Band 43, Heft 11-12

A fraction of poplar bud exudate was analyzed by GC/MS and the novel aliphatic hydrocarbon esters 2-methylpropyl-, 2-methyl-2-propenyl-, 2-methylbutyl-, 3-methylbutyl-, 2-methyl-2- butenyl-, 4-methylpentyl-, (5)-methylhexyl- and 3,7-dimethyl-2,6-octadienyl(geranyl)- caffeate were identified.

The epicuticular leaf waxes from E. characias, E. nicaeensis and E. peplus were found to contain pentacyclic triterpenoids in great amounts in addition to the common lipid wax constituents: alkanes, wax esters, aldehydes, primary alcohols and fatty acids. The gross amount of triterpenoids were triterpenols, however, their acetates and fatty acid esters as well as the corresponding ketones were found, too. Distinctive variations in the occurrence of 13 triterpenoids α- and β-amyrin, lupeol, germanicol, α-fernenol, simiarenol, β-amyrinone, lupenone, lupeol acetate, lupeol esters, α- and β-amyrin esters and α-fernenol esters were observed. Therefore all Euphorbia species showed very species specific compositions concerning these triterpenoids.

Epicuticular waxes from various organs of Cistus albidus L. showed always an organ specific composition. Leaf wax contained homologous series of very long chained and saturated wax components, such as hydrocarbons, wax esters, fatty acids and alcohols. Additionally in this wax extract triterpenoids and great amounts of resin were found. Sepal wax had nearly the same wax composition like that of leaves with a trend to shorter chain lengths and not so great amounts of resin. Petal wax contained the common wax lipids, too, but the homologous series showed a shift to shorter chain lengths, and great amounts of unsaturated lipids were found in hydrocarbons, wax esters and fatty acids. In addition triterpenol esters were found but no resin. Stamen wax was similar to that of petals. The shift to shorter chain lengths and the presence of great amounts of unsaturated lipids showed a characteristic distribution for stamens. Seed wax contained only saturated wax lipids. In contrast to leaf waxes, the short chain compounds were dominating, and triterpenoids and resin were missing. The chemical wax composition of the various organs of C. albidus was correlated to the surface structures of these organs by SEM pictures.

Weakly phytotoxic, water-soluble pigments from culture filtrates of the potato early blight organism Alternaria solani have been identified by 1 H NMR, UV, mass spectrometric, and acetylation studies as the pair of rotamers represented by structure 1a.

The flavanones 2 S-lupinifolin and 2R:3R-lupinifolinol have been isolated as major components from a methanolic extract of Lonchocarpus guatamalensis root bark. These known compounds occur together with a minor flavanone (2R:3R-3-O-methyl-lupinifolinol) not previously recognized as a natural product.

Rigid layer (sodium dodecylsulfate(SDS)-insoluble cell wall) and peptidoglycan fractions were obtained from the Chromatiaceae (Thiocysüs violacea and Chromatium tepidum) and from Ectothiorhodospira vacuolata. Chemical composition of rigid layers from all three species indicated the presence of peptidoglycan-bound protein. Qualitative and quantitative composition of isolated peptidoglycan indicate an Aly-type. meso-Diaminopimelic acid was the only diamino acid found. Direct cross-linkage of peptide sidechains was confirmed by separation of respective dipeptides (M-A;pm-D-Ala) from partial acid hydrolysates of peptidoglycan. GlcN and MurN of the sugar strands are completely N-acetylated. All peptidoglycan fractions contained small amounts of Gly.

Two different hexose-phosphorylating enzymes, hexokinase and fructokinase, were partially purified from suspension-cultured Catharanthus roseus cells. One of the enzymes, hexokinase, catalyzed the phosphorylation of both glucose and fructose. The K m values for glucose and fructose were 0.06 mM and 0.23 mM, respectively. The V max of the enzyme with fructose was approximately three times higher than with glucose. This enzyme was specific in its requirement for ATP and its K m value for ATP was 52 μM. The optimum pH was 8.0 and Mg 2+ or Mn 2+ was required for the activity. The activity was inhibited by considerably higher concentrations of ADP (i.e., 4 mM ADP was required for 50% inhibition). The second enzyme, fructokinase, was specific for fructose, and no activity was detected with glucose as substrate. This enzyme used UTP or CTP as phosphate donor. The K m values of this enzyme for fructose and UTP were 0.13 mM and 0.15 mM, respectively. The pH optimum was 7.2, and Mg 2+ or Mn 2+ was required for the activity. These divalent cations could be partially replaced by Ca 2+ . The activity was inhibited noncompetitively by ADP and AMP. 90% inhibition of the activity by 0.5 mM ADP was observed in the presence of 2 mM UTP and 5 mM MgCl 2 . Fructose-2,6-bisphosphate, glucose-1,6-bisphosphate, glucose-6-phosphate, and fructose-6-phosphate had little or no effect on the activity of both the hexokinase and the fructokinase. Based on these results, a discussion is presented of the role of hexokinase and fructokinase and their involvement in the regulation of the metabolism of sugars in Catharanthus cells.

A soluble enzyme catalyzing the transfer of the glucose moiety from UDP-glucose to the nitrogen atom of nicotinic acid was detected in protein preparations from heterotrophic cell suspension cultures of parsley (Petroselinum hortense Hoffm.). Enzyme activity was enriched 22-fold by ammonium sulfate precipitation, gel filtration and ion exchange chromatography. The UDP-glucose : nicotinic acid-N-glucosyltransferase showed a pH-optimum at pH 7.8-8.2 and a temperature optimum at 30 °C. The apparent K M values were determined to be 170 μM for nicotinic acid and 1.2 mM for the cosubstrate. The native enzyme had a molecular mass of about 46 kDa. The glucosyltransferase reaction was shown to be reversible. The transfer of the glucose molecule from nicotinic acid-N-glucoside to uridinediphosphate yielding uridinediphosphoglucose and nicotinic acid could be demonstrated indicating that nicotinic acid-N-glucoside has a high group-transfer potential.

A degradation product of nicotinic acid representing the pyridine carbon skeleton was isolated and purified from parsley cell suspension cultures after incubation with [6- 14 C]nicotinic acid for 70 h. The catabolite was identified as glutaric acid by means of spectroscopic (GC-MS and 1 H NMR) and chromatographic (TLC, HPLC) techniques. Glutaric acid when applied to parsley cell cultures was readily degraded to CO 2 but intermediate products could not be identified.

Acetolactate synthase (EC 4.1.3.18; ALS) has been extracted from etiolated barley shoots (Hordeum vulgare L.) and purified to near homogeneity. Purification was made possible by a fivestep procedure using hydrophobic interaction, gel filtration, anion-exchange and hydroxylapatite chromatography, the last two steps performed with an HPLC- and FPLC-system, respectively. A 300-fold purification was achieved representing 13% of the initial activity in the crude extract; only small amounts of pure acetolactate synthase could be isolated. Although the enzyme was found labile during the chromatographic steps, purified ALS maintained its activity for several hours and could be stored at 70 K for weeks with a 15-30% loss. The apparent molecular weights of the enzymatically active species as determined by gel filtration were about 440 kDa and 200 kDa, respectively. We assume these species are no isozymes but different polymeric forms of a basic unit of ALS. SDS-PAGE analysis showed one polypeptide with an apparent molecular weight of 58 kDa. Preliminary enzymatic characterization of the purified enzyme confirms a marked synergism in the feedback control by branched-chain amino acids. The combination of valine plus leucine exhibited the most co-operative inhibition.

3-Aminoalkylidene-2H-pyran-2,4(3H)-diones (APs), possessing a conjugated enamino moiety which is common to cyanoacrylates and 2-aminoalkylidenecyclohexane-1,3-diones (ACs), were found as a new class of photosynthetic electron transport inhibitors. Although the structural requirement of APs for photosynthetic electron transport inhibition was very similar to that of cyanoacrylates and ACs, thermoluminescence measurements indicated that the binding manner of APs to D 1 protein was totally different from that of other inhibitors.

A cell strain originally derived from floral meristem tissues of Carthamus tinctorius (dyer’s saffron) produced substantial amounts of a novel red pigment under controlled culture conditions. The pigment isolated f r om alcoholic extracts of C. tinctorius cultures was compared with authentic carthamin, anthocyanins, betain, and carotenoids. It differed markedly from carthamin and showed none of the characteristic properties of the glycoside or chloride forms of authentic delphinidin, cyanidin, and pelargonidin. Analytic data indicated that this pigment also differs from betanin and from α- and β-carotene. The name “Kurenamin” was tentatively given to this newly isolated red pigment. Effect of the culture media, micro-elements, macro-elements, and putative substrates on the kurenamin production were investigated during cell suspension culture.

The effect of photoinhibition on the primary (Q A ) and secondary (Q B ) quinone acceptors of photosystem I I was investigated in isolated spinach thylakoids by the methods of thermoluminescence and delayed luminescence. The amplitudes of the Q (at about 2 °C) and B (at about 30 °C) thermoluminescence bands which are associated with the recombination of the S 2 Q A - and S 2 Q B charge pairs, respectively, exhibited parallel decay courses during photoinhibitory treatment. Similarly, the amplitudes of the flash-induced delayed luminescence components ascribed to the recombination of S20A and S2OB charge pairs and having half life-times of about 3 s and 30 s, respectively, declined in parallel with the amplitudes of the corresponding Q and B thermoluminescence bands. The course of inhibition of thermoluminescence and delayed luminescence intensity was parallel with that of the rate of oxygen evolution. The peak positions of the B and Q thermoluminescence bands as well as the half life-times of the corresponding delayed luminescence components were not affected by photoinhibition. These results indicate that in isolated thylakoids neither the amount nor the stability of the reduced OB acceptor is preferentially decreased by photoinhibition. We conclude that either the primary target of photodamage is located before the O b binding site in the reaction center of photosystem II or QA and OB undergo simultaneous damage.

A measuring system was designed for simultaneous recording of modulated chlorophyll fluorescence and light scattering changes. The kinetic relationship was investigated between lightinduced changes in non-photochemical fluorescence quenching, as determined by the saturation pulse method, and in light scattering, as measured via the apparent absorbance change at 543 nm. Very similar, but not identical kinetics were observed, reflecting a close non-linear relationship between these two indicators of thylakoid membrane energization. Fluorescence was found more sensitive at low levels of energization, while scattering continued indicating further increases in energization when quenching already was saturated. A general relationship between quenching and scattering is demonstrated which holds irrespective of whether energization is varied during induction or via changes in light intensity or CO 2 concentration. In the light-off responses, only part of fluorescence quenching was found to relax with the same kinetics as scattering. It is suggested that at high levels of energization slowly reversible membrane changes may be induced which have the potential of non-photochemical quenching at a low level of energization, and which are not accompanied by scattering changes. Neither quenching nor scattering changes displayed kinetics sufficiently fast to be taken as a direct expression of internal thylakoid acidificati on in intact leaves. This conclusion is drawn from comparative measurements of proton-uptake, as reflected by CO 2 -solubilization upon light-induced stroma alkalization, and of chlorophyll luminescence. Both, the initial CO 2 - gulp and the pH-dependent luminescence rise were found to clearly precede the development of energy-dependent quenching.

It has been shown that various furocoumarins are able to cause dark hemolysis in red blood cells (RBC). However, this effect is evident only at relatively high furocoumarin concentrations (4.6 x 10 -4 M) - much higher than those used in photosensitization experiments or photochemotherapeutic treatments. Among the various furocoumarins examined in this study, only psoralen (Ps) and 3-carbethoxypsoralen (3-CPs) showed strong photohemolytic effects, while the other compounds revealed little or no activity. This fact indicates that Ps and 3-CPs are able to induce selective damage to the cell membrane of RBC. By pre-irradiating furocoumarin in ethanol or isotonic saline solutions and adding the irradiated solutions to a RBC suspension, hemolysis was observed in various compounds. The products of photolysis which form during pre-irradiation may be responsible, in terms of hemolysis, for toxic effects on RBC.

Several chlorooxygen compounds, hydrogen peroxide and reducing molecules in the presence of chelated iron (Fenton systems) are oxidants of biological relevance. These compounds are either produced in living tissues or are in use as desinfectants or drugs. Tetrachlorodecaoxide as the active principle in the drug OXOFËRIN® can be differentiated from the above mentioned oxidants by means of simple biochemical test systems where different activators and detector molecules are used.

The retention, tissue distribution, and localization of [22,23- 3 H 2 ]dihydroazadirachtin A was investigated in the female locust, Locusta migratoria. A constant quantity of 0.4 to 0.5 μg of dihydroazadirachtin A per gram body weight is recovered in the unchanged form 5 days after injection of any of the physiologically effective doses in the range of 1.5 to 3.0 pg/g. Malpighian tubules account for 73.7% of the amount of dihydroazadirachtin A which is retained in the whole body. In the Malpighian tubules it is localized in the basal and inner regions. It is inferred from these results, that dihydroazadirachtin A acts in its unchanged form through a high-affinity binding to organ-specific membrane receptors.

The effects of azadiraehtin A , given orally, on the reproduction of mature females of Rhodnius prolixus were studied. Azadiraehtin A caused a reduction in oocyte growth and consequently in the egg production in a dose-dependent manner. Eggs layed had no alteration in their viabilities. A significant correlation between these effects and the titers of both vitellogenin in the haemolymph and vitellin in the ovaries was observed. Ecdysteroid titers in haemolymph and ovaries, as determined by radioimmunoassay, were decreased by this treatment. In vitro analysis suggested that azadiraehtin may directly interfere in ovarian ecdysteroid production. The significance of these findings in relation to the mode of action of azadiraehtin is discussed.

The scent gland secretions of Dumeril’s ground boa (Acrantophis dumerili), pooled from two adult males and a female, were analyzed by gas chromatography-mass spectrometry. 2-Hydroxypropanoic acid, hexadecanoic acid, as-9-octadecenoic acid, octadecanoic acid, cholesterol, and 5-cholesten-3-one were indicated. These results are compared with those obtained in analyses of the scent gland secretions of other snakes.

The alkenyl acetates E9-12:Ac, Δ11-12:Ac and Zll-14:Ac were identified as minor components of the C. murinana female pheromone blend by chemical analyses of volatile pheromone gland constituents and potential pheromone precursors, electrophysiological recordings from single receptor cells, and field trapping tests. Gland washes from virgin females contained these compounds at 3%, 10% and 5%. respectively, the amount of the primary pheromone component Z9-12:Ac already reported. A 0.3% addition of either Δ11 - 12 : Ac or Z11-14:Ac significantly raised trap captures over Z9-12:Ac alone and a 3-30% addition of either minor component revealed maximum captures, not increased further by including both synergists within the same blend. A functional role of the E9-12: Ac could not be established during this study; 3% of it when combined with the pheromonal ratio mixture of the three other components tended to increase trap captures further whereas in various other mixture combinations the E9-12:Ac strongly reduced captures. These inhibitory effects were more pronounced with attractant blends based on Δ11-14:Ac rather than Δ11-12:Ac. Each blend component activated its own type of antennal receptor cell.

The effect of hydrostatic pressure on the calcium-dependent hydrolysis of dinitrophenyl phosphate by the sarcoplasmic calcium transport enzyme has been studied. The magnesium dinitrophenyl phosphate complex is the true substrate of the enzyme (K - 7000 M -1 ) by which it is hydrolyzed at 20 °C with a turnover rate of 4 s -1 . Activation by calcium ions occurs between 0.1 and 1 μM as observed for ATP hydrolysis. The activation volume of the enzyme saturated with both ligands exhibits pronounced pressure-dependence, rising from 25 ml/mol at atmospheric pressure to 80 ml/mol at 100 MPa. The apparent binding volumes for magnesium dinitrophenyl phosphate and calcium are likewise pressure-dependent. The volume changes connected with the binding of magnesium dinitrophenyl phosphate is quite small approaching zero at 100 MPa. The apparent binding volume for calcium greatly increases with pressure from 35 ml/mol at atmospheric pressure to 150 ml/mol at 70 MPa. A nearly constant binding volume of approximately 40 ml/mol results if the effect of pressure on the respective rate constants that contribute to the apparent binding constant, is taken into account. The pressure-dependence of enzyme activity at subsaturating calcium concentrations yields an activation volume of 250 ml/mol related to the rate of calcium binding indicating the occurrence of a transient large volume expansion of the enzyme complex. The volume changes observed for the calcium-dependent interaction of the enzyme with magnesium dinitrophenyl phosphate well agree with that found for magnesium p-nitrophenyl phosphate (W. Hasselbach and L. Stephan, Z. Naturforsch. 42c , 641-652 (1987)) indicating that the found volume changes are intrinsic properties of the transport enzyme, independent of the respective energy donor.

The spontaneous formation of giant (diameter > 10 μm) vesicles from a number of phospholipids in excess aqueous solution has been studied by light-microscopy. Electrically neutral as well as charged phospholipids swell to form giant vesicles only if the lipids are in the fluid phase. This shows that electrostatic repulsion alone cannot explain the spontaneous formation of giant vesicles. The results confirm the suggestion that steric forces between extended membranes play a significant part in this process.

A preparation, staining and measuring protocol for high resolution flow cytometry of chromosomes was developed. This method allows us to identify all chromosome types and is suited for characterization of permanent cell lines and cell clones by establishing their flow karyotypes. In cell clones this procedure can be used for the detection of chromosomal aberrations which appear spontaneously or are induced by mutagen treatment and persist in the cell population.

Flageilated swarmers representing an alternative mode of asexual reproduction are budded off as hollow spheres at the dorsal surface. They are covered by cells of the dorsal epithelium and the interior cavity is lined by ventral epithelium. Fiber cells are in the interspace. Early primordia in the interspace between both epithelia of the mother animal are cavities filled with granular material and surrounded by non-flagellated epithelium cells. No mitoses are seen during growth of the bud whose interior becomes densely flagellated as is characteristic for the ventral epithelium. Fiber cells are passively included. The bud at first forms a bulge at the dorsal surface which is then pinched off. The flattened shape of Trichoplax is attained via cup-shaped and C-shaped intermediate stages after opening of the hollow sphere.

Ipsenol, Ipsdienol, and E-myrcenol are male specific compounds of Ips schmutzenhoferi. Overall 46 compounds could be identified from males and females. The diversity of oxygenated terpenes is particularly high in males.

Male E. uncula showed a synergistic response to lures containing Z3-10:Ac and Z5-10:Ac in a Z3/Z5 ratio of 1/10. The dodecenyl compounds Z5-12:Ac, Z7-12:Ac and Z7-12:OH, perceived via further types of antennal receptor cells, acted inhibitorily on this binary lure. Two species of Euxoa also responded to Z3-10: Ac as a single compound or lure component, respectively. This is the first report of a Δ3-alkenyl compound being involved in sexual attraction in the Noctuidae.

The relative stabilities of different ferricinium cations were studied in buffer or serum-containing solution and in vivo and compared with their cytostatic effects in cultures of Yoshida ascites tumor cells. Although the ferricinium cation itself was the most unstable cation compared with the dimethyl-ferricinium cation and the deuterated ferricinium derivatives it showed the highest cytostatic effect.

A group of 19 anti-class II monoclonal antibodies produced in different laboratories were tested in ELISA for their ability to bind to a panel of synthetic peptides selected from HLA-DQ alpha and beta chains. No one of the antibodies tested was found to react with the synthetic fragments, thus confirming the common finding that MoAbs generally fail to recognize fragments of the native antigen. The possibility that this result might be partly due to the procedure used for screening hybridoma supernatants is discussed.