Volume 52, Issue 9-10

Reactive oxygen species are involved in many diseases where the radical species OH . peroxynitrite and the non-radical, hypochlorous acid, play an outstanding role. The formation of OH -type oxidants is essentially confined to a few types of reactions. The most prominent ones are the one-electron reduction of hydrogen peroxide by Fe 2+ or Cu + - ions (Fentontype reactions), reaction of hypochlorite with superoxide and finally formation and decay of peroxynitrite (ONOOH), formed from superoxide and NO. In this communication we wish to report on a simple model system allowing to differentiate between these ROS: ethene formation from ACC is only detectable in the presence of hypochlorite (v. Kruedener et al., 1995) and not detectable with Fenton-type oxidants or SIN-1 (3-morpholinosydnonimine, a peroxynitrite generator by releasing sequentially superoxide and NO) at 10 |.im concentrations. On the other hand, ethene formation from KMB is negligible in the presence of hypochlorite but proceeds rapidly with Fenton-type oxidants (4 μᴍ H 2 O 2 ; 4 μᴍ Fe 2+ ) as well as with 1 μᴍ SIN-1. Stimulation of Fenton-type oxidants and not of SIN-1 by EDTA and characteristic patterns of inhibition by SOD, catalase. hemoglobin and uric acid allow a differentiation between these two potential precursors of OH-radicals. Synthetic ONOOH shows different reaction kinetics as compared to SIN-1. Inhibition of ONOOH-dependent ethene formation by different compounds occurs more or less “random” indicating an unspecific influence of proteins and also small molecules. Comparism of the individual inhibition types of several selected compounds allows a differential analysis as to the generation pathway of the final oxidants, OH- radical or peroxynitrite

Representatives of the genera Odixia and Ozothamnus, Australian Asteraceae belonging to the tribe Gnaphalieae, have been analyzed for the presence of flavonoid aglycones accumulated on leaf and stem surfaces. Fiftytwo more or less lipophilic flavonoids have been identified. Methyl ethers of apigenin, luteolin, kaempferol and quercetin are abundant, while rare methyl ethers of isoscutellarein, 8-hydroxy galangin, herbacetin and gossypetin appear to be characteristic. The distribution of rare flavonoids is discussed

Leaf extracts of Erythroxylum coca var. coca Lam. (E. c. var. coca) yielded six O-eonjugates of Eriodictyol flavonoids, while the equivalent extracts from Erythroxylum novogranatense var. novogranatense (Morris) Hieron (E. n. var. novogranatense) contained five flavonoids, two of which were O-conjugates of Luteolin and three were O-conjugates of Kaempferol. All six of E. c. var. coca methanolic extracted peaks (resolved by HPLC) were found to have a 2, 3 single bond, which in E. n. var. novogranatense is replaced by a 2- hydroxy allene. The other primary difference in the predominant flavonoids between these taxa is the chemical composition of the sugar and/or acyl O-conjugation and site(s) at which this conjugation occurred. The results suggest that the most abundant O-conjugated flavonoids of E. c. var. coca and E. n. var. novogranatense may be used as chemotaxonomic markers for the two taxa. Therefore, the O-conjugated peaks of Eriodictyol , are distinct chemotaxonomic markers for E. c. var. coca and the O-conjugated peaks Luteolin and Kaempferol for E. n. var. novogranatense. These taxa are two of the four cultivated Erythroxylum taxa that contain commercial quantities of the cocaine alkaloid in their leaves, this entity also sets apart the taxa from other members of Erythroxylum. We suggest that the biochemistry of flavonoids of other Erythroxylum taxa may also be species selective

Two-year-old seedlings of Picea abies L., P. glauca L., P. mciriana L., Pinus sylvestris L. and Pseudotsuga menziesii L. were planted in a sample plot influenced by high concentrations of cement dust, and in an unpolluted (control) area in 1990. In 1994, the six-year-old trees were dug up in the pre-bud-break period. A comparative analysis of soluble sugars (glucose, fructose, sucrose, maltodextrines), starch and hemicelluloses contents was carried out and the changes in carbohydrate partitioning were estimated. A decrease in the total content of soluble sugars, mainly of glucose, fructose and maltodextrines, was observed in polluted trees, especially in roots, stems and buds, while the sucrose content increased in different organs. Fluctuations in glucose and fructose contents in roots were in good correlation with these sugars in needles and maltodextrines in stems and shoots. Changes in starch and hemicellulose partitioning between different organs of trees were modest and their directions were highly dependent on species

Culture filtrates of different fungi of the genus Rhizopus forming tempe (i.e. traditional Indonesian food) were grown on a soybean protein-raffinose-phytate medium and investigated for protease activity using soyprotein as substrate. All isolates belonging to the species R. oryzae, R. stolonifer, R. oligosporus, and R. microsporus var. chinensis, formed the wellknown Rhizopus-pepsin (aspartic proteinase, 35 kD, isoelectric points: 5.9, 5.0, <4) and an additional protease mainly active under alkaline conditions. The new protease (33 kD, isoelectric points: variable and isolate specific) was purified approximately 300-fold and shown to be a serine protease (inhibitor studies). During fungal culture (12 -135 h) the aspartic proteinase is expressed first followed by the serine protease. Both proteases are insensitive to the soybean Kunitz and Bowman-Birk inhibitors. The best rate of soyprotein degradation is achieved by the coordinate action of both proteases at pH 6.5. The examined Rhizopus isolates differ in the time course and intensity of the expression of the alkaline protease

The absorption spectra and the corresponding molar absorption coefficients of the fluorophores umbelliferone, FAD and FMN and of the FAD and FMN containing flavoprotein NADPH-cytochrome P450 reductase of different oxydation-reduction states are documented. Binding spectra of the ligand umbelliferone with the CYP2B1: NADPH-cytochrome P450 reductase-complex were determined by difference spectroscopy. The Scatchard plot of the equilibrium ligand binding shows a high affinity part and a low affinity part of 12 and 34 umbelliferone binding sites per CYP2B1: reductase-complex molecule, respectively The fluorescence excitation and emission spectra of the donor molecule umbelliferone and the acceptor molecules FAD and FMN are given. The fluorescence spectra of the reaction components under test conditions of CYP2B1-dependent 7-ethoxycoumarin-O-deethylase are measured. The excitation energy transfer from the donor umbelliferone (λ E =380 nm; λ F = 460 nm) to the acceptor molecule FMN (λ E =465 nm; λ F =525 nm) was examined under assay conditions. The results dem onstrate that a radiationless Förster-type energy transfer takes place in the presence of the CYP2B1: reductase-complex. It turned out that this effect is a function of the protein complex-concentration. The data presented here combined with previously made observations by Müller-Enoch (Müller-Enoch D. (1994), Z. Naturforsch. 49c, 763-771) support the finding that the umbelliferone molecules, n = 12-34, bound per mole of CYP2B1: reductase-complex, transfer their absorbed light energy radiationless to the FAD binding domain. The complex formed containing 12 or 34 molecules of umbelliferone provides absorption coefficient values at λ= 380 nᴍ of 78 and 221 mᴍ -1 · cᴍ- 1 , respectively. The Förster-type energy transfer from the donor umbelliferone to the acceptor FAD not only leads to a light activation of the singlet state of FAD but also to a conformational change of the amino acids close to the FAD binding side to favour the encaging of the FAD* triplet state which reacts with the NADPH to form the FADH 2 reductase. Due to this process the overall reaction can start with the unquenched excited FAD* triplet state as an interm ediate which is about 30 kJ/mol lower in energy than the dark reaction

A new mechanism for alkane functionalization by iron(III)-peroxide adducts was proposed, and the importance of electrophilic nature of the metal-peroxide adduct with η 1 - coordination mode was emphasized. This idea suggests that formation of high-valent ironoxo species occurs most likely when the metal-peroxide intermediate is activated through electronic interaction with both the peripheral organic group and substrate; the latter two act as an electron donor to the peroxide adduct

NAD + kinase was isolated by chromatography steps from asynchronous cultures of the achlorophyllous ZC mutant of Euglena gracilis. A non Ca 2+ -calmodulin dependent form, whose activity was stimulated by EGTA, was selected for its large quantity and high specific activity. Studies of the kinetic parameters revealed two kinds of NAD + binding site, depending on NAD + concentrations, and changes induced by EGTA, Ca 2+ and Ca 2+ -calmodulin. The search for effectors, soluble (S) and membrane-bound (P), in Euglena gracilis synchronously grown (in a light-dark regime of 12h:12h), and collected at circadian times (CT) - corresponding to the maximum, CT 17, and to the trough, CT 09, of the circadian rhythm of NAD + kinase activity - was also undertaken by testing the modulations of the kinetic parameters of the prepared NAD + kinase. The results suggest: (i) structural changes of NAD + binding sites depending on NAD + concentrations; (ii) possible binding of the Mg-ATP -2 (or Ca-ATP -2 ) on the NAD + sites, because of their common ADP motif; and (iii) different and specific modulations of the kinetic parameters of the two types of NAD + binding site by the Ca 2+ -calmodulin complex. In addition, the results indicate, in pelletable fractions isolated at CT 09 and CT 17, the presence of two kinds of effector: (i) the first one, possibly Ca 2+ , which increases the Umax’s while decreasing the binding of NAD + ; (ii) the second one, possibly the Ca 2+ -calmodulin complex, which provokes a complete reverse effect. Each of these two effectors seems to be, alternatively and rhythmically (eight circadian hours apart), partially released from the membranes

The recently isolated and characterized unicellular photosynthetic prokaryote Acaryochloris marina (Miyashita et al., 1996) contains chlorophylls a, d , and traces of a chlorophyll c-like pigment as well as phycocyanin. a type of allophycocyanin, zeaxanthin and cx-carotene, chlorophyll d being the predominant chlorophyll component. Quantum requirement measurements of the photosynthetic oxygen evolution resulted in about 12 quanta for excitation of chlorophylls a and d and 18 for phycocyanin. The data also revealed that these pigments are involved in energy absorption for photosynthetic oxygen evolution. Energy is transferred efficiently and equally well between the chlorophylls. Light absorbed by phycocyanin which is organized in phycobiliprotein aggregates (M arquardt et al., 1997), results in a less efficient energy transfer to the reaction center chlorophylls

The adsorption of catalase on two types of soot differing in their structure has been characterized. The adsorption of this enzyme obeys the Tyomkin adsorption isotherm. It has been established that the catalase immobilized on soot and graphite takes part in the electrochemical oxidation of phenol. The enzyme activity of catalase immobilized on both types of soot was studied on the decomposition of hydrogen peroxide. The kinetic and activation param eters of the processes studied have been determined

We found, by circular dichroism and Raman spectroscopy measurements, that the secondary structure of the native ovalbumin and of its heat-stable form, called S-ovalbumin, is a probe of the structural differences between the two proteins. Small angle X-ray scattering and circular dichroism measurements performed on the two proteins under denaturing conditions, with different concentrations of guanidine hydrochloride, show the changes of the tertiary and secondary structure and a different pathway in the unfolding process. These experimental data confirm that the conversion of native ovalbumin into S-ovalbumin is irreversible and reveal that the response of the two proteins to the same chemical environment is different

We elucidated the effects of substituting seven amino acids for Ala at residue 251 of the Chlamydomonas reinhardtii D1 protein on herbicide resistance and photoautotrophic growth. Ala 251 has been suggested to play a key role in the structural integrity and function of the stromal loop between transmembrane helices IV and V of D1 and has previously been shown to affect resistance to “classical” PSII specific herbicides. Sensitive and rapid microtiter assays were employed to compare herbicide resistance and photoautotrophic growth in the various mutants. Substitution of Ala 251 by Ile, Leu or Val conferred resistance to the PSII herbicides atrazine, bromacil and metribuzin but not to DCMU, and impaired photoautotrophic growth in high and low light. Compared to an otherwise isogenic wildtype strain, the lie and Val mutants exhibited nearly identical levels of herbicide resistance and reduced growth while the Leu mutant had even slower growth and higher levels of herbicide resistance. In contrast Cys, Pro, Ser and Gly mutants were phenotypically indistinguishable from wildtype in terms of herbicide sensitivity and photoautotrophic doubling times. Collectively the seven Ala 251 mutations differed markedly from an Ala mutant (dr-1) at the well characterized Ser 264 D1 residue in terms of herbicide resistance and photoautotrophic growth

Methanolic extracts of 11 Rumex L. species (Polygonaceae) were examined against brine shrimp and R. scutatus L. has shown significant brine shrimp lethality (LC 50 =0.96 μg/ml). Methanolic extracts of the roots of R. scutatus were fractionated and the active fraction led to the isolation of 4 anthraquinone aglycones: emodin, chrysophanol, physcion and aloe emodin. Their toxic sequence was found as chrysophanol> aloe emodin> emodin> physcion. These anthraquinone aglycones showed a significant activity with LC 50 =0.00, LC 50 =0.01, LC 50 =0.05, LC 50 =0.15 μg/ml, respectively

Partially purified samples of thymidine phosphorylase were obtained from four preparations of human uterine leiomyomas and uteri using the method of Yoshimura et al. (1990), Biochim. Biophys. Acta 1034, 107-113. Among the studied twelve pyrimidine derivatives, 5- bromouracil. 5-nitrouracil, 5-fluorouracil, 6-aminouracil, 4,6-dihydroxy-5-nitropyrimidine are competitive inhibitors, while allyloxymethylthymine is an uncompetitive inhibitor of thymidine phosphorylase activity. 6-benzyl-2-thiouracil inhibits the activity of the enzyme in a mixed way. The most potent inhibitor of the thymidine phosphorylase activity is 5-bromouracil and uracil the weakest one. Stronger inhibition of these compounds on the activity of thymidine phosphorylase was found in uterine leiomyomas than in uteri

The main phenolic constituents from Brazilian propolis, originating from Sao Paulo State, were isolated and identified: three flavonoids, a prenylated coumaric acid and two new benzopyranes, E and Z 2,2-dimethyl-6-carboxyethenyl-8-prenyl-2H-benzopyranes

A comparative study of the in vitro effects of the coumarin compounds, scopoletin and aflatoxin B 1 (AFB 1 ), on bovine (Bos indicus) hepatic mitochondrial respiration was carried out polarographically, using isocitrate -NAD + (3 - site), succinate (2- site), and reduced cytochrome c (1 - site), as respiratory substrates. Both scopoletin and AFB 1 elicited a substrate - dependent stimulation or inhibition of the mitochondrial states 4 and 3 respiration. The results suggest that AFB 1 has a higher tendency to inhibit the mitochondrial respiration than scopoletin. while scopoletin showed higher uncoupling effects than AFB 1 . The effects of scopoletin and AFB 1 on mitochondria were more pronounced on the electron transport than on phosphorylation reaction. The extent (3-35% ) of AFB 1 induced inhibition of bovine mitochondrial respiration observed in this study, was appreciably lower than the values indicated in other animal species (rats and guinea fowls) reported in previous studies using equivalent concentrations of the toxin. These results were discussed in terms of the susceptibility of the animal species to the toxic effects of scopoletin and AFB 1

Zolpidem is an imidazopyridine sedative-hypnotic which interacts with central benzodiazepine- receptors. To examine its effects on uterine smooth muscle we have compared with those obtained by diltiazem, papaverine and diazepam on different experimental models. The IC 50 values obtained indicate similar behaviour of zolpidem and diazepam. They showed more active against the spontaneous contractions and those induced by KCl (60 mᴍ) or by CaCl 2 (0.01-10 mᴍ) in Ca 2+ -free depolarizing medium than against acetylcholine (0.1 mᴍ)-induced contractions. Both of them also showed more effectiveness against the tonic component of the acetylcholine-evoked contraction than against the phasic one. All the drugs tested were less powerful against contractions induced by oxytocin than against those induced by other agonists. This observation let us speculate that the mechanism of action of zolpidem may be related to an action on Ca 2+ influx through voltage-dependent Ca 2+ channels due to an interaction with low affinity receptor located at the plasmalemma as has been suggested for diazepam

The action of sodium hypochlorite (NaOCl) on bovine nasal cartilage was studied by proton nuclear magnetic resonance ( 1 H-NMR) spectroscopy in order to model degradation processes of cartilage caused by neutrophil-derived hypochlorous acid. Nasal cartilage was chosen as a mean of comparison because it differs from articular cartilage in its composition. It contains some more proteoglycans, i.e. polymeric carbohydrates and less collagen than articular cartilage. This is important for studying the influence of hypochlorous acid on cartilage components (collagen and polysaccharides). Cartilage samples were incubated at 37 °C with phosphate buffer in the presence or absence of NaOCl. Supernatants were collected and assayed by NMR-spectroscopy. In the presence of pure phosphate buffer, the supernatants of bovine nasal cartilage were less rich in low molecular mass metabolites (e.g. amino acids, lactate) than articular cartilage. However, intense signals for highly mobile N acetyl groups of cartilage polysaccharides were detectable in nasal cartilage. NaOCl caused an increase in signals for acetate and formiate. Signals for N-acetyl groups rose only during the first 25 minutes of incubation with NaOCl. Then, their concentration decreased markedly. These changes were related to an enhanced release of Chondroitinsulfate from nasal cartilage

The methanol extract of Brazilian propolis was fractionated by HPLC, based on HuH13 (human hepatocellular carcinoma) cell cytotoxicity assay. Two isomers of diterpenoid with a molecular formula of C 20 H 30 O 3 (MW: 318.46) were isolated. The structures of these colorless compounds were determined as clerodane diterpenoids (I, 15-oxo-3, 13Z-kolavadien-17-oic acid; II, 15-oxo-3Z, 13E-kolavadien-n-oic acid)

Propionibacteria have been reported as being oxygen sensitive bacteria. Nevertheless, in this work, we intend to show that Propionibacterium shermanii CDB 10014 is able to grow even at high volumetric oxygen transfer coefficients (K L a). Similar final cell concentrations were achieved in experiments at different K L a values, with increasing cell yield from 0.08 g/g cells in anaerobic conditions to 0.22 g/g at K L a 61 h -1 . The organic acid production pattern changes depending on the K L a. At K L a higher than 36 h -1 , propionic acid was not produced and acetic acid was the main product. At K L a higher than 20h -1 , lactic acid began to be produced. In further studies, no important differences in the growth yield were observed in the pH range 5.5-7.0, while the cell production was enhanced by tem peratures in the range 30-35 °C

The antioxidant properties of extracts from seeds of white bean, pea, lentil, everlasting pea, faba bean, and broad bean were investigated by enhanced chemiluminescence (ECL) and photochemiluminescence (PCL) methods. All extracts exhibited antioxidative activity. The antioxidative efficacy of the extracts investigated varied markedly and did not dependent upon their content of phenolic compounds. Results of TLC analysis of the extracts indicated that the antioxidant activity originated from several phenolic compounds, partly similar in their polarity