Volume 52, Issue 7-8

Four new botrydial derivatives, hymendial (1), 7α-hydroxydihydrobotrydial (2), 7α-hy­droxy-10-O-methyldihydrobotrydial (4), and 7α-acetoxy-15α-methoxy-10-O-methyl-dihydro-botrydial (5) were isolated together with dihydrobotrydial (3) from the culture fluid of the ascomycete Hymenoscyphus epiphyllus. In addition, cytochalasin H (6a), 18-deoxycytocha-lasin H (6b) and (+)-mellein (7) were produced by this fungus. Hymendial (1), possessing an a, ß-unsaturated dialdehyde functionality, exhibits antimicrobial and cytotoxic activities and is mutagenic in the Ames Salmonella assay.

Hyphae of Cyathus striatus and C. olla in culture and on infested plant debris were heavily encrusted with crystals. Scanning electron microscopy revealed that raphide-and styloid­ shaped crystals were associated with the hyphae of C. olla in canola stubble and in culture. Bipyramidal crystals were also present in culture. Distinct raphide druses developed on C. striatus hyphae colonizing wood chips, but in culture most crystals were bipyramidal or other shapes. Energy-dispersive X-ray microanalyses, FT/IR spectroscopy, and 13C NMR spectros­ copy determined that these crystals were calcium oxalate. This is the first report of calcium oxalate crystal production by these fungi. This characteristic has implications towards decomposition of organic matter, biomineralization, nutrient cycling, and soil genesis.

By repeated selection of pigment portions of tissue the red callus induced from root seed­lings of Penstemon serrulatus Menz. was chosen for suspension culture, which was maintained in Schenk and Hildebrandt medium supplemented with naphthaleneacetic acid (0.2 mg/l), 6-benzylaminopurine (2 mg/l) and sucrose (50 g/l). From the cultured cells eight phenolic compounds were isolated. They were identified as cyanidin 3-O-glucoside, delphinidin 3-O-glucoside, luteolin, luteolin 7-O-glucoside, norartocarpetin 7-O-glucoside, verbascoside, martynoside and leucosceptoside A. The kind of cell line, its age and light irradiation were important factors in flavonoid production, but production of phenylpropanoid glycosides was found to be unaffected by these factors. The phenolic composition found in the cell culture was compared with those in the flowers and leaves of original plants of P. serrulatus.

Naturally grown two-month-old seedlings of Avicennia marina contain high concentrations of Na+ and Cl - .+ Our NMR studies revealed an accumulation of glycinebetaine, asparagine and stachyose in A. marina. The highest concentration of glycinebetaine was observed in young leaves, while the distribution of stachyose was restricted in stems and roots. A sparagine comprised more than 96% of total free amino acids in roots and 84% in leaves from two-year-old plants. Little or no accumulation of proline or polyols, which are proposed as compatible solutes in other plants, could be detected in A. marina. The activities of phosphofructokinase, pyrophosphate:fructose-6-phosphate 1-phosphotransferase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase (decarboxylating), phosphoenolpyruvate carboxylase and NAD:malate dehydrogenase from young leaves of A. marina were inhibited by NaCl, while the activity of fructose-1,6-bisphosphate aldolase was activated by 50-200 m M NaCl. There was little or no effect of high concentrations (up to 500 mᴍ ) of glycinebetaine on the activities of any of these enzymes. No significant protection by glycinebetaine was detected against NaCl inhibition of these enzymatic activities. Based on these results, possible mechanisms for the salt-resistance of A. marina cells are discussed.

The intra-and interspecific acting allelochemicals of Kalanchoe daigremontiana, K. tubiflora and K. pinnata (Crassulaceae) were isolated and could be identified as p-hydroxybenzoic-, protocatechuic-, gallic-, p-coumaric-and coffeic acid. By measuring length of stems and primary roots of Kalanchoe-daughter plants the intra-and interspecific inhibitory activities of authentic compounds could be demonstrated.

Dieldrin is an organochlorine insecticide highly toxic for human beings. Although its exact mechanism of action is not well known, there is evidence that it acts at the cell membrane level. In fact, the lipophilicity of the pesticide as well as that of the phospholipid bilayer present in biological membranes makes the latter a most likely target for the interaction of dieldrin with living organisms. In order to evaluate its perturbing effect upon cell membranes the pesticide was made to interact with human erythrocytes and molecular models. These studies were performed by scanning electron microscopy on erythrocytes, fluorescence spectroscopy on dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles and X-ray diffraction on multilayers of dimirystoylphosphatidylcholine (DMPC) and dimyristoyl-phosphatidylethanolamine (DMPE). It was observed that dieldrin particularly interacted with DMPC liposomes and multilayers perturbing its molecular arrangements. However, no effect was noticed on erythrocytes, which might be due to its high cholesterol content.

A complete inactivation is observed after a 3 min pre-incubation at 70 °C with mitochondrial F 0 F 1 -ATPase complex depleted of the ATPase natural inhibitor protein (ammonium-Sephadex submitochondrial particles) and activated MgATP-submitochondrial par­ticles (particles that after a 4 h-pre-incubation at 42 °C released the endogenous inhibitor protein). However, latent MgATP-submitochondrial particles (particles containing the inhibi­tor protein) pre-incubated under the same conditions are totally inactivated only after 15 min of pre-incubation. When ammonium-Sephadex particles are reconstituted with 20 μg/ml of purified ATPase inhibitor protein there is an increase of 15-fold in the half-time for ther­mal inactivation (t 0.5 ), showing that the inhibitor protein protects the mitochondrial F 0 F 1 -ATPase complex against thermal inactivation.

Chicken embryo cells were treated with caffeine (0.5-8.0 mᴍ) alone or combined with various chemical and physical DNA - and/or chromatin-interactive agents. Analytical procedures comprised scheduled (SDS) and unscheduled (UDS) DNA synthesis, RNA synthesis (RNS), the activities of O 6 -alkylguanine-DNA alkyltransferase (AT) and poly (ADP-ribose) polymerase (PARP) as well as nucleoid sedimentation. Additional investigations were done in rat thymic and splenic cells. The effect of caffeine on DNase-I activity served as an in vitro-model system.- When present in the PARP-, SDS-, UDS- and RNS-assays, caffeine inhibited the corresponding tracer ( 14 C-NAD, dT- 3 H, 3H-U) incorporation in a dose-dependent manner. The AT activity was slightly stimulated. At concentrations of 0.06-0.3 mᴍ, caffeine inhibited DNase-I activity by excess substrate. No specific effects of caffeine could be shown by nucleoid sedimentation.- Besides the reduced permeability of the cells to nucleic acid precursors, the results obtained with the PARP- and DNase-I assays give evidence for the formation of a DNA-caffeine adduct as a prominent mechanism of cellular caffeine effects including DNA repair inhibition.

Two different membrane perturbing agents -cholesterol and benzyl alcohol were applied to modify the fluidity of pea thylakoid membranes. Well pronounced decrease of membrane fluidity was observed upon increasing of cholesterol added. Rigification of thylakoid membranes was accompanied by reduction of cation-induced increase of light-scattering intensity and results in lower extent of increase of spillover upon cation induced stack-unstack transi­tion. Thylakoid membranes, treated with local anesthetic benzyl alcohol, showed an increase of membrane fluidity, but approximately the same behavior as untreated thylakoids in respect to the light scattering intensity dependence on cation concentration. Upon stack-unstack transition the observed changes of energy distribution for benzyl alcohol treated thylakoids are similar to those for controls. The data are discussed in terms of influence of membrane fluidity on lateral reorganization of pigment-protein complexes.

Plastids contain a NAD(P)H-plastoquinone-oxidoreductase which is homologous to the eubacterial and mitochondrial NADH-ubiquinone-oxidoreductase (complex I), but the meta­bolic function of the enzyme is still not yet understood. The enzyme consists of at least eleven subunits (NDH-A-K), which are all encoded in the plastid chromosome. In this study we have investigated the tissue-specific and light-dependent expression of the subunits NDH-H and NDH-K in maize, rice and mustard by western blot analysis. No NDH-proteins were found in root tissue, indicating that the presence of the enzyme is confined to leaf plastids. Analysis of the expression during the light-dependent development from etioplasts to chloro­plasts showed that high amounts of NDH-H and -K are present in etioplasts. The same result was found for subunits of the ATPase. In contrast, components of the photosynthetic electron transport chain (PSII-B, cytochrome f and PSI-D) accumulated only after illumination. In an second investigation, the expression of NDH-proteins along the natural chloroplast develop­ mental gradient from proplastids to chloroplasts in light-grown maize leaves was analysed. NDH-H and NDH-K as well as the ATPase were present at the youngest stages of chloro­plast development, while the massive accumulation of subunits of the photosystems and the cytochrome b 6 /f-complex took place in older leaf sections. We conclude from these studies that a functional NAD(P)H-plastoquinone-oxidoreductase is present in etioplasts and devel­oping plastids. We suggest that the enzyme serves the generation of a proton gradient across the prothylakoid membrane that is necessary for protein integration into the membrane at developmental stages where a functional photosynthetic electron transport chain is not yet operating.

The cellular localization of the betaine lipids diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and diacylglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA) was investi­ gated by a) chemical analysis of subcellular fractions and b) immunochemical methods using specific antisera and either fluorescence microscopy or electron microscopy for detection of the label. A homogenate of Lycopodium annotinum (Pteridophyta) was fractionated by differential and density gradient centrifugation. The particulate fractions obtained were analyzed for chlorophyll, cyt c oxidase, NADH-cyt c reductase and DGTS. Non-plastidial fractions were enriched in DGTS and only minor amounts of this lipid could be attributed to chloroplasts. Anti-DGTS and anti-DGTA sera were produced by immunization of rabbits. The monospecificity of the antisera was examined with cells of Chlamydomonas reinhardtii (Chlorophyceae) containing DGTS, Pavlova lutheri (Haptophyceae) containing DGTA and Ochromonas danica (Chrysophyceae) containing both DGTS and DGTA. Euglena gracilis which is free of betaine lipids, was used as a control. For the test, a FITC-coupled goat anti-rabbit antibody was used and detected by fluorescence microscopy. Thin sections of Ochromonas and Pavlova were incubated first with the anti-lipid sera and subsequently with a gold-coupled anti-rabbit serum and then examined in the electron microscope. With O chro­ monas, anti-DGTS as well as anti-DGTA sera gave an accumulation of gold label in the cytoplasmic space but not in the chloroplasts. Similar results were obtained with Pavlova using anti-DGTA serum. These results describe for the first time the cytochemical localiza­ tion of DGTS and DGTA strongly suggesting both these lipids to be associated mainly with non-plastidial structures.

Ralstonia (formerly Burkholderia or Pseudomonas) solanacearum ATCC 11696 - a plant deleterious bacterium - was shown to produce under iron limited conditions of growth an iron complexing compound which facilitated iron uptake into iron-starved cells. The structure of the siderophore was elucidated as 4-[[(3-acetylhydroxyamino)-propyl]amino]-2-[2-[[3-(acetylhydroxyamino)-propyl]amino]-2-oxoethyl]-2-hydroxy-4-oxo-butanoic acid, a compound known under the trivial name of schizokinen. Its partially cyclized form, schizokinen A , could also be isolated from the R. solanacearum culture supernatant and was characterized.

Plums attacked by the fungus Taphrina pruni produce great amounts of catechins and caffeoyl-ᴅ-quinic acids. This reduces - as we show in this paper - the degradation of indole-3-acetic acid. The increased indole-3-acetic acid concentration causes enhanced growth of infected plums in comparison to not infected ones.

We have investigated the physicochemical and paramagnetic properties of the new gado­linium complexes of N-(pyrid-2-yl-methyl)-ethylenediam ine-N,N',N'-triacetic acid (Gd-PED TA) and N -tris(2-aminoethyl)amine-N',N',N",N",N''',N'''-hexaacetic acid (Gd-TTAHA). The relaxivities as well as the thermodynamic and conditional stability constants of these complexes with respect to the physiological relevance were determined and discussed in comparison with the commercially available gadolinium(III) diethylenetriaminpentaacetic acid/ gadopentate dimeglumine (G d-DTPA, Magnevist ® ). In case of Gd-TTAHA a twofold higher relaxivity and a complex stability similar to Gd-DTPA were determined. It is shown, that lower concentrations of Gd-TTAHA are sufficient for the same signal enhancement in the T 1 -weighted MR image compared with Gd-DTPA and, thus, the use of the new contrast agent Gd-TTAHA should diminish risks for health. Therefore, Gd-TTAHA might be used potentially as a new contrast agent for clinical MRI application.

Steroid esters of cynnamic acid derivatives have been synthesized by a heterogeneous Wittig reaction under sonochemical conditions from the corresponding triphenylphosphonium bro­mides and unprotected phenolic aldehyds using K 2 CO 3 as a base. 5β-Cholan-3α, 7α, 12α, 24-E-ferulate (11') exhibited a marked inhibitory effect on influenza virus A. The synthetic 3α, 24-E-diferulates of 5β-cholan-3α, 24-diol, 5β-cholan-3α, 12α, 24-triol and 5β-cholan-3α, 7α, 12α, 24-tetrol (8, 9 and 12) showed antitumor activity on leukemia P-388 in mice.

Colchicine (CC) is an antimitotic and antitubular agent which is currently used in medicine as a drug to treat various diseases. In spite of its widespread use the interaction of this drug with membranes is not yet known clearly. To characterize alterations in membrane molecular dynamics produced by CC, human erythrocytes were spin-labeled with 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA), and membrane fluidity was quantified by meas­uring the changes in the order parameter (S) which reflects the anisotropy due to restricted motional averaging, correlation time (τ c ) representing the spin label tumbling rate in the sample and phase transitions derived from ESR measurements. CC induced significant changes in both order parameter and correlation time at the surface of 5-DSA labeled membrane, but this was not the case at the core of 16-DSA labeled membrane. Order parameter, S and rotational correlation time, τ c , showed a biphasic character in the temperature range of 5−50 °C. This fact was used to calculate phase transition temperatures and activation energies of untreated (control) and treated erythrocyte cells with CC.

In Central Europe and other temperate regions the lipophilic bee hive product propolis normally originates from the bud exudate of poplar trees that is collected by the bees. Based on bee observations, various other sources have been discussed in particular for tropical regions, but in only few cases the origin has been proved by analytical methods. We have analyzed propolis samples from managed honey-bees as well as from feral bee colonies in the Sonoran Desert. Propolis collected in hives out of flight reach of poplars contained flavonoid aglycones and other phenolics that point to specific plants as the source of propolis in this area, namely Ambrosia deltoidea and Encelia farinosa.

Light-evoked intracellular electrical responses were measured simultaneously with the changes in intracellular free Ca 2+ or Sr 2+ concentration by means of arsenazo III. The response-versus-stimulus intensity characteristics was measured in two states of relative light adaptation in strontium-containing saline (10 mᴍ Sr 2+ ; 0.25 mᴍ Ca 2+ ). Supralinear parts of the curves with slopes > 1 were found for the receptor current amplitude and the current­-time integral as well as for the amplitude of the arsenazo signal within at least 0.3 log units of identical stimulus intensity range. Maximum slopes of response-versus-intensity curves are on average approximately 1.7 for the receptor current and the arsenazo signal in both adaptational states. The rise in time of the arsenazo signal evoked by a light flash shows two phases. In strontium saline we observe a fast increase at the onset of the arsenazo signal which is detectable within the first 20-100 ms of the signal rise and a slow further increase to the maximum, with a duration of 0.7-6 s. In the detailed analysis of both phases we observed that the amplitude of each phase increases supralinear, i.e. disproportionate to the stimulus intensity. The fast rising phase of the arsenazo signal is not visibly voltage-dependent. This fast rising phase might be a monitor of an IP 3 -sensitive calcium or strontium release. Since the amplitude of the fast rising phase increases supralinear with the stimulus intensity, we propose the interpretation that more than one IP 3 -molecule must bind to the IP 3 -receptor to open this channel of the intracellular calcium store. Only the slow rising phase is voltage-dependent. The kinetic of this slow rising phase was accelerated with more negative membrane potentials. This predominant slow phase correlates with the receptor current-time integral. Therefore the slow rising phase of the arsenazo signal might be a monitor for influx of calcium or strontium through light activated ion channels in the plasma membrane. The supralinearity of the amplitude of the slow rising phase might indicate the supralinear increase in calcium or strontium, which is carried by the receptor current. Additionally, we calculated the fraction of strontium and calcium ions of the total receptor current. When calcium was replaced by strontium in the superfusate about 3-30% of the receptor current are carried by strontium and calcium ions. In physiological saline calcium contributes about 1-1.5% to the receptor current.

By ozonolytic degradation of the chromophore of isopyoverdin from Pseudomonas putida BTP 1 ʟ-2,4-diaminobutyric acid was obtained. This proves that the C-3 of the chromophore is S-configurated.

From cultures of Corynebacterium glutamicum ATCC 14067 (Brevibacterium sp. DSM 20411) a catecholate sid­ erophore could be isolated. It comprises three identical units consisting of L-Thr, Gly and 2,3-dihydroxybenzoic acid which form a tri-lactonic macrocycle -the second example of this structural type.