Volume 44, Issue 11-12

The broad application of chemical crop protectants and intensive plant breeding for the development of agriculture and the management of plant disease have created conditions of environ­ mental concern. Molecular genetic m ethods are being applied to the analysis of phytopathogenic fungi and their corresponding diseases. Rapid development of this research field will permit the isolation and characterization of genes involved in pathogenic processes. A detailed understanding of the molecular events of disease onset and progression will facilitate the design of improved procedures for disease prevention and control. Invited Trends Article

1 Δ g → 3 ∑ g - near infrared chem iluminescence from the peroxidation reaction of dopa, dopamine, noradrenaline and adrenaline was measured. The spectrophotometric method based on the bleaching of p-nitrosodimethylaniline was applied to check the generation of singlet oxygen catecholamines.

The viscid exudate found on fronds of the fern, Cheilanthes kaulfussii, consists of terpenoids in which flavonoid aglycones are dissolved. One of the terpenes is shown to be em-kaur-16-en-19-oic acid. The farinose frond exudate of Notholaena pallens contains this acid as the major constituent and its 3 R-hydroxy derivative, together with flavonoid aglycones as minor components. In N.incana, two of the diterpenes that form the farinose exudate along with flavonoid aglycones have been identified as 16R-ent-kauran-17-oic acid and 16R,17-dihydroxy-3-oxo-ent-kaurane (abbeo-kutone).

A methanolic extract of Piscidia erythrina root bark has been found to contain various isofla-vonoids including rotenone (rotenoid), lisetin (coum aronochrom one) and six known isoflavones (ichthynone, piscidone, piscerythrone, 2′-deoxypiscerythrone, 6′-prenylpiscerythrone and 3′,5′-diprenylgenistein). The extract additionally yielded three new 5-hydroxyisoflavones (piscery-thrinetin, 2′-hydroxypiscerythrinetin and isow ighteone) and a previously unreported coum arono­ chrom one (8-prenyl-lisetin). All four com pounds were identified using a com bination of spectro­ scopic (UV , MS, 1 H NMR) and chemical methods. Although several other 5-hydroxyisoflavones were also isolated from the root bark extract, the quantities of each were sufficient only to permit their partial characterization. Structure 2 for piscidone has been confirmed by 1 H NMR spectros­ copy.

The demosponge Pleroma menoui (order Lithistida, suborder Trienosina (= Desmophorina), family Pleromidae), collected in the Coral Sea south-east of Noum ea at a depth of 500 m, is proven here to contain the novel alkaloids ethyl 6-brom o-3-indolcarboxylate and 3-hydroxyacetyl-6-brom oindole.

Uptake of apomorphine and elimination kinetics in brain and muscle tissue of the weakly electric fish Gnathonemus petersii (Mormyridae) was determined by HPLC. 20 min exposure of the fish to apomorphine in the aquarium water (0.4 mg/1) resulted in a concentration factor of 1.09 for brain and 0.55 for muscle tissue. Elimination from brain tissue can be described with first order kinetics (t 1/2 = 2.4 h).

The sex pheromone compound [6,7-3H]-6,ll-hexadecadienyl acetate is enzymatically converted to its corresponding fatty alcohol, aldehyde and acid in living and homogenized olfactory hairs and in other parts of the antennae of the male silk-moth Antheraea polyphemus. The pheromone is metabolized in vitro mainly in the inner hydrophilic compartments, but scarcely in the outer hydrophobic cuticle of the olfactory hairs.

Citrate synthase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius was purified 365-fold to electrophoretic homogeneity. At 40 °C and pH 8.1 the homogeneous enzyme shows a specific activity of 73 units per mg, which corresponds to a turnover number of 44 sec-1. Citrate synthase from S. acidocaldarius shows pro-S stereospecificity, as is found with a partially purified preparation of the enzyme from Thermoplasma acidophilum, another thermoacidophilic archaebacterium.

, leaf protoplasts and cell suspension cultures of Lupinus polyphyllus and isolated vacuoles were studied for cellular and subcellular localization of peroxidase isoenzymes. Isoelectric focusing revealed 16 peroxidase isoenzymes. The basic peroxidase isoenzymes are predominantly localized in the vacuole and, to a minor degree, unbound in the intercellular space. The acidic isoenzymes are cell wall-bound in plants and not detectable in suspension-cultured cells. Large amounts (up to 11.0 U/ml) of a single basic isoenzyme are detectable in the spent medium of cell suspension cultures.

Diphosphatase (inorganic pyrophosphatase) activity was localized within compartments of cotyledons of germinating cucumber seeds during the stage of maximal conversion of fat into carbohydrates. At this stage, almost 2 mol pyrophosphate are produced during the formation of one mole sucrose from 0.28 mol triglyceride. When organelles of the 2000 x g pellet or 10,000 x g pellet were separated by density gradient centrifugation and gradient flotation, the diphosphatase activity paralleled the profiles of markers of the plastid stroma but was virtually absent from the glyoxysomes. Within the fraction of small vesicles and membranes, diphosphatase was attributed to the plasma membrane. The main portion of diphosphatase, contained in the plastids, was partially purified by chromatography on anion exchange resin and molecular sieving, leading to a 75-fold enrichment compared to the stroma fraction. Trace amounts of diphosphatase observed in the glyoxysomal fraction were analyzed in the same way. Comparison of the isoelectric points and the activity profile at different pH values and the inhibitory effect of the various cations indicated that the trace amounts of diphosphatase activity in the glyoxysome fraction represented contaminations originating from the plastids. The plasma membrane form of diphosphatase is an integral membrane protein which was solubilized with octylglucoside. It was shown to differ from the plastid form in pH optimum and sensitivity towards bivalent cations. All forms of diphosphatase were clearly distinguished from other phosphohydrolytic activities.

Filamentous Cyanobacteria. Hydrogen Peroxide, Photosystem II. Nitrogen Metabolism By means of mass spectrometric analysis we have been able to demonstrate H 20 2-production and its decomposition by photosystem II in thylakoids of the filamentous cyanobacterium Oscil­ latoria chalybea. This H 2 O 2 -production and its quasi simultaneous decomposition by the S-state system can be readily demonstrated in flash light illumination (K. P. Bader and G. H. Schmid, Biochim. Biophys. Acta 936, 179-186 (1988)) or as shown in the present paper in continuous light at low light intensities. These light conditions correspond essentially to the culturing condition of the organism on nitrate as the sole nitrogen source. Under these conditions, however, electron transport between the two photosystems seems to be mostly disconnected and respiratory activity practically non existent. Under these conditions, on the other hand, nitrate reductase is induced and nitrate reduced. The present paper addresses the question how this organism might solve the metabolic problems of nitrate reduction with such an electron transport system. Tested under high light intensities under which the organism would not grow at all, electron transport between the two photosystems is optimally linked and the system funnels part of its photosynthetically pro­duced electrons into a conventional cyanide-sensitive respiratory electron transport chain and even into an alternative Sham-sensitive (cyanide-insensitive) respiratory chain. This is made possible by the overweight of photosystem II capacity in comparison to photosystem I activity as reported in this paper. Under the conditions described, the cyanobacterium grows also on ar­ginine as the sole nitrogen source. Most interestingly under these conditions nitrate reductase induction is not shut off as is the case with other aminoacids like ornithine or alanine in the medium. Nitrite reductase is not induced in these bacteria, if grown on arginine as the sole nitrogen source. This observation is discussed in context with the fact that arginine is a major storage product (cyanophycin) in this organism and that the observed photosystem II mediated H 2 O 2 -production might be correlated with arginine metabolism.

The synthesis of the 3′-arylazido-2-azido ATP derivative 3′-O-{3-[N-(4-azido-2-nitrophenyl)-amino]propionyl}2-azido-adenosine 5′-triphosphate (2,3′-DiN3ATP) is described. The bifunc­ tional photoreactive ATP analog is characterized spectroscopically. Photoaffinity labeling of F, ATPase from Micrococcus luteus by this analog results in the inactivation of the enzyme and in the formation of higher molecular weight cross-links,

The occurrence of β-carotene-5,6-epoxide in higher plant photosynthetic tissue is described. The compound is found in isolated chloroplasts, thylakoids and other subchloroplast particles but can only be detected in intact leaves or cotyledons of higher plants when these are exposed to very high light intensities or to inhibitors such as monuron or paraquat. The distribution of the epoxide within the individual pigment-protein complexes is given. It is particularly associated with the PS I reaction centres (C P I and CP la) and less so with the PS II reaction centre (CPa). Circular dichroism shows that the β-carotene-5,6-epoxide isolated from photosynthetic tissue is optically inactive. It is therefore not produced enzymically but is a product of photooxidative events in the photosynthetic apparatus. Its presence in photosynthetic tissue is a reliable indicator of photooxidative damage to the thylakoid membrane involving oxidation of β-carotene.

By combining a high frequency modulation system for measurement of fluorescence with a phosphoroscope type apparatus for measurement of luminescence, recordings of fluorescence and luminescence induction kinetics under identical conditions were obtained. Both measuring sys­tems tolerated the application of saturating pulses of white light for rapid, transient elimination of photochemical quenching at photosystem II reaction centers, thus allowing determination of the non-photochemical quenching component. The saturation pulse induction curves of luminescence are well correlated with the corresponding curves of fluorescence, suggesting that luminescence yield is lowered by the same type of non-photochemical quenching (mostly “energy dependent quenching”) as fluorescence. Hence, in order to evaluate luminescence signals in terms of the rate of charge recombination at photosystem II reaction centers, knowledge of fluorescence quenching is required.

Photosystem II complexes from the cyanobacterium Anacystis nidulans have been investigated by Western blots with antisera raised against four photosystem II peptides from plants and with an antiserum raised against the soluble L-amino acid oxidase protein from/1. nidulans to achieve an iden­tification of the polypeptides - especially of the L-amino acid oxidase related protein - in isolated photosystem II complexes. Anacystis photosystem II complexes which were solubilized with lauryldimethylamine N-oxide and purified by sucrose cushion and sucrose gradient centrifugation, contained as major Coomassie brilliant blue stained polypeptides a 71 kDa band of unknown identity, a 62 kDa band, which partly contained D-l, a 55 and 49 kDa band which were immuno-reactive with an antiserum to the 47 kDa peptide of tobacco PS II complexes, and three distinct bands in the 30 kDa region. These latter bands could be identified as the extrinsic Mn stabilizing peptide (27-30 kDa), D-l (30-33 kDa) and a 36 kDa peptide (35 - 38 kDa) which crossreacted with the antiserum raised against the soluble L-amino acid oxidase protein of 50 kDa. These results suggest that the 36 kDa peptide present in purified photosystem II complexes from A. nidulans might be a processed form of the soluble 50 kDa L-amino acid oxidase protein.

Two natural antibiotics, cerulenin and thiolactomycin, were tested for their inhibitory efficacy on de novo fatty-acid biosynthesis of chloroplasts isolated from oat and spinach seedlings and compared with that of known herbicides. With both antibiotics a strong dose-dependent inhibition of the incorporation of [l- 14 C]acetate into the fatty-acid fraction of the isolated plastids was detected. The l 50 -values for the inhibition of acetate incorporation into fatty acids are about 4 µM in the case of thiolactomycin and about 50 µM in the case of cerulenin for both mono-and dicotyledonous plants. These values are much higher than those of the particular graminicides cycloxydim and diclofop (0.15 and 0.1 µM), which were developed to control grass weeds in dicotyledonous crop cultures.

A Synechocystis 6714 mutant resistant to the phenol-type herbicide ioxynil was isolated and characterized. Ioxynil was shown to inhibit both the donor and the acceptor sides of photosystem II, but at different concentrations. The mutation found in the psbA gene (encoding the D, protein) at codon 266 (asparagine to threonine) [G. Ajlani, I. Meyer, C. Vernotte, and C. Astier, FEBS Lett. 246, 207-210 (1989)] gives a ten-fold resistance of the acceptor side to ioxynil without any modification of the sensitivity of the donor side. Electron transfer between the primary and the secondary acceptor of photosystem II was identical in the mutant and the wild type. The mutant remains sensitive to atrazine and is even more sensitive to DCMU than the wild type.

Hepatoma, Acyclonucleoside Metabolism , dN and dTMP Kinases. dNTP Pool l-(l,3-D ihydroxy-2-propoxym ethyl)-5,6-tetram ethylene-uracil (DHPTU) is a newly synthe­ sized acyclonucleoside which shows cytostatic properties. It was tested in Syrian hamster 6 days after heterotransplantation of Kirkm an-Robbins hepatoma. A reduction of tumour weight by 61% was found 48 h after its intraperitoneal (i.p .) administration in doses of 20 mg per kg of body weight. Inhibition of tumour growth is accompanied by a reduction of dThd, dGuo and dTMP kinase activities in tumourcytosol (by 91% and 74% and 55% , respectively) and decrease in contents of dTTP, dGTP and dA TP (by 92% , 77% and 67% , respectively) in dNTP pool. DHPTU is not phosphorylated by any tumour dN kinases, but undergoes cleavage with TU release in reaction catalyzed by the tumour cell enzyme, com petitively inhibited by FA . After [ 14 C]DHPTU or [14C]TU had been given i.p. to the animals with the tumour, 90% of the subcellu­lar fraction labelling fell into the nuclear fraction. How ever, if [l4C ]DHPTU was administered with FA and DCF, 27% of radioisotope was found in the nuclear fractions and 68% in cytosol. Since D C F which prevented FA deamination to FB (which is not an inhibitor of the mentioned enzyme) reduces DHPTU-induced changes in activity of dN kinases and dTMP kinase in hepato­ ma cells, the cytostatic activity of DHPTU seems to be connected to an enzyme which releases TU from DHPTU .

Com pound Eye, Pigment Migration, Adrenaline, Octopamine, Noradrenaline The light-induced expansion of the screening pigment in the com pound eyes of the sphingid moth Deilephila and the owlfly Ascalaphus is counteracted by local application of noradrenaline or octopamine. Ata critical concentration, similar for both drugs, the expanding effect caused by a light stimulus is completely neutralized, and at higher drug concentrations light stimulation induces a contraction of the screening pigment. The contracting effect of noradrenaline and octopamine is counteracted by adrenaline. None of the other putative neuroactive substances tested (acetylcholine, GABA , histamine, melatonin, serotonin and taurine) has a com parable effect on light-induced pigment movement. It is hypothesized that pigment migration is controlled by a system of antagonistically acting catecholamines, similar to the noradrenaline/adrenaline system present in vertebrates.

The light energy dependence of the Limulus ventral nerve photoreceptor current was deter- mined for two reproducible states of adaptation. In the double logarithmic plot the energy dependence of the current amplitude and that of the current-time integral, which represents the total Charge transport through the membrane during the response, show several sections with different slopes: 1)a linear section (slope about 1) 2)a supralinear section (slope 2-4) 3)a sublinear section (slope < 0.5) and 4)a second sublinear section (slope > 0.5, only in current-time integral curve) A significant deviation from slope 1 indicates that the single events (bumps) which constitute the macroscopic receptor current, are not independent of each other in size and/or number. It may indicate a positive or negative cooperativity. Light adaptation reduces the size of the receptor current and shifts the response vj. energy characteristics towards higher light energies. For light- and dark-adapted cells the transitions between the sections occurat same amplitude or area values, only the light energy values are different. The energy dependence of the time parameters, which characterize the shape of the light response, shows the same partition in 4 sections as the receptor current. The time parameters are shortened whith increasing stimulus energy and light adaptation. The amount of the shortening is greater in the supralinear section of the amplitude or area vs. energy characteristics than in the other sections (except for latency and time-to-peak with greatest shortening in the linear or first sublinear section). Section 4 is correlated with a new increase of response duration and simultaneously with the appearance of the second response component. The results can be inierpreted sufficientiy in terms of the bump-speck-model and of an automatic gain control mechanism.

Exposing native calf thymus DNA (in 0.1 м NaClO 4- , 5 mм cacodylic acid buffer, pH 6.81, 25 °C) to increasing concentrations of Hg(C 104)2 produces dramatic changes in its circular dichroism (C D ). Let r = [mol of added Hg(II)*/[mol DNA base]: the conservative CD spectrum of the DNA B-form, consisting of the 273 nm major ( + ) C D band, the 245 nm major ( -) C D band, the 219 nm minor ( + ) CD band, and the 208 nm minor ( -) CD band, becom es non-conservative in appearance at 0.01 < r < 0.12 and assumes the spectral characteristics of a left-handed D N A double helix at 0.12 < r > 1.0. The presence of a number of isoellipticity points shows that welldefined equilibria exist between the various chiroptical forms of mercurated DNA . The CD changes are totally reversible upon the removal of H g(II), at least up to r = 1.0, demonstrating that H g(II) keeps all base pairs in register.

, Allgem eine Botanik und Pflanzenphysiologie, Auf der Morgenstelle Suspension cultured cells of an anthocyanin-containing cell line (DCb) from Daucus carota L. ssp. sativus respond very rapidly to treatment with a soluble carbohydrate elicitor from the wall of the oom ycete Pythium aphanidermatum by synthesizing 4-hydroxybenzoic acid which is incorpo­ rated into the plant cell wall. Enzym es of phenol m etabolism , phenylalanine am m onia-lyase (P A L) and chalcone synthase (C H S), respond to elicitor treatment in different ways. Both the catalytic activity of PAL and its subunit concentration, measured by means of immunoblotting, show a transient increase upon elicitation, whereas CHS the initial enzyme of the flavonoid pathway, is inhibited after administration of the elicitor. and consequently anthocyanin accum ula­ tion ceases. In protoplasts derived from the cultured cells a very similar elicitor-induced response has been observed. Lacking a cell wall, the protoplasts secrete the 4-hydroxybenzoic acid into the culture fluid. The carrot protoplasts isolated by wall degrading enzym es retain their responsive­ ness to the fungal carbohydrate elicitor. The value of the protoplast system for studying the im m ediate events following elicitor treatment is discussed.

The use of intergenic spacer (IGS) fragments of plant ribosomal DNA (rDNA) for the differ­ entiation between genera and species is tested by cross-hybridization experiments with different IGS probes of two Cucurbitaceae, Cucurbita pepo (zucchini) and Cucumis sativus (cucum ber). Hybridization with cloned fragments of different parts of the IGS of ribosomal DNA exhibit a different degree of conservation within and between the Cucurbitaceae genera. In general, Cucur­ bita species seem to be closer related to each other than the Cucumis species. A repetitive element of the external transcribed spacer (ETS) shows a more genus-specific pattern, reacting only with the respective genera; the region preceding the ETS is conserved between the Cucurbita species but also cross-hybridizes weakly with the Cucumis species. AGC-rich element of the Cucumis sativus IGS (“Cfo-cluster”) is present in small amounts in Cucumis melo (melon) and even less represented in other genera of the Cucurbitaceae.

The analogical representation of the genetic code on base “e ” is described. The possible use of the “e ” base in codification processes is em phasized.

The heat production of E. coli K12 growing aerobically in glucose limited chemostat cultures is determined in the range of specific growth rates μ ( = dilution rates D) from 0,058 h -1 to 0.852 h -1 for two different glucose concentrations S e in the instream of the chemostat. namely S e1 =0.3182 g·1 -1 and S e2 = 0.6364 g·1 -1 . Heat production Q and biomass production P per unit of culture volume show well correlated patterns for S e1 and S e2 . For S e1 the highest value Q actually measured is 443-10 -3 W·1 -1 at D = 0.74 h -1 with P = 0.068 g·1 -1 ·h -1 and for S e2 593·10 -3 W·1 -1 at D = 0.497 h -1 with P = 0.108 g·1 -1 ·h -1 . Heat production Q B per unit of biomass appears to be independent of S e at least up to D - 0.5 h -1 .At higher D there is strong indication that Q B possesses a real maximum. The highest value of Q B actually measured is 4.8 W·g -1 at D = 0.74 h -1 . For S e1 and S e2 there were significantly higher specific growth rates verified in chemostat culture than μ max Batch= 0.717 h -1 which is the maximum specific growth rate in comparable, unlimited batch cultures. The real maximum of Q B is estimated to be in the vicinity of μ max Batch . This suggests the hypothesis of a maximum principle for the growth in batch culture. For S e1 a closed analytical expression is derived for the relationship between μ and the substrate concentration S. μ[S] features a S-shaped characteristic with μ max Chemostat= 0.905 h -1 ; 1/2 μ max Chemostat is reached at S = 2.85·10 -3 g·1 -1 . Three basic parameters which characterize the overall metabolism of the cells, namely the heat released per unit of substrate consumed, (Q s , the effective yield of biomass, Y eff , and μ max Chemostat are identified to depend on S e .

Ethanolic extract of propolis (EEP) was tested as a protective agent against gamma irradiation in mice. The mice were exposed to 6 Gy gamma irradiation from a 60 Co source, and were treated intraperitoneally with EEP, administered before and after their irradiation. While the non-treated mice expired within 12 weeks, the mice that received a series of EEP treatments survived the irradiation, and their leucocyte count as well as their spleens’ plaque-forming activity returned to normal. It is suggested that an antioxidant and a free radical scavanger in the EEP are responsible for the radiation protective effect of the extract of this natural product.

E. coli suspended in D 2 O showed a better survival than in H 2 O when the bacteria were treated with various damaging agents like UV , heat and freezing. On the contrary E. coli grown in D 2 O - containing media - therefore being fully deuterated - were more sensitive than normally grown bacteria.

The swarming phenomenon of 18 strains was tested with the intention to identify motile, swarming bacteria. Not all bacteria which are motile in liquid are able to swarm on semisolid nutrient broth, but all swarming bacteria show characteristic swarming developments, resulting in different swarming zones which could be scanned densitometrically. The densitograms are fully reproducible for the same strain and show similarities between strains of one species.

Contrary to an earlier report, male B. pinicolella are shown to respond to synergistic blends of (Z)-5-decenyl acetate with its corresponding alcohol analogue. Four other, decenyl or dodecenyl, com pounds are reported as potent trapping inhibitors. This pattern of pheromonal attraction and inhibition conforms with that found in some species groups of Coleophoridae, supporting the view of a close phylogenetic origin of the two families.

Antitum oral effect of ethanolic extract of propolis (EEP) was demonstrated in mature mice-bearing Ehrlich carcinoma. Survival rate after EEP treatment was compared to that of bleomycin, given alone or in combination every two days for 36 days and follow ed up for 14 additional days. The survival rate at 50 days was 55% after EEP and 40% after bleomycin, while all the mice-treated with EEP + bleomycin combination demonstrated shorter survival than the controls. It is concluded that while the in vivo activity of bleomycin is reduced in the presence of cytochrome-C-reductase inhibitors (like some of the EEP components are), the antitumoral property of EEP in the tumored animal model studied is significant and lasting.

Cell suspension cultures of Petroselinum crispum (Mill) Nyman cultivars “Paramount” and “Plain-leaved” were capable of biotransforming exogenously supplied geraniol largely into nerol and minor quantities of neral and geranial. Maximal conversions into nerol (32-36%); and neral: < 1% (cult. “Plain-leaved”), <5% (cult. “Paramount”) were usually recorded by 24 h. Over the incubation period a low proportion of the substrate, 32-40% was involved in biotransformation. Cyclic and other acyclic monoterpenoids were not biotransformed.