Band 54, Heft 11

The structures of the two fengycins, lipopeptides from Bacillus subtilis, were elucidated by spectroscopic methods and chemical degradation. They show a close structural relationship to the plipastatins from Bacillus cereus differing only in the stereochemistry of the Tyr residues.

The structure and stereochemistry of the siderophore isolated from the culture medium of Ralstonia eutropha (formerly Alcaligenes eutrophus) could be elucidated as staphyloferrin B by spectroscopic methods and chemical degradation. The relationship of the three species of the new genus Ralstonia is reflected in the observation that all three form citrate sidero-phores.

Above-ground parts and roots from four Stachys species (S. germanica L., S. sylvatica L. and the Balkan endemics S. thracica Dav. and S. plumosa Griseb.) as well as o f three Betonica species (B. officinalis L. and the Balkan endemics B. bulgarica Deg. et Neic. and B. scardica Griseb.) were screened for phenols (phenylethanoid glycosides, flavonoid glycosides and the phenolic diterpene betolide). Three phenylethanoid glycosides, a flavonoid glycoside and the phenolic diterpene betolide were isolated and identified, most of them for the first time in the investigated species. The results obtained support the view that Stachys and Betonica are well separated genera.

Leaf extracts of Erythroxylum ulei O. E. Schulz, produced six O-conjugated flavonoids. Three of the flavonoid aglycones were isoflavones, two were isoflavanones and the remainder a flavonol (quercetin). The major glycosides of these flavonols included mono-and dirham-nosides, mono-glucosides and galactosides with either 3̕ 4̕, or 7 linkage or a combination thereof. The two isoflavanones, in addition to being glycosylated, possessed a methyl group at position 2 of the C-ring. These flavonoids may be used as chemotaxonomic markers for the taxon.

The changes of aroma ingredients during the process of flower opening from Bulgarian rose were monitored by head space method and solvent extraction.W e also analyzed contents of glycosidic alcoholic aroma together with activities of the hydrolytic enzymes throughout the flower development and the opening. At flower petal opening time, the total amount of aromas in the head space gas reached the highest level. The concentration of citronellol was abundant in the head space gas at this stage, whereas the concentration o f 2-phenylethanol became higher than that of citronellol 4 hr after the opening stage. In the volatile extracts, higher accumulation was observed in 2-phenylethanol than those of monoterpenoids at this stage, and the content of the former still increased after flower opening. Glycosidic citronellol, geraniol, and other m onoterpenes started their accumulation ju st before flower opening stage and then reached the maximum level. The amount of these glycosidic compounds were less than those in the volatile extracts. In contrast to the monoterpenes, 2-phenylethyl glycosides accumulated in a higher level than in the volatile extracts starting at least 12 hr before the opening stage. The amount of the glycosidic precursors of 2-phenylethanol detected in the rose petals before flower opening always was higher than the amount of 2-phenylethanol which was released later. The decline of glycosidic 2-phenylethanol at flower opening stage may be due to partial enzymatic hydrol­ysis. Thereafter a drastic decline was observed, indicating that rapid enzymatic hydrolysis occurred during these stages.

A theoretical molecular modelling study has been conducted for cutin, the biopolyester that forms the main structural component of the plant cuticle. Molecular dynamics (MD) simulations, extended over several ten picoseconds, suggests that cutin is a moderately flexible netting with motional constraints mainly located at the cross-link sites of functional ester groups. This study also gives structural information essentially in accordance with previously reported experimental data, obtained from X -ray diffraction and nuclear magnetic resonance experiments. MD calculations were also performed to simulate the diffusion of water mole­cules through the cutin biopolymer. The theoretical analysis gives evidence that water perme­ation proceedes by a “hopping mechanism”. Coefficients for the diffusion of the water molecules in cutin were obtained from their mean-square displacements yielding values in good agreement with experimental data.

Appressorium formation in germinating conidia of Magnaporthe grisea was induced on a hydrophilic (noninductive) surface by antifungal compounds. Respiratory inhibitors or un­ coupling agents such as strobilurins, antimycin A , myxothiazol, rotenone, pterulone A , and oligomycin A were particularly effective whereas sodium cyanide had no effect. Cyclosporin A was effective only at high concentrations. These differentiation-inducing effects were only observed at subfungicidal concentrations at which more than 50% of the germinating conidia formed appressoria. Cycloheximide, nystatin, amphotericin B, and papulacandin A did not induce appressoria. Different strains of M. grisea displayed the same overall response to the inhibitors, varying merely in the percentage of appressoria formed. A combination of the respiratory inhibitors with 2-phenyl-4H -1-benzopyran-4-one (flavone), diphenyleneiodonium (DPI), or salicylhydroxamic acid (SHAM), compounds which interfere with the cyanide-resistant respiration, resulted in a higher sensitivity of the strains towards the respiratory inhibitors, but had no effect on appressorium formation.

In eight metribuzin-resistant photoautotrophic cell cultures of Chenopodium rubrum (Thiemann and Barz, 1994 a, b) sequence analyses of a part of the psbA gene coding for the photosystem -II D1 protein had revealed different double and triple mutations within the herbicide binding niche of the protein (Schwenger-Erger et al., 1993). Two pairs of the exam­ined cell lines carried identical mutations within this part of the protein, although their growth performance and their herbicide resistance patterns were different, both at the cellu­lar and the thylakoid level. Starting from the known part of the psbA gene we have amplified the remaining psbA sequences using inverse polymerase chain reaction. Thus the complete sequence of the coding part of the gene was elucidated. After sequence analyses we found an additional amino acid exchange at the position 184 (ile → asn) of the D1 protein in cell line L1. Metabolic consequences of this mutation are discussed. Partial sequence analyses of the psbD gene of the herbicide resistant cell culture lines revealed no mutation within that part o f the D2 protein, which is in direct contact with the D1 protein.

Changes in the photosynthetic apparatus and relative antenna sizes of photosystem (PS) I and PS II were measured in the rice canopy. We used juvenile rice seedlings to examine light utilization and its absorption in the bottom leaves and obtained the following results: (1) When referred to chlorophyll (Chl), levels of the electrochromic shift at 550 nm and cytochrome ƒ decreased from the sixth to the third leaves, but there was no loss of pigment (P)-700. As a consequence, the PS II/PS I ratio significantly decreased from 1.5 in the sixth leaves to 0.9 in the third leaves. (2) The electron transport capacity in the sixth leaves was 1.5-times larger than that in the third leaves. (3) The levels of cytochrome b 6 referred to Chl were almost constant from top to bottom. (4) The photosynthetic performance of the leaf de­creased concomitant with the depth, whereas the respiration was slightly increased. From these results, we hypothesize that there are maintenance mechanisms when the imbalances of light absorption and electron transport capacity occur in the bottom leaves.

The influence of 14 acyclonucleosides 1 , derivatives of adenine, guanine, uracil and thymine on the phosphorylation of dAdo, dGuo, dCyd and dThd occurring in the cytosol of growing amelanotic melanoma transplanted to Syrian hamsters, as well as on inhibition of tumor growth were studied. From among the studied ACNs eight were tested earlier (Modrzejewska et al., 1996, The influence of alkoxymethyl purine and pyrimidine acyclonucleosides on growth inhibition of Kirkman-Robbins hepatoma and possible mechanism of their cytostatic activity, Z. Naturforch. 51c, 75 - 80 ) ; from among the newly synthesized ACNs, 1,3-N, N-diallyloxymethylthymine (AMT2), 1-N-allyloxymethyl-5,6-tetramethyIeneuracil (AMUTM ), and tested previously 1-N-allyloxymethylthymine (AMT1), administered i.p. in a dose of 0.2 mmol /kg body weight reduce the tumor mass from 0.98 g to 0.64 g ± 0.11 g (i.e. 35% ± 12% ). 48 hours after i.p. administration of the mentioned ACNs in the same dose a reduction of tumor mass is accompanied by the inhibition of dAMP, dGMP and dTM P synthesis. AMT1 inhibits dThd phosphorylation from 6.2 to 4.22; AMT2 suppresses dAdo, dGuo and dThd phosphorylation by, correspondingly, from 2.8 to 1.7, from 10.8 to 7.5 and from 6.2 to 4.2; AMUTM depresses dAMP synthesis from 2.8 to 1.6 (all data: μmol of 2’dNMP formed per mg of protein per min. × 10 -4 ). None of the 14 studied acyclonucleosides influences dCMP synthesis. In vivo, after hydration of allyloxymethyl group to hydroxypropoxymethyl residue (having -CH 2 OH group), AMT1, AMT2 and AMUTM undergo phosphorylation to corresponding triphosphates. Phosphorylated ACNs are not incorporated into tumor DNA, however they inhibit dAdo, dGuo and dThd incorporation into DNA. It is concluded that ACN triphosphates are not substrates for DNA polymerase but, competing with dATP dGTP and dTTP, inhibit incorporation of these 2’dNTP into DNA and, in consequence, reduce tumor growth, which is presumed to be the main mechanism of cytostatic activity of the studied ACNs.

9-(3,4-Dimethyl-5-pentyl-furan-2-yl) nonanoic acid [diMeF(9,5)] and 9-(3,4-dimethyl-5-propyl-furan-2-yl) nonanoic acid [diMeF(9,3)] and its corresponding methyl esters have been assayed for inhibitory activity on horseradish peroxidase (E C 1.11.1.17) by measuring the peroxidase-catalyse decomposition of indole-3-acetic acid. Both compounds and their meth-ylates are com petitive inhibitors to horseradish peroxidase with inhibitor constants (K 1 ) of 50 ± 0.9 × 10 -5 ᴍ respectively 5.2 ± 0.8 × 10 -5 ᴍ. Development of inhibitory effect requires not only the presence of the furan heterocycle but also of a polar side chain.

We have produced a chloroform extract from Achillea which includes stigmasterol and sitosterol. By comparing it with the pure compounds an anti-inflammatory effect (with mouse ears) is assumed. The topical anti-inflammatory effect of the chloroform extract from Achillea ageratum (Asteraceae)and of stigmasterol and β-sitosterol, isolated of this extract has been evaluated, against to 12-0-tetradecanoylphorbol acetate (TPA )-induced mouse ear edema, using simple (acute model) and multiple applications (chronic model) of the phlogistic agent. Myeloperoxydase activity also was studied in the inflamed ears. In the acute model the extract exerted a dose-dependent effect. All the doses assayed (1, 3 and 5 mg/ear) significantly reduced the edema (50% , 66% and 82 % , respectively). The isolated sterols stigmasterol and β-sitosterol (with doses of 0.5 mg/ear) had similar effect as the extract with doses of 1 and 3 mg (59% and 65% respectively). In the chronic model the anti-inflammatory effect generally was a more moderate one. The highest dose of the extract decreased the edema reduction to 26% with the highest dose of the extract applied. With the compounds the effect decreased to 36% with stigmasterol, and 40.6% with β-sitosterol. Myeloperoxydase activity (MPO ) was reduced by the extract and the compounds in the acute model, however, in the chronic edema, the enzyme inhibition was very weak with all treatments even with the standard substance. These results indicate that the chloroform extract of Achillea ageratum and some of the its components stigmasterol and β-sitosterol are more effective as topical anti-inflammatory agents in acute than in the chronic process and their action is markedly influenced by the inhibition of neutrophil migration into inflamed tissue.

Internode explants of grape vine were used to produce proliferating callus cells in vitro. The tissues were grown on a modified M/S -medium supplemented with different concentra­tions of boron (H 3 BO 3 ) in the range of 0 to 600 μᴍ and aluminium (AlCl3) in the range of 0 to 85 μᴍ . With increasing concentrations of boron the content of the following flavanols declined: procyanidin B3 (catech in -(4α → 8) catechin), procyanidin B1 (epicatechin-(4β → 8) catechin), procyanidin B2 (epicatechin-(4β→8) epicatechin), and B2 -3 -O -gallate , cate­chin and epicatechin. ECG (epicatechingallate) showed increased values in dependance on boron supply. Procyanidin B5 (epicatechin-(4β → 6)-epicatechin) showed an indifferent behaviour. In the case of aluminium the concentrations of flavanols were generally increased up to a maximum of 46% with the exception of ECG and B5 where no significant change was observed. While the total sum of flavanols was decreased by boron up to about 30% in comparison to the control (no boron addition) the content of flavanols was basically increased by aluminium up to about 25% . We conclude that the addition of watersoluble boron and aluminium compounds to the culture can significantly modify the synthesis of special monomeric and oligomeric flavanols.

Flagellin gene sequence polymorphisms were used to discrimate amongst 53 strains of Campylobacter jejuni and C. coli. The Campylobacter strains were made up of forty-three strains of Campylobacter jejuni and 10 strains of Campylobacter coli. The results were analysed in relation to Penner serotyping. Twenty DNA PCR -RFLP patterns (genotypes) were identified by analysis of Dde I fragment length polymorphisms in flagellin gene (fla A and fla B) polymerase chain reaction (PCR) products. Flagellin gene 13 genotype was a feature of 15% of strains, followed by flagellin gene 8 (9 %). Differences in fragment patterns were observed not only between members of two species, but also between individual strains of the same species. The strains that were non-typable by the Penner serotype were distributed into 6 flagellin gene types. In conclusion, Dde I fla typing is reproducible and offers high typability. However, when the scheme is used in combination with the Penner serotype it provides improved discrimina­tion for the characterizing and subtyping of isolates.

The interaction of pyridinium salts (PS) with red blood cells and planar lipid membranes was studied. The aim of the work was to find whether certain cationic surfactant counterion influence its possible biological activity. The counterions studied were Cl - , Br - , I - , ClO 4 - , BF 4 - and NO 3 - . The model membranes used were erythrocyte and planar lipid membranes (BLM). At high concentration the salts caused 100% erythrocyte hemolysis (C 100 ) or broke BLMs (CC). Both parameters describe mechanical properties of model membranes. It was found that the efficiency of the surfactant to destabilize model membranes depended to some degree on its counterion. In both, erythrocyte and BLM experiments, the highest efficiency was observed for Br - , the lowest for NO 3 - . The influence of all other anions on surfactant efficiency changed between these two extremities; that of chloride and perchlorate ions was similar. Some differences were found in the case o f BF 4 - ion. Its influence on hemolytic possibilities of PS was significant while BLM destruction required relatively high concentration of this anion. Apparently, the influence of various anions on the destructive action of PS on the model membrane used may be attributed to different mobilities and radii of hydrated ions and hence, to different possibilities of particular anions to modify the surface potential of model membranes. This can lead to a differentiated interaction of PS with modi­fied bilayers. Moreover, the effect of anions on the water structure must be taken into ac­count. It is important whether the anions can be classified as water ordering kosmotropes that hold the first hydration shell tightly or water disordering chaotropes that hold water molecules in that shell loosely.

Oxidation of low density lipoprotein (LDL) by copper ions is not influenced by glucose in a concentration range between 1 and 20 mᴍ . LDL-oxidation by peroxynitrite or the simulta­neous action of nitrogen monoxide and superoxide, produced by morpholino-sydnonimine (SIN-1) is considerably enhanced by typical hydroxyl-radical scavengers such as formate or mannitol and by glucose. Since both free radicals, nitrogen monoxide and superoxide, are produced by activated leukocytes and endothelial cells the presented reaction might repre­sent a simple model for the cooperative function of reactive oxygen species (ROS) and glucose in certain diabetic blood vessel diseases such as atherogenesis and retinopathy.

A gene coding for a soluble protein with homology to the β subunit of the nicotinic acetyl­choline receptor from goldfish was isolated from a cDNA library of Haementeria ghilianii salivary glands. Comparison of the leech protein sequence with the database showed that the N terminus has high homology with the extracellular portion of acetylcholine receptor β subunits, whilst the C terminus, highly charged, has homology to proteins which may be involved in chelating divalent cations. The leech protein was expressed in mammalian cells and the product compared to the native protein. Both proteins are glycosylated and form polymers which are disrupted by heat but not by reducing agents. A role for this protein in salivary gland secretion is suggested.

The sterol composition of Hymeniacidon sanguinea and Halichondria panicea from the Black Sea was investigated. Both sponges contain similar mixtures of stanols and of dietary Δ 5 -sterols. Main sterols appeared to be C 27 -sterols, which could be connected with a common diet for the both sponges. Saturated short side chain sterols have been found in Hymeniaci­don sanguinea. Three of them were novel for sponges. A possibility for the transformation of some dietary sterols into stands is discussed.

Live Trypanosoma cruzi amastigotes hydrolyzed p-nitrophenylphosphate (PNPP), phos-pho-amino-acids and 32 P-casein under physiologically appropriate conditions. PNPP was hy­drolysed at a rate of 80 nmol ·mg -1 ·h -1 in the presence of 5 mм MgCl 2 , pH 7.2 at 30 °C. In the absence of Mg 2+ the activity was reduced 40% and we call this basal activity. At saturating concentration of PNPP, half-maximal PNPP hydrolysis was obtained with 0.22 mм MgCl 2· Ca 2+ had no effect on the basal activity, could not substitute Mg 2+ as an activator and in contrast inhibited the PNPP hydrolysis stimulated by Mg 2+ (I 50 = 0.43 mм ) . In the absence of Mg 2+ (basal activity) the stimulating half concentration (S 0. 5 ) for PNPP was 1.57 mм , while at saturating MgCl 2 concentrations the corresponding S 0.5 for PNPP for Mg 2+ -stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.99 mм . The Mg-dependent PNPP hydrolysis was strongly inhibited by sodium fluoride (NaF), vanadate and Zn 2+ but not by tartrate and levamizole. The Mg-independent basal phosphatase activity was insensitive to tartrate, levamizole as well NaF and less inhibited by vanadate and Zn 2+ . Intact amastigotes were also able to hydrolyse phosphoserine, phos-phothreonine and phosphotyrosine but only the phosphotyrosine hydrolysis was stimulated by MgCl 2 and inhibited by CaCl 2 and phosphotyrosine was a competitive inhibitor of the PNPP hydrolysis stimulated by Mg 2+ . The cells were also able to hydrolyse 32 P-casein phosphorylated on serine and threonine residues but only in the presence of MgCl 2 . These results indicate that in the amastigote form of T. cruzi there are at least two ectophosphatase activities, one of which is Mg 2+ dependent and can dephosphorylate phospho-aminoacids and phosphoproteins under physiological conditions.

Scattered light intensities measurements of the nuclear part of adult bovine lenses are reported. In the investigated samples the existence of a binary liquid phase separation from a metastable state is demonstrated by the phenomenon of hysteresis. Fluctuations near spino­dal are studied by means of a tentative theoretical approach. Accordingly the scattered light intensities were analyzed reduced by the intensity obtained for the stable state of the system. Fluctuations in the stable state are ascribed to protein aggregates and are analyzed by means of random density fluctuation theory. To evaluate the correlation length of the fluctuations near spinodal the Ornstein-Zernike theory is adopted. Temperature dependence of the corre­lation length ξ of the fluctuations near spinodal can be described by equation for critical fluctuations . For the investigated lenses the exponent ν varies from 0.65 to 0.74 and the parameter ξ 0 varies from 1.6 nm to 3.6 nm. The spinodal temperature T s for the investigated samples is evaluated.

A new mechanism of cell damage by alternating mag­netic field with hematoporphyrin is described. C6 gli­oblastoma cell suspensions were exposed to an alternat­ing magnetic field with frequency 180 kHz up to 60 min in the presence of hematoporphyrin in H 2 O and in D 2 O . The results presented suggest that an alternating mag­netic field is able to activate hematoporphyrin, and this method may be a basis for cancer treatment.