effects of copper ion (Cu 2+ ) on the photosynthetic electron transport func­tion was investigated both in NaCl washed (depleted in 17 and 23 kDa polypeptides) and native (unwashed) photosystem II membrane preparations from spinach (Beta vulgaris) chlo-roplasts. Copper in the range of 2.0 to 15 μᴍ strongly inhibited the electron flow from water to 2,6-dichlorobenzoquinone in NaCl washed particles in a concentration dependent manner. Com plete inhibition was noticed at 15 μᴍ Cu 2+ . Oppositely in native membranes, 15 μᴍ C u 2+ inhibited only 10-12% of control activity. It was found that calcium ion (Ca 2+ ) significantly reduced the Cu 2+ inhibition of electron transport activity. The Ca 2+ supported prevention of Cu 2+ toxicity was specific to Ca 2+ . Further analysis indicated that both Cu 2+ and Ca 2+ act competitively. Since Ca 2+ is known to have stimulating/stabilizing effect at the donor side of photosystem II, it is therefore suggested that Cu 2+ in NaCl washed particles exerts its inhibi­tory effect(s) at the oxidizing side of photosystem stimulates/stabilizes the oxygen evolution.

The chemical reduction of the formyl group of pheophorbide b with sodium cyanoborohy­ dride in methanol leads to 7 1 -methoxy-and 7 1 -hydroxy-pheophorbide a. The same reaction with zinc pheophorbide b yields in addition zinc pheophorbide a. This was characterized by mass and 1 H -NMR spectroscopy. Infiltration of zinc pheophorbides a and b and of zinc 7 1 -hydroxy-pheophorbide a into etiolated oat leaves yielded phytylated products. The best yield in the esterification was obtained with 7 1 -hydroxy-pheophorbide a. Analysis of the products revealed the formation of zinc pheophytin a from all infiltrated compounds. The significance for the transformation of chlorophyll b into chlorophyll a is discussed.

Photosystem I monomers from wildtype cells of Synechocystis PCC 6803 and from a mu­ tant deficient in the psaL gene were crystallized. PsaL encodes for the hydrophobic subunit L, which has been proposed to constitute the trimerization domain in the PS I trimer. The absence of subunit L facilitated crystallization of the PS I monomer. The unit cell dimensions and the space group for the crystals from this preparation could be determined to be a = b = 132 Å , c -525 Å, α = β = 90°, y = 120°, the space group is P6 1 or P6 5 . The results show the potential of using specifically designed deletion mutants of an integral membrane protein for the systematic improvement of crystal structure data.

The investigations presented here focus on the CO 2 /H 2 O gas exchange in damaged and undamaged spruce trees while using open-air measurements as well as measurements under defined conditions in the laboratory. The studies were performed at two different sites in the Hunsrück and the Westerwald mountains. In the laboratory the CO 2 /H 2 O gas exchange was measured on detached branches under controlled conditions in the course of two years. CO 2 saturation curves were also generated. In addition CO 2 compensation points were deter­ mined employing a closed system. In the natural habitat diurnal course measurements of photosynthesis and transpiration as well as light-saturation curves for photosynthesis were performed. In parallel with the photosynthesis and transpiration measurements, measurements of the water potential were taken at both locations. The photosynthetic capacity and transpiration rate show a typical annual course with pronounced maxima in spring and late summer and minima in summer and winter. The needles of the damaged trees exhibit higher transpiration rates and a distinct reduction in photosyn­ thesis than the needles of the undamaged trees during two seasons. The diurnal course measurements of net photosynthesis and transpiration show a maximum in photosynthesis and transpiration in the afternoon in May and September, but a characteristic midday depression in July. Photosynthesis was markedly lower and transpiration higher in the needles of the damaged trees. The damaged trees show a lower increase in the light and CO 2 saturation curves and higher CO 2 compensation points as compared to the undamaged trees. The water potential reaches much lower values during the course of the day in needles of the dam­ aged trees. The reduction of the photosynthetic rate on one hand and the increase in transpiration on the other hand result in an extreme lowering of the water use efficiency in photosynthesis. The damage to the thylakoid membranes and to the guard cells obviously results in a pro­ found disturbance of the physiological homeostasis of the needles and could thus lead to premature needle loss.

Radioadapted chicken embryo cells (X-irradiation in ovo with 10 cGy at the 14th day of development with priming periods of 24 h) were treated in vitro by challenge doses of 14 different DNA -and/or chromatin-interactive agents, including X-rays. A decrease in the cel­lular damage, as measured by scheduled DNA synthesis, was only observed with X-irradia­tion. Sedimentation of nucleoids as well as viscosity of alkaline lysates from ethidium bro­mide (0.35 - 400 (μg/ml)-, novobiocin (125 -1800 μg/ml)-, and hyperthermia (30 min at 43° and 45°)-treated cells suggest a higher tendency of radioadapted cells to undergo positive DNA supercoiling. When DNA from adapted and non-adapted chicken embryo cells was used as substrate, neither its digestion by DNase I nor the inhibition of DNase I activity by various DNA-interactive agents was changed in primed cells. From the previous investigations as well as from the present results it is concluded that an increase of tightening of protein-DNA interactions within the nuclear matrix is a molecular determinant of the elevated radiation resistance in radioadapted chicken embryo cells.

The effect of four amphiphilic cationic compounds that differ in their polar head properties on the kinetics of calcium ion desorption from unilamellar lecithin liposom e membranes has been studied. The compounds under investigation were: N-benzyl-N,N-dimethyl-N-tetradecy-lammonium chloride (BDTA), N-methyl-N-tetradecylmorpholinium bromide (MTM), N-methyl-N-dodecyloxym ethylenem orpholinium chloride (MDOM) and N,N,N-trimethyl-N-te-tradecylammonium bromide (TMTA). Kinetic constants were determined using the three-compartmental analysis for isotopic labels. For the above mentioned compounds electronic structure calculations were performed and point charges and dipole moments of the molecules determined by using quantum chemis­try methods. It was found that the rate of desorption increases with increasing concentration of the compounds studied, and effectiveness of the compounds follows the sequence: BDTA >MTM >TMTA >MDOM . For dipole moments the sequence obtained is somewhat different, namely: BDTA>MTM >MDOM >TMTA. Apparently, the differences in the effectiveness of action of the compounds in the calcium desorption process follow not only from the values of dipole moments of their polar heads but also from the values of point charges on some atoms and from steric properties.

Anion Transport, Erythrocyte Membrane. Organophosphorus Insecticides, Methylparathion, Pesticides The effect of the organophosphorus insecticide methylparathion and its main metabolite methylparaoxon on chloride and sulfate equilibrium exchange in pig erythrocytes was investi­ gated using an isotope labelling technique. Efflux of both radioactive isotopes with time follow ed a single exponential. Methylparathion and methylparaoxon inhibited the chloride equilibrium exchange in erythrocyte ghosts in a dose-and time-dependent manner. There was no difference between effects evoked by these two compounds. Methylparathion and methylparaoxon inhibited sulfate efflux from resealed ghosts. The effect was also dose-and time-dependent. Again, there was no difference between the action of both agents. Dixon analysis revealed a non-competitive character of the inhibition of the exchange of both anions with apparent K i values 183 and 184 μᴍ for methylparathion and methylparaoxon, respec­tively in the case of chloride transport; for sulfate exchange these values were 675 and 648 μᴍ. It was suggested that structural similarity between the parent agent and its metabolite accounts for their identical effects. Methylparathion and methylparaoxon might inhibit the anion exchange indirectly by changing the fluidity of the erythrocyte membrane or directly by binding to the band 3 protein and evoking conformational changes that lead to the inhibi­tion of the anion transport. The insecticides, due to their ability to phosphorylate, might also disturb some regulation processes in the band 3 protein and affect anion transport in this way.

Bioassay-guided fractionation of the methanolic extract of Tamarindus indica fruits led to the isolation of L -(-)-di-nbutyl malate which exhibited a pronounced cytotoxic activity against sea urchin embryo cells. In order to study structure-activity relationships, close-structure relatives of di-n-butyl malate were synthesized using d -(+) -and L -(-)-malic acid as starting materials, and their cytotoxic activities were exam in ed for the sea urchin em bryo assay. L -(-)-D i-n-pentyl malate was the most effective inhibitor to the development of the fertilized eggs. Significant inhibitory activity was not seen in the esters of D -(-)-isomer .

Penicillin G (PEN) is a widely used antibiotic whose mechanism of action is related to the interference with the synthesis of bacteria cell wall. In order to evaluate its perturbing effect upon human cell membranes PEN was made to interact with human erythrocytes, isolated resealed human erythrocyte membranes and molecular models. The latter were multibilayers of the phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidyl-ethanolamine (DMPE) as well as DMPC large unilamellar vesicles. These studies were per­ formed by scanning electron microscopy, fluorescence spectroscopy and X-ray diffraction methods. The observed results coincide in that PEN did not exert any significant effect upon the structures of the red cell membrane neither on its molecular models. This is in agreement with its reported lack of major toxicity and hem atological reactions.

Hydroxylated anthraquinones have been found to accumulate in different developmental stages of the chrysomelid beetle Trirhabda geminata. Eggs, larvae and adults were analyzed by HPLC and GC-MS. Each developmental stage analyzed contained 1,8-dihydroxy-3-methyl-anthraquinone (chrysophanol) and 1,8-dihydroxyanthraquinone (chrysazin). No anthraquinones were detected in the faeces of T. geminata. The level of stored anthraqui­ nones did not change during starvation. In the host plant of this specialist herbivore, the brittlebush Encelia farinosa (Asteraceae), anthraquinones were not detected. Possible biolog­ical functions of anthraquinones stored in T. geminata are discussed.

A rapid FISH (fluorescence in situ hybridization) technique (Fast-FISH) for quantitative microscopy has been recently introduced. For highly repetitive D N A probes the hybridization (renaturation) time and the number of necessary washing steps were reduced considerably by omitting formamide or equivalent denaturing chemical agents. Due to low stringency conditions major and minor binding sites of the probes used showed visible FISH signals well suited for quantitative image-microscopy. The discrimination of minor and major binding sites was possible by automated image-processing. Here, a further, quantitative optimization of the Fast-FISH technique is described that allows to clearly discriminate major and minor binding sites of a-satellite probes by an easy image classification parameter. With respect to the optimization it was necessary to verify two sensitive parameters (hybridization time and temperature) of the given rapid FISH protocol. A s examples the systematic optimization for the two probes D 12 Z 2 (major binding site on the centromere of chromosome 12) and D 8 Z 2 (major binding site on the centromere of chromosom e 8) are shown. The optimal hybridiza­tion conditions concerning rapidness and quality of chromosome morphology were obtained using a hybridization temperature of 70 °C and a hybridization time of 60 min. For these conditions major and minor binding sites were clearly discriminated by the intensity maxi­ mum S max of the corresponding FISH-spots.