Volume 53, Issue 3-4

Highly sensitive and specific recognition systems for microbial pathogens are essential for disease resistance in plants. Proteinaceous elicitors activating plant pathogen defense have been identified in numerous antagonistic plant/fungus interactions. Precisely defined signal structures required for elicitor-mediated activation of plant defense are indicative of the involvement of receptors in elicitor perception and subsequent signal generation. Use of pure elicitor preparations has helped to establish a functional link between binding of elicitors to high-affinity binding sites in plant plasma membranes and activation of plant defense. Thus elicitor binding sites appear to function as physiological receptors. Currently, isolation and molecular characterization of elicitor receptors is under way. Transfer of new recognition specificities into plants is supposed to be a key strategy for engineering pathogen resistance in economically important crops.

Three months old plants of the Chinese tung-oil tree Aleurites montana (Euphorbiaceae) were cultivated for 4 months in air containing 700 ppm CO 2 . These plants, which grow substantially better in the CO 2 -enriched atmosphere, were analyzed by mass spectrometry for photosynthesis and photorespiration together with control plants grown all the time in normal (350 ppm CO 2 ) air. Thereafter part of the plants was subjected for two weeks to 0.3 ppm SO 2 in the atmosphere and again analyzed for photosynthesis and photorespiration. Aleurites montana exhibits a strongly CO 2 -dependent photosynthesis which partially explains the observed stimulatory effect of 700 ppm CO 2 on growth of the plant. In control plants grown in normal air, photorespiration measured simultaneously with photosynthesis via the uptake of l80 2 in the light, is much lower than in C3-plants like tobacco (H e et al., 1995, Z. Naturforsch. 50c, 781-788 ). In Aleurites grown in 700 ppm CO 2 , however, photorespiration is completely absent in contrast to tobacco when grown under 700 ppm CO 2 . In tobacco, photorespiration is not inhibited to the extent of the in vitro experiments in which plants grown at 350 ppm CO 2 are measured under the increased CO 2 content of 700 ppm. Gas exchange measurements carried out by mass spectrometry show that the ratio of O 2 evolved to CO 2 fixed is about 0.5. Apparently, part of the CO 2 fixed is channelled into a metabolic path without concomitant O 2 -evolution. Although the plant has no succulent appearance (its leaves somehow resemble maple leaves) apparently a Crassulacean type metabolism is performed. When Aleurites plants grown all the time in normal air with 350 ppm, are exposed for two weeks to 0.3 ppm SO 2 the treatment completely inhibits this CO 2 -fixing portion which is tentatively attributed to a Crassulacean type of metabolism. This is demonstrated by a normal C3-type ratio O 2 evolved /CO 2 fixed of 1. When Aleurites plants, grown for 4 months in a CO 2 -enriched atmosphere of 700 ppm CO 2 , are subjected for two weeks to 0.3 ppm SO 2 , the features of control plants show up again. When these plants are tested under 350 ppm CO 2 the Crassulacean type CO 2 -fixation apparently is not inhibited by SO 2 . Photorespiration, although low, is present in the same activity as in the controls. Seemingly, an increased level of CO 2 in air tends to alleviate the impact of the SO 2 at least in the Chinese tung-oil tree.

It has been demonstrated that cyclic polyether, K -picrate-18-crown-6 inhibited photosyn­ thetic electron transport (Sabat et al., 1991, Z. Naturforsch. 46c , 87-92) . We further analyzed the alterations induced in the fast chlorophyll a fluorescence and thermoluminescence pattern of pea thylakoids by calcium-18-crown-6 (crown-picrate). The results indicate that the site of action of calcium crown-picrate is at the acceptor side of photosystem II.

The effect of different L-phenylalanine (Phe) concentrations (0 .1-12.1 mᴍ) , on acetylcholinesterase (AChE ) and Na + ,K + -ATPase activities of brain homogenate and pure enzymes, was investigated at 37 °C. AChE and Na + ,K + -ATPase activities were determined according to Ellman G. L., Courtney D., Andres V. and Featherstone R. M. (1961), Biochem. Pharmacol. 7, 88 - 95 and Bowler K. and Tirri R. (1974), J. Neurochem. 23, 611-613 ) respectively, after preincubation with Phe. AChE activity in brain homogenate or in pure eel E.electricus enzyme showed a decrease, which reached up to 18% with concentrations of 0.9-12.1 mᴍ. Brain homogenate Na + ,K + -ATPase activity showed an increase 16-65% with 0.24-0.9 mᴍ of Phe, while an activity increase of 60 -65% appeared with 0.9-12.1 mᴍ of Phe. Pure enzyme activity (from porcine cerebral cortex) was not affected by high Phe concentrations, while it was increased by low concentrations. The above results suggest: a) A direct effect of Phe on AChE, b) A direct effect of low Phe concentrations and an indirect effect of high ones on Na + ,K + -ATPase.

Acetylcholinesterase (AChE), Na + , K + -ATPase and Mg 2+ -ATPase activities were esti­mated in homogenised rat pituitary and hypothalamus of 4-and 22-month-old rats. AChE activity was not altered in the pituitary of aged com pared to adult rats, while it was found decreased by about 40% in the hypothalamus. Na + ,K + -ATPase activity remained stable in the hypothalamus, while it was decreased by about 38% in the pituitary. Mg 2+ -ATPase activ­ity remained unchanged in the hypothalamus, but was increased by about 83% in the pitu­itary. This pituitary Na + , K + -ATPase inactivation may result in pathological mood and de­creased neural excitability and metabolic energy production in aged animals.The age-related alterations of AChE , Na + , K + -ATPase and Mg 2+ -ATPase activities may reflect changes in secretion and responses of some hormones of pituitary and hypothalamus.

The inhibitory effect of a series of 5′-O-amino acid and oligopeptide derivatives of uridine on rat liver UDP-glucuronosyltransferase (UGT) activities was investigated using two assay systems. A quantitative structure-activity relationship (QSAR) study was performed. The compounds include a lipophilic residue linked to the nucleoside by a variable spacer. More­ over, half of the derivatives have two spacers linked to the uridine moiety. Compound 1, a serine derivative of isopropylideneuridine, was found to be the most potent inhibitor of both 4-nitrophenol (4-NP) and phenolphthalein (PPh) glucuronidation, with an I 50 of 0.45 mᴍ and 0.22 mᴍ , respectively. Kinetic studies with this substance revealed a mixed type of inhibition towards 4-NP and UDP-glucuronic acid, with apparent Ki values of 150 μᴍ and 120 μᴍ , respectively. The dipeptide derivatives 11-14 exhibited a low activity against 4-NP conjuga­ tion. However, a marked suppression of PPh glucuronidation was found with compounds 11 and 13. Generally, compounds with two spacers are more inhibitory against the UGT activi­ties studied. The QSAR analysis outlined the significance of the spacers with a minimum length of 5 atoms and lipophilic residues linked to them for the inhibitory effect of the compounds. The most significant contribution to this effect is given by the six-atom spacer for both, 4-NP and PPh substrates. 4-NP converting UGT isoforms seem to respond more specifically to the inhibitors: a five-atom for the first and a six-atom for the second spacer enhance binding to both 4-NP and PPh conjugating isoenzymes, while a long second spacer contributes to inhibitor binding to UGT isoforms only converting PPh.

Tamoxifen, Anticancer Drug, Erythrocyte Membrane, Phospholipid Bilayer Tamoxifen is a non steroidal antiestrogen drug extensively used in the prevention and treatment of hormone-dependent breast cancer. To evaluate its perturbing effect upon cell membranes it was made to interact with human erythrocytes and molecular models. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and of dimyristoylphospha-tidylethanolamine (DMPE), representative of phospholipids classes located in the outer and inner leaflets of the erythrocyte membrane, respectively. Experiments by fluorescence spectroscopy showed that tamoxifen interacted with DMPC vesicles fluidizing both its polar head and acyl chain regions. These results were confirmed by X-ray diffraction which indi­ cated that tamoxifen perturbed the same regions of the lipid. However, it did not cause any significant structural perturbation to DMPE bilayers. The examination by electron micro­ scopy of human erythrocytes incubated with tamoxifen revealed that they changed their normal discoid shape to stomatocytes. According to the bilayer couple hypothesis, this result means that the drug is inserted in the inner leaflet of the erythrocyte membrane. Given the fact that tamoxifen did not interact with DMPE, it is concluded that it interacted with a protein located in the cytoplasmic moiety of the erythrocyte membrane.

Changes of dynamic and structural parameters of egg yolk lecithin (EYL) liposome mem­branes in the heating-cooling cycles have been studied using the E S R spin probe method. The investigations were conducted in the range of temperatures from -18 °C to +60 °C. It has been found that in the range of temperatures -15 °C to +45 °C in both the heating and the cooling run the spectroscopic parameters changed practically along the same curve (re­versible changes). However, after exceeding this range of temperatures one of the parameters (partition coefficient of the spin probe 2,2,6,6 -tetramethylpiperidine -1-oxyl; TEMPO) changed along a closed curve, showing the phenomenon of thermal hysteresis. In the heating process the TEMPO content in liposome membranes was smaller than this in the cooling process. We assume that during the heating, the lipid molecules of the outer liposome layers dissolve in the aqueous medium. In the cooling process they can aggregate and form new liposomes, what in turn increases the surface of liposome membranes, accessible for the TEMPO probe (active surface).

A sample from Egyptian propolis was investigated by TLC and GC/MS. 39 compounds were identified, 8 being new for propolis. Partial structures of four new esters of caffeic acid have been proposed.

The paracloacal gland secretions of immature and adult American alligators (Alligator mississippiensis) from Louisiana were analyzed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy to investigate age-class differences in lipid composition. The secretions of both immature and adult alligators contain saturated C 14 and C 16 free fatty acids and decyl, undecyl, dodecyl, tetradecyl, and hexadecyl acetates. Com­pounds observed only in immature alligators include dodecanoic acid, C 12 and C 14 alcohols, C 10 -C 14 butanoates, an array of C 8 -C 18 3-methylbutanoates, C 12 - C 15 hexanoates, C 12 and C 14 octanoates, a C 12 decanoate, C 10 -C 14 dodecanoates, C 10 - C 16 tetradecanoates, C 12 and C 14 hexadecanoates, a C 12 octadecanoate, and a diterpene hydrocarbon identified as (E ,E )- 7,11,15-trimethyl-3-methylenehexadeca-1,6,10,14-tetraene (β-springene). Compounds observed only in adults include several C 7 -C 16 free fatty acids and certain C 10 - C 18 acetates. The age-class differences we observe in the paracloacal gland lipids of alligators from Louisiana are similar to those previously reported for alligators from Texas.

Most of the basic knowledge about radical reactions comes from radiation chemical studies in vitro. In view of the rapidly increasing knowledge on radical reaction in vivo, it is important to reconcile the fundamental physico-chemical reaction characteristics of radicals with the need to explain their alleged biological effects. Severe problems in the understanding of their in vivo action remain unsolved. An example is phagocytosis, which seems to be a paradigm of a ‘deleterious’ radical process. The exact mechanism is not clear; so it is an open question whether the intruder is eventually killed by radicals (like OH) or by endproducts of radical reactions (like H 2 O 2 and/or HOCl). It is even more difficult to understand signalling by radicals: owing to their chemical nature they are ‘unspecifically’ reacting species -they with­ draw or add electrons-and thus their reactions are governed by redox-properties. Since all radicals have different redox characteristics and different molecular shapes, the usual key-and-keyhole picture for molecular interaction does not apply, as there, is no reactive site conceivable which has the property of reacting with radicals ‘specifically. Our intent in this article is: (i) to briefly review some fundamental characteristics of in vitro radical reactions, (ii) to extrapolate from this to the conditions in vivo, and (iii) to discuss current hypotheses concerning the redox-regulation of cellular signalling. This leads us to the tentative conclusion that radicals per se must be tolerated by the cell and do not threaten its life, if they stay below a certain concentration limit. The main biologi­ cal implication of radical-reactions seems to be that the cell derives signals from the balance of oxidative versus reductive processes and that radicals may interact with pathways of intra-and intercellular communication.

Cellular defense against excessive peroxynitrite generation is required to protect against DNA strand-breaks and mutations and against interference with protein tyrosine-based sig­ naling and other protein functions due to formation of 3-nitrotyrosine. We recently demon­ strated a role of selenium-containing enzymes catalyzing peroxynitrite reduction. Glutathione peroxidase (GPx) protected against the oxidation of dihydrorhodamine 123 (D H R) by perox­ ynitrite more effectively than ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a selenoor-ganic compound exhibiting a high second-order rate constant for the reaction with peroxynit­ rite, 2 × 10 6 M -1 S -1 . The maintenance of protection by GPx against peroxynitrite requires GSH as reductant. Similarly, selenomethionine but not selenomethionine oxide exhibited inhibition of rhodamine 123 formation from DHR caused by peroxynitrite. In steady-state experiments, in which peroxynitrite was infused to maintain a 0.2 μᴍ con­ centration, GPx in the presence of GSH, but neither GPx nor GSH alone, effectively inhib­ ited the hydroxylation of benzoate by peroxynitrite. Under these steady-state conditions peroxynitrite did not cause loss of ‘classical’ GPx activity. GPx, like selenomethionine, pro­ tected against protein 3-nitrotyrosine formation in human fibroblast lysates, shown in West­ern blots. The formation of nitrite rather than nitrate from peroxynitrite was enhanced by GPx , ebselen or selenomethionine. The selenoxides can be effectively reduced by glutathione, establishing a biological line of defense against peroxynitrite. The novel function of GPx as a peroxynitrite reductase may extend to other selenoproteins containing selenocysteine or selenomethionine. Recent work on organotellurium compounds revealed peroxynitrite reductase activity as well. Inhibition of dihydrorhodamine 123 oxidation correlated well with the GPx-like activity of a variety of diaryl tellurides.

Oxidation of low density lipoprotein (LDL) by copper ions is strongly inhibited by dif­ ferent aqueous extracts (Salix spec (SE); Fraxinus-Solidago-Populus (Phytodolor R )(PD)) in a concentration range between 4 and 7 μg/ml. 10 to 50 μᴍ salicylic acid (SA) stimulate LDL-oxidation whereas higher concentrations (10 to 500 μᴍ) show no effect. Likewise ethene release from 2-keto-4-methylthiobutyrate (KMB) is strongly inhibited by the above extracts in a reaction driven by dihydroxyfumarate (DHF) in the presence of copper ions. This system may represent som e features of the diabetic situation where DHF as an endiole may stand for certain Amadori products. In order to find out whether the inhibitory effects are due to copper chelation we tested the copper-dependent conversion of photodynamic ethane release from a-linolenic acid into ethene formation. Copper chelation is apparently only partially involved in inhibition of copper-dependent oxidations and only at a certain concentration of extracts from Salix spec (SE) or extracts from Fraxinus-Solidago-Populus (Phyto-dolorR)(PD).

Reactive oxygen species such as OH·, peroxynitrite and the non-radical, hypochlorous acid, play outstanding roles in many diseases. The formation of OH·(Fenton-)-type radicals is catalyzed by enzymes such as xanthine oxidase (XOD) via one-electron reduction of mo­lecular oxygen producing superoxide radical anions (O 2 · - ). Subsequent transfer of one elec­ tron to hydrogen peroxide by Fe 2+ or Cu + -ions yields OH-radicals measurable as ethene release from 1-keto-4 methylthiobutyrate (KMB). Xanthine oxidase or activated neutrophils are prominent sources of this strong oxidant produced at inflammatory sites. Many natural compounds such as salicylates or flavonoids interfere either with the pro­ duction of these activated oxygen species or function as radical scavengers and thus as antiox­ idants. Extracts from willow-bark (Salix spec.) and also other species such as ash-tree (Fraxi-nus spec.) or poplar (Populus spec.) have been used as antiinflammatory drugs since a long time. In this communication we wish to report on model reactions to demonstrate a) the radical scavenging activities of such plant extracts inhibiting ethene release from KMB induced by Fenton-type oxidants and b) the inhibition of the formation of nitrogen monoxide (NO) from hydroxylamine includ­ing XOD either in the presence or absence of myoglobin (MYO) measurable as nitrite forma­tion: In the absence of MYO, superoxide dismutase is an excellent inhibitor of nitrite forma­tion but is inactive in its presence. Extracts from the willow-bark or the drug Phytodolor R , however, are inhibitory both in the presence and absence of MYO. As active principle, the flavonoid rutin included in these extracts is likely to function as one inhibitor of the XOD-mediated reaction.

Ubiquinone and α-lipoic acid are natural constitutents which are involved in mitochondrial energy metabolism. Their bioenergetic activities require redox-cycling. In the case of a-lipoic acid redox-cycling leads to dihydrolipoic acid which occurs in multienzyme com plexes in­ volved in the citric acid cycle while UQ recycles through semi-and divalently reduced ubiqui­ nones in the respiratory chain. We have proved the validity of the concept about the antioxi­ dant function of these natural compounds in their reduced form. Ubiquinol was found to interfere with lipid peroxidation of liposomal membranes being itself degradated by two consecutive oxidation steps. Dihydrolipoic acid was found to totally recycle ubiquinone to the antioxidant active divalently reduced form. In contrast to the antioxidative derived reaction products of ubiquinols which in turn promoted lipid peroxidation, the antioxidant derived reaction product of dihydrolipoic acid was the unreactive two electron oxidation product a-lipoic acid. Our experim ents demonstrate the existence of an dihydrolipoic acid driven recycl­ ing of UQ to the antioxidative-active UQH 2 . The efficiency of the antioxidative capacity of the latter was found to be diminished through prooxidant activities of the antioxidant-derived metabolites.

Culture supernatants of the arthritogenic bacteria Mycoplasma pneum onia, Mycoplasma arthritidis, Borrelia burgdorferi and Erysipelothrix rhusiopathiae stimulated primary cultures of human fibroblasts to release reactive oxygen species into the environment, whereas cell walls and membranes of these bacteria had no effects. Lipopolysaccharides of various gramnegative bacteria and lipid A , the lipid moiety of endotoxines, also failed to stimulate the release of reactive oxygen species by fibroblasts. The stimulatory fractions of the culture supernatants of Mycoplasma arthritidis and Erysipelothrix rhusiopathiae exhibited a molecular weight of about 9.5 kDa. After an induction period of 5 min the presence of the stimulant was not necessary any more. The primary radical released by the fibroblasts was the superoxide anion O 2 - . Radical formation took place continuously over some hours. Additionally, low-level chemiluminescence of fibroblasts was increased upon stimulation with culture supernatants of Mycoplasma arthritidis and Erysipelothrix rhusiopathiae. No irreversible injury of the fibroblast was caused upon stimulation and the cells exhibited normal proliferation pattern after replacing them to the culture medium.

Air pollutants are supposed to modulate physiological responses of alveolar macrophages (AM ). This study was addressed to the question whether at neutral pH sulfur(IV) species in comparison to sulfur(VI) species cause AM to release proinflammatory mediators and which pathways are involved in their generation. Supernatants obtained from canine AM treated with sulfite (0.1 mᴍ to 2 mᴍ) enhanced the respiratory burst of canine neutrophils, measured by lucigenin-dependent chemiluminescence, whereas supernatants derived from AM treated with sulfate (1 mᴍ) did not. The neutrophil-stimulating activity released by sulfite-treated AM consisted of platelet-activating factor (PAF) and leukotriene B 4 (LTB 4 ) as shown by desensitization of the corresponding receptors. Inhibitors of phospholipase A 2 substantially suppressed release of neutrophil-stimulating activity by sulfite-treated AM. Inhibition of 5- lipoxygenase in sulfite-treated AM also reduced neutrophil-stimulating activity, while inhibition of cyclooxygenase had no effect. In conclusion, sulfite induces AM to release lipid mediators via phospholipase A 2 - and 5-lipoxygenase-dependent pathways. These mediators activate neutrophils via the receptors for PAF and LTB 4 .

Under certain pathological conditions such as cerebral ischemia and reperfusion the occur­rence of free radicals is remarkably increased. However, only very little information is avail­ able on their quantitative relevance for the pathophysiology and final outcome of diseases. The aim of the present study was to evaluate the contribution of oxygen radicals in the pathogenesis of a stroke. For this purpose a rat model for stroke was used. Two of three vitamin E deficient groups were repleted with different dosages of DL-a-tocopherylacetate. N o signs of vitamin E deficiency could be observed. However, the weight gain during reple­tion was increased in the vitamin E repleted groups. Brain infarction was created by occlusion of the right middle cerebral artery (MCAO) for two hours. After 24 hours the measurements of infarct volumes were taken. The infarct volume of the group with the highest repletion dosage was significantly reduced by 81%. This was also expressed in a higher rate of gait disturbances after MCAO of the deficient animals. The control of vitamin E status exhibited a similar repletion-dependent level in plasma and brain. These results strongly support the hypothesis that the generation of oxygen radicals occurring during reperfusion is an impor­tant aspect of the pathophysiological mechanism in brain infarction.

The molecular mechanism of the anthracycline-dependent development of cardiotoxicity is still far from being clear. However, it is generally accepted, that mitochondria play a significant role in triggering this organ specific injury. The results presented in this study demonstrate that, in contrast to liver mitochondria, isolated heart mitochondria shuttle single electrons to adriamycin, giving rise to oxygen radical formation via autoxidation of adria­mycin semiquinones. This one electron reduction of anthracyclines is catalyzed by the exoge­nous NADH dehydrogenase associated with complex I of heart mitochondria, an enzyme which is lacking in liver mitochondria. Upon addition of NADH heart mitochondria generate significant amounts of adriamycin sem iquinones while liver mitochondria were ineffective. Adriamycin semiquinones undergo both autoxidation leading to superoxide radical release and complex reactions under formation of adriamycin aglycone. Due to the high lipophilicity adriamycin aglycones accumulate in the inner mitochondrial membrane where they interfere with electron carriers of the respiratory chain. Adriamycin aglycone sem iquinones emerging from an interaction with complex I were found to trigger homolytic cleavage of H 2 O 2 which results in the formation of hydroxyl radicals. As demonstrated in this study the activation of adriamycin by the exogenous NADH dehydrogenase of cardiac mitochondria initiates a cas­cade of reaction steps leading to the establishment of oxidative stress. Our experiments sug­ gest the exogenous NADH dehydrogenase of heart mitochondria to play a key role in the cardiotoxicity of adriamycin. This organ-specific enzyme initiates a sequence of one electron transfer reactions ending up in the establishment of oxidative stress.

The leaf exudate of Ozothamnus ledifolius, previously reported to contain a series of flavonoid aglycones, mostly consists of a mixture of terpenoids. From this resinous material we now identified four sesquiterpenes, a diterpenediol and two pentacyclic triterpene acids. In the phenolic portion we found three phenylethyl esters.

From the broth of submerged cultures of Verticillium rexianum (teleomorph: Nectriopsis exigua), a mycophilic fungus growing on myxomycetes, monacolin K was iso­lated as a weak inhibitor of pectate lyase. Monacolin K is the first secondary metabolite described from V. rexianum.

Acetylcholinesterase (AChE) is a significant compo­nent of the membrane contributing to the permeability changes during synaptic transmission and conduction. Phenylketonuria is a group of metabolic disorders in which phenylalanine (Phe) is highly elevated in blood (up to 0 .1 ᴍ) resulting in*mental retardation etc. AChE activity was measured spectrophotometrically after incu­ bation with various Phe concentrations. Phe interaction with DNA was evaluated with an established HPLC method. Phe was found to inhibit AChE almost 40% , at a concentration of 5 mᴍ, whereas a 62.5% DNA peak exclusion (molecular interaction) was observed when Phe was incubated with DNA at a concentration of 3 mᴍ. In addition the ratio of DNA: Phe determined the potency of the observed molecular effect.