Band 50, Heft 5-6

The criterion that pure natural science can only investigate objective phenomena which can be observed by independent observers sets certain limits to our scientific understanding of brain functions. The methods and the present state of brain research and of computer development are described. The limitations of brain research are discussed by comparing the properties of brains and computers. At least for the time being we do not know of any natural scientific -i.e. physical or chemical -method which allows the objective measurement of consciousness, sensations, and emotions.

It is shown that Pseudomonas fluorescens ATCC 13525 and Pseudomonas chlororaphis ATCC 9446 produce identical pyoverdins and ferribactins. As the structures of these sidero­ phores are usually species or even strain specific this exception should be kept in mind in view of the reclassification of the genus Pseudomonas. From Pseudomonas chlororaphis an additional siderophore could be obtained which has the same peptide chain as the co-occur­ ring pyoverdins and ferribactin, but a chromophore which is typical for azotobactins from Azotobacter vinelandii.

From Frullania teneriffae 5 known flavone aglycones and 6 flavone glycosides were isolated, among them the new 6-OH-luteolin-6-O-β-ᴅ-glucoside-7-O-β-ᴅ-glucoside-6′′′-(3-hydroxy-3-methylglutaroylester). F. cesatiana contains 2 flavone-di-C-glucosides and the new 6-OH-luteolin-7-O-β-ᴅ-glucoside-6′′-(3-hydroxy-3-methylglutaroylester)-3′-O-glucuronide. The phenolic pattern of F. eboracensis was compared to that of F. muscicola. The known 6-OH-luteolin-7-O-β-ᴅ-glucoside-6′′-(3-hydroxy-3-methylglutaroylester) was identified from F. gibbosa and F. arecae.

Several species of the large genus Artemisia have been analysed for the flavonoid aglycone composition of their leaf exudates. These species belong to different taxonomic groups within Artemisia. Flavone and flavonol aglycones were found as the major flavonoid constituents of the leaf exudates. Many of these compounds were 6-methoxylated, with additional substitutions at the 7-, 3′-and 4′-position of the molecule. In addition, 7,4′-substituted apigenin and luteolin derivatives and rarely also coumarin derivatives were encountered in some species. The observed substitution trends are in accordance with literature data. Taxonomic and eco­logical aspects are briefly discussed.

Two antimicrobial metabolites were isolated from submerged cultures of Cladobotryum rubrobrunnescens, a mycophilic fungus growing on a Inocybe species. One of the compounds proved to be identical to flavipucine (2), an antibiotic previously isolated from Aspergillus flavipes (Casinovi et al., 1968) and from a Macrophoma species (Sassa T. and Onuma Y. (1983), Agric. Biol. Chem. 47, 1155-1157). The other metabolite, brunnescin (1), is a new tetrasubstituted furan derivative which exhibits antibacterial, antifungal and cytotoxic effects.

Grown under anaerobic conditions in presence of nitrate, Haloferax volcanii shows nitrate reduction and accumulation of nitrite in the culture medium. We found a membrane-associated nitrate reductase, which could easily be solubilized by gently stirring of isolated membranes. Surprisingly, this nitrate reductase requires no NaCl for its activity. A medium pH of 7.5 and high temperatures up to 80 °C are necessary for optimum activity. Kinetic studies showed that the apparent K ᴍ was 0.36 mmol/l for nitrate and 80 μmol/l for dithionite-reduced methyl viologen. The respiratory chain inhibitor cyanide effects nitrate reduction noncompe-titively with respect to nitrate with a K i of 0.3 mmol/l. Azide was a strong inhibitor: The concentration required for half maximal inhibition was 60 μmol/l, whereas thiocyanate and chlorate were much weaker inhibitors. The isolated enzyme was partially purified by frac­tionated precipitation using polyethylene glycol. SDS gel electrophoresis resulted in three putative subunits of the nitrate reductase of molecular masses of about 100, 61, and 31 kDa.

N-(3,4-Methylenedioxyphenyl)carbamates and related compounds were prepared to develop flower-inducing compounds in Asparagus seedlings and to assay binding to microsomal P-450 that has been postulated to be a possible triggering site for flowering. Some of the compounds had flower-inducing activity as effective as that of the previous potent com­ pounds. The microsomal P-450 formed a type I spectrum with a representative of the present compounds as well as with previous, structurally different classes of flower-inducing compounds; which are carbamate and amidine derivatives and piperonyl butoxide, a methylene-dioxy compound known as broad-spectrum P-450 inhibitor. Upon addition of NADPH , the spectra of these compounds changed to those with a peak centered at 460 nm, indicating that they all form a reactive, high affinity species to produce a complex with P-450. The target species of the compounds that induce flowering in asparagus seedlings was thought to be cytochrome P-450.

The intrinsic polypeptide D1, isolated from photosystem (PS) Il-particles of the cyanobacterium Oscillatoria chalybea, was obtained by electroelution and fractionated extraction with organic solvents. Purification was demonstrated by Western blotting and amino acid sequenc­ing. By carrying out D1 -immunization in rabbits a polyclonal monospecific D1-antiserum was obtained. For the qualitative characterization of D1 as a lipid-binding peptide, the effect of the lipids phosphatidylglycerol (PG), monogalactosyldiacylglyceride (MGDG) and phosphatidylcho­ line (PC) on PSII-oxygen evolution was analysed in reconstitution experiments. In these experiments purified photosystem II (PSII)-particle preparations were treated with the en­zyme phospholipase A 2 and supplemented with lipid emulsions. We were able to show that the inhibition of electron transport, as the consequence of this lipase treatment, was only relieved, if phosphatidylglycerol was added to the preparation. A model was proposed, in which phosphatidylglycerol is a functional effector for the optimal conformation of D1 in the PSII core complex. Phosphatidylglycerol molecules are unusually tightly bound to the D1 peptide by hydrophobic interactions. A covalent binding seems not probable. The localisation of phosphatidylglycerol binding sites was found by trypsin treatment of D1 and analysis of the obtained oligopeptides with HPLC and immunoblotting. The binding sites could be confined to the hydrophobic amino acid section between arginine 27 and arginine 225, which is known to be the membrane anchor of D1. This has led us to the conclusion that the phospho­lipid phosphatidylglycerol plays an important role for anchoring the D1-peptide and for its orientation in the thylakoid membrane. Phosphatidylglycerol with its high amount of palmitic acid has in prokaryotic cyanobacteria apparently a role in stabilization and orientation. The high turn-over of D1 and the spatial separation of the synthesis-and incorporation-site in the membrane, developed during evolution in eukaryotic organisms, might have changed the requirement on the mobility and the orientation of D1 in photosynthetic membranes.

Oligomers (dimer to heptamer) of chitosan and their partially/completely N-acetylated derivatives were examined for (+)-pisatin-inducing activities in pea epicotyl elicitor assay at concentrations ranging from 6.25 to 100 μg/ml. The structures of the oligomers were analyzed by 1 H NMR and MALDI TOF MS spectrometers. The chitosan oligomers from dimer to tetramer were inactive, while the pentamer exhibited a moderate activity at 100 μg/ml. The hexamer showed a pronounced activity at 100 μg/ml, whereas the oligomer had no activity at the higher concentrations. Although the chitosan heptamer induced a moderate amount of (+)-pisatin (ca. 60 μg/g fr. wt.) at 50 μg/ml, the oligomer did not elicit pisatin formation at 100 μg/ml. The dimer to tetramer of both partially N-deacetylated chitin (DAC) and chitin were totally inactive. The DAC pentamers (d.a. 31%, 52%), DAC hexamers (d.a. 26%, 41%), and DAC heptamers (d.a. 26%, 36%) exhibited significant elicitor activities at lower concentrations (6.25-25 μg/ml) than the native chitosan oligomers. The chitin pentamer to heptamer had weak or no activity. Both glucosamine and N-acetylglucosamine were inactive in the assay. These facts suggest that the DAC pentamer to heptamer may act as elicitors for pisatin induction in pea-fungus interactions.

In order to investigate new metabolites which are only induced in a plant callus infected by a pathogenic fungus, dual cultures with combinations of 10 species of fungi and 6 plant cell lines from different species were established. Among the combinations tested, the methanolic extract of a dual culture consisting of a plant cell line, Phytolacca americana and a fungus, Botrytis fabae showed a marked antifungal activity to Cladosporium herbarum. The main active constituent of this extract was identified to be phytolaccoside B (Pls B) by the spectroscopic analyses.

5-Hydroxy-3-vinyl-2(5H)-furanone, a potent and selective inhibitor of human synovial phospholipase A 2 was isolated from fermentations of a Calyptella species. Its structure as identified by spectroscopic methods is identical to PA 147, an antibiotic previously isolated from a streptomycete. 5-hydroxy-3-vinyl-2(5H)-furanone inhibits the aggregation of human and bovine platelets stimulated by different inducers and exhibits weak antimicrobial activities.

The dynamic properties of water molecules in the rabbit lens were studied by proton nuclear magnetic resonance line shape analysis, measurements of relaxation times as a function of temperature and calorimetric measurements. The experiments prove, as already suggested by other authors, that there are two types of water in the lens of rabbit eyes, namely bound unfreezable hydration water and bulk freezable water. Line shape analysis and relaxometry showed, that this two types of water exist in two different environments, which may be identified as the nucleus and the cortex of the lens. The line shape analysis showed furthermore that water molecules in the rabbit lens has a common spin lattice relaxation time (T 1 ), but two different transverse relaxation times (T 2A and T 2B ). The tentative model of fast water exchange on the T 1 time scale and slow water exchange on the T 2 time scale, was used to explain experimental proton relaxation data of the rabbit lens. An estimation for this exchange rate K ex by comparing it to the relaxation times is given (T -1 1 ≪ k ex ≪ T -1 1 ). It has also been shown by a calorimetric measurements, that the lenses can be easily undercooled to temperatures well below the freezing point of water. The achievable maximum undercooling temperature of the lens is a function of the cooling rate K C , therefore it has to be considered as an experimentally adjustable parameter which is not characteristic for the investigated sample. Thus it must be noted that any previous discussions about the specific value of the temperature of water crystallisation in biological systems need to be carefully reconsidered.

After incubation with DNA human lymphocytes release neutral and acid DNase activities into the culture medium; the release depends on DNA concentration and time of cultivation. The electrophoretic mobility of the released neutral DNase activity is in accordance with DNase I and the electrophoretic mobility of the released acid DNase activity with DNase II. The released DNase activities do not originate from dead cells and are not influenced by blast cell formation. The anesthetic halothane can inhibit the released neutral and acid DNase activities. Inhalation anesthesia can possibly disturb the correlation between DNA and DNases in human blood.

Methanolic extracts from corpora cardiaca of three moth species, Hippoteon eson (Sphingidae), Imbrasia cytherea (Saturniidae) and Bombyx mori (Bombycidae) show adipokinetic activity in conspecific bioassays. Haemolymph carbohydrates in these moths are not affected. These extracts are also active in heterologous bioassays: haemolymph lipids are increased in Locusta migratoria, whereas a small effect on haemolymph carbohydrates was observed in Periplaneta americana. Therefore, locusts can be used to monitor adipokinetic activity in corpora cardiaca from moth extracts during isolation. The three moth species possess an adipokinetic peptide with the same retention time on reversed phase high performance liquid chromatography (RP -HPLC) as a peptide isolated previously from Manduca sexta, which was code-named Mas-AKH. H. eson contains a second active peak with a similar retention time on RP-HPLC as the hypertrehalosaemic peptide isolated previously from Helicoverpa zea , code-named Hez-HrTH. Both synthetic peptides, Mas-AKH and Hez-HrTH, produce an adipokinetic effect in the three experimental moth species. In H. eson, the haemolymph concentration of Mas-AKH or Hez-HrTH needed to elicit a maximum hyperlipaemic response is about 20 to 30 nᴍ. Flight behaviour in the three moth species is quite different: H. eson is a good hovering flyer, I. cytherea is a comparatively bad flyer and B. mori males show only degenerate flight movements during their mating dance. Haemolymph lipid levels in H. eson decrease drastically during 15 min of flight and return to pre-flight levels in a subsequent rest period. The amount of lipids metabolized during flight is 10.9 mg/gxhr. Haemolymph carbohydrate levels drop during flight, but remain low during the 45 min of recovery. Haemolymph lipids in “dancing” males of B. mori remain constant. In individuals, however, which have low initial lipid levels in the blood, lipid concentrations increase significantly in a subsequent 15 min rest period after “dancing”. Metabolic changes during flight in I. cytherea were not investigated due to this species’ poor flight performance.

A new method is described which allows to image the steady-state distribution of m em ­ brane potential of single neurons in situ. The method consists of staining the tissue with an extracellular voltage-sensitive dye (Di-4-ANEPPS) and impaling a single neuron with a microelectrode. After focusing the imaging system onto the cell a large series of images are taken with a CCD camera at the appropriate excitation wavelength of the voltage-sensitive dye while the neuron’s membrane potential is shifted by a periodic current injection (PCI). Afterwards two groups of images are averaged separately: those images while the cell was at rest and those images while the cell was hyperpolarized. After subtraction of these averaged images, the resulting difference image shows only the membrane potential of the cell which was altered periodically. The success of the method is demonstrated on leech cells in intact ganglia. If applied to cells with a basically two-dimensional arborization pattern, the decrease of activity in the difference image in areas further away from the injection site should relate to a decrease in membrane potential according to the passive electrotonic properties of the cell under study.

The technique of ex vivo 31 P NMR spectroscopy has been used for the investigation of metabolic turnover in 15 cadaveric human kidneys obtained from brain-dead donors for transplantation. Measurements were carried out at 1.5 T time-dependently during regular hypothermic storage in histidine-tryptophan-α-ketoglutarate solution. The minimum delay between explantation and spectroscopy was 2 h 41 min. In one case of a discarded organ the measurements could be extended over a period of 161 h 35 min. A detailed kinetic model describing monoexponential ischemic phosphate turnover, which accounts for various interrelations of the NMR visible metabolites, has been derived. Averaged velocity constants of the decays of nucleotides and phosphomono- and -diesters ranged from 0.0047 to 0.294 h -1 at approximately 4° C. Intracellular pH decreased monoexponentially with an averaged velocity constant of 0.31 h -1 .

Leaves and other aerial parts of several succulent species each of Kalanchoe and Senecio exhibit more or less obvious wax coatings. The major components of these waxes were identified to be pentacyclic triterpenes. Some of these are rare natural products, while others are rather widespread.

From the methanolic extract of fresh leaves of the Kenyan Vemonia auriculifera Hiern., a lettuce seeds growth regulator was isolated and identified as 8-desacylvernodalol (1) by spectroscopic analysis.

Activities of the enzyme glutathione S-transferase were determined in leaves of sorghum varieties of differential susceptibility to infection with sugarcane mosaic virus. Inoculation with the virus resulted in significant induction of the enzyme in the leaves of the immune host, while hitherto unpublished dramatic reductions were detected in the leaves of the systemic host sorghum varieties.

The percentage of anthraquinone glycosides in the roots of Rumex crispus L. (Polygonaceae) is significantly greater than in other parts of the plant. The roots of R. crispus in different vegetation stages were investigated with regard to anthraquinone glycoside content. Their toxicity towards shrimp were also tested during the vegetation period. The data showed that the percentage of anthraquinone glycosides as well as the toxic effect on the shrimp increased during the vegetation period.

Osmundalin, a glucoside of osmundalactone, isolated from the royal fern, Osmunda japonica, stimulated the deterrent cell (“R receptor” cells, associated with deterrency) in the medial sensilla styloconica of Bombyx mori. From the dose-response curve, the lower limit of detection was 3.6 x l0 -5 - 3.6 x l0 -4 ᴍ. This is the first report that glucoside lactone elicits the impulses from the deterrent cell of Bombyx mori.