Band 39, Heft 6

The esters of p-coumaric, ferulic and caffeic acid with malic acid were isolated from leaves, the ester of sinapic acid with malic acid from cotyledons of Raphanus sativus plants via preparative HPLC. The esters were identified by hydrolyses and following capillary GC , by H-NMR and FAB-MS. Their concentrations in leaves and tubers were determined by gradient HPLC on reversed phase material.

Epicuticular waxes were extracted with hexane from seed coats of 16 species from the genus Cistus (Cistaceae) in amounts corresponding to an average of 0.07% per dry weight. All cuticular waxes obtained from the various Cistus species consisted of hydrocarbons, wax esters, triterpenol esters, aldehydes, primary alcohols and free fatty acids. These compounds were present as homologues in changing patterns. In addition two unidentified acids and two unknown alcohols were found. Epicuticular waxes from seed coats differed qualitatively and quantitatively from those of Cistus leaves and petals and confirmed the existence of organ specific compositions of epicuticular waxes. The wax components secreted in this composition result in a cover which in scanning electron microscopic pictures resembles a smooth layer of solidified fluid lacking any crystalline structure.

Photomixotrophic cell suspension cultures of Ruta graveolens were qualitatively and quantita­tively analyzed by gaschromatography and mass spectroscopy for volatile compounds. The terpenoid hydrocarbons geijerene and pregeijerene, the C 9 -C 13 methylketones and a series of aliphatic esters, respectively, were found as main constituents. The esters consisted of acetic acid, 2-methylbutyric acid and 3-methylbutyric acid which were esterified with straight chain or branched C 8 -C 11 alcohols. The data are discussed in comparison to previous studies on callus cultures.

Ethyl acetate extracts of diffusates from the fungus-inoculated leaflets of Desmodium gangeticum have been found to contain six isoflavonoid phytoalexins including the isoflavones genistein and 2′-hydroxygenistein, and the isoflavanones dalbergioidin, diphysolone and kievitone. These known phytoalexins occur together with a new antifungal isoflavonoid (desmocarpin) for which the structure (-)-(6aR; 11aR)-1,9-dihydroxy-3-methoxypterocarpan is proposed.

Both, the flavonolignane silybin and the decapeptide antamanide, protect animals against the mushroom poison phalloidin from Amanita species. The X-ray structures of silybin and anta­manide have been solved in previous studies. Here an attempt has been made to explain the antagonistic antihepatotoxic protection by similar chemical structure elements in the two compounds. It can be shown that two aromatic rings in a definite arrangement are essential for the attachment to the target structure of the liver cell membrane. Comparable pharmacological tests are discussed.

The essential oil from leaves of Chrysanthemum balsamita L. was analyzed by GC and GCMS techniques. About 30 components have been identified. The oil revealed insecticidal properties against aphids. Very little phytotoxicity was observed. The insecticidal activity could not be attributed to one of the single main substituents of the oil.

Growth of 6 bacteria (Serratia marcescens, Bacillus megaterium, Bacillus subtilis, Streptococcus viridis, Micrococcus luteus, and Mycobacterium phlei) was inhibited by 50% if the growth medium contained sparteine at concentrations between 0.5-10mᴍ. Total growth inhibition, which was bacteriostatic in nature, was achieved at 20 mᴍ. The growth of 6 phytopathogenic fungi was also affected: at a sparteine concentration of 15 mᴍ the growth of Alternaria porri was reduced by 40% as compared to the untreated control. Respective values were 18% inhibition for Piricularia oryzae, 33% for Helminthosporium carbonum, 15% for Rhizoctonia solani, 5% for Fusarium oxysporum, and 42% for Asperquillus oryzae. Since the concentrations of quinolizidine alkaloids range from 1-200 mᴍ (roots, leaves, or stems) or 10-200 mmol/kg (seeds) in Leguminosae, it is discussed whether quinolizidine alkaloids are involved in the antimicrobial defense of lupins, in addition to their potential role as allelopathic or herbivore repellent defense compounds.

Polyphagous molluscs such as Helix pomatia and Arion rufus generally do not feed on plants containing alkaloids. Of 19 species tested 10 species were totally avoided, the other 9 species were less attacked than Lactuca sativa, which was readily taken. Plants containing quinolizidine alkaloids were studied in detail. Those species with the α-pyridone alkaloids cytisine and N-methylcytisine were avoided to a higher extent than plants with lupanine as the major alkaloid. Since the repellency observed could be due to other natural products present in the plants besides the alkaloids, the feeding response of Helix pomatia was tested on artificial diets containing quinolizidine alkaloids in various concentrations as the only variable. If the snails had the choice they clearly preferred alkaloid-free food or a diet with only low alkaloid concen­trations. Half-maximal repellency of cytisine is less than 2 mᴍ, of sparteine 1-5 mᴍ, and of lupanine 1-8mᴍ. Since the in vivo concentrations of sparteine, cytisine, and lupanine are equal to or higher than the inhibitory concentrations required it is concluded that quinolizidine alkaloids constitute a potential antimolluscan principle of legumes.

In an attempt to distinguish between the UDP-glucose: flavonol 3-O-glucosyltransferase (3GT) and the UDP-glucose: anthocyanidin 3-O-glucosyltransferase in flower buds of Petunia hybrida, several properties of these activities were determined. The 3-glucosylation of anthocyanidin had a pH-activity optimum of 7.2, that of flavonol pH 9.2 to 9.5. Anthocyanidin 3GT activity was lowered in the presence of EDTA or β-mercaptoethanol, but this was due to an effect on the anthocyanidin substrate. The two 3-glucosylating activities were to a similar extent inhibited by an increasing ionic strength in the enzyme assay and showed an identical iso-electric point (5.2) as determined by chromatofocusing. Molecular weights were identical: 26000, 52000 or 78000 daltons as determined by gel-filtration. Antiserum raised against partially purified 3GT gave identical immunoprecipitation curves with flavonol 3GT and anthocyanidin 3GT. Special attention was given to the 3-O-glucosyltransferase in mutants with low levels of 3GT activity. These mutants are unable to form significant amounts of anthocyanins but contain wild- type amounts of flavonols. The enzyme of such mutants had the same iso-electric point and identical titration-curves with antiserum as the enzyme from wildtype plants. 3GT from wildtype or mutant plants glucosylated flavonols at higher rates than anthocyanidins.

Two further loci can be added to the three already known (g, gl and fg) in Silene controlling the 7-O and 2″-O-glycosylation of the C-glycosylflavone isovitexin (6-C-glucosylapigenin). These loci, O7g for the 7-O-galactosylation and D6a for the 2″-O-arabinosylation, appear only to be expressed in cotyledons and rosette leaves and control the biosynthesis of isovitexin 7-O- galactoside and isovitexin 7-O-galactose 2″-O-arabinoside in these parts of plants in which the recessive alleles of the loci g, gl and fg are homozygous (Steyns et al. [9]). The possibility that locus g, whose dominant alleles gG and gX control the 7-O-glucosylation and the 7-O-xylosylation of isovitexin respectively, controls the synthesis of isovitexin-7-O- galactoside was excluded by demonstrating the presence of isovitexin-7-O-galactoside and the 7-O-galactosyltransferase catalyzing its biosynthesis in addition to isovitexin-7-O-glucoside and the 7-O-glucosyltransferase in cotyledons of gGgG seedlings. The synthesis of the 2″-O-arabinoside is neither controlled by locus gl, whose dominant alleles glR and glA control the 2″-O-rhamnosylation and 2″-O-arabinosylation of isovitexin respectively, nor by locus fg, whose dominant allele Fg controls the biosynthesis of isovitexin 2″-O-glucoside. This was shown by the presence of isovitexin-7-O-galactose 2″-O-arabinoside and the 2″-O-arabinosyltransferase catalyzing the synthesis of the 2″-O-arabinoside in addition to isovitexin-7-O-galactose 2″-O-rhamnoside in cotyledons of glRglR seedlings and by the presence of isovitexin-7-O-galactose 2″-O-arabinoside and the 2″-O-arabinosyltransferase in addition to isovitexin-7-O-galactose 2″-O-glucoside and the 2″-O-glucosyltransferase in cotyledons of Fg Fg seedlings.

The kinetic of the reversible dissociation of glycerol dehydrogenase from Bacillus megaterium in slightly alkaline medium was measured by biochemical, chemical and physical methods. The dissociation is followed by changes in the secondary structure and can be prevented by addition of NAD or increased potassium chloride concentration. Crosslinking by suberimidate, but not by monofunctional imido esters, shows a high stabilization against alkali, urea or heat inactivation caused by hindrance of dissociation.

Sexual differentiation of the reproductive cells (gonidia) of the green alga Volvox carteri f. nagariensis Iyengar is triggered by a glycoprotein inducer which is released into the medium by sexual male spheroids. If added, it induces the next generation cycle to become sexual. About 3000 pheromone molecules per spheroid will give full induction. Specific binding sites for the pheromone have been identified in the extracellular matrix in which characteristic events take place. Inducer binding and action are reversible for a certain time period until the start of cell division; thereafter, determination of the gonidia is final. Extent of sexual induction is proportional to the duration of inducer pulses. Inhibition of sexual induction reveals as essential extracellular components of the induction system high molecular acidic glycoproteins, cyclic nucleotides, Ca 2+ /calmodulin, and pheromone-controlled gene products. Low matrix levels of cyclic AMP and cyclic GMP seem to stimulate induction; high levels to inhibit.

1.In isolated chloroplasts which are provided with essential exogenous substrates for glycerolipid biosynthesis (sn-G3P and UDPgal) the incorporation of fatty acids into lipids shows the same pH dependence as the fatty acid synthesis itself with a stromal pH optimum close to 8.5. 2.Furthermore high rates of glycerolipid biosynthesis appear to be accompanied by a preferred oleate incorporation as compared to palmitate. 3.Reinvestigations of the sn-G3P requirement of plastid lysophosphatidic acid formation with rapidly prepared substrate-free chloroplast extracts under approximately physiological conditions reveal a lower specificity of the primary sn-G3P acylation for oleate, as recently found for the fatty acid transfer from purified acyl-ACP fractions on to sn-G3P, catalyzed by purified acyl transferase 1. 4.A comparison of calculated stromal sn-G3P levels under physiological conditions (0.1- 0.3 mᴍ) with those, required for half saturation of the primary acylation reaction either with oleate (K m (sn-G3P) = 0.3 mᴍ) or palmitate (K m (sn-G3P) = 0.6 mᴍ) in chloroplast extracts suggests, that both fatty acids to be involved in lysophosphatidic acid formation within chloroplasts, although oleate would be preferred. 5.The latter observation facilitates the understanding of a palmitate accumulation in chloroplast lipid fractions, induced by increasing sn-G3P concentrations in chloroplast sus­pensions. 6.Although stimulating fatty acid synthesis from acetate in intact chloroplasts, acyl-CoA- synthesizing-conditions (presence of CoA and ATP) in the applied chloroplast extracts appear to inhibit fatty acid incorporation into sn-G3P and thus to exert a regulatory function between the plastidary and extraplastidary glycerolipid biosynthesis.

To get further insight into the causes of differences between rat splenic and thymic cells with regard to DNA repair synthesis, scheduled (SDS) and unscheduled (UDS) DNA synthesis as well as nucleoid sedimentation of the cells were investigated under the influence of ethidium bromide (EB, 1 -1000 μg/ml). - At concentrations of ≳ 25 μg/ml, EB inhibited SDS of both cell species and UDS of thymic cells; much higher additions of the drug (>200 μg/ml) were needed to diminish UDS of splenic cells, lower EB-concentrations (25- 175 μg/ml) stimulating the UDS of the splenic cell preparation. - The sedimentation rate of splenic and thymic cell nucleoids within neutral sucrose gradients had a biphasic dependence on the EB-concentrations. As compared to thymic cells however, preincubation of splenic cells with 50-250 μg EB/ml resulted in a significant greater (15-30 percent) sedimentation distance. - The results suggest that a relation­ship exists between the stimulation of UDS and the ability of cells to establish a greater DNA compactness in the presence of EB.

The influence of aggregation and temperature on the excited state kinetics of C-phycocyanin from Mastiqocladus laminosus has been studied. Polarized fluorescence decay curves have been recorded using a synchronously pumped dye laser in conjunction with a synchroscan streak camera. The experimental data for all samples can be fit satisfactorily assuming a biexponential decay law. Fluorescence depolarization times have been interpreted in terms of energy transfer among the different chromophores. The influence of temperature is only moderate on the intra­molecular relaxation, but pronounced on the rates of energy transfer. Both are dependent on the size of the aggregate. The biexponential decay of the α-subunit containing only one chromophore, indicates the presence of different subsets of chromophores in these samples. The results are discussed in terms of variations of the chromophore arrangements upon temperature induced changes in the protein conformation.

Ethoxyethyl 3-octylamino-2-cyanoacrylate and related compounds in which the amino group was replaced by N-CH 3 , S, O and CH 2 were synthesized and their activity as inhibitors of the Hill reaction in isolated pea chloroplasts determined. All compounds showed moderate to high activity but there was no obvious correlation between activity and the electronic character of the ester carbonyl group. The stereochemistry of the various inhibitor molecules was deduced from the PMR spectra and the possible influence of stereochemistry on Hill inhibitory activity discussed. Replacement of the olefinic proton in the 2-cyanoacrylates with a β-alkyl substituent was examined and a specific relationship between the length of the alkyl chain and activity was observed.

An antibody prepared against purified cytochrome c-553 from Nostoc muscorum inhibits the redox function of cytochrome c-553 as checked by a diaphorase assay. 20 to 30% inhibition of NADPH-driven respiratory and light-induced oxygen uptake by Nostoc thylakoids is observed when cytochrome c-553 specific antibodies were applied. However, only 30 to 50% of the total cytochrome c-553 content is released from isolated membrane material, thus being accessible to antibodies. Supplementing the isolated membrane material with excess Nostoc cytochrome before adding the antibody abolishes inhibition. The data provide further evidence for soluble cytochrome c-553 being a link between photosynthetic and respiratory electron transport in blue- green algae.

The effect of aureomycin on photosynthesis was investigated. This antibiotic which has been reported to stimulate photosynthesis at very low concentrations is an effective inhibitor at higher concentrations. In mesophyll protoplasts and isolated chloroplasts from spinach, 50% inhibition of CO 2 reduction required about 20 μᴍ aureomycin. The reduction of 3-phosphoglycerate and of oxaloacetate by intact chloroplasts was also inhibited, but not that of nitrite and methylviologen which was actually stimulated. NADP reduction by broken chloroplasts and methylviologen- dependent photophosphorylation were also sensitive to aureomycin. The electrochromic shift at 518 nm which indicates formation of a light-dependent membrane potential was suppressed in the presence of 200 μᴍ aureomycin and the transthylakoid proton gradient was decreased. The data confirm reports that aureomycin has uncoupling properties, and they indicate that it also acts as an inhibitor of ferredoxin/NADP reductase.

Several species of marine diatoms were grown at + 18 °C and + 22 °C under normal air condi­tions (0.035 vol.% C0 2 ) at a light/dark alteration of 14: 8̄ h. Intensity of white light was 1 mW (~ 5000 lux). An artifical nutrient solution of 35%o salinity was used. Algae - harvested during exponential growth - were exposed to different intensities of UV-B radiation (439, 717 and 1230 J · m -2 · m -1 ) for 2 days. UV-B radiation depressed the growth of all tested marine diatoms. Low levels of UV-B resulted in a slight increase of the biomass production (dry weight) compared to not UV-B treated cells. Enhanced UV-B doses caused a diminution of the primary productivity in all species. Algae exposed to UV-B stress showed a marked decrease in the protein and pigment content (chlorophyll a, chlorophyll c 1 + c 2 and carotenoids). In + 22°C grown cells of Lauderia annulata and Thalassiosira rotula were more sensitive to UV-B radiation than those cultures grown at + 18 °C. Bellerochea yucatanensis cells grown at +22 °C were less affected after UV-B exposure than at +18°C grown algae. The UV-B sensibility and growth of the individual species varied in a mixture of several marine diatoms. Results were discussed with reference to the UV-B effect on metabolic processes.

A method is described for the partial purification of a granulomonopoiesis inhibitor derived from 2001-batches equivalent to 1 kg or 2 · 10 12 leukocytes of porcine blood. Serum-free condi­tioned media were concentrated and separated via ammonium sulfate precipitation, acetone partitioning, ultrafiltration and gel chromatography. The active substance revealed a molecular mass of 500-700 Da and was not thiol-dependent, thus contrasting it with previously-reported granulopoiesis inhibitors. Inhibition of colony formation in an in vitro myeloid stem cell assay using various colony-stimulating factors showed that mature granulocytes and monocytes were equally affected. Apparent lack of cytotoxicity was suggested using a new reversibility test.

The work reported here concerns the specificty of four antibodies elicited by synthetic peptides corresponding to three domains of the HLA-B7 glycoprotein, and one of the β 2 microglobulin. One of the four peptides had been previously investigated, but the data obtained by its elicited antibodies were uncomplete. Of the three HLA-B7 peptides assayed, one was unable to raise an immune response. The other two produced antibodies in good titer that reacted with the antigen peptide, but were unable to recognize antigenic determinants when tested on membrane glycoproteins solubilized from human lymphoblastoid cells. In contract, the antibody elicited by the only fragment from the β 2 microglobulin recognized the native β 2 microglobulin molecule as well as HLA/β 2 m com­plexes, and reacted with intact human cells, as determined by ELISA and by FACS analysis. This peptide, which contains one of the most hydrophylic fragment of the whole molecule, is likely an important antigenic site, since it is also recognized by traditional antisera raised against native β 2 microglobulin.

The plasmid pHFK 206 consisted of plasmid pBR 322 and an 1.3 × 10 -6 D insert, a cloned seg­ment of a soybean rRNA repeating unit with a preferential binding region for soybean RNA polymerase I, was coupled to cyanogen bromide activated agarose (Sepharose 4B) and used for affinity chromatography of RNA polymerases. It could be shown by elution profiles and sodium dodecylsulphate/polyacrylamide slab gels that highly purified RNA polymerase from Escherichia coli MRE 600 bound to pHFK 206- Sepharose as holoenzyme with an apparently full complement of σ-subunit. With initially purified soybean RNA polymerase I from chromatin of 2,4-D treated hypocotyls it was demonstrated that pHFK 206-Sepharose could be used as an affinity chromatography method for purification of soybean RNA polymerase I in high degrees and economy of time.

Accumulated experimental information is used to assess the possible significance of thermal diffusion to mass transport in living matter. Possible thermal gradients across membranes, a single living cell, and an ensemble of such cells (e.g. an organ, tumor, etc.) are estimated. The corresponding model calculations, although not describing the biological process in detail, lead to conclusions about the possibilities for thermal diffusion as follows. Adequate thermal gradients to support substantial thermal diffusion could exist across biological membranes. Thermal diffusive flow would become significant when ordinary Fickian diffusion is sufficiently sup­pressed, e.g. in more concentrated systems near critical points of solution (i.e. near incipient phase separations). Conditions favorable to thermal diffusion functioning as a mechanism for active transport appear possible. Thermal diffusion appears much more important for transport into and out of an ensemble of cells than into or out of a single cell. Such mass transport by thermal diffusion could assume a sizable magnitude for an ensemble of cells with the dimensions of an organ or a tumor.

Receptor potentials of Limulus ventral photoreceptors were recorded in two defined states of moderate light- and considerable dark adaptation (LA, DA) by a repeated stimulus sequence consisting of a conditioning 2 s illumination (white light, response saturating intensity) followed by two 10 ms test flashes at fixed intervals evoking LA and DA responses (intensity varied from threshold to saturation of response amplitude). The half saturating intensity I 50 was determined from response height vs log stimulus intensity curves for LA and DA, while the photoreceptor was superfused either by reference saline (physiological ion concentrations, including 10 mmol/l Ca 2+ ) or by test salines in which the [Ca 2+ ] was varied between 40 μmol/l and 100 mmol/l. The sensitivity of the dark-adapted receptor does not significantly depend on the [Ca 2+ ] ex , but the sensitivity shift due to LA (measured by / 50 ) is reduced when the [Ca 2+ ] ex is lowered, and augmented when the [Ca 2+ ] ex is increased. Additional reduction of the [Na 2+ ] ex from 463 mmol/l to 46 mmol/l or increase of the [Mg 2+ ] ex from 50 mmol/l to 100 mmol/l does not counteract the effect of lowered [Ca 2+ ] ex on LA. The results confirm the assumption that a transient increase of the intracellular [Ca 2+ ] supplied from extracellular sources during the light response is the main cause for LA This calcium effect on light adaptation is neither characterized by a calcium/sodium antagonism, nor mimicked by magnesium, in contrast to the calcium effect on the gating of the light-activated ion channels.

The quantitative analysis of the alkaloids in Cinchona tissue cultures by means of a reversed-phase ion pair HPLC method is reported.

Strains of the phytopathogenic species Botrytis cinerea, Ceratocystis coerulescens, C.fimbriata, Fusarium oxysporum, and Rhizoctonia solani were cultivated on a de­fined synthetic culture medium. The plant hormone abscisic acid (ABA) was identified by gas chromato- graphy/mass spectrometry and gas chromatography data, and the contents determined in mycelia (15.9-31.9 ng/g mycel) and culture media (1.0-4.6 ng/ml) by scintillation counting.

Effects of temperature on the spectral properties of chlorophyll in solution and in micelles are reported. After correcting for the volume expansion coefficient of the solvent, it is observed that temperature has no detectable effects on the spectral properties of chlorophyll (between 14 °C to 35 °C). Solutions examined include acetone, chloroform and ethyl alcohol. It is concluded that the temperature induced change in refractive index, of the solvent, has no significant affect on the chlorophyll spec­trum. In micelles containing chlorophyll there are significant temperature induced spectral changes. Heating and cooling results in an irreversible redistribution of monomeric and oligomeric forms of chlorophyll.

The concept of concerted and synergistic feedback inhibition of the regulatory enzyme of a branched bio­synthetic pathway is critically discussed on the basis of data obtained with the threonine-lysine-sensitive asparto- kinase of Rhodospirillum rubrum. It is proposed that only one name (concerted feedback) should be used to specify allosteric feedback inhibitions where at least two end- products of the branched pathway are required for complete inhibition of the regulatory enzyme.

Mature female gametes of several Laminariales excrete a substance which acts interspecifically to release and to attract spermatozoids from ripe antherids. This pheromone was identified by chemical degradation and by synthesis of the natural compound as 1-(1′,2′-cis-epoxi-but-3′-enyl)- cyclohepta-2,5-diene (3). An oxirane is a novel feature among the mating substances of thallophytes. It is there­fore suggested to name the active hormone “Lamoxirene”.

Osmotic shock was applied to phage T2 in such a manner that the compact-mass of DNA was released from phage. The shape of this compact-mass of DNA was studied under electron microscope. It appeared that the DNA was packed into an elongated icosahedron similar to the phage head.