Band 45, Heft 9-10

From the aerial parts of Phebalium elatius ssp. beckleri six novel coumarins have been isolat­ ed. These com pounds are all based on a 6,7-dimethoxycoumarin nucleus with a modified C-8 isoprenyl substituent. The prenyl side chains were identified, primarily on the basis of NMR studies, as 1,2-epoxy-3-methylbut-3-enyl = (+)-6 -methoxyphebalosin (1), 1-acetoxymethyl-2-methylprop-2-enyl = (+)-6 -m ethoxyisomurralonginol acetate (2), 1,2-epoxy-3-hydroxy-4-(3-m ethylbutanoyloxy)-3-m ethylbutanyl = 1 ′,2′-epoxy-6-methoxycasegravol-4′-(3-methyl)-butanoate (3), 1-(S)-hydroxy-3-methyl-2-oxobutanyl = (+)-6 -methoxymurranganon (4), 3-methyl-2-oxotetrahydrofuran-4-yl = 6-methoxy-3′.4′-dihydroisomicrominutin (5) and (E)-3-hydroxy-3-methylbut-l-enyl = 6 -methoxymurraol (6).

Bud Exudate, GC-MS Bud exudates from twelve clones of Populus nigra originating from seven countries were analyzed by gas chromatography-mass spectrometry and the principal compounds identified. Specimens of P. nigra var betulifolia differed in bud exudate com position from the other specimens analyzed, and were more similar in exudate com position to P. deltoides than were the other specimens. The range of bud exudate com position was greater than that which would be expected to occur within a single coherent species.

Pentacyclic Triterpenoids, G lycyrrhiza glabra, 1 H and 13 C Chemical Shifts Five pentacyclic triterpenoids have been isolated from the minor constituents of local liquorice roots, one of them (8) has not been isolated before from liquorice root. Their struc­ tural formulae and stereochemical configuration was determined by spectroscopic methods. 13 C and 1 H NMR data have been compiled.

Tissue cultures of Pimpinella anisum and P. major were used to study the biosynthesis of pseudoisoeugenols. The putative precursors were labelled with 13 C with one exception that was labelled with 14 C. The incorporation of the label was controlled by 13 CNMR and liquid scintil­ lation, respectively. The biosynthetic sequence found was L-phenylalanine, p-cinnamic acid, p-coumaric acid, p-hydroxycoum aric alcohol, p-methoxycoumaric alcohol and anethol. The incorporation rates ranged from 0.5% to 25%.

Latex vacuoles of Chelidonium majus (Papaveraceae) naturally sequester protoberberine and benzophenanthridine alkaloids. Isolated vesicles take up a number of other heterologous alkaloids, such as 9,10-dihydroergocryptine, vinblastine, strychnine, nicotine, colchicine, lupanine, and 13-hydroxylupanine, with a strong preference for the indole alkaloids and for nicotine. Kinetic data clearly show that the uptake proceeds via simple diffusion and not by carrier-mediated transport. The alkaloids are concentrated in the vesicles against a concentra­ tion gradient. MgATP has no influence on alkaloid uptake. It is likely that alkaloids are trapped in the vesicles by chelidonic acid (concentration in latex 58 mM, in vesicles 661 mM) which com plexes these alkaloids and thus prevents their rediffusion out of the vesicles. Uptake of these alkaloids by leaf protoplasts and vacuoles of Ch. majus, by vacuoles of suspension-cultured cells of Lupinuspolyphyllus and by rabbit erythrocytes was studied for comparison.

In the Vicia faba-Rhizobium leguminosarum symbiosis defence reactions are established by a variable phytoalexin concentration in the nodules. No differences were observed between effective (fix + ) and ineffective (fix - ) nodules. In nodules of field grown plants the average phytoalexin concentration was about 3 times higher than that from sterile pot-cultured plants infected with different Rhizobium leguminosarum strains. A great variability of phytoalexin accumulation in nodules of single plants was observed. In field grown plants about 60% of the nodules of each plant had very low levels (< 50 µg wyerone g nodule fresh weight -1 ) the other 40% had levels between 100 and 600 µig w yerone-g nodule fresh weight -1 indicating different defence reactions in the nodules distributed over the root system of one plant. Phytoalexin accumulation was more affected by the cultivar of the host plant than by the infecting Rhizobium strain. Two inbred lines had the lowest concentration in nodules

A 3 β-hydroxysteroid oxidoreductase was isolated and characterized in the microsomes of Digitalis lanata cell cultures. The enzyme catalyzes the conversion of 5α-pregnane-3,20-dione to 5a-pregnan-3 β-ol-20-one and requires NAD(P)H 2 . The enzyme was found to have a pH optimum of 80. The reaction had an optimum incubation temperature of 25 °C with linear reduction for the first 4 h, reaching maximum enzyme activity after 7 h. Substrate kinetics for 5a-pregnane-3,20-dione and NADPH 2 resulted in apparent K m -values of 18.5-20 (µM for 5a-pregnane-3,20-dione and 50-120 µM for the co-substrate NADPH 2 . In order to localize 3β-hydroxysteroid oxidoreductase differential centrifugation as well as linear sucrose density gradient centrifugation were performed. The results obtained lead to the conclusion that 3β-hydroxysteroid oxidoreductase is not associated with a single cell compartment, but consists of a major soluble part and a markedly smaller part of endoplasmic reticulum-associated activity

HM GCoA reductase was identified in seedlings, callus cultures, cell suspension cultures and in needles of spruce ( Picea abies) (L.) (Karst). Activity was found in both the 18 K pellet and in the 105 K pellet with different ratios between the two fractions from the various sources. The enzyme has a pH-optimum of 7.9 and an absolute requirement for NADPH . The presence of a thiol reagent such as dithiothreitol is required for activity. K m for HM G CoA is 20 -25 μM. Detergents have differential effects on the activity. In seedlings, enzyme activity was considerably higher in the hypocotyls than in the cotyledons. Enzyme activity was high in dark-grown and low in light-grown seedlings. When the light conditions were reversed, levels of activity adapted to the respective new conditions (increase or decline of specific activity). Aerobic incubations of seedlings, callus cultures or needles in medium containing a carbon source, resulted in a large (up to 20-fold) transient increase of HMGCoA reductase activity. Transfer of stationary phase cell suspension cultures into new medium caused a similarly large increase of activity. A number of carbohydrates induced the enzyme, glucose, fructose and sucrose being most effective. The increase of activity was prevented by cycloheximide. All changes of activity were much more pronounced in the 18 K pellet HMG CoA reductase

Absorption spectra and fluorescence properties of 2,3-dihydro-4(1 H)-quinolinone derivatives were studied at room temperature in different solvents. It has been found that the fluorescence quantum yields and fluorescence decay times strongly depend on the molecular structure and solvent polarity. The character and the energy of excited states were determined by PPP and CNDO/S quantum-chemical calculations.

The amino acid sequences of phytochrome from Avena sativa, Oryza sativa, Curcurbita pepo, Pisum sativum and Arabidopsis thaliana have been analyzed with a variety of computer programs, with a view to identifying areas of the protein which contribute to the properties of this photoreceptor. A region at the C-terminus has been shown to be amphiphilic, and by ana­logy with surface-seeking peptides, may be responsible for interaction of phytochrome with lipid bilayers. Possible targeting sequences in phytochromes have been identified, including a series of four basic residues which correspond to those responsible for transport of nuclear-located proteins. Sites capable of post-translational modification have been found in monocot sequences, but not in dicot sequences. Areas of the phytochrome molecule which are exposed on the surface of the portein, and which are therefore capable of interaction with other cellular macromolecules, have been identified. Analogies with other biliproteins have been used to define minimum chromophore-protein interactions. Possible enzymic activities associated with phytochromes have been discussed with respect to local amino acid sequence similarity with enzymes.

Regulation of acetolactate synthase (ALS, EC 4.1.3.18) in the phototrophic prokaryote Rhodospirillum rubrum was studied. In cell free extracts of 5 strains investigated, enzyme activity was very labile (about 80% loss of activity within 12h during storage at 4 °C) but was stabilized to some extent by 10 (μM FAD and 20 vol.% glycerol. By filtration of extracts through Superose 6 HR gels (FPLC technique), ALS activity of all strains was separated in two fractions of 200 and 600 kDa, respectively. The enzyme fractions had about the same affinity to pyruvate (ATm = 1.6 - 1.8 m M) , the same sensitivity to L-valine (50 and 65% inhibition by 0.1 m M valine in the standard test mixture) and the herbicide sulfometuron methyl (90 and 92% inhibition by 1 μM herbicide), but differed greatly in their sensitivity to inhibition by 0.4 m NaCl. In culture media with 2-oxobutyrate (2-OB), growth began only after a lag-phase of several days (5 days with 1 mM of the inhibitor). Cells grown in the presence of 2-OB had a reduced total ALS activity and did not contain the 200 kDa fraction. The inhibition of ALS by valine was noncompetitive in respect to pyruvate (K 1 = 0.l m M ) . From other branched-chain amino acids tested (L-leucine, L-isoleucine, norvaline, norleucine) only isoleucine was inhibitory (K 1 ; = 3.1 m M )

Magnetic Interaction, Manganese C om pounds, Photosystem II, Manganese Clusters Based on the investigations for the magnetic interaction in the synthetic binuclear man­ganese com pounds of (μ-oxo bridge and for results by magnetic measurements and EXA FS studies on the native enzyme, it was concluded that there are two dimeric manganese units in the oxygen-evolving center of the photosystem II. One of them is a di-(μ-oxo-bridged dimer with M n -M n distance of 2.7 Å, and it has a Mn 2 (III/III) oxidation state in both the S 1 ,-and S 2 -states. A second binuclear unit, which we will call as X-pair, is assumed to have a Mn 2 (II/III) oxidation state in the S 1 -state, and is oxidized in the S,-to S2-state transition. The multiline ESR signal observed for the S 2 -state has been attributed to be due to a partially oxidized (Mn 2 -(III/IV) oxidation state) of the 2.7 A-pair, which should be formed through the interac­ tion between the 2.7 A-pair with Mn-,(III/III) state and the X-pair of Mn 2 (III/III) which is gen­ erated in the S 1 -to S 2 -state transition.

The timing of changes in the protein synthesis pattern of elicitor-treated, [35S]methionine-labelled parsley cells (Petroselinum crispum) was analyzed by two-dimensional gel electropho­ resis. Five groups were distinguished from a large number of elicitor-responsive as well as un­ responsive proteins. Two groups were synthesized de novo either early or late after elicitor ap­ plication; two other groups were strongly reduced in their rates of synthesis either early or late after elicitor application; and one group was not appreciably affected at all. The elicitor-in-duced changes altered the total protein com position considerably. A few selected, induced pro­ teins were functionally identified. These included two early induced enzymes, phenylalanine amm onia-lyase (PAL) and 4-coum arate:C oA ligase (4C L), and a late induced enzyme, a ber-gaptol O-methyltransferase (BM T) which is specifically involved in the biosynthesis of furano-coumarin phytoalexins. The biological significance of the observed differential timing of changes in protein synthesis rates is discussed.

The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent nonaethylene glycol lauryl ether and then reconstituted in spherical egg phosphatidylcholine bilayers as described earlier (U. Scheuring, K. Kollewe, W. Haase, and D. Schubert, J. Membrane Biol. 90, 123-135 (1986)). The resulting paucilamellar proteoliposom es of average diameter 70 nm were transformed into smaller vesicles by French press treatment and fractionated according to size by gel filtration. The smallest protein-containing liposomes obtained had diameters around 32 nm; still smaller vesicles were free of protein. All proteoliposome samples studied showed a rapid sulfate efflux which was sensitive to specific inhibitors of band 3-mediated anion exchange. In addition, the orientation of the transport protein in the vesicle membranes was found to be “right-side-out” in all samples. This suggests that the orientation of the protein in the vesicle membranes is dictated by the shape of the protein’s intramembrane domain and that this domain has the form of a truncated cone or pyramid

Flow cytometric 1-parameter DNA analysis and 2-parameter DNA /protein analysis have been performed with cell material of the diptera species Chironomus thummi, Drosophila melanogaster, Calliphora vicina and Musca domestica using an impulse cytophotometer with a new quartz objective, that was especially manufactured for cytofluorometric investigations. The occurrence of heterogenous cell populations with aneuploid and polyploid DNA content within the cell material of different developmental stages of diptera species have been determined, whereby in larvae polyploid cell populations and in imagos aneuploid cell populations predominate. Partially separation of 2C cells from other cell populations with higher DNA content can be done by Ficoll-Hypaque centrifugation as demonstrated with cell material from Chironomus larvae. For flow cytometric DNA analysis of insect cell material a simple and rapid cell preparation and staining technique is presented by using the DNA-specific fluorochrome DAPI in combination with the protein fluorochrome sulforhodamine 101. Employment of flow cytometry in diptera genetics might be a new tool for cytological and cytogenetic investigations as shown with the classical genetic objects Chironomus and Drosophila

Hydroxyl radicals were generated radiolytically in N 2 O -and N 2 O / O 2 (4: 1)-saturated aqueous solutions of hyaluronic acid. The hydroxyl radicals react rapidly with hyaluronic acid mainly by abstracting carbon-bound H atom s. As a consequence of subsequent free-radical reactions, chain breakage occurs the kinetics of which has been followed using the pulse radio­ lysis technique. In the absence of oxygen, strand breakage was followed by the change in conductivity in­ duced by the release of cationic counterions condensed at the surface of hyaluronic acid which is a polyanion consisting of subunits of glucuronic acid alternating with N-acetyl-glucosamine. It appears that strand breakage is not due to one single first-order process, however, the con ­ tributions of the different com ponents cannot be adequately resolved. At pH7 the overall half-life is 1.4 ms, in both acid and basic solutions the rate of free-radical induced strand breakage is accelerated (at pH 4.8, t 1/2 = 0.6 ms; at pH 10, t 1/2 = 0.18 ms). In the absence of oxygen there is no effect of dose rate on the kinetics of strand breakage. In the presence of oxygen in addition to conductom etric detection, strand breakage was also followed by changes in low-angle laser light-scattering. These two techniques are complementary in that in this system the conductometry requires high doses per pulse while the light-scat­ tering technique is best operated in the low -dose range. In the presence of oxygen a pro­ nounced dose-rate effect is observed, e.g. at pH 9.7 after a dose of 9.4 Gy the overall half-time is approx. 0.5 s, while after a dose of 6.6 Gy the half-time is approx. 0.23 s. Both the yield and the rate of strand breakage increase with increasing pH, e.g. at pH 7 G(strand breaks) = 0.7 × 10 -7 mol J -1 and at pH 10.4, 4.8 × 10 -7 mol J -7 . The radiolytic yields of CO 2 , H 2 O 2 , organic hydroperoxides, O 2 ·- and oxygen consum ption have been determined in y-irradiated N 2 O/ 0 2(4: 1)-saturated solutions of both hyaluronic acid and β-cyclodextrin.

Lactate dehydrogenase from pig heart is inactivated by the NAD + -analog P 1 -N 6 -(4-azidophenylethyl)adenosine-P 2 -[4-(3-azidopyridinio)butyl]diphosphate (6) upon irradia­tion with UV light of wavelengths in the range from 300 to 380 nm. The decrease in enzyme activity can be prevented by the addition of NAD + and oxalate. The modified enzyme shows a reduced binding capacity for its coenzyme as compared to native lactate dehydrogenase. The amount of incorporated coenzyme is deduced from the ribose content of inactivated enzyme. Tryptic digestion of the modified protein and separation of the peptides by HPLC yields 5 ribose-containing fractions. One of them, fraction 6 6 , is split by treatment with nucleotide pyrophosphatase into two subfractions, 63 and 58. Only subfraction 63 contains ribose. Whereas peptide 58 shows a UV absorption spectrum similar to that of 4-(3-aminopyridinio)-butyl phosphate (3). Amino acid analyses of the peptides indicate that the inactivator forms covalent bonds with different parts of the protein: Peptide 63 is characterized by a great por­tion of hydrophobic amino acids whereas peptide 58 shows a high degree of hydrophilicity.

The fate of injected [ 14 C]serotonin (0.1 μCi/1.6nmol per animal) was investigated in the crayfish Orconectes limosus after injection. Specific radioactivities reached highest values in the green gland, followed by intestine, gills, nervous tissue and digestive gland. Label accumulation was lowest in the tail muscle. Cold serotonin injected into the hemolymph (50 nmol/100 g wet wt) was cleared with a mean half life of 12.9 +/- 2.6 min (15°C; N = 5). By HPLC separation of hemolymph samples taken at intervals after the injection of [ 14 C]serotonin, the formation of a metabolic product could be demonstrated. The formation of the metabolite was not, however, detectable by native fluorescence, suggesting the metabolic breakdown of the indole ring. The combined amounts of acid soluble radioactivity recovered from hemolymph, green gland, digestive gland and intestine yielded 80-100%, 50-70% and 30-50% of the injected dose after 7 min, 1 h, and 4 h, respectively. Since other tissues did not account for the balance and less than 5% of the injected label had been excreted into the medium after 4 h the results suggest the gradual incorporation of the metabolites into the tissues

The order parameter, S, of the plasma membrane of in toto human peripheral blood lym­ phocytes was obtained by electron spin resonance spectroscopy in the temperature range 25-41°C. This membrane is completely in the liquid crystalline state above 31 °C. In presence of the antigen ETB from Staphylococcus aureus at the concentration of 4 μ/3 × 10 7 cells an overall decrease of the order parameter for this membrane is observed. The decrease of 5 is followed by an upwards shift at about 35 °C of the temperature of the liquid crystalline state.

Native Fluorescence, Tumor, Lung Illumination of unstained 9 |am cryosections of lung tissue with 365 nm results in visible fluorescence light with a maximum intensity at about 460 nm. These fluorescence tomographical studies can be used for detecting carcinoma of the lung. The fluorescence pattern obtained can be matched nicely with histological findings. Since it takes less than 5 min for getting the fluorescence images, the fluorescence tomographical technique might be used in addition to established methods for determining the histology of a biopsy sample.

The approach of male Lobesia botrana (Lepidoptera: Tortricidae) in the wind tunnel to a source of sex pheromone was studied using two video cameras connected to image processing equipment. Three-dimensional records demonstrate that the flight to calling females is quite direct compared to the zigzag flight reported for other species. Convoluted paths displaying bimodal distribution of track and course angles were only obtained with a synthetic pheromone blend consisting of E-7,Z-9-dodecadienyl acetate, E-7,Z-9-dodecadien-l-ol and Z-9- dodecenyl acetate at the proportions of 1 /0.2/0.05. This suggests that this blend is still suboptimal and that zigzag flight may be due to a lack of precision in upwind motion caused by inadequate composition of the stimulus

From the seed exudates of Vicia faba L. (cv. Muchamiel) the flavonoid aglycones 7,3′,4′-trihydroxyflavone, 7,4′-dihydroxyflavone, quercetin and kaempferol, and the flavonoid glycosides quercetin 7-glucoside and kaempferol 7-glucoside were identified. This is the first time that the flavonoids present in Vicia seed exudates are described. The study of the flavonoids present in legume seed and root exudates is especially important since these substances may act as chemical signals activating or inhibiting Rhizobium nodulation genes. In fact, the activating effect on Rhizobium nod genes of 7,3′,4′-trihydroxyflavone and 7,3′-dihydroxyflavone has previously been reported. It is remarkable, that these compounds increase dramatically in mature pods, and these tissues might have an additional ecological role in the signal function on Rhizobium to establish the symbiosis

13 CNMR , GC and GC/MS analysis of the steam distillate from leaves and stems of alfavaca, Ocimum gratissimum, from the Brazilian Amazon resulted in the identification of methyl eugenol (46.83%) and eugenol (19.26%) as the main components, and another 34 compounds, mainly monoterpene and sesquiterpene hydrocarbons

A cDNA clone for NADPH: protochlorophyllide oxi­doreductase from barley was subcloned for production of antisense-mRNA. This enabled heterologous hybridi­ zation with RNA from cress seedlings (Lepidium sativum L.). The m RNA level for NADPH: protochlorophyllide oxidoreductase did not decrease in cress seedlings during irradiation with continuous far-red or white light up to 12 h. The amount of NADPH: protochlorophyllide oxi­ doreductase protein, identified by Western blot de­ creased 5-fold after continuous irradiation with white light for 12 h. Species differences for light regulation of RNA are discussed.

We consider the strongly nonplanar hydrogen bond­ ing of nitrous bases in single Watson-Crick pairs to be due to pyramidal structure of their NH,-groups. 1080