Band 51, Heft 1-2

Flavonoid aglycones were detected as minor constituents of the leaf wax of the Myrtaceae Leptosperm um laevigatum. They belong to the rarely encountered C-methyl flavonoids. One of them is a new natural product, 5-hydroxy-3,7,3′,4′-tetramethoxy-6-C-methyl flavone.

Amino acids of propolis samples from different origins were analysed and quantified. Seven of them presented almost the same percentage in all samples, indicating that bees may have obtained amino acids from three sources: plants, pollen contamination and from their own metabolism. The regeneration properties of propolis in relation to the presence of some amino acids are discussed.

A great deal of an elicitor-active oligosaccharide (1.47 g) was obtained from 40 g of poly­ saccharide laminaran with the guidance of elicitor activity in the bean cotyledon assay. Physicochemical means suggested that the oligosaccharide was a β-1,3-and β-1,6-linked triglucoside. This was a partial structure of LN-3, having elicitor activity to alfalfa and bean. The triglucoside exhibited a specific elicitor activity to bean, not to alfalfa and pea. The highly digested hydrolysate containing monom er and dimers as major components did not show elicitor activity in the bean cotyledon assay. The structural feature was essential for elicitation of phytoalexin accumulation in bean cotyledons. These results suggested that this triglucoside had a specific elicitor activity and a minimum elicitor-active entity to bean cotyledons.

In chemostat cultures of Rhodobacter capsulatus, growing aerobically in darkness, in situ nitrogen fixation occurred at significantly lower oxygen concentrations than the acetylene reduction activity of nitrogenase, as determined with samples from the culture under opti­ mum assay conditions. In contrast to nitrogenase of cultures growing diazotrophically at low oxygen, nitrogenase present in inactive form at the higher oxygen concentrations could not be activated to fix nitrogen by increasing i.) the energy supply by illuminating the cultures, ii.) the supply of cells with electron donor and iii.) cellular respiration. These results suggest that oxygen controls cellular nitrogen fixation directly rather than indirectly by interfering with the general metabolism. On this basis, the sensitivity toward oxygen of nif gene expression as well as of nitrogenase polypeptide accumulation was studied. The nifH promoter was active up to about 40% air saturation, exhibiting a biphasic sensitivity to oxygen, i.e. steep decreases in activity between 1 to 2% and between 10 to about 20% air saturation. A similar behaviour was observed with respect to cellular levels of both polypeptides of component 1 (Rc1) of nitrogenase, which were accumulated up to about 20% air saturation. Component 2 (Rc2) was less oxygen-sensitive than Rc1, its steady state level reached a maximum at 2% air saturation. A sudden increase in the ambient oxygen concentration, i. e. oxygen stress, revealed that, in vivo, nitrogenase was fairly stable against oxygen damage. Modification of Rc2 by ADP-ribosylation occurred under ammonium shock conditions, regardless of whether the cells fixed nitrogen or not. But it was not observed with cultures growing aerobically in the dark at steady state or under oxygen stress. Results obtained with mutant W107I of R. capsulatus lacking the modifying/demodifying enzymes supported the conclusion that modifi­ cation of Rc2 was not required for the inactivation of nitrogenase by oxygen.

Isolated membranes of the moderate halophilic bacterium Haloferax volcanii are able to hydrolyze ATP via an ATPase, which needs the presence of Mg 2+ or Mn 2+ , high concentra­ tions of NaCl, a pH value of 9, and high temperatures with an optimum at 60 °C. We have not found any phosphatase activity in the preparations. We developed a purification method for the isolated enzyme with an enrichment factor of 90. SDS-gel electrophoresis of the partially purified enzyme of Haloferax volcanii showed putative ATPase subunits of 63, 51, 37, and 12 kDa. N-ethylmaleimide (NEM) a specific inhibitor for V-ATPases, which alkylates cysteines, inhibited the enzyme slightly. Binding of tritiated NEM to the isolated ATPase fractions resulted in labelling of the 63 and 51 kDa peptides. Using PCR with degenerate oligonucleotides, we could clone and sequence a gene cluster encoding the Aj part of the halophilic ATPase. The described genes are organized in an operon in the order D. C, E, B, A , named alphabetically according to their decreasing size. The deduced products of 64.5, 52, 38.7, 22, and 11.6 kDa confirm the results of the partial purification of the ATPase. Biochemical characterization of the Haloferax volcanii ATPase gave the following results: In presence of Mn 2+ higher rates of ATP hydrolysis could be observed than in presence of Mg 2+ , but free manganese ions inhibited the enzyme activity of the ATPase. Calculation of the true concentrations of the complex between ATP and the respective divalent metal ion led to determination of Michaelis-Menten constants for ATP in the hydrolysis direction of 1 mм in presence of MgCl 2 and 0.24 mм in presence of MnCl 2 . Sodium chloride concentrations in the molar range induce changes in K M by a factor of about 10. The enzyme is specific for ATP; other nucleotides including GTP and ADP are competitive inhibitors of ATP hy­ drolysis.

Quantum requirements of photosynthetic oxygen evolution at 679 nm, fluorescence emis­sion spectra at liquid nitrogen temperature (77 K) and fluorescence induction kinetics in the presence of DCM U, were measured in the cyanobacteria Anabaena variabilis M3, Anabaena variabilis ATCC 29413 and A nacystis nidulans R2, each grown under low-or high-CO 2 conditions. Low -CO 2 grown cells of the cyanobacteria showed a higher quantum requirement of photosynthetic oxygen evolution and a higher ratio o F 710-740 to F 680-700 fluorescence and a lower variable fluorescence in the presence of DCMU than high-CO 2 grown cells. These findings indicate a change in excitation energy distribution in favour of photosystem I. The result might be an enhancement in ATP formation caused by cyclic electron flow which in turn provokes dissolved inorganic carbon (DIC) accumulation in these low-CO 2 grown cells.

In our previous study (Gruszecki et al., 1995) we have postulated that the mechanism of cyclic electron transport around photosystem II, active under overexcitation of the photosynthetic apparatus by light is under control of the xanthophyll cycle. The combination of dif­ferent light quality and thylakoids having various levels of xanthophyll cycle pigments were applied to support this hypothesis. In the present work photosynthetic oxygen evolution from isolated tobacco chloroplasts was measured by means of mass spectrometry under conditions of high or low levels of violaxanthin, being transformed to zeaxanthin during dark incubation in an ascorbate containing buffer at pH 5.7. Analysis of oxygen evolution and of light-induced oxygen uptake indicate that the de-epoxidation of violaxanthin to zeaxanthin results in an increased cyclic electron transport around PS II, thus dimishing the vectorial electron flow from water. An effect similar to de-epoxidation was observed after incubation of thylakoid membranes with specific antibodies against violaxanthin.

Within a project that applied biochemical criteria to the diagnosis of damage to Norway spruce, 43 sites in western and eastern Germany showing only moderate tree damage were screened for the amounts of methanol soluble phenolic compounds in spruce needles. The concentrations of most of the main compounds-especially catechin-positively correlated with needle loss and the altitude of the site. It was also found that it is necessary to differenti­ate between trees younger and older than 60 years of age. The correlations between the increase of the phenolic compounds studied and the needle loss or the damage class are stronger in the younger trees, possibly implicating differences in metabolic state or distur­bances in protectective mechanisms in the older trees.

Feeding experiments carried out with cattle and horses could prove the toxic effects of P. marcgravii (Rubiaceae) in all cases. The typical symptoms of “sudden death”, however, are observed in ruminants only. This difference could not be explained so far. A part from fluoroacetate, two more substances also have influence the toxic effects and have been isolated from P. marcgravii for the first time: N-methyltyramine and 2-methyltetra-hydro-β-carboline (2-Me THBC). Structure elucidation of these compounds is mainly accom­ plished by 1 H-NM R, 13 C-NMR and MS techniques. Due to the small quantity of fluoroacetate (5.4 μg/g plant), the main toxic effect obviously lies in the two discovered substances. In contrast to the slow death of horses (monogastriers), the “sudden death syndrome” of cattle (ruminants) can be explained as a result of the higher resorbility of these two substances in the gastro-intestinal system. Given orally, both substances influence the monoamine oxidase type A (MAO-A): N-methyltyramine acts as a competitive substrate, and 2-Me THBC is one of the most effective MAO-A-inhibitors. Thus, the decomposition of the specific MAO-A-substrates noradrenaline and adrenaline as well as of N-methyltyramine itself is inhibited. The α-and β-receptors of the sympathetic system are stimulated more strongly, which leads to a drastic rise in blood pressure and thereby to a more rapid distribution of fluoroacetate in the body. This results in a reinforced input of fluoroacetate in the cells of especially active organs of the body (heart etc.). Thus, even smaller quantities of fluoroacetate are lethal.

The effect of Mg 2+ -ATP on purified acetylcholinesterase (AChE) from electric tissue of Electrophorus electricus (L.) was studied. The enzymatic activities were measured with acetylcholine and acetylthiocholine as substrates. The kinetic parameters V max , K m and Hill coefficient (n H ), for acetylcholine and acetylthiocholine were modified with Mg 2+ -ATP. It was shown that acetylcholinesterase presents an apparent activation at high concentration of substrates and an inhibition in the presence of Mg 2+ -ATP at low concentration of acetylcho line and acetylthiocholine. In addition, the data suggest that Mg 2+ -ATP induced an allosteric modulation of the acetylcholinesterase obtained from Electrophorus electricus (L.), and indi­cate an active adenosine triphosphate participation during cholinergic activity.

We describe results of computer simulation calculations of mixed and homochiral oligoribotides showing that it is possible to bind ʟ( D )-monomers at the term in al positions of chains built from D (L )-ribotides, but insertion at internal positions produces a large in stability of the chains. Coupled with aproposed new mechanism for the growth of these lin ear polymers in prebiotic conditions, our results imply that enantiomericcross-inhibition could have been bypassed to make all- D (ʟ) oligoribotidesdominant. We also show that racemic amino acids can form stable peptide β-strands, which rules out a similar origin for modern L -amino acids. Alternative schemes are proposed to account for the homochiralities of the amino acids and of other bio molecules.

Newly synthesized allyloxymethyl purine and pyrimidine acyclonucleosides [Fig. 1, comp. 1-6] were tested in Syrian hamster, six days after heterotransplantations of Kirkman-Robbins hepatoma and compared with Th5, Th5P and PMT [Fig. 1, comp. 7 -9 ]. 48 hours after intraperitoneal (i.p.) administration of AMT and Th5 in a dose of 80 mg per kg body weight, these compounds reduced tumor weight by 42%, while AMU (in the same dose) by 30%. The inhibition of tumor weight is accompanied by a decrease in dThd and dGuo kinase activities in tumor cytosol by AMU (36% and 33%, respectively) by AM T (59% and 53%, respectively) and by Th5 (58% and 55%, respectively). AMU, AMT and Th5 are phosphorylated in vivo by kinases present in cytosol of growing hepatom a to mono, di and triphosphates, but allyloxymethyl residue of AMU and AMT is first hydrated to hydroxypropoxymethyl residue, having CH 2 OH group. The lack of phosphorylation of PMT in vivo (having saturated propoxymethyl residue) and phosphorylation of Th5P (when used as a substrate for dNMP kinase) only to Th5 diphosphate suggested that AMU, AM T and Th5 triphosphates are responsible for the inhibition of dTMP and dGMP synthesis.

In this work, we propose a relaxation model for the interpretation of NMR proton spinlattice and spin-spin relaxation times of mammalian lenses. The framework for this model is based on nuclear magnetic spin-lattice relaxation measurements as a function of tem perature at different Larmor frequencies for whole rabbit lenses and fragments of the lens. According to this model, two different dynamic processes of the water molecules determine the relaxation behaviour, namely rotational diffusion and translational surface diffusion. These dynamic processes in conjuction with a two site exchange model give a good explanation of all the measured relaxation data. From the experimental data, we were able to obtain the activation parameters for rotational and translational diffusion of bound lens water. Correlation times of 2.1×10 -11 sec and 2.5×10 -9 sec and activation energies of 20.5 kJ/mol and 22.5 kJ/mol respectively were found at 308K. At low Larmor frequencies (≤ MHz) the longitudinal relaxation is mainly determined by translational surface diffusion of bound water with a mean square displacement of 1.5 nm, whereas at higher frequencies (≥300 MHz), rotational diffusion is the main relaxation mechanism. The spin-spin relaxation is determined by translational diffusion over the whole frequency range and therefore shows only a very small dispersion. By our model it is possible to explain: 1) the strikingly large difference between the T 1 value and the T 2A and T 2B values observed in the lens and 2 ) the different values of the activation energies measured at different fields for the lens.

The effects of different diets (high carbohydrate, high protein, high fat) and diets contaminated with polychlorinated biphenyls (PCBs) and/or γ-hexachlorocyclohexane (lindane) on the levels of serum triglycerides, cholesterol and phospholipids were investigated in Wistar rats. Serum triglyceride levels differed significantly among the diets, while those of cholesterol and phospholipids were much less affected by the diet composition. A change in diet composition resulted in a gradual adaptation to the lipid levels characteristic of the new diet with major variations including oscillations. There was, however, no specific component of a diet that could be associated with any specific change in serum lipids. While feed deprivation decreased the serum lipids (40-65% in 3 days), refeeding the starved animals caused pronounced increases of the lipids that were different among the diets. The response of the triglyceride levels was the strongest (up to 10 times the starvation levels) followed by those of the phospholipids (4-fold) and cholesterol (2.5-fold). Response of the triglyceride levels peaked within 1 or 2 days of refeeding, whereas those of cholesterol and phospholipids took 4 days to reach the maximum. Feeding PCB-contaminated diets increased the serum lipids in a dose-dependent manner (15-250 ppm). Higher PCB concentrations were increasingly inhibitory (350 ppm) or overtly toxic (> 400 ppm). Elevated lipids returned to the starting levels immediately after peaking (triglycerides) or only after several days (cholesterol, phospholipids) but with an earlier onset at lower PCB concentrations. Refeeding starved animals with PCB- contaminated diets also increased the serum lipids dose-dependently. Feeding lindane-containing diets (50-150 ppm) as well as refeeding animals with lindane diets resulted in a considerable increase of the triglyceride levels, while cholesterol and phospohlipids increased much less. Higher lindane concentrations ( 250 ppm) were inhibitory. The outcome on serum lipid levels on feeding diets contaminated with both PCBs and lindane was basically additive.

1. Light-evoked electrical responses were measured in Limulus ventral nerve photoreceptors simultaneously with changes in the cytosolic free calcium concentration, by means of arsenazo III. 2. It has been shown here for the first time that the rise of the arsenazo signal consists of two phases. Only the slow phase in the rise of the signal depends on the membrane voltage. The reversal potential of the amplitude of this slow rising phase was about +196 mV. After removal of external calcium the reversal potential was about +20 mV. 3. When Na + in the superfusate was replaced by Li + , the amplitude of the fast rising phase was reduced on the average to 50%, while the slow rising phase was not affected. 4. We conclude that the fast rising phase is caused by release of calcium from internal stores while the slow increase in [Ca 2+ ] i is due to influx across the plasma membrane, possibly through light-activated ion channels.

A study was made of the hatching process of the lancelet, Branchiostoma lanceolatum (Amphioxus), under laboratory conditions. Embryos in the early neurula stage (5 to 6 pairs of somites) escape through a hole made in the fertilization envelope, following intensive rotatory movements and partial digestion of the envelope. Using the chromogenic substrate Hide Powder Azure (HPA) at pH 8.0, proteolytic activity was detected in the hatching medium. The effect of various protease inhibitors was investigated. Whereas the activity was only slightly affected by inhibitors of trypsin, chymotrypsin or thiol proteases, chelating agents such as EDTA and 1,10-phenanthroline prevented the degradation of HPA, as well as the breakdown of the vitelline membrane of alcohol fixed oocytes by the protease from hatching fluid. These data suggest the presence of a metalloprotease in the hatching medium. How far this enzyme can be compared with hatching enzymes found in echinoderms and fishes (where they have been identified as matrix degrading metalloproteases), awaits further study of the lancelet enzyme, including its isolation and purification.

8-Oxo-2′-deoxyguanosine, 8-Oxoguanine, 8 -Hydroxyguanine, Base Modification, DNA Oxidative Damage The influence of 2′-deoxyguanosine (dG) oxidation at the C-8 position on N-glycosidic bond stability was in­ vestigated. A kinetic analysis of dG and 8-oxo-2′-deoxy-guanosine (8-oxodG) depurination reactions was carried out in water solutions at pH ranging from 2 to 7.4 and temperature of 100 °C. The results indicate that N-glyco­ sidic bond of 8-oxodG is significantly more stable in comparison with dG at any pH applied. At pH 5.1 hy­drolysis rate of dG is 4.5-fold higher than that for 8-oxodG. The chemical stability of the modified nucleo­ side in oxidatively damaged DNA is one of important factors contributing to its mutagenic potential. Results of our experiments indicate that 8-oxodG, potentially mutagenic and carcinogenic nucleoside, is hardly suscep tible to spontaneous depurination and its removal from cellular DNA depends mostly on the activity of DNA repair enzymes.