Band 52, Heft 3-4

The enzyme activity of magnesium chelatase was determined in intact etioplasts of barley (Hordeum vulgare L.) seedlings. Irradiation of isolated plastids with white light for 15 min does not lead to any activation of the enzyme but to a decrease in activity, especially in etioplasts. The enzyme was inhibited by chlorophyllide and zinc pheophorbide only to a certain extent. Strong inhibition was observed with the metal-free pheophorbide (K i = 0.92 μM) but not with pheophytin or chlorophyll. Penetration of chlorophyllide through the envelope membrane was confirmed by the chlorophyll synthase reaction that occurs in the inner membranes of etioplasts and chloroplasts. The possible role of inhibition of magnesium chelatase by pheophorbide in senescent leaves and tetrapyrrole transport across the plastid envelope are discussed.

The yellow colour of Chrysanthemum segetum petals is due to the presence of the 7-O-glucosides of quercetin and particularly gossypetin (8-hydroxyquercetin). In petal extracts of C. segetum an enzyme was demonstrated which catalyzes the transfer of the glucosyl moiety of uridine 5'-diphosphoglucose (UDPG) to the 7-hydroxyl group of flavonols with gossypetin and quercetin as the best substrates. Besides flavonols flavanones and flavones were found to be glucosylated in the 7-position. The pH-optimum of the reaction highly depended on the substrate used. With quercetin as substrate, maximal enzyme activity occurred at a pH of 8.25 and a temperature of 25 °C, but 7-O-glucosylation also proceeded at low temperatures. Studies on temperature stability revealed, that there was no influence on the glucosylation reaction up to 40 °C. Higher temperatures led to a loss of enzyme activity. Using gossypetin as a substrate a similar course of temperature stability was observed. Addition of Mg 2+ , Ca 2+ and KCN slightly stimulated 7-O-glucosylation, whereas Co 2+ , Cu 2+ , Fe 2+ , Hg 2+ , p-hydroxymercuribenzoate and N-ethylmaleimide showed a strong inhibitory effect. Additional enzymatic studies were performed with the commercial strain " Stern des Orients" where gossypetin 7-O-glucoside is restricted to the inner parts of the petals. For enzyme extracts from both parts of the petals gossypetin was found to be the most attractive substrate. In comparison to quercetin (133.4 μkat / kg protein) an about three times higher specific activity of the 7-O-glucosyltransferase(s) was determined with gossypetin (382.1 μkat/ kg protein) as substrate, indicating that hydroxylation of quercetin in 8-position to gossypetin precedes 7-O-glucosylation.

An optimum way of immobilizing xanthine oxidase on graphite was found where a redox transformation of the enzyme was observed. The nature of the redox maxima was hypothe­ sized on the basis of the dependence of the half-wave potential (E p/2 ) on the pH of the solution. The enzymatic activity of xanthine oxidase adsorbed on two kinds of soot was studied by the oxidation of xanthine. The kinetic and activation parameters of the enzyme reaction were determined.

The photodynamic action of hypericin on photosynthesis and respiration were monitored in Synechocystis sp PCC 6803 and Synechoccus ATCC 6311 (Anacystis nidulans). An oxygen electrode was used to measure net oxygen evolution or consumption. The amount of hypericin required to inhibit photosynthesis was quantitively determined. Photosynthesis was completely inhibited in Synechocystis when the molar ratio of chlorophyll/hypericin was about 70:1. Higher concentrations of hypericin did not stop the uptake of oxygen, but rather stimulated the process in the light. Hypericin was readily washed out of the cells forming no permanent associations in the bacterial cell. Hypericin inhibited electron transfer and consequently oxygen production by PSII particles. For half maximum inhibition of oxygen evolution, with PSII membrane particles, the molar ratio of chlorophyll/hypericin was about 10:1.

It has been postulated that the oxygen-evolving centers of photosystem II do not operate independently in the cyanobacterium Synechococcus leopoliensis in contrast to those of the chlorophyte Chlorella vulgaris and the diatom Phaeodactylum tricornutum (Mauzerall and Dubinsky (1993), Biochim. Biophys. Acta 1183, 123-129). Dependence would mean the existence of charge transfer among adjacent units and would be manifested by different saturation curves for the individual flashes of a sequence (different cross-sections), stronger damped oscillations and oxygen formation under the first flash, independently of the length of dark adaptation. We show in the present publication that in the filamentous cyanobacterium Oscillatoria chalybea the O 2 -evolution pattern which shows an O 2 -signal under the first flash (despite dark adaptation) can be explained within the heterogeneous Kok-model, assuming a non-standard initial S-state distribution (Bader, Thibault and Schmid (1983), Z. Naturforsch. 38c. 778-792).

Photosynthetic oxygen evolution from photosystem II particles was analyzed as consequence of a train of short (5 μs) flashes of different light quality and different intensities to study cyclic electron flow around photosystem II. Damped oscillations of the amplitudes of O 2 -evolution corresponding to a flash sequence were fitted numerically and analyzed in terms of a nonhomogeneous distribution of misses, represented by the probability parameter α i . Application of red light, known to promote cyclic electron flow around photosystem II (Gruszecki et al., 1995) results in a considerable increase of all α i, indicating that at the molecular level the misses may be interpreted as resulting from a competition for the reduction of oxidized P680 between cyclic electron flow and the electron flow coming from the water splitting enzyme. In accordance with previous findings, application of light flashes of the spectrum covering the absorption region of carotenoids resulted in an inhibition of cyclic electron flow and a pronounced decrease of the level of the miss parameter. Possible molecular mechanisms for the activity control of this cyclic electron transport around photosystem II by carotenoids are discussed.

Proteins cross-reacting with antibodies directed against a-and β-spectrin were recently detected in plant cells. In this report we have studied the ability of these proteins to interact with other components of membrane skeleton such as ankyrin. f-actin and calmodulin. It was found that the polypeptide of high molecular weight reacting with anti-a-spectrin antibody binds calmodulin in Ca 2+ -dependent manner. Protein complexes containing polypeptides cross-reacting with anti-spectrin antibodies interact with muscle f-actin (in co-sedimentation assay) and with erythrocyte ankyrin (ELISA -type assay). These data further substantiate a possibility of occurrence of spectrin-based membrane skeleton in higher plant cells.

Respiration and related aspects of metabolism were investigated in the roots and leaves of 2-year-old trees of the mangrove plant, Avicennia marina in the presence of 100, 250 and 500 mᴍ NaCl. The rate of respiration of leaves increased with increasing concentrations of NaCl in the incubation medium, but respiration of roots was not similarly affected. In order to examine the relative rates of catabolism of glucose by the glycolysis-tricarboxylic acid (TCA) cycle and the oxidative pentose phosphate pathway (PP pathway), we determined the rates of release of 14 CO 2 from [1- 14 C]glucose and from [ 6 - 14 C]glucose in segments of roots and leaves. The ratios of rates (C 6 /C 1 ) in roots varied from 0.30 to 0.44, while ratios of 0.85 to 0.99 were obtained when leaves were incubated in the presence of various concentrations of NaCl. It appeared that the PP pathway was more involved in sugar catabolism in the roots than in the leaves of A. marina. Uniformaly 14 C-labelled sucrose, incubated with segments of roots and leaves for 18 h, was converted to CO 2 , amino acids (mainly glutamine), organic acids (mainly malic acid), sugars and ethanol-insoluble macromolecules. The incorporation of radioactivity into most of these components was not significantly affected by NaCl. However, in leaves (but not in roots) the release of 14 CO 2 from [ U - 14 C]sucrose was en­ hanced by NaCl at 250 mᴍ and 500 mᴍ, while the rate of incorporation of radioactivity into macromolecules was reduced by high concentrations of NaCl. Incorporation of radioactivity from [ U - 14 C]sucrose into malic acid was enhanced in both roots and leaves by an increase in the concentration of NaCl from 100 mᴍ to 500 mᴍ (this concentrations is similar to that in sea water). Independent of the concentration of NaCl, more than half of the radioactivity in the neutral fraction from leaves was incorporated into an unidentified sugar, while in the same fraction from roots, the radioactivity was associated with glucose, fructose and sucrose. On the basis of these results, a discussion is presented of the characteristics of catabolism of sugars in A. marina in relation to salt resistance.

Production of propionic acid by Propionibacterium shermanii CDB 10014 was enhanced by a pH value of 6.5 and by temperatures in the range 35-37 °C. Depending on the type of yeast extract, succinic acid can be produced in higher proportions, with decreasing propionic acid yields. With respect to propionic acid production, Difco yeast extract has shown the best results when yeast extract preparations from other different suppliers were compared. To replace yeast extract by a cheaper vitamin-nitrogen source, corn-steep liquor was tested. A complete depletion of glucose was achieved, yielding a final propionic acid concentration of over 35 g/l. These results are even better than those obtained with Difco yeast extract and suggest the possibility of an economical process based on corn-steep liquor.

Ascochyta rabiei, the causal agent of Ascochyta blight on chickpea plants, secretes a cutinase in the culture filtrate when it is induced by cutin or hydroxylated fatty acids. This cutinase is the main esterase in the culture fluids. The enzyme was purified to homogeneity by three successive chromatographic steps. It showed an apparent molecular weight of 22 kD in SDS-PAGE and cleaved ester bonds of 3 H-labelled cutin or p-nitrophenylbutyrate with maximal activities around pH 8. As a serine esterase, cutinase is strongly inhibited by organophosphorous compounds and the most effective inhibitor 2,3,5-trichloropyridine-6-(0-methyl-O-n-butyl)-phosphateester (MAT 9564) shows a K 1 value of 0.8 nᴍ. The cutinase gene was cloned from a genomic cosmid library by screening with two oligonucleotides directed against cutinase consensus peptides. The gene was subcloned to a 1.7 Kb Sa1l/Hindlll-insert and sequenced. The cutinase gene codes for a 223 amino acid protein with strong homology to other fungal cutinase sequences. The purified cutinase is encoded by a single copy gene.

The present study was conducted to clarify the mechanism of toxicity of organic compounds using lipid model membranes (liposomes and planar lipid membranes). The compounds studied were trialkyltin and trialkyllead chlorides, dialkyltin dichlorides and some inorganic forms of those metals. Two different (anionic and cationic) detergents were also used in the experiments to change the surface properties of liposomes. As a measure of interaction between the compounds studied and model membranes were the release of liposome bound praseodymium and the change in stability of planar membranes under the influence of those compounds. On the basis of the results obtained it was postulated that the mechanism of interaction between tin-and leadorganics and model lipid membranes is a combination of different factors featuring interacting sides. The most important properties determining the behaviour of organic compounds in the interaction were lipophilicity and polarity of different parts of the organics and the steric arrangement they can take in the medium. On the other hand, the surface potential of the lipid bilayer and the environment of the lipid molecules, that play a significant role in the availability of the lipid bilayer to the organics, were important factors in the interaction.

Streptomyces ghanaensis (ATCC 14672) produces the phosphoglycolipid antibiotic moenomycin consisting of several components. A solid phase extraction procedure was developed which allowed a rapid isolation of both moenomycin and its biosynthetic intermediates from culture filtrates. Semi-preparative high performance liquid chromatography followed by high performance liquid chromatographv-mass spectrometry provided structural data on the different moenomycin components. In order to obtain initial information on the biosynthetic pathway, moenomycin non-producing mutants were isolated. They were shown to release intermediates with shorter lipid chains suggesting that the lipid chain synthesis probably takes place at a later stage of the moenomycin biosynthesis. Based on the biological activity and the analytical data, we assume that a modification and in particular a shorter lipid portion drastically influences the inhibitory activity of this antibiotic.

Hydration indexes of protein α-amino acids were measured by the new method of absorption millimeter spectroscopy (AMS) at 10 mm wavelength (1.05 cm -1 ; 31.42 GHz). This region of electromagnetic waves, located between the region of high-frequency dielectric spectroscopy and that of far-infrared spectroscopy, allows to measure hydration on the basis of different rotational and reorientational mobilities of water in hydration shell. Contribution to hydration of the methylene group is shown to be significantly greater than that of polar -OH , -CONH 2 and -COOH groups, while the contribution of the charged -CH(N + H 3 )COO - group is even negative. The high sensitivity of AMS method to hydrophobic hydration allows to build up the hydrophobicity scale which gives an acceptable correlation (r=0.95) with the heat capacities of the aqueous solutions of amino acids. Thus, millimeter absorption spectroscopy, the method of hydration determination at a molecular level (determination of V-structure of water with lifetime ~50 ps), allows to quantitatively distinguish hydration of polar and non-polar groups as well as calorimetry does at a macroscopic level.

Saponins isolated from Medicago sativa L. appeared to stimulate lipolytic activity and did not influence amylolytic or proteolytic activity of Neopancreatinum (extract of pancreatic enzymes: lipase, proteases and amylases). Saponins isolated from the aerial part of Medicago sativa L. were demonstrated to stimulate lipase activity more effectively than saponins sepa­ rated from the root of this plant. When saponins were treated with 0.02 m HC1 at 37 °C for 1 hour their lipase stimulation ability did not change. The saponins stimulated Neopancreatinum lipolytic activity more in­ tensively in the presence of sodium cholate.

The cytotoxicity of 22 natural and semi-synthetic simple coumarins was evaluated in GLC 4 , a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC 50 values > 100 μᴍ , following a continuous (96h) incubation, most coumarins exhibited only low cytotoxicity. Several compounds, however, displayed significant potencies. As far as the structure -cytotoxicity relationship is concerned, it is conspicuous that all the potentially active natural compounds possess at least two phenolic groups in either the 6,7-or 6,8-positions. In addition, the 5-formyl-6-hydroxy substituted semi-synthetic analogue was found to be potent, reflecting the importance of at least two polar functions for high cytotoxicity.

Targeting studies using the anti-cancer agent neocarzinostatin (NCS), conjugated to anti­ bodies have shown relatively poor specificity. From the literature, it is unclear whether NCS mediates its effects either in conjugated or unconjugated form. In the present work we have used a conjugate of NCS with transferrin, a biological ligand with a well defined endocytic route, to probe these mechanisms. NCS was covalently coupled to transferrin using the heter­ obifunctional reagent sulfo-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and 2-iminothiolane to give a stable thioether-linked conjugate with a ratio of 1.6 mol of NCS per mole of transferrin. The binding activity of transferrin was completely retained. Conjugation of NCS to transferrin resulted in an apparent enhancement of cytotoxicity. However, incubation with excess transferrin had no influence on the observed enhanced toxicity, indicating that endocytosis is not responsible. Further experiments demonstrated that the apparent enhancement was dependent on incubation conditions and not an effect due to endocytosis of ligand. Studies where apo-NCS competed with holo-NCS and transferrin strongly indicated that the cytotoxicity of both NCS and conjugate is mediated by direct entry of the dissociated chromophore into the cell.

In the butterfly Inachis io, a pupal melanization reducing factor (PMRF) which is located throughout the entire central nervous system controls the intensity of pigmentation of pupal cuticle depending on the background color of the pupation site. PMRF does not only reduce melanization but, in addition, enhances lutein incorporation in a dose-dependent manner to form pupae with yellow color on bright backgrounds. The present paper reports on the effects on pupal pigmentation caused by cyclic nucleo­ tides and phosphodiesterase (PDE) inhibitors which prevent degradation of cyclic nucleo­ tides. The injection of cAMP did not alter pupal coloration whereas its membrane-permeable analog dibutyryl-cAMP mimicked dose-dependently PMRF activity. Thus, pupae of reduced melanization and, in addition, enhanced yellow coloration were formed. This indicates that an increased intracellular cAMP level is capable of mediating PMRF effect. Also, the injection of the PDE inhibitor isobutylmethylxanthine (IBMX) caused dose-dependently pupae of reduced melanization and enhanced lutein incorporation. Theophylline (another PDE inhibitor) was only slightly effective (23% inhibition of melanization) at the highest dose compared to IBMX. The injection of cGMP and its analog dibutyryl-cGMP exhibited no melanization reducing effect. Extracts of abdominal ganglia (AG) which contained PMRF activity caused significantly brighter pupae when injected in combination with IBMX. However, this stimulation by IBMX became no longer effective at higher AG doses. Therefore, the present results are suggestive of an involvement of cAMP as a second messenger in the action of PMRF on pupal color adaptation.

Fifty-eight Campylobacter strains were isolated from children with diarrhoea at various health centres in Lagos and from healthy chicken. Twenty -nine strains of Campylobacter were isolated from humans, while the same number were isolated from chicken. The strains were biotyped using the modified Preston biotype scheme. The Preston biotyping results have been compared with the results of Penner serotyping. Out of fifty-eight strains studied, the technique identified ten strains (17%) as C. coli, three (5%) as C. lari and fourty-five (78%) as C. jejuni, by the coding system. This technique identified twenty-eight Campylo­ bacter species. This method highlights the usefulness of this technique in the biotyping of local strains, however, when the two schemes are used in combination they give excellent typing results suitable for epidemiological purposes.

Eight urs-12-ene triterpenoids, β-sitosterol, (+)-catechin, and apigenin 7-O-glucoside were isolated from the leaves of Acaena pinnatifida R. et P. The triterpenoids were characterized as pomolic acid, pomolic acid-3-acetate, tormentic acid, 2-epi-tormentic acid, euscaphic acid, tormentic acid glucoside, niga-ichigoside F1, and niga-ichigoside F2.

The isolation of well formed crystals of the biomineral whewellite (monohydrated calcium oxalate) from Opuntia microdasys, a cactaceae species found in central Mexico, is described. The morphology of the crystals was investigated by means of electron microscopy. Infrared spectroscopic measurements allow an unambiguous characterization of the nature of the crystals. This is the first report of the presence of a biomineral in this species.

The effects of phosphatidylcholine, phosphatidyletha-nolamine and phosphatidylserine on the thermal decom­position of methyl 13-hydroperoxyoctadecadienoate were investigated in a chloroform/methanol mixture. Among the phospholipids tested, phosphatidylserine in­ hibited the thermal decomposition of methyl 13-hydro­ peroxyoctadecadienoate to about 30% after exposure to 60 °C for 48h wheareas only 10% remained in the absence of the phospholipid for the same heating period. Addition of eugenol with phosphatidylserine showed high synergistic activity which prevented the decomposition of the hydroperoxide to a higher extent under the same conditions. Phosphatidylserine was also found to inhibit iron -accelerated decomposition of methyl 13-hydroperoxyoctadecadienoate. Moreover, the decomposition of the hydroperoxide accelerated by ascorbic acid was effectively prevented by a combination of eugenol and phosphatidylserine.

A mouse monoclonal antibody reacting in ELISA with a synthetic peptide representing a linear amino acid stretch of the protein antigen was tested on all overlap­ ping 5-mer to 9-mer fragments of the peptide, as prepared by multi-pin synthesis. Analysis of the binding data suggests that several residues in the peptide might be relatively unrelevant for recognition, while few others seem to play a critical role as key residues. On the basis of such observations, we attempted to reconstruct an alternative essential epitope by introducing multiple amino acid substitutions in the 9-mer peptide exhibiting the best binding activity, and then tested its ability to be recognized by the monoclonal antibody.

Levels of a wide range of biogenic amines and related metabolites were determined in the brain of the silk­ worm, Bomby mori, during pupal and adult development using a three-dimensional HPLC system with multiple coulometric electrochemical detection. In the brain of the female adults, metabolic pathways such as tyrosine (TYR-4)->dihydroxyphenylalanine (L -DOPA)-dopamine (DA), TYR-4->tyramine (TYRA), and tryptophan (TRP)->5-hydroxytryptamine (5-HT) were identified. At this stage, 3,4-dihydroxyphenyleth-ylene (DOPAC) was also detected. Metabolic pathways of biogenic amines in the brain from pupal to adult stages are discussed.

Two sulfur compounds have been identified in the anal gland secretions of captive-raised adults of the striped polecat (Ictonyx striatus), an African mustelid. 3-Ethyl-1,2-pentanedithiolane was observed in the secretions of an adult male and an adult female. 1,3-Pentanedithiol was observed in the male; this compound has not previously been reported from mustelid anal glands.