Volume 40, Issue 7-8

Comparative studies on the essential oil of the whole plant and callus-and suspension cultures of Pimpinella anisum L. were carried out. Both the whole organism and the cell cultures were shown for the first time to be similar in their constituents e.g. anethole, pseudoisoeugenol-(2-methyl-butyrate), epoxy-pseudoisoeugenol-(2-methyl-butyrate) and β-bisabolene. In difference to the plant, the callus-culture contained myristicine.

From Bryum capillare Hedw. a variety of flavone glucosides and their 6″ malonyl esters were isolated. Diosmetin 7-O-β-D-glucoside-6″-malonylester, luteolin 7-O-β-D-glucoside-6″-malonyl-ester and 6-OH-luteolin 7-O-β-D-glucoside-6″-malonylester are new flavone malonyl esters. This is the first report of flavone glucoside malonylesters in a non vascular plant. The flavonoid pattern of the gametophyte is different from that of the sporophyte. The chemotaxonomic relevance of these results is discussed.

Pseudomonas, Chorismic Acid Metabolism (Alternative Route), Phenazine Biosynthesis From Pseudomonas cepacia cultures 3-acetamino-4-hydroxy-benzoic acid has been isolated. This compound is of biogenetic interest as it suggests an alternative route in the chorismic acid metabolism initiated by an attack of an equivalent of NH 3 at C-5 rather than at C-2 as usually observed. Implications on the phenazine biosynthesis have been discussed.

During the incubation of soybean cotyledons with Pmg-elicitor for 22 hours the pH of the diffusion droplets increases from 7.2 to 8.3. This pH-shift is a precondition for the formation of the typical red colour of the diffusion droplet. After inhibiting the pH-shift by the use of 100 mм phosphate or Tris buffer instead of 10 mм buffer as solvent for the elicitor, the red colour is no longer formed with the exeption of 100 mM Ammediol buffer. However, the normal pattern of pterocarpan induction can be measured in the absence of the red colour in the diffusion droplet. Tris and Ammediol buffers exhibited a smaller pterocarpan induction as compared to phosphate buffer as solvent for the Pmg-elicitor.

Following inoculation with the fungus Helm inthosporium carbonum , excised leaflets of the tropical papilionate legume Calopogonium mucunoides have been found to produce isoneorautenol, a new dimethylpyrano-substituted isoflavonoid (pterocarpan) phytoalexin. This compound accumulates together with dem ethylm edicarpin, neodunol, tuberosin, sophorap-terocarpan A, and a sixth highly antifungal isoflavonoid (calopocarpin) characterised as 3,9-dihydroxy-2-(3,3-dimethylallyl)pterocarpan. Chromatographic and spectroscopic examination of ‘hom oedudiol’ (a pterocarpan from N eorautanenia edulis roots previously considered to possess the structure now assigned to calopocarpin) has shown that in reality this substance is identical with sophorapterocarpan A. The systematic relationship of Calopogonium to other genera within the legume tribe Phaseoleae is briefly discussed.

The new α-pyrone derivative II was isolated in crystalline form from extracts of dormant seeds of Rosa canina. It inhibits germination of seeds of Amaranthus caudatus completely, but reversibly, at conc. > 2.5 × 10 -5 м . Its chemical structure was elucidated by mass spectrometry, 1 H-and 13 C-NMR spectroscopy to be 3-methyl-1-oxa-bicyclo (4,1,0) hept-5-en-2-one-4,6-dicarboxylic acid di­methylester (II). It is derived from 3-methyl-2H-pyran-2-one-4,6-dicarboxylic acid by cyclo-propanation with diazomethane. The parent compound which is a natural product of the seeds of Rosa canina was isolated as its dimethyl ester I the structure of which was elucidated by mass and NMR spectroscopy. I shows less inhibition of seed germination than II. The reference a-pyrones dimethyl-6-methyl-2H-pyran-2-one-3,5-dicarboxylate (III) , 3-methoxycarbonyl-5-methoxycar-bonylmethyl-6-methyl-2H-pyran-2-one (IV), and isodehydracetic acid methylester (V) did not show any inhibitory activity. The relationship of II with threo-dihydrohematinic acid (VII) is discussed.

The enzymatic activities of the pyruvate dehydrogenase complex (PDC) and acetyl-CoA synthetase (ACS) have been compared in extracts of plastids isolated from spinach leaves and from both green and etiolated pea seedlings. A ll plastid preparations were shown to be capable of synthesizing acetyl-CoA, not only via acetyl-CoA synthetase, but also via the pyruvate dehydroge­ nase complex, though, with different activities. Both pathways are apparently under metabolic control. Thus, the substrate levels in photosynthetically active spinach chloroplasts appear to favor acetyl-CoA synthesis via ACS (apparent K m for acetate of 0.1 mм) , because calculated stromal pyruvate levels (0.1 m M) appear to limit its formation via the PDC (apparent K m for pyruvate of 0.2-0.3 nм) . In spinach chloroplasts, therefore, the PDC pathway seems to be predominantly involved in providing precursors for branched-chain amino acid biosynthesis (vali­ne, leucine and isoleucine). Acetyl-CoA, synthesized via ACS, may additionally function as an inhibitor of the chloroplast PD C , because, as in mitochondria, relatively low concentrations of the end products NADH and acetyl-CoA strongly inhibit the PD C in chloroplast extracts. On the other hand, comparatively high concentrations of MgATP, a cofactor for ACS, inhibited the PDC complex. The pH optimum of about 8 and the high Mg-requirement distinguishes both enzymes from mitochondrial PDC and reflects an accomodation to stromal conditions in photosynthetically active chloroplasts.

5-Carboxymethyl-2-hydroxymuconate isomerase and 5-carboxymethyl-2-oxo-hex-3-ene-1,6-dioate decarboxylase in the 4-hydroxyphenylacetate meta-cleavage pathway have been purified to over 98% homogeneity. The native enzymes, which appear to be monomers, have apparent molecular weights of 37,000. The isomerase shows a pH optimum 8.0 and does not require ions for its catalytic activity; Mg 2+ is required for the decarboxylase reaction. Apparent K m values for their respective substrates are 2.8 × 10 -4 м for the isomerase and 1.4 × 10 -4 м for the decarboxy­ lase. The highly purified enzymes were used to study the spectral characteristics of the decarboxy­ lase substrate.

Cyanogenesis, β-Glucosidase, Linamarase, Trifolium repens Linamarase was purified by a three-step procedure from leaves of a clone of Trifolium repens. The partially purified enzyme was used to produce antilinamarin. The antiserum contained anti-linamarase as well as antibodies against one or more other β-glucosidases. By incubation with an extract of a plant without linamarase activity pure antilinamarase could be obtained. Comparison of the titration curves of crude and purified antilinamarase shows that linamarase is an enzyme antigenically different from other β-glucosidases, and that this enzyme is absent from plants recessive for the gene Li.

A high yielding production of the trichothecene mycotoxin 3-acetyldeoxynivalenol (3-AcDON) in cultures of Fusarium culmorum is described. By supplying [ 14 C]acetate, 14 C-labelled 3-AcDON suitable for further metabolic studies has been obtained. The pattern of labelling has been ascertained by using 13 C-labelled acetate precursors, and is in line with established biosynthetic data. A second trichothecene produced in significant amounts by F. culmorum has been identified as 3α, 15-diacetoxy-7α, 8α-dihydroxy-12, 13-epoxytrichothec-9-ene (7α, 8α-dihydroxycalonec-trin).

From the seeds of Swietenia mahagoni two limonoids have been isolated and identified as swietenine and the hitherto not described methyl-2,3,6-trihydroxy-melia-C-8(30)-enate-3-tiglate (2-hydroxy-swietenine) by spectroscopic methods.

Extracts of S. asoca bark and pure compounds isolated from the bark were tested for properties that might inhibit the conversion of arachidonic acid by the PGH 2 synthetase. They were assayed spectrophotometrically with adjrenaline as cofactor. Methanol- and ethyl acetate extracts inhibited the conversion. The observed inhibition was confirmed in an oxygraphic assay. Two procyanidin dimers from the ethyl acetate extract showed enzyme catalyzed oxidation in our assay. The ether extract of the bark was also found to contain yet unknown substances which were capable of being oxidised by the PGH 2 synthetase. The combined action of the components of the bark may explain the mode of action of the drug Asoka Aristha, the main ingredient of which is the bark of S. asoca. The drug is traditionally used in Sri Lanka to treat menorrhagia.

The marine diatoms Bellerochea yucatanensis and Thalassiosira rotula were grown at different salinities (20/25, 35, and 40/45‰ salinity (S), respectively) under normal air (0.035 vol.% CO 2 ). No significant variations in the percentage of gross photosynthetic products (e.g. total amino acids, sugar phosphates) were found as a function of salinity during growth. The bulk of the soluble 14 C-radioactivity was detected in amino acids. 14 C-labelling of glutamine increased markedly with salinity. Low salt - grown algae are characterized by enhanced amino acid pools, mainly of aspartic acid, asparagine and glutamine. It was found that the tested amino acids are not involved in osmoregulation.

Dimethyl or diethylamine reacts very smoothly in ethanol solution with catechols to the corresponding aminoquinones. The paramagnetic intermediates, the semiquinones, are investigated by ESR and ENDOR spectroscopy using the spin stabilisation technique with diarylthallium cations. The spectra of these radicals show clearly the thallium and nitrogen coupling and the hydrogen hyperfine structure. For some examples the signs of the proton coupling constants are determined by TRIPLE resonance experiments. The results indicate a negative spin density in one position of the unsymmetrically substituted radical 3. With the results obtained for the monocyclic aminosemiquinones the hyperfine structure of the adrenochrome semiquinone is analysed.

ESR-spectra were recorded during the oxidation of N-acetyldopamine and N-β-alanyldopamine in aqueous solutions. Semiquinone radicals were detected under conditions of spin stabilization by Zn 2+ ions. The appearance of the spectra was the same in the presence or in the absence of proteins. No evidence was obtained for the formation of products that could have arisen eventually from intermolecular Michael-type addition of nitrogen nucleophiles.

The labeling of immunocomplexes for scanning electron microscopy (SEM) is a fairly new technique, and the various procedures, that have been proposed, have not yet been compared. Such comparative evaluation was performed with Candida protease as a target antigen. This secretory enzyme of the opportunistic yeast Candida albicans can be localized on the surface of fungal blastopores and mycelia, both after growth in proteinaceous medium and upon infection of murine peritoneal macrophages. The presence of the protease antigen was confirmed by immunofluorescence and by immunoperoxidase-light microscopy. The decoration of protease - anti protease complexes for SEM was attempted with colloids derived from the immunoperoxidase reaction, by the immunogold technique, and by antibodies linked to beads of synthetic polymers (polystyrene, polymethacrylate, polyacrolein). In addition, inactivated Staphylococcus aureus was used, which binds to antibodies through its protein-A. The high resolution by SEM of surface structures was matched only by the colloid based decoration techniques. All conjugates with beads suffered from inconsistent binding, which did not correspond with the distribution of the surface antigen. The comparatively best result with beads was obtained with polystyrene (Latex). Colloid based techniques in addition allow for critical point drying, which cannot be applied to synthetic beads in the usual manner.

The kinetics of thermal denaturation of ceruloplasmin in water and in water with different percentage of D20 in the temperature range 25 - 85 °C, following the optical density change of the 610 nm charge transfer band of the protein has been investigated. A temperature T t ≃ 65 °C above which an irreversible denaturation process of the protein active site takes place has been found. The kinetics of the denaturation process of the protein are fitted by two first order reactions, which have been assigned to a different thermal denaturation behaviour of the two Cu 2+ type I sites existing in the protein. Addition of D 2 O to the protein solution affects the two kinetics in a different way, i.e. the rate of one of them is increased whilst that of the other is decreased. The different effect of D 2 O on the kinetics of disruption of the two copper sites is discussed in terms of different location and degree of hydrophobicity of the two Cu 2+ type I sites.

Similar density values of q = 1.39- 1.46 g/cm 3 and similar repeating periodicities were deter­ mined for murein and pseudomurein. Periodicities of 4.5 and 9.5 Å were measured in the planes of the sacculi of both cell wall polymers. Vertical to these planes periodicities of 42 to 45 A were obtained. These data may be interpreted as functions of an oblique-angled elementary cell with an area of about 4.5 Å × 10 Å and a height of 21 to 22.5 Å. This elementary cell includes one disac-charidepeptide subunit. As a consequence of this lattice periodicities the glycan strands of murein and pseudomurein perform twofold screw axes. Therefore the peptide chains, linked at distances of about 10 Å to every second sugar residue, point in identical directions. The parallel array of the polysaccharide chains associated with periodicities of 4.5 Å causes, therefore, a separation of the carbohydrate and peptide moieties of murein and pseudomurein. The head-to-head or tail-to-tail packing of such layers provides periodicities of 42-45 Å. Because of the twofold screw axis of the glycan strands, the secondary structure of the polysac­ charide strands of murein resembles the secondary structure of chitin. Since all glucosidic oxygen atoms occupy equatorial positions, if the β-1,3 linked N-acetyl-D-glucosamine residues are in the 4 C 1 , conformation and the β-1,3 linked N-acetyl-L-talosaminuronic acid residues in the 1 C 4 conformation, the polysaccharide chains of pseudomurein may also perform a twofold screw axis.

Total lipids from liver, head, skin and muscle of Bogue were separately isolated and their composition was investigated by a combination of analytical determinations, and column and thin layer chromatography. The major components of the neutral lipid fractions from all tissues studied were triglycerides, followed by cholesterol. The triglyceride fraction of skin and head contains significant amounts of glyceryl ether analogs. Low contents of free fatty alcohols were also identified, decreasing in the order: head, muscle, skin and liver. The major components of all phospholipid fractions was phosphatidylcholine (viz. 36-59% of total phospholipids) followed by phosphatidylethanolamine (viz. 23-34% of total phos­ pholipids). Low amounts of sphingomyelin and phosphatidylserine were also identified in all cases. All the tissues studied were found to contain plasmalogens, as well as glyceryl ether analogs in both, the depot fats and the phospholipid fractions.

The possible interaction of DDT with the lipids dimyristoyl lecithin (DML), dipalmitoylphos-phatidylethanolamine (DPPE) and tripalmitin (TP) was studied. The work was carried out on oriented films and crystalline powders of DDT-lipid mixtures at different molar ratios by X-ray diffraction techniques. The diagrams showed only the patterns of pure DDT and that of the corresponding lipid. It is concluded that new phases were not formed and, therefore, no interactions occurred.

The decline of the transport ratio of the sarcoplasmic calcium pump observed in a recent study (A. results from the retardation of calcium oxalate precipitation at low calcium/protein ratios. The prevailing high internal calcium level supports a rapid calcium backflux and a compensatory ATP hydrolysis during net calcium uptake which reduces the transport ratio. Yet, the determined calcium back­ flux does not fully account for the decline of the transport ratio. A supposed modulation of the stoichiometry of the pump by external calcium (0.1 μм) is at variance with results of previous studies showing a constant transport ratio of two in the same calcium concentration range.

Higher activity of ethanol production due to higher density of yeast cells, was observed in yeast cells immobilized with artificial polymer carriers of higher molecular motion. These polymer carriers were prepared by radiation-induced polymerization below 0 °C. Yeast cells were­ immobilized with these carriers by adsorption method during multiplication. Molecular motion of polymer carriers in swollen state was evaluated by the line width of nuclear magnetic resonance spectrum. A possible reason for higher activity was discussed. This study of relation between molecular motion of polymer carriers and activity of yeast cells immobilized with these carriers, is very useful to design the chemical and physical structure of polymer carriers for immobilization of microorganisms.

The technique of positive fast atom bombardment mass spectrometry combined with collisional activation and metastable ion data has been shown to provide molecular mass and useful fragmentation information for discrimination between the coumaric acid isomers. In addition to the occurrence of an ortho-effect, also para and meta positions could be distinguished.

Thymus membranaceus is an endemism of southeastern Spain, which belongs to section Pseudothymbra of genus Thymus. In this work we have achieved an investigation of the flavonoid glycosides present in the aerial parts of this plant. Among the compounds identified are the rare luteolin-7-0-β-D-xyloside, luteolin-7-0-β-D-[rhamnosyl (1→2)glucoside] and luteolin-7-0-β-D-[xylosyl (1→2)glucoside] previously not known to occur in the Labiatae.

Echinacea angustifolia and E. purpurea contain in all plant parts chichoric acid as well as chichoric acid derivatives: chichoric acid methyl ester and chichoric mono and dimethylether.

Spatial pattern formation during oscillating glycolysis in a thin layer of extract from yeast cells is investigated by two-dimensional spectrophotometry using video techniques and by dark-field detection of refractive index gradients. The time intervals of repetitively reappearing trans­ mission patterns coincide with the phases of maximum metabolic turnover during each glycolytic cycle. The spatial arrangement of the patterns is strongly correlated with a network of convection cells which permanently exists in the liquid layer. The patterns are generated by the coupling of reaction with Marangoni type convection due to gradients in surface tension which are mainly caused by evaporative cooling and influenced by chemical composition and reactivity.

DDT, Dieldrin DDT and Dieldrin produced a significant increase in the hemolymph sugar level of intact crab of Oziotelphusa senex senex apparently by triggering the release of hyperglycemic hormone (HGH).