Band 55, Heft 7-8

Several species of freshwater green algae belonging to the order Zygnematales (Spirogyra crassa (Ktz.) Czurda, S. longata (Vauch.) Ktz., and Mougeotia viridis (Ktz.) Wittr.) were found to have a specific composition of the volatile fraction, which confirms an earlier proposal for the existence of two groups in the genus Spirogyra. Antibacterial activity was found in volatiles from S. longata.

Hexane and methanol extracts of Paspalum scrobiculatum seeds have yielded several known fatty acids, sterols, unusual straight chain hydrocarbons which are well known for their insect pheromone activity along with the antitumour glyceride, α-palmitin.

A comparison of the pigments in Fuchsia extorticata pollen with the pigments extracted from the unusually coloured blue-black “fuchsia” bee pollen confirms the New Zealand tree fuchsia as the source of the bee pollen. The pigments were identified as the p-trans-coumaroylated derivatives of delphindin-, petunidin and malvidin-3-O-glucosides and delphinidin-3-O-(p-cis-coumaroyl-glucoside). The anthocyanins are accompanied by several flavonol glycosides; kaempferol-3-sophoroside, quercetin-3-sophoroside and kaempferol 3-neohesperidoside.

Nine flavonoids have been isolated from Ulex jussiaei and U. minor (Leguminosae). From both species the isoflavonoids ulexin A and the new naturally occurring ulexin B have been identified, together with isoderrone, the pterocarpans (-)-maackiain and (-)-4-methoxymaackiain, and the chalcone isobavachromene. The pterocarpan (-)-2-methoxymaackiain was only present in the first species and the isoflavones isolupalbigenin and ulexone A have been identified in the second one. 13 C NMR data of isobavachromene, isolupalbigenin and ulexone A are also included. The antifungal activity of the isolated compounds was tested by the bioautographic method against Cladosporium cucumerinum. The most active compounds were the pterocarpans, the chalcone and the isoflavones with non-hydroxylated open chain prenyl substituents.

A collection of Eupatorium semialatum from Guatemala was investigated for the occurrence of sesquiterpene lactones. In addition to the sesquiterpene cinnamoyloxyoplopanone (5), lactones of the eudesmanolide type were found and identified by NMR as derivatives of 8 β-hydroxyreynosin and 8 β-hydroxybalchanin, esterified with 2-substituted acrylic acids. The acid moieties were determined to be the enantiomeric (R)- and (S)-(2-hydroxy-1-methoxyethyl) acrylic acid, in 1, 2 and 4 respectively, and (1-hydroxy-ethyl) acrylic acid in 3.

The new bishomoflavone ononin and its glucoside have been isolated from Ononis vaginalis. The structures were determined primarily by NMR spectroscopy. The assignment of NMR signals was performed by means of 1 H - 1 H COSY, HMQC and HMBC experiments.

Two new triterpenoid saponins, 3-O-{[ β-ᴅ-4-O-sulfonylglucopyranosyl-(1→3)]-α-ʟ-ara-binopyranosyl}-ursolic acid -28-O-[β-ᴅ-glucopyranosyl] ester (indicasaponin C) and 3-O-{[β-ᴅ-4-O-sulfonylglucopyranosyl-(1→3)]-[β-ᴅ-xylopyranosyl-(1→2)]-α-ʟ-arabinopyranosyl}-ursolic acid -28-O-[β-ᴅ-glucopyranosyl] ester (indicasaponin D) have been isolated from Fagonia indica. The structures were determined primarily by NMR spectroscopy. The assignment of NMR signals was performed by means of 1 H - 1 H COSY, NOESY, ROESY, TOCSY, HMQC and HMBC experiments.

This paper describes a comparative study by using different chromatographic supports (silica gel, chitin and chitosan) to separate biflavonoids from Rheedia gardneriana by column chromatography. The results indicated that chitin can be used as alternative method, but the yield of the compounds is lower than when silica gel is employed. In contrast, chitosan is not a good chromatographic support for the separation of the biflavonoids under the same experimental conditions.

The coumarin patterns of Pelargonium sidoides DC. and Pelargonium reniforme CURT., forming the origin of the herbal medicine “umckaloabo”, were analysed and compared for therapeutic equivalence. For both species, members of tri- and tetraoxygenated coumarins almost exclusively were present in the respective metabolic pools. However, the roots of P. sidoides and P. reniforme expressed conspicuously distinct coumarin variations, with umckalin, its 7-O-methyl ether, 7-acetoxy-5,6-dimethoxycoumarin, 6,8-dihydroxy-7-methoxycoumarin, 6,8-dihydroxy-5,7-tetramethoxycoumarin, artelin and three unique coumarin sulfates as uncommon metabolites of this class of secondary products of P. sidoides. Furthermore, the highly oxygenated but known coumarins fraxinol, isofraxetin and fraxidin were associated with the new 8-hydroxy-5,6,7-trimethoxycoumarin as representatives of P. reniforme. Of the twelve identified coumarins only the two species shared the ubiquitous scopoletin and the unique 6,7,8-trihydroxycoumarin. From the oxygenation patterns it is evident that the majority of these Pelargonium coumarins match the recently established basic structural requirements for marked antibacterial activity, i.e. the presence of a methoxy function at C-7 and an OH group at either the C-6 or C-8 position. The current data on the coumarin profiles of each Pelargonium species also indicate a previous erroneous identification of the plant material claimed to be P. reniforme. Absence and presence of umckalin and its 7-O-methyl ether defines P. reniforme and P. sidoides, respectively.

The aerial parts of Centaurea achaia afforded, in addition to several known sesquiterpene lactones and sesquiterpene hydroxyesters, a new germacranolide and a new elemanolide. Their structures were determined as the 8 α-O-(4,5-dihydroxy-tigloyloxy) esters of salonitenolide and of 11,13-dihydromelitensin, respectively. The in vitro antifungal activity of most compounds was tested against nine fungal species using the micro-dilution method. All the tested compounds showed strong antifungal activity.

Exopolysaccharides from the lactose-negative yeast Rhodotorula glutinis 16P were synthesized by co-cultivation with the yeast Kluyveromyces lactis MP11 or with the homofermentative lactic acid bacteria Lactobacillus helveticus 9A in a cheese whey ultrafiltrate. Exopolysaccharides were produced by lactose hydrolysis, performed by two pathways: with β-galactosidase from Kluyveromyces lactis MP11 which assimilates glucose and galactose; with β-galactosidase and Lactobacillus helveticus 9A which uses lactic acid. By growing the two mixed cultures maximum yield was obtained as follows: 11.4 g/l and 15.8 g/l, respectively. Structural units of the carbohydrate composition of the two polymers are mannose (72.4-63.5%), glucose (2.0-15.9%), galactose (25.3-19.8%) and xylose (3.6-4.3%). Mannose dominated in the polysaccharide compositions.

Two novel resorcinol derivatives were isolated from the mycelium of Lasiodiplodia theobromae IFO 31059 as potato micro-tuber inducing compounds. Their structures were established by spectroscopic methods as ethyl (6′R)-2,4-dihydroxy-6-(6′-hydroxyheptyl)benzoate (8) and isobutyl (6′R)-2.4-dihydroxy-6-(6′-hydroxyheptyl)benzoate (9). The absolute stereochemistry of the asymmetric center of (8) was determined by an advanced Mosher method.

Resistance inducing effects of an extract of Lychnis viscaria L. seeds, containing different brassinosteroids (BR), were investigated. Application of aqueous solutions in concentrations from 0.5 to 10 mg/l (dry wt. of extract) resulted in an enhanced resistance of tobacco, cucumber and tomato to viral and fungal pathogens of up to 36% compared with water-treated control plants. No direct anti-fungal effects in mycelium growth assays with Phytophthora infestans could be observed. After treatment and inoculation with powdery mildew a stimulation of different PR-proteins (ca. + 20% for peroxidase, + 30% for chitinase and up to + 68% for β-1,3-glucanase) in cucumber was found. A chitinase in gel-electrophoresisassay showed a stronger induction of a distinct isoform under the same conditions. Time course of peroxidase induction and changes of apoplastic protein patterns revealed by SDS-PAGE indicated an earlier triggering of defence responses after plant-extract treatment and pathogen attack, probably being responsible for the increased resistance. Involvement of the brassinosteroids in the plant extract is discussed to elicit or mediate the activation of defence-mechanisms.

The wild-type yeast strain Zygosaccharomyces bisporus CBS 702 produced α-hydroxyketones (acyloins) from amino acid precursors after transamination to the corresponding 2-oxo acids. The key enzyme of the subsequent decarboxylation and C − C bond forming reaction, pyruvate decarboxylase (PDC ), was examined for its substrate- and stereo-specificity. A wide variety of saturated and unsaturated aliphatic and aromatic aldehydes was successfully converted to acyloins. 19 of the biotransformation products identified had not been reported as natural substances before. Product yields were strongly affected by substrate structure.

Chloroplasts isolated from three populations of the tropical marine Chlorophyte Anadyomene stellata collected off the coast of Greece were analyzed for their fatty acid composition. Following the preparation of fatty acid methyl esters, GC-MS (El) was utilized to identify the fatty acids present in each population. Including isomers, the fatty acid profile of all three algal populations was comprised of 19 fatty acids (4 saturated, 6 monounsaturated, 9 polyunsaturated) with palmitic acid present in the highest amounts (25-27% of total fatty acids). Analysis of variance revealed significant differences amongst the three populations in the percent of total fatty acids for twelve of the fatty acids. High levels of C 20 PUFAs, an atypical observation in Chlorophytes, were observed in all three populations comprising approximately 17% of total fatty acids. Furthermore a 14:2 PUFA , apparently rare in marine macrophytic Chlorophytes, was identified in significant quantities. Surprisingly, we did not find any of the conjugated tetraene containing fatty acids that we previously identified in the A. stellata populations studied from the Florida Keys.

Alkaline treatment of the Ectothiorhodospira sp. light harvesting system II induces monomerisation of the bacteriochlorophylls and a bleaching of the carotenoid absorption bands in the visible region. Concomitantly, the maximum of absorption observed around 373 nm shifts towards 354 nm. This shift does not result from the Soret band but from a change of the absorption properties of the carotenoids. Furthermore, these pigments are not modified chemically but the spectral conversion results from environmental changes. It is assumed that the dissociation of the bacteriochlorophylls in alkaline medium is accompanied by a structural reorganisation of the complex which reinforces the interactions between the polypeptides and the carotenoids.

The enzyme, β-glucogallin: 1,2,3,6-tetra-O-galloyl- β-ᴅ-glucose 4-O-galloyltransferase, which catalyzes the last common step in the biosynthesis of the two subclasses of hydrolyzable tannins, i.e. gallotannins and ellagitannins, was purified 868-fold from leaves of pedunculate oak ( Quercus robur, syn. Q. pedunculata) to apparent homogeneity. Polyclonal antibodies against this pivotal enzyme were raised in rabbits and purified by protein-A chromatography, gel-filtration and affinity complexation. They were found to react specifically with acyltransferase from oak, displaying no cross-reactivity towards analogous enzymes from other plants synthesizing hydrolyzable tannins along the same biogenetic route, e.g. Rhus typhina or Tellima grandiflora.

An alkaline phosphatase (ALPase) from Saccharomyces cerevisiae strain 257 was purified 345-fold with specific activity of 54 533 nmol × min −1 × mg protein −1 . It was shown to be a dimeric protein (apparent mol. wt. approx. 130 kDa) with optimum activity at pH 8.6 - 8.8 and good stability at 50 °C. The ALPase was a non-specific enzyme hydrolyzing a wide variety of monophosphate esters. The enzyme showed protein phosphatase activity and this activity was not Mg 2+ - dependent in contrast to p-nitrophenyl phosphate (pNPP) activity. The K m value for pNNP hydrolysis was determined to be 2.2 × 10 −5 м. Orthophosphate inhibited the enzyme in a competitive mode with the K i of 2.3 x 10 −4 м. Phosphate transfer of the ALPase is almost zero with all alcohols tested except for Tris.

Organisms and cellular systems are required to adapt to stress conditions like high temperature, often responding by accumulating organic solutes, such as sugars. This accumulation is associated with the effectiveness of these osmolytes in minimizing protein denaturation and membrane damage under stress conditions. In this work, we have studied the effect of sugars on the protection against thermal inactivation of mitochondrial F 0 F 1 -ATPase complex, in preparations of submitochondrial particles containing or depleted of inhibitor protein. We observed that after 15 min of pre-incubation at 70 °C of latent MgATP-submitochondrial particles (with inhibitor protein) in the presence of 1.5 м of sucrose or trehalose, or 3.0 ᴍ of glucose or fructose, about 80% of enzyme activity remained active. In the same conditions, but in the absence of sugars, the activity of the particles was completely abolished. Submitochondrial particles depleted of the inhibitor protein (AS-particles) were almost completely inactivated after 3 min of pre-incubation at 70 °C in the absence of sugars and more than 60% of the enzyme activity remained active when these particles were pre-incubated in the presence of sugars. In such condition, the enzyme acquires a more compact and heat-stable conformation. Sugars, as well as the inhibitor protein, inhibit reversibly F 0 F 1 -ATPase complex activity and protect this enzyme against inactivation by high temperature. Interestingly, the protection, promoted by sugars, of particles containing inhibitor protein is higher than of particles depleted of inhibitor protein, suggesting a synergism between sugar and inhibitor protein.

A new and simple enzymatic method was developed for preparative isolation of anthraquinone pigments from Dermocybe sanguined. The endogenous β-glucosidase of the fungus was used to catalyze the hydrolysis of the O-glycosyl linkage in emodin- and dermocybin-1-β-ᴅ-glucopyranosides. The developed enzymatic method was found to be effective for the pigment isolation, as the hydrolysis occurred virtually completely, thus leading to a high pigment yield. Two fractions were obtained by the method: Fraction 1 (94% of the total pigment amount), containing almost exclusively the main pigments emodin and dermocybin, and Fraction 2 (6% ), containing the anthraquinone carboxylic acids. A 10.5 kg amount of fresh fungi yielded 56 g of Fraction 1 and 3.3 g of Fraction 2 anthraquinones. The anthraquinones in each fraction were separated by thin-layer chromatography using toluene-ethyl acetateethanol- formic acid (10:8:1:2, v/v/v/v) as eluent. The components on the chromatograms were detected and characterized by measurements on a densitometer-spectrophotometer. Combined gas chromatography-mass spectrometry was applied to determine the anthraquinone derivatives of Fraction 1 after methylation and acetylation.

The enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) and its functional subunits from barley (Hordeum vulgare L.) leaves were purified to homogeneity by activitydirected sequencial steps of chromatography. Based on the molecular mass estimation by SDS-PAGE, the large subunit (LS) had an apparent molecular weight of ca. 55 kDa, whereas the small subunit (SS) was ca. a 14 kDa polypeptide chain. The N-terminal sequences, established by automated Edman degradation analysis of the purified subunits, showed very close sequence homologies (52-92%) with the subunits of other rubisco enzymes reported from several photosynthetic species. In order to establish the chemical heterogeneity in the rubisco from barley, the amino acid composition of purified native enzyme was analyzed and the results systematically compared with other known type-I rubisco enzymes from spinach, maize, tobacco and pea. Major differences have been observed in the amino acid composition of barley rubisco, the concentration of cysteine, serine, threonine, isoleucine, leucine, arginine and tryptophan residues were found quite variable as compared to other higher plants. The thermostability of the native rubisco was also investigated using circular dichroism and fluorescence spectroscopy. The critical ( T c ) and melting ( T m ) temperatures were determined to be 60 °C and 57 °C, respectively, and at this temperature the enzyme not only retains its structural integrity but also its enzymatic activity. Results of these studies were discussed in the light of structural and functional adaptation of this bifunctional enzvme in C 3 and C 4 plants to their environments.

Several N-alkanoyl derivatives (4-9 and 13-16) of the potent mitochondrial complex I inhibitor TaClo (1-trichlorom ethyl-1,2,3,4-tetrahydro-β-carboline, 2) have been synthesized in order to elucidate the role of hydrophobic portion in the inhibitory action. Using rat brain homogenates or submitochondrial particles, the inhibitory effects of these compounds towards NADH-ubiquinone reductase (complex I) activity indeed appeared to correlate quite strongly with their lipophilic character. An X-ray structure analysis, exemplarily performed for N-acetyl-TaClo (4), revealed the N-substituent of such chlorinated agents to be dramatically pushed out of the β-carboline ring ‘plane’ due to the high steric demand of the huge trichloromethyl group at C-1.

The effects of 9β-benzoyloxy-la, 2α, 6β, 8α, 15-pentaacetoxy-dihydro-β-agarofuran and 9β furoyloxy-1α, 6β, 8α-triacetoxy-dihydro-β-agarofuran, major phytogrowth inhibitors isolated from the aerial parts of Maytenus disticha (Celastraceae) and seeds of Maytenus boaria (Celastaraceae), respectively, on different photosynthetic activities of isolated spinach chloroplasts have been investigated. Photophosphorylation and electron transport (basal, phosphorylating and uncoupled) were inhibited in a concentration dependent manner by both compounds, therefore acting as Hill reaction inhibitors. The site of action of these natural compounds was located in the span from P 680 to Q A . 9β-benzoyloxy-1,2,6,8,15-pentaacetoxydihydro-β-agarofuran was one order of magnitude more potent (I 50 = 2.6 μм) than 9β-furoyloxy-1,6,8,-triacetoxydihydro-β-agarofuran, suggesting that the substitution at C-9 and the acetoxy groups at carbons 2 and 15 are important structural requirements for the displayed inhibitory activity.

The rhythmic leaflet movements of the plant Desmodium gyrans (L.f.) DC slow down in the presence of a static magnetic field. The leaflet positions were digitally retrieved from sequential CCD camera images of the moving leaflets. The experiments were performed under constant light (ca. 500 lux) and temperature (about 20 °C) conditions. The period of the leaflet was then around 5 min. Leaflets moving up and down in a magnetic field of approximately 50 mT flux density increased the period by about 10% due to a slower motion in the “up” position. Since during this position a rapid change of the extracellular potentials of the pulvinus occurs, it is proposed that the effects are mediated via the electric processes in the pulvinus tissue.

In the course of the lipoxygenase-catalyzed transformation of linoleic acid to 13S-hydroperoxy- 9Z,11E-octadecadienoic acid, iron ions are liberated. This iron release has been determined using a spectrophotometric assay based on the complexation of ferrous iron by 3-(2-pyridyl)-5,6-bis-(4-phenylsulfonic acid)-1,2,4-triazine disodium salt (ferrozine). Further comparative measurements demonstrated that iron release correlates to deficient oxygen supply. We speculate that release of iron ions is caused by modifications of histidine residues located at the active site of the enzyme. Liberation of iron ions may be responsible for increased generation of lipid peroxidation (LPO) products observed after a myocardial infarction since iron ions induce nonenzymic LPO processes.

Investigation on the immunoprotective activity of Cu/Zn superoxide dismutase from Humicola lutea 103 AL (HLSOD) in hamsters with transplanted myeloid tumor was performed. Survivability, tumor growth and tumor transplantability were followed. The immune status of tumor-bearing animals, injected with the optimal protective HLSOD dose, was examined during 27 days after tumor transplantation by the following parameters: (i) the number, migration and phagocytic activity of peritoneal macrophages, (ii) the phagocytic activity of blood polymorphonuclear leukocytes (PMNs), (iii) the responsibility in vitro of spleen lymphocytes to T and B cell mitogens. It was established that intraperitoneal inoculation of HLSOD produced a protective effect on the development of tumors. Elongation of the latent time for tumor appearance and inhibition of the tumor growth were observed. The decreased percentage of mortality in early stage of tumor progression was established. Immunological studies on tumor-bearing hamsters (TBH) induced a tem porary immunorestoring effect on the suppressed phagocytic activities of peritoneal macrophages and blood PMNs during the first 14 days of tumor development. Moreover, HLSOD showed an expressed stimulating effect on proliferative activity in vitro of spleen B lymphocytes from healthy and TBH as well. The immunorestoring and protective effect of the enzyme was probably due to improve of the oxidant-antioxidant balance in peritoneal phagocytes. The tem porary character of the effect can be explained with the interference of immunosuppressing factors produced by tumor tissue as well as by the presence of tumor antigens, tumor cells and antigen-antibody complexes in the circulation.

A novel inhibitor of angiotensin - converting enzyme (ACE) identical to a sequence part of human muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chemically synthesized. Amino acid sequence was as follows Pro - Glu - Asn - Ile - Lys - Trp - Gly - Asp. This peptide inhibited rabbit lung ACE with a K 1 value of 1.6 μм. The inhibitor of ACE acts in a non-competitive manner. The amino acid sequence of the new inhibitor was com- pared of GAPDH from other sources.

The fatty acid composition of the willow leaf beetle Chrysomela vigintipunctata (Scopoli) (Coleoptera: Chrysomelidae) is presented in this paper. Fatty acids in the total lipid extract, triacylglycerols, free fatty acids and polar lipids were compared. One hundred and fifteen fatty acids were identified in the total lipids. The mixture comprised compounds with normal and branched-chains of 12-30 carbon atoms and zero to six double bonds in different positions in the carbon chain. Substantial amounts of unsaturated eicosanoic fatty acids known as important precursor of eicosanoids in insects were detected in the lipids as were biologically significant positionally isomeric dienes, trienes and tetraenes of the series (n-3) and (n-6) of C16, C18, and C22 fatty acids. Also present was a mixture of hydroxy-FA. Triacylglycerols contained mostly saturated and monounsaturated fatty acids. Polyunsaturated fatty acids were found mostly in free fatty acids and especially in polar lipids.

On further phytochemical investigation of the aerial parts of Verbascum undulatum, two new acylated iridoid glycosides, 6-O-[3-O-(trans-3,4-dim ethoxycinnamoyl)-α- ʟ-rhamnopyranosyl] aucubin (1) and 6-O -[3-O-(trans-p-methoxycinnamoyl)-α-ʟ-rhamnopyranosyl]aucubin (2) were isolated. These structures were determined by spectral methods, mainly by 1D and 2D NMR spectroscopy.