Volume 46, Issue 7-8

Two new triterpenoids, isolated from the frond exudate of the fern Notholaena rigida, have been identified as 3 β, 12 β,25-trihydroxy-(20 R-24 R)-epoxy-dammarane- 12-acetate (1), and 24 R,25-diO-isopropyliden-9(19)-cyclolanostan-3 β-ol (5), based on NMR spectroscopic studies.

Further investigation of polar constituents in white lupin roots revealed the presence of genistein 7-O-β-glucoside (1 a), genistein 4′-O-β-glucoside (1 b), 2′-hydroxygenistein 7-O-β-glucoside (2 a) and 2′-hydroxygenistein 4′-O-β-glucoside (2 b) as simple isoflavone gluco­ sides; luteone 7-O-β-glucoside (3 a), licoisoflavone A 7-O-β-glucoside (4 a) and licoisoflavone A 4′-O-β-glucoside (4 b) as prenylated (= 3,3-dimethylallyl-substituted) isoflavone glucosides; and genistein 7-O-β-(6′-O-malonyl)glucoside (1 am) and 2′-hydroxygenistein 7-O-β-(6″-O-malonyl)glueoside (2 am) as malonylated isoflavone glucosides. Six compounds (2 a, 2 b, 3 a, 4 a, 4 b, and 2 am) are new naturally occurring isoflavone glucosides.

Tropane alkaloids are typical natural products of solanaceous plants. The patterns of these alkaloids from hairy roots of Hyoscyamus albus were determined by GC-MS analysis. 18 alka-loidal compounds were detected, six of them only in trace amounts. Some of these alkaloids, namely hygrine, 3a-acetoxytropane, 3 β-acetoxytropane and 6-hydroxylittorine as well as the trace compounds N-methylpyrrolidinylhygrine, phenylacetoxytropane and two isomers of feruloyloxytropane were not previously reported to be components of this species. The major compounds formed were found to be hyoscyamine and littorine.

From a strain of Pseudomonas fluorescens pseudobactin and several related compounds were isolated and their structures were elucidated. In this way a reference compound (5) could be obtained for the unambiguous determination of the absolute configuration of C-1 of the pyoverdin chromophore in newly isolated representatives of this class.

From ferribactins which accompany pyoverdins in iron deficient cultures of fluorescent pseudomonads a subunit has been obtained formed by condensation of D-tyrosin with L-2,4-diaminobutyric acid to give 2-(1-R-amino-2-p-hydroxyphenylethyl)-1,4,5,6-tetrahydropyrimidine-4-S′-carboxylic acid (1). Evidence is presented that 1 is the precur or of the typical pyoverdin chromophore 7 a.

The structure elucidation of two pyoverdins from the strain R. of Pseudomonas aeruginosa is described.

The chlorophyll synthesis in the brown algae Desmarestia aculeata is affected by light quality and by the nutrient state in the medium before the illumination. Pulses of 5 min of red, green and blue light together with 200 μM nitrate in plants growing under natural conditions deter­ mined similar induction of chlorophyll synthesis. However, when the plants were incubated previously under starvation conditions the light effect was different. The induction of chlorophyll synthesis was greater after blue and green light than after red light pulses. Red-light photoreceptor was only involved in the chlorophyll synthesis under no nutrient limitations and under starvation conditions after previous illumination with blue light followed by far-red light. The induction of chlorophyll synthesis by green and blue light pulses applied together with nitrate was greater when the algae were incubated in starvation conditions than in natural conditions (normal nutrient state). Because all light effects were partially reversed by far-red light the involvement of a phyto-chrome-like photoreceptor is proposed. In addition, a coaction between blue-and a green-light photoreceptors and phytochrome is suggested.

Fluorescence quantum yields, singlet lifetimes and triplet yields for 2-methyl-2,3-dihydro-4(1 H)-quinolinone and N -m ethyl-2,3-dihydro-4(1 H)-quinolinone were determined as a function of temperature. In non-polar solvents, an efficient non-radiative process identified as an indirect singlet →triplet transition occurring through S 2 (n,π*) was found to depopulate the lowest S 1 (π,π*) state. In polar solvents, direct singlet → triplet intersystem crossing appears to dominate. Photochemical dehydrogenation produced the corresponding 4(1 H)-quinolinone with a yield of few times 10 -2 . This was shown to be the only traceable reaction route.

A new contact site between the two reaction center polypeptides D 1 and D 2 of photosystem II close to arg 238 and arg 234 respectively is proposed. The amino acid sequences involved are between the 4 th transmembrane and a connecting parallel helix. The sequence includes a tryp­ sin sensitive site in both polypeptides, the likely cleavage site in the rapid turnover of the D 1 polypeptide and part of the herbicide binding site. The contact site is oriented towards both quinone binding sites Q A and Q B . A folding of the backbone of the amino acid sequences involved is proposed.

The responses of photoautotrophic (PA) cultured cells of tobacco (Nicotiana tabacum cv. Samsun NN) and liverwort (Marchantia polymorpha L.) to thirty-eight cyclohexanedione derivatives were surveyed. Each derivative was also tested for inhibitory activity on photosynthetic electron transport (PET), using isolated thylakoids, and herbicidal activity, using seed­ lings and mature plants. Comparison of the results from the different assays showed that the responses of PA cells to each com pound correlated more closely with the responses of seed­ lings and mature plants than did the results of the Hill reaction assays. Our findings suggest that PA cultured cells would be a suitable screening material for identifying potential herbicides with PET-inhibiting activity.

The selection of plants resistant to photosynthetic herbicides is limited by the low rate of success in obtaining a photosynthetic callus. An attempt was made to induce diuron tolerance with chlorophyllous tissue strains of Bromus erectus: growth, chlorophyll contents and total fatty acids were studied during four months of photoheterotrophic growth in the presence or absence of 6 μM diuron. The presence of the herbicide, which reduced the fresh weight by 40%, provoked increases in chlorophyll levels by 2-or 3-fold, and in galactolipids (especially DGDG) which were enriched in linolenic acid content. These results, closely related to already described characteristics of herbicide-resistant material, suggest that callus of Bromus erectus could constitute an interesting photosynthetic in vitro material.

The reversible binding kinetics of atrazine, diuron and ioxynil were measured via their binding and release parameters during steady state inhibition of electron transport. The parameters were determined in isolated chloroplasts of peas and of triazine-resistant and -susceptible bio­ types of Chenopodium album using a kinetic model. This model is based on the flash-induced oxygen evolution patterns of isolated broken chloroplasts. It was found that the binding parameters were always significantly higher in the case of an oxidized acceptor quinone complex as compared with a semi-reduced complex. Triazine resistance seems to originate from a significant increase of the release kinetics. The release parameters could be used to calculate the residence times of the herbicides at the D 1 protein. The values of these residence times were always much higher for the herbicides than for Q B ; this explains the inhibition of electron transport. The only exception was the residence time of atrazine in the resistant biotype, where the value was close to that of Q B . It is concluded that the “on” kinetics of a compound to its binding environment at the D 1 protein are determined principally by the accessibility of the niche to the compound. The differences in activity between herbicides are mainly due to variations in the release kinetics.

By means of GC -FTIR , GCMS and GC analysis more than 70 components of the steam distillate of seeds and leaves from Crotalaria ochroleuca (Fabaceae, Papilionaceae) were found and partially identified, most of them being aliphatic hydrocarbons, aldehydes and fatty acids. Weak, but significant repellency of the seeds, its steam distillate as well as of the seed oil com ponent 2-isopropyl-3-methoxypyrazine, 2-isobutyl-3-methoxypyrazine and 2-pentylfuran was observed with the curculionid beetle Sitophilus zeamays. Furthermore, no attractivity of fresh leaves was found for a number of test insects.

o-Succinylbenzoate: coenzyme A ligase, an enzyme involved in menaquinone biosynthesis, was purified from Mycobacterium phlei and characterized with respect to isoelectric point, molecular weight, pH optimum, temperature optimum and kinetic data. The enzyme hydrolyses ATP to AMP. The substrate and cofactor specificity of the enzyme was tested with analogues of o-succinylbenzoic acid, different nucleotides, thiols and divalent cations. The enzyme appears to possess broad specificity for substrates and cofactors.

In Oscillatoria chalybea, as in other cyanobacteria, nitrate reduction depends directly on photosynthetic activity. Hence, nitrate reduction occurs only in the light and appears inhibited when photosynthesis is inhibited by the herbicide DCMU . Growth of Oscillatoria cells is possible on a variety of exogenous nitrogen sources in the medium and appears largely independent on the type of nitrogen source. However, if citrulline is the exogenous nitrogen source or if no exogenous nitrogen source is given in the medium, growth appears almost fully inhibited. Nitrate reductase activity measured in French-press particles of nitrate-grown cells is depend­ent on the age of the culture with maximum nitrate reductase activity being reached on the 5th day. Thereafter activity decreases steeply to less than 20% of the maximal activity within 10 days. Besides the growth stage it is the type of exogenous nitrogen source used in the medium which is important for the development of nitrate reductase activity. It appears that in the presence of nitrate, nitrite and arginine, nitrate reductase activity is induced whereas in the presence of ammonia or amino acids like alanine nitrate reductase activity is not induced, as already reported in the literature. Nitrate reductase is also induced if arginine and ammonia are simultaneously offered as exogenous nitrogen source. Arginine metabolism in Oscillatoria cells is characterized by the fact that thylakoid preparations of Oscillatoria catalyze the trans­ formation of arginine to give ornithine and ammonium. The arginine-metabolizing enzyme differs from the usual arginine-induced arginase. The enzyme seems to be constitutive, not manganese-dependent, exhibiting an approximately 5 times higher substrate affinity to arginine than the known arginase. In the present paper we propose that in Oscillatoria it is arginine which induces the synthesis of nitrate reductase.

Activities of the glycolytic enzymes were determined in seedlings, callus cultures and cell sus­ pension cultures of spruce (Picea abies) (L.) (Karst). The rate-limiting enzymes of the pathway were the hexokinases, ATP: phosphofructo-kinase, fructose-1,6-bisphosphatase and pyruvate kinase. Two phosphofructokinases were found: ATP : fructose-6-phosphate 1-phosphotransferase (PFK) and pyrophosphate :fructose-6-phosphate 1-phosphotransferase (PFP). In the presence of its activator fructose-2,6-bisphos-phate, PFP had a 4 -5-fold higher specific activity than PFK. PFP could be activated about 20-fold by fructose-2,6-bisphosphate at saturating concentrations of the substrates (fructose-6-phosphate and pyrophosphate). The increase of V max was accompanied by a strong increase in the apparent affinity of the enzyme for the substrates. Km for fructose-6-phosphate and pyrophosphate was 0.44 mM and 24 μM, respectively. K a for fructose-2,6-bisphosphate was 24 nM. In seedlings, specific activity of the glycolytic enzymes was 30-300 percent higher in the hypocotyls, except for fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase, their activity being 100-150percent higher in the cotyledons, This distribution remained unchanged during periods of 2 -16 weeks of cultivation of the seedlings. In callus cultures and in cell suspension cultures, grown mixotrophically with different car­ bohydrates, all enzymes were between 1-and 7-fold higher than in autotrophically grown seed­ lings. Incubation of seedlings in mineral salt mixture containing a carbohydrate resulted in a rapid coordinate increase of the activities to the levels of callus-or cell suspension cultures. This induction required a carbohydrate and oxygen. During prolonged cultivation of cell suspension cultures, when carbohydrate became limiting, activity of the enzymes slowly declined.

Recent advances in studies on plant ADP -glucose pyrophosphorylase (AGP), the key enzyme of starch biosynthesis, are presented. AGP constitutesthe First committed and highly regulated step of starch synthesis in all plan ttissues. The importance of AGP in carbohydrate metabolism and several of its features, such as potent regulation by cellular effectors (3-phosphoglycerate and P i ), an unusual two subunit-types structure, tissue-specific and developmentally-regulated expression, and presence of the AGP -deficient mutants, make it an attractive, but complex, target forbiotechnological manipulations. Some strategies for future research on AGP are discussed.

Vacuolar type ATPases have been found on various endomem branes of eukaryotic cells, e.g. lysosomes, chromaffin granules, vesicles derived from the Golgi apparatus, endosomes and vacuoles. Although this ATPase type is targeted to different compartments in one cell, only one gene for each subunit had been found per genome. Using PCR across intron-exon boundaries we show that two different genes encode the cat­alytic subunit of the V-ATPase in Psilotum nudum and Equisetum arvense. The substitution rates for the three codon positions and the intervening sequences show that in Psilotum both genes are transcribed and are under selection pressure, however, this seems not to be the case for Equisetum. The relatively high similarity between the two genes found in each species as compared to the interspecies similarities suggest that for some time after the gene duplication had occurred the two copies were subject to gene conversion mechanisms. An unexpected degree of conservation of the intervening sequences themselves is noted and statistically verified, however, no structural constraints that could explain these findings were detected.

The effect of azide on M g 2+ -and Ca 2+ -dependent ATPase of differently activated CF 1 isolated from spinach chloroplasts was studied. It is shown that Mg 2+ -ATPase activity is sensitive towards azide irrespective of the applied method of enzyme activation. Heat-or trypsin-acti-vated Ca 2+ -ATPase is also inhibited, whereas methanol-and octylglucoside-stimulated or DTT-activated Ca 2+ -ATPase is not affected by azide. Preincubation of the DTT-activated enzyme with low concentrations of Mg 2+ induces azide susceptibility of the Ca 2+ -dependent ATPase.

In tobacco chloroplasts hydrazine-dependent dinitrogen formation measured by mass spectrometry as the consequence of short saturating light flashes is always linked to a substantial oxygen uptake (G. Renger, K. P. Bader, and G. H. Schmid, Biochim. Biophys. Acta 1015, 288, 1990). However, in thylakoids of the filamentous cyanobacterium Oscillatoria chalybea this dinitrogen formation is not linked to an apparent O 2 -uptake, even at the high concentration of 1 mм hydrazine. Whereas in tobacco chloroplasts Tris-treatment does not affect hydrazine dependent dinitrogen formation up to a concentration of 3 mм hydrazine, Tris-treatment of thylakoids of O. chalybea affects strongly both oxygen evolution and dinitrogen evolution under a single turnover flash as well as under ten flashes. In contrast to tobacco chloroplasts, the presence of hydrazine up to concentrations of 3 mм does not substantially affect photosynthetic O 2 -evolution. The observed dinitrogen evolution is affected by DCMU regardless whether induced by a single turnover flash or by ten flashes, whereas in tobacco dinitrogen evolution and the O 2 -uptake linked to it (which is not observed in the cyanobacterium) were clearly not affected by DCMU in the single turnover flash. In Oscillatoria the earlier described Photosystem II-mediated H 2 O 2 formation and decomposition is influenced by hydrazine. In the presence of 300 μм hydrazine the usually present O 2 -uptake leading to H 2 O 2 formation appears diminished.

Quenching analysis of chlorophyll fluorescence by the saturation pulse method is used to investigate the pH-dependency of O 2 -dependent electron flow in intact spinach chloroplasts with high ascorbate peroxidase activity. When carboxylase/oxygenase activity is blocked, pho­tochemical and non-photochemical quenching are initially low and increase with illumination time. Quenching shows a pH-optimum around pH 6.5, but only when ΔpH-formation is al­ lowed. It is suggested that overall O 2 -dependent electron flow involves two major components, namely O 2 -reduction (Mehlerreaction) and reduction of the H 2 O 2 formed in the Mehlerreaction, involving enzymic activity of ascorbate peroxidase and monodehydroascorbate reductase. The separated pH-dependencies of light driven O 2 -reduction (presence of KCN) and of H 2 O 2 -reduction (anaerobic conditions) reveal contrasting pH-optima around pH 5 and 8.5, respectively. Energy-dependent, dark relaxable non-photochemical quenching is not observed with O 2 -reduction but with H 2 O 2 -reduction, and only at pH-values above 6.5. The relevance of these findings with respect to regulation of photosynthetic electron flow is discussed. It is suggested that upon limitation of assimilatory electron flow O 2 -dependent non-assimilatory flow is responsible for ΔpH-formation, by which it is autocatalytically stimulated. It is proposed that this autocatalytical reaction sequence is the basis of the so-called “Kautsky effect” of chlorophyll fluorescence induction.

The disaccharides trehalose and sucrose, and to a lesser extent the monosaccharides glucose and fructose, decrease the apparent K m of the Ca 2+ ,Mg 2+ -ATPase of sarcoplasmic reticulum for Pi. This effect is more pronounced at pH 7.4 than at pH 6.2. The enzyme is not phosphorylated by Pi when the temperature of the medium is decreased to 0 °C, but when 1.5 ᴍ trehalose or sucrose is present phosphoenzyme formation increases to 0.5 nmol E-P/g protein.

Chloramphenicol is a widely used antibiotic with low levels of toxicity. However, scanning electron microscopy revealed morphological changes in human erythrocytes when they interacted in vitro with therapeutical concentrations of chloram phenicol. To explain this shape change, a study concerned with the possible interactions of this antibiotic with bilayers built-up of phospholipids located in either side of the red cell membrane was performed by X-ray diffraction. Results indicated that chloramphenicol was unable to perturb in any significant extent the structure of the phospholipids under study. The only noticeable effects were phase transitions produced to dim yristoylphosphatidylethanolamine bilayers.

We concentrate on instances in which the phenotypic expression of information encoded in an RNA primary sequence might be revealed by the folding of the RNA itself. We have discov­ered that this situation finds concrete realization in the design of RNA molecules capable of maximizing the rate of autocatalytic synthesis when incubated with viral Qβ-replicase. This requires that we introduce the notion of phenotypic traits at the molecular level. Thus, the problem of finding RNA sequences whose phenotype favorably influences propagation amounts to finding RNA sequences which fold so as to optimize enzymatic performance and are in addition endowed with the proper recognition sites. The proof that these two problems are indeed equivalent has two steps: First we predict the metastable folded structures formed as a template RNA chain grows by sequential incorporation of nucleotides. The transient folded states appear to be involved in the regulation of the enzyme activity and they occur in a manner which is “oblivious” of thermodynamic time scales. Secondly, we compute the time-dependent activation energy for relaxation of each intermediate structure. This is done to establish constraints necessary for optimization of the regulatory role of RNA folding. The search for prospective template sequences is subject to such constraints. Our results aim at elucidating an optimization process realized by molecular selection in de novo (template-free) RNA synthesis by Qβ-replicase. We argue that the phenotype which mediates selection is given by metastable folding which emerges together with the printing of the genotype, that is, within the time span of a replication turnover.

Polycyclic aromatic hydrocarbons and aromatic amines exercise a highly specific influence on the crystallization of cholesterol. The strength of these non-covalent, presumably epitaxial interactions correlates with the carcinogenic activity of these substances. The presented results are in support of the assumption that a specific process of adsorption and crystallization with cholesterol of the plasma membrane takes place during the initial phase of the carcinogenesis by aromatic compounds.

The comparative anthelmintic activity of a possible prodrug, 2,2′-dicarbomethoxyamino-5,5′-dibenzimidazolyl methanol (2) with its parent compound 2,2′-dicarbomethoxyamino-5,5′-dibenzimidazolyl ketone (1) and the reference drug mebendazole (3 a) is reported. At a dose of 25 mg/kg, compound 2 was 100% effective against Ancylostoma ceylanicum in hamsters. Compound 2 also exhibited a similar order of activity against Syphacia obvelata, Hymenolepis nana and H. diminuta at a dose of 100 mg/kg. The drug exhibited lethal effects against metamorphic forms of A. ceylanicum at a dose of 100 mg/kg. However the trichostrongylids, Nippostrongylus brasiliensis remained unaffected up to a dose of 250 mg/kg of 2. Both 1 and 3 a exhibited inferior activity than 2 except against adult A. ceylanicum. The activity of 1 and 2 has been explained on the basis of their ability to resist systemic hydrolysis resulting in higher concentration of the active drug in biophase.

Four constituents of the hair pencil secretion of the male African sugarcane stalk borer, Eldana saccharina, having a molecular mass of 312 and peculiar El mass spectra with an exceptionally abundant base peak at m /z 103, were isolated preparatively from an extract of the secretion. Using 1 H and 13 C NMR spectral analysis, these constituents were identified as fiveand six-membered cyclic glycerol acetals of Z-9-hexadecenal, viz. cis- and trans-2-(Z- 8-pentadecenyl)-4-hydroxymethyl-1,3-dioxolane, and cis- and trans-2-(Z-8-pentadecenyl)- 5-hydroxy-1,3-dioxane. These compounds are related to the 2-alkenyl-4-hydroxymethyl-1,3- dioxolane dihydrogen phosphate esters, known to be the active constituents of the smooth muscle contracting acidic phospholipid (Darmstoff) which was isolated from the intestine of mammals. The presence of these acetals in the tail brush secretion of E. saccharina could possibly be the first evidence that com pounds related to the active principle of Darmstoff, may also be present in the insect kingdom. The possibility that these four compounds or their dihydrogen phosphate esters might play a part in the eversion or retraction of the tail brushes of the male insect, is briefly discussed.

Propagation of electrical signals is studied in Retzius cells of the leech in culture using voltage-sensitive fluorescent dyes at a spatial resolution of 8 × 8 μm 2 and 14×14 μm 2 and at a sam pling interval of 0.12 ms. The neurons are stimulated by a microelectrode impaled in the soma. Action potentials of a halfwidth of 2 -3 ms are triggered close to the end of the primary neurite dissociated from the leech. They propagate back to the soma at invariant halfwidth at a veloci­ ty of 50-230 μm/ms. They pervade extended arborized secondary neurites which are grown on extracellular matrix protein. Their width is enhanced up to a factor two. The velocity is around 100-150 μm/ms such that delays up to 3.5 ms are observed. Accordingly the neuritic trees are not isopotential. The features of propagation are found to be incompatible with passive spread.

The space-dependent density of cells is evaluated for the following situation: (i) The cells are forced to make a directed movement (ii) the space for the cellular migration is restricted. The steady state distribution density is obtained when the drift current density equals the diffusion current density. The analogy to the Boltzmann statistics is shown. In a further step the cellular volume is introduced. For this case the density distribution is described in analogy to the Fermi statistics. The necrotactic response of granulocytes is used to verify the model.

Patterns of endogenous plasmids and nitrate reductase activities were analyzed in the phototrophic bacterium Rhodobacter (Rb.) capsulatus. From 10 strains investigated (including a UV-induced plasmidless nit - mutant), 4 were unable to grow photosynthetically with nitrate as N-source and lacked nitrate reductase activity (nit strains). Irrespective of the nit phenotype, all wildtype strains contained at least one large plasmid with a size ranging from 93 to 134 kb. Thus, other than in plasmid- cured mutants (J. C. Willison, FEMS Microbiol. Lett. 66, 23-28[1990]), in wild-type strains of Rb. capsulatus the nit - character was not related to lack of endogenous plasmids.

Cell growth inhibitions on human cancer cell cultures were determined for the indole sulphur-containing phytoalexins cyclobrassinin, brassilexin (previously isolated from vegetables of the Cruciferae family) and their synthetic analogues 5-methoxybrassilexin and homocyclobrassinin. The most biologically active of these products is brassilexin (LD 50 = 8 μg/ml).