Volume 50, Issue 3-4

Four samples of Brazilian propolis were investigated by GC/MS of different fractions. 32 volatile compounds, (10 of them new for propolis), as well as 12 more polar compounds (one of them new for propolis) were identified. Antibacterial activity was found in some propolis fractions.

A new antibiotic was isolated from fermentations of an american strain of Collybia confluens. Its structure was elucidated by spectroscopic methods as 2,10,10-trimethal-4-oxo-tricyclo[7.2.0.0 2.5 ]undec-6-en-carbaldehyde (with the relative stereo chemistry IS, 2R, 5R, 9R) (1). The inhibitor, which was named collybial, is structurally related to koraiol, a sesquiterpenoid isolated from Pinus koraiensis (Khan V. A. (1979), Khim. Prir. Soedin 5, 652-658). Collybial inhibited the growth of Gram-positive bacteria at concentrations starting from 21.5 μm . The propagation of vesicular stomatitis virus (VSV) in baby hamster kidney (BHK-21) cells was inhibited by 21.5 μm collybial. Cytotoxic effects on BHK cells were observed at 5 fold higher concentrations.

The biosynthesis of the phytotoxins solanapyrone A , B and C produced by the phytopathogenic fungus Ascochyta rabiei has been investigated. To optimize feeding conditons for the tracer experiments the growth of the fungus and the accumulation of the toxins in submers culture were determined. The accumulation kinetics revealed that formation of the toxins occurs in the stationary phase of the growth indicating that synthesis of solanapyrones follows a typical pattern of secondary metabolism. Incorporation experiments with sodium [1- 14 C]- and [2- 13 C]acetate were performed and the NMR-spectroscopically determined labelling pattern of the 13 C-enriched solanapyrone A compound confirmed that the carbon skeleton of this compound is formed via the polyketide pathway. The biosynthetic route to solanapyrone B is discussed.

Eight 5-arylimino-3,4-tetramethylene-1,3,4-thiadiazolidine-2-thiones and eight 4-aryl-1,2- tetramethylene-1,2,4-triazolidine-3,5-dithiones were synthesized and their phytotoxic activities were investigated using sawa millet (Echinochloa utilis), green microalgae (Scenedesmus acutus) and protoporphyrinogen-IX oxidase isolated from etiolated corn (Zea mays) seedlings. 5-Arylimino-3,4-tetramethylene-1,3,4-thiadiazolidine-2-thiones showed strong phytotoxic activities and the same herbicidal mode of action as known for peroxidizing herbicides. 5-Arylimino-3,4-tetramethylene-1,3,4-thiadiazolidine-2-thiones were not or very little converted into 4-aryl-1,2-tetram ethylene-1,2,4-triazolidine-3,5-dithiones either with E. utilis seedlings present for 7 days, with S. acutus cells, or using glutathione 5-transferase (GST) and glutathione (GSH). The phytotoxic activities of 4-aryl-1,2-tetram ethylene-1,2,4-triazolidine- 3,5-dithiones were stronger than those of 5-arylimino-3,4-tetram ethylene-1,3,4-thiadiazolidine- 2-thiones [cf. Sato, Y., et al., Z. Naturforsch. 49c, 49-56 (1994)].

Exogenous gibberellin A 3 (GA 3 ; 30 μm) promoted shoot elongation of germinated seeds of Dioscorea nipponica, an East Asian species of a taxonomic section Stenophora of the genus Dioscorea (yams). Endogenous gibberellins (GAs) were analyzed by combined gas chromatography-mass spectrometry (GC-MS) from growing shoots of three East Asian Stenophora species. Gibberellin A 4 , GA 12 , GA 19 , GA 24 and GA 53 were identified by Kovats retention indices and by full spectra of GC-MS from D. quinqueloba. Gibberellin A 19 , GA 24 and GA 53 were also identified from D. nipponica and D. septemloba. These results suggest that two separate biosynthetic pathways of GAs, early 13-hydroxylation and non-13-hydroxylation pathways are functioning in the growth of shoots of these thee species.

By means of mass spectroscopic measurements in an artifical gas atmosphere containing the stable nitrogen isotope 15 N 2 we were able to demonstrate nitrogen fixation capacity in the filamentous cyanobacterium Oscillatoria chalybea. Our technique proved to be wellsuited also for investigations on the light-induced nitrogen fixation in the purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus. Oscillatoria chalybea grown without combined nitrogen showed a substantial 15 N 2 -uptake which could clearly be correlated with nitrogen fixation. Nitrate grown cultures did not show this nitrogen uptake or only to a minimal extent. Addition of ammonium chloride resulted in a rapid deactivation of the nitrogenase system. Similar observations have been made with other so-called switch-off effectors like phenazine methosulfate. The structural integrity of the filaments appeared to be a prerequisite for nitrogen fixation also in this organism, as even mild mechanical homogenization strongly inhibited the N 2 -uptake signals. Illumination of the assays under conditions where the photooxidition of water is not operational (Bader, K. P. (1994), Biochim. Biophys. Acta 1188, 213 -219) did not affect the nitrogen fixation in Oscillatoria chalybea. Illumination of cultures with concomitant release of oxygen from the water splitting reaction resulted in strong inhibition of 15 N 2 -uptake.

Iron(III)-NTA (nitrilotriacetic acid) solution shows high activity for oxidative degradation of 2′-deoxyribose in the presence of hydrogen peroxide, whereas its activity of Fe(III)-TFDA (2-aminomethyltetrahydrofuran-N,N-diacetic acid) is negligible under the same experimental conditions; however the latter solution exhibits abnormally higher reactivity for oxygenation reaction at 8-position of 2′-deoxyguanosine than other iron(III) chelates examined. These results suggest that oxidative degradation of deoxyribose and the oxygenation of deoxyguanosine are caused by a different iron(III)-peroxide species.

Selenite and selenocystamine [(CyaSe) 2 ] efficiently activate the decomposition of H 2 O 2 y GSH and by other thiols, as demonstrated using a leuco crystal violet POD-based H 2 O 2 assay which is applicable (unlike other assays) also in presence of thiols. The GPx-like activities were estimated to be 3.6 and 2.7 μmol H 2 O 2 /min per μmol SeO 3 2- and (CyaSe) 2 , respectively. Both selenium compounds also activate reduction of the heterocyclic N-oxide resazurin (RN→O) to resorufin (RN) by GSH; H 2 O 2 competes with reduction of this dye. GSSeH and CyaSeH, formed by interaction of GSH with SeO 3 2- and (CyaSe) 2 , respectively, are likely to be the active reductants. CyaSeH, generated γ-radiolytically from (CyaSe) 2 , exhibits an absorption peak at 243 nm and is removed by H 2 O 2 with a rate constant of 9.7x10 2 ᴍ -1 s -1 , and slightly slower by hydroperoxides. We have no evidence for one-electron interactions between GSSeH or CyaSeH and H 2 O 2 , with formation of free radical intermediates, as previously proposed in the case of selenium-activated reduction of cytochrome c by GSH (Levander et al., Biochemistry 23, 4591-4595 (1973)). Our results can be explained by O-atom transfer from the substrate to the active selenol group. RSeH + H 2 O 2 (RN→O)→RSeOH + H 2 O (RN), and recycling of RSeOH to RSeH (+ H 2 O) by GSH, analogous to the selenenic acid pathway of GPx. The substrate specificity appears to be different, however, in that GPx is unable to catalyse RN→O reduction, and GSSeH hardly catalyses the decomposition of cumene- or t-butyl-hydroperoxide; CyaSeH, on the other hand, is active also with the hydroperoxides. RN→O is reduced to RN also by certain oxidizing free radicals, e.g. by the thiyl CyaS · ; O -atom transfer may in this case lead to the generation of reactive oxyl radicals.

Trivalent lanthanide cations are suitable probes for Ca 2+ -binding sites in photosystem II (PS II). PS II membranes prepared from Nicoticinci tabacum, intact and depleted of the extrinsic polypeptides were exposed to lanthanide ions (Dy 3+ and Eu 3+ ). Small concentrations of dysprosium and europium ions enhance oxygen evolution under short saturating flashes. Higher concentrations of the rare earth cations cause the release of the three extrinsic peptides (17, 23 and 33 kDa) and reduce O 2 yield. The reactivation of the PS II membranes, thus depleted of the 33 kDa subunit, by Ca 2+ ions is not possible. Comparing Eu 3+ with Dy 3+ in this effect shows that Eu 3+ is more effective than Dy 3+ , because a lower Eu 3+ -concentration in comparison to that of Dy 3+ inactivates O 2 -evolution. The differences between europium and dysprosium can be explained by their different ionic radius. Our results suggest the existence of two Ca-binding regions: one with a low affinity for calcium would be located on the contact surface of the 23 and 33 kDa proteins and the second one with a high affinity, should be located close to the Mn-cluster and to tyrosine-161 (Z ). The more tightly-bound calcium would be responsible for the activity of the PS II system.

Light-induced phosphorylation of a 114 kDa protein in plasma membranes isolated from the tips of maize coleoptiles was investigated in the presence of several thiol reagents at the concentration of 1 mᴍ. Dark phosphorylation of the protein was not affected but lightinduced phosphorylation was inhibited 50% with iodoacetamide, 75% with N-ethylmaleimide and 93% with N-phenylmaleimide. Previous incubation of the inhibitors with mercaptoethanol abolished the inhibitory activity completely. N-phenyl-maleimide showed the same inhibition whether it was applied before or after irradiation of the sample. Involvement of thiol group(s) in processes after photoexcitation is discussed.

The liverwort Ricciocarpos natans has been cultivated on Gamborg B5 medium with different levels of ammonium, nitrate, phosphate and sucrose. The formation of the cell wall pigments riccionidin A and B was shown to be dependent on the level of these nutrients as well as on the intensity of light. A decrease in nitrogen supply (mainly nitrate) induced the formation of both wall pigments, whereas phosphate and high levels of sucrose inhibited the pigment synthesis. Decreasing the illumination rate led to lower anthocyanidin contents. Dedifferentiated cultures also produced wall pigments, but their amount was much lower compared to differentiated cultures.

The enzyme aspartate aminotransferase has been isolated from the halophilic bacterium Haloferax mediterranei in its apoenzyme form. The interaction with its coenzyme (pyridoxal phosphate) has been investigated. For concentrations up to 0.05 mᴍ, the incubation with pyridoxal phosphate reconstituted the active complex (holoenzyme) following a second order kinetic with a k 2 of 5.2 min -1 mᴍ -1 . This active complex showed a dissociation constant (K d ) of 7.8 x 10 -6 ᴍ. For concentrations higher than 0.1 mᴍ, pyridoxal phosphate produced an inactivation process with a complex second order kinetic. This inactivation is partially reverted by dialysis or by lysine treatment. Thus, after 80% of inactivation, 55% of the original activity is recovered by a long-time dialysis, and with 50 mᴍ lysine also a partial reactivation (among 20-33%) is observed. The enzyme treated with 1 mᴍ pyridoxal phosphate has a different behavior in Sepharose chromatography indicating that the modified enzyme presents a smaller size due to a conformational change.

Several hypotheses link the molecular mechanism of action of the antiarrhythmic drugs (AAD) that belong to class I to non-specific interactions with phospholipids sited in the neighborhood of sodium channels in the membrane of the myocardium. Procainamide (PROC ), one of the least lipophilic drugs of this group, was induced to interact with bilayers of dimyristoylphosphatidylcholine (DMPC) and dimirystoylphosphatidylethanolamine (DMPE), liposomes of DMPC and human erythrocytes. The perturbing effects of PROC upon these systems were respectively determined by X-ray diffraction, fluorescence spectroscopy and scanning electron microscopy. It was found that PROC exerted very little effect upon DMPC and DMPE even at such a high concentration as 10 mᴍ . However, at therapeutical plasma concentrations, PROC induced shape changes in vitro to red cells.

The 7-oxosterols 1-2 and the 7-hydroxysterols 3-6 induce resistance toward the fungal pathogens Puccinia striiformis West, and Puccinia hordei Otth in barley and wheat. Primary leaves of the plants were sprayed with solutions of the compounds ( 10 -4 mol/l in 1% aqu. ethanol) followed, 2 days later, by challenge inoculation with the fungal pathogens. The results indicate that 7α- and 7β-hydroxylated epimers of β-sitosterol and cholesterol show the highest value of induced resistance (39-49% reduction of infection sites). No enhanced resistance toward the fungi Erysiphe graminis DC f.sp. tricitic and hordei and Cochliobolus sativus Ito & Kuribayashi was observed.

The dodecanucleotide d(CGCGATATCGCG) 2 was characterized by thermodynamic and UV-spectrophotometric measurements. A van’t Hoff enthalpy of ΔH uv v.H. ~ - 190 kJ/mol was determined for the thermal transition using UV spectroscopy. This value was confirmed by differential scanning calorimetry (DSC). In addition we obtained the thermodynamic data ΔH DSC = -405.1 kJ/mol, ΔS DSC = -1290 J/mol·K and ΔG DSC = -53.2 kJ/mol for the helix to coil transition of the dodecanucleotide. The association of berenil and the oligonucleotide was accompanied with a stabilization of the host duplex (increase in T m ) and an increase in the van’t Hoff enthalpy. The berenil binding parameters (ΔΔH DSC = -32.6 kJ/mol, ΔΔS DSC = -72 J/mol·K and ΔΔG DSC = -11.1 kJ/mol) revealed significant differences compared to those of the pentamidine aggregation (ΔΔH DSC = -23.7 kJ/mol, ΔΔS DSC = -53 J/mol·K and ΔΔG DSC = -7.8 kJ/mol). The transition of the pure oligonucleotide was characterized by a substantial amount of intermediate states (σ DSC = 0.43) which decreased significantly upon binding of the drugs (σ DSC ~ 0.80). The structural features of the complexes were analyzed by FT-IR spectroscopy. From these experiments we conclude that the configurations in the berenil and pentamidine complexes are different.

Chemotactic cell motion in aggregation patterns of the slime mould Dictyostelium discoideum is analyzed by a computerized cross-correlation method. In this excitable medium the movement of amoebae is selforganized by signals of cAMP that propagate as rotating spirals or expanding concentric circles (target patterns). A vortex-like rotation of cells is found close to the core of spiral waves, with a maximum velocity of 15 μm/min. Cell motion and spiral tip orbiting follow an opposite sense of rotation. The calculation of streamlines of the most probable trajectories reveals the exsitence of a closed curve (radius ≈ 130 μm) corresponding to the boundary of the spiral core. This spatial-limit cycle attracts the amoebae on rotational paths and leads to the formation of a cell-free disk in the centre of the pattern. In contrast, pacemakers of target patterns organize cell movement in a radial, star-shaped motion, leading directly to the formation of central mounds.

The fiber cells of the middle layer of Trichoplax adhaerens are interconnected by slender extensions. Newly formed connections after mechanical disruption of the tissue studied in ultrathin sections revealed cytoplasmic continuity between the cell bodies, suggesting a syncytial organisation of the fiber cell layer. The slender extensions connecting the cell bodies are traversed by microtubules and microfilaments. The structure of rare osmiophilic cell contacts suggests a stage in the fusion of adjacent cell membranes.

In female adults of Gryllus bimaculatus, ovaries as well as the abdominal integument produce free and conjugated ecdysteroids in vitro (Hoffmann et al., 1992). The aim of the current study was to determine the influence of various potential ecdysteroid biosynthesis effectors (RH 5849, KK 42, diflubenzuron, ketoconazole, azadirachtin, acetylenic and allenic cholesteryl derivatives B1, B6, AL2), and also of the protein synthesis inhibitor cycloheximide, on net release of moulting hormones in vitro by the adult ecdysteroid sources. All the compounds examined can be divided into four groups due to their different effectiveness on both the ecdysiosynthetic tissues. The non-steroidal ecdysteroid agonist RH 5849 (group 1) enhanced ecdysteroid synthesis in ovaries, but inhibited hormone production in the abdominal integument. Treatment in vitro with diflubenzuron (dimilin) and cycloheximide (group 2) strongly reduced ovarian ecdysteroid synthesis whereas they were hardly effective on integumental hormone production. The group 3 compounds (ketoconazole, B1, B6, AL2, azadirachtin A) demonstrated a stronger inhibitory effect on the abdominal integument than on the ovary. The imidazole compound KK 42 (group 4) was a very potent effector of ecdysteroid biosynthesis in both hormone sources.

The cytochrome b gene of 6 South American screech owls of the genus Otus (O. choliba, O. atricapillus, O. usta, O. sanctaecatarinae, O. guatemalae, and O. hoyi) and two Old World species (Otus scops and Otus leucotis) was amplified by polymerase chain reaction (PCR) and partially sequenced (300 nucleotides). Otus atricapillus, O. guatemalae, O. hoyi and O. sanctaecatarinae which are morphologically very similar, have been treated as belonging to a single species, A. atricapillus (Sibley and Monroe, 1990). Nucleotide sequences differ substantially between these taxa (6.3 to 8.8% nucleotide substitutions) indicating that they represent well established and distinct species which had been implicated already from ecological and bioacoustical analyses (König, 1991, 1994). The importance of vocal and ecological characters for the taxonomy of nocturnal birds is thus confirmed by our molecular analysis. Phylogenetic relationships were reconstructed between Old and New World owls using character state (“maximum parsimony”; PAUP 3.1.1) and distance matrix methods (neighbour- joining; MEGA).

The dependence of spermatogenesis function on murine age shall be investigated. Thus, testicular samples of at least 10 NMRI mice per group aged 0 to 26 months are analysed by flow cytometry after staining the DNA with DAPI. The aim of this study is to be able to account for the influence of age on mice. There are no changes in spermatogenesis in mice aged 11 weeks up to 16 months with respect to testis weight and to the frequency of different testicular cell types. From 16 months onwards, there is a tendency to a reduced spermatogenesis function; The frequency of round spermatids is decreased. In addition, there is an increased chromatin dispersion in elongated spermatids with age. Thus, older mice (>16 months) should not be used for experiments e.g. on radiation and drug effects any more. The frequency of abnormal diploid spermatozoa does not increase with age.

From Bartramia ithyphylla the following five biflavonoids were isolated: philonotisflavone, 2,3-dihydrophilonotisflavone, dicranolomin, 5′,3′′′-dihydroxyamentoflavone and 5′-hydroxyamentoflavone.

In vitro a one-step selection for glyphosate tolerance of two Bulgarian tobacco cultivars Zlatna Arda and Nevrokop A24 was carried out. After treatment with glyphosate of the control plants and their regenerants polyphenol and alkaloid changes were determined. A decrease in the polyphenol concentrations and an increase in the alkaloid concentrations were registered only for Zlatna Arda.