Band 53, Heft 5-6

From the culture medium of the phytopathogenic bacterium Pseudomonas corrugata, a lipopeptide siderophore was isolated that comprises interesting structural elements such as cyclic condensation products of the two amino groups of 2,4-diamino butyric acid with the carboxyl group of a second amino acid, and the rarely encountered L-threo-β-hydroxy histidine.

The seed lipids of the Chinese tung-oil tree Aleurites montana are composed of 97.2% triglycerides, 2% phospholipids and of 0.8% glycolipids. In the triglycerides 67% of all fatty acids are α-eleostearic acid (configuration C 18:3 , ∆ 9 cis, ∆ 11 trans, ∆ 13 trans). This acid is bound in the triglycerides in the 1,3 position. Via mercury Il-acetate adducts and subsequent chromatography on silica gel a separation of this fatty acid from the seed fatty acids is possible. By this procedure an a-eleostearic acid-free tung-oil mixture is obtained, which consists by one half of C 18:2 , 25% of C 18:1 and by 25% of the saturated fatty acids C 16:0 , C 18:0 and C 14:0 .

Three new piperidine alkaloids were isolated from stems, leaves and flowers of Lobelia laxiflora L. (Campanulaceae). The structures of racem. cis-8,10-diethyl-3,4-dehydrolobelidiol (1), racem. trans-8-ethyl-10-phenyl-3,4-dehydrolobelidiol (2) and racem. cis-8-ethyl-10-phenyl-3, 4-dehydrolobelidiol (3) were established by spectral analyses. The residues obtained from the ethanol extracts from stems (S), leaves (L), and flowers (F) were applied in carrageenan (Car)-and cobra venom (CV)-induced acute inflammation in mice. A suppression of paw edema formation at a dose of 100 mg kg -1 was established. In this study the antiinflammatory potential of Lobelia I. was regarded in connection with the complement system. The sequential activation and assembly into functional units of complement components can proceed via two different pathways, named classical (CP) and alternative (AP). The ability of the residues, nonalkaloid fractions, alkaloid fractions and the three alkaloids at a concentration from 0.125 to 1.0 mg ml -1 to inhibit complement activation and thus to prevent inflammatory process was estimated in vitro in human serum via both pathways. All of them inhibited complement activity with a predominant action on CP.

Collybia nivalis, Favolaschia sp. 87129, Pterula sp. 82168 and Omphalotus olearius were cultivated on natural substrates. The antibiotic metabolites oudemansin A, strobilurins A, D. illudin S and pterulone were isolated and identified. A new antifungal metabolite, pterulone B, was described from cultures of Pterula sp. 82168 on wood. Collybia nivalis was found to be the first species of this genus to produce strobilurins and oudemansin A. As compared to rich media the cultivation on natural substrates resulted in the production of fewer metabolites. The concentrations of the antibiotics, however, were sufficient to inhibit other saprophytic fungi.

Acriflavine treatment on Streptomyces tendae generated a bald mutant (bid-1) with an altered antibiotic pattern. The parental strain produced nikkomycins and juglomycins, whereas the mutant bid-1 was only capable of juglomycin synthesis. The existence of a mutant defective in morphogenesis and in nikkomycin biosynthesis suggests a common regulation of these processes. An interesting finding of this study is that mutant bld-1 produced two carbazole derivatives, hitherto never seen in cultures of the parental strain. It seems likely that the DNA intercalating dye acriflavine, by mutagenesis, had activated cryptic genes which are involved in carbazole synthesis. The two carbazole derivatives were identified as the neuronal cell protecting compounds CS-79B and carquinostatin A, recently isolated from a wild-type of S. exfoliatus. We found that both substances showed antibacterial activity.

Pinus sylvestrish., Catechin, Ferulic Acid, p-Coumaric Acid, Picein Within a series of regular projects investigating the suitability of biochemical parameters as biomarkers for the early recognition of environmental pollution, the levels of selected phenolic compounds were determined in Scots pine needles (Pinus sylvestris L.) from young and adult trees at three field sites with different SO 2 pollution (Rösa>Taura>Neuglobsow). Young trees showed no differences in the levels of soluble phenolics in case of increasing loads of SO 2 , whereas the concentration of the soluble phenolic component catechin in previous needles of adult trees altered significantly. In current year’ needles, differences were only found in the presence of high SO 2 deposition. In contrast to catechin, picein and total phenolics remained unaffected. The concentration of catechin in the previous year’s needles of adult trees correlated positively with needle necroses. The phenolic components of the cell walls of pine needles were also analysed. The main phenolic compounds were identified as p-coumaric acid and ferulic acid. Differences in the levels of p-coumaric acid were detected in the needles of adult trees between the three sites, with the highest levels being measured at the site with the lowest pollution (Neuglobsow). However, changes in the p-coumaric acid content in young trees were low. No site-related differences were found regarding ferulic acid in adult and young pines. The findings are discussed and compared with data reported in the literature.

The mitochondrial F 1 ATPases from two cereal crops, wheat and pearlmillet, were purified and studied. The wheat F 1 ATPase could be purified to homogeneity and is apparently com­posed of six subunits with apparent molecular weights of 55 kDa (α and β), 35 kDa (γ), 26 kDa (δ’) and 22 kDa (δ). The e subunit was barely detectable. Both enzymes reveal typical non-linear kinetics but show variability in their response to bicarbonate and chloride. While the wheat F 1 ATPase is stimulated by bicarbonate and chloride, the pearlmillet F 1 ATPase is inhibited by both anions. The two enzymes are Mg 2+ dependent ATPases and are competitively inhibited by Ca 2+ , unlike maize, pea and turnip ATPases. Both the enzymes also possess a GTPase activity which is two fold higher than the ATPase, unlike rice, sorghum and oat root F 1 ATPases.

The biochemical and ultracytochemieal localization of alkaline phosphatases in permeabilized cells of Saccharomyces cerevisiae 257 has been studied. The treatment with non-ionic surfactant Triton X-100 allows the penetration of the substrate into intact yeast cells and thus provides detailed detection of the enzyme activity in ultracytochemical studies.

Tissue type (type 2) transglutaminase (TGase, EC 2.3.2.13) has been implicated in various cellular processes including cell death. In order to better understand the role of this enzyme in cell death, human melanocytic A375-S2 cells were treated with sphingosine, a cell-signaling mediator. During the rapid onset of cytotoxicity caused by this lipidic agent, tissue TGase was translocated from the cytoplasm to the cell nuclei. This observation was further remarked in relevance to its previously undescribed activity for DNA degradation. The DNA hydrolytic activity associated with tissue TGase was dependent on Mg 2+ in contrast to the Ca 2+ requirement for the classical cross-linking acrivity of TGase, and was inhibited by Zn 2+ . Based on the results shown here, we propose a novel aspect of tissue TGase in cell death.

The effects of acridine derivatives (proflavine and 2,7-dialkyl derivatives, diacridines and triacridines, 9-aminoacridine carboxamides, and 9-anilinoacridine, amsacrine and its congeners) on overall RNA synthesis in vitro, on synthesis of initiating oligonucleotides and the binding of the enzyme to DNA were studied. The primary mechanism of action is related to inhibition of the enzyme binding to DNA. The acridines (intercalating or non-intercalating and bis-intercalating ligands) assayed here differ in the properties of their complexes with DNA. Correlation is generally observed between inhibition of RNA synthesis in vitro and cytotoxicity in cell cultures for di- and triacridines and 9-aminoacridine carboxamide derivatives. No relationship was found between the effect on RNA polymerase system and biological effects for amsacrine and its derivatives in contrast to the other series of acridines studied here. The aniline ring seems to decrease the inhibitory potency of a ligand. The discrepancy between the biological effect and RNA synthesis inhibition may be due to a different mechanism of cytotoxicity action of amsacrine which is a potent topoisomerase II poison.

When Spirulina platensis filaments were exposed to 0.75 mW.m -2 .s -1 of ultraviolet-B radiation (the ultraviolet-B radiation under clear sky condition is ~1.0 mW.m -2 .s -1 ), an inhibition in photosystem II activity was observed, the inhibition being 90% after 90 min exposure. Upon exposure to ultraviolet-B, the room temperature emission characteristics of Spirulina cells were altered when excited with light primarily absorbed by chlorophyll a or phycobilisomes. When the cells were exposed for 3 h the emission at 685 nm (F 685 ), when excited at 440 nm (primarily chlorophyll a absorption), was enhanced compared to 715 nm (F 715 ) band of photosystem I suggesting a decrease in energy transfer from photosystem II to photosys­ tem I. Similarly, when the cells were excited at 580 nm (primarily the phycobilisomes), the ratio of emission intensity at 685 nm (F 685 ) to that of 655 nm (F 655 ) was decreased in the exposed cells. This change in emission characteristics seems to be linked with the uncoupling of the energy transfer from allophycocyanin to chlorophyll a of photosystem II. A small shift in emission peak positions was also indicated when excited either at 440 nm or 580 nm. Analysis of the fast induction of chlorophyll a transients in the presence and absence of 10 μm 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU) indicated that ultraviolet-B expo­ sure initially affects Qᴀ, the primary stable acceptor of photosystem II, and then the plastoquinone (PQ) pool. Our results on the loss in photosystem Il-catalyzed Hill activity with p-benzoquinone or dichlorobenzoquinone as electron acceptors also supports the contention that ultraviolet-B, even at low dose, initially alters the Qᴀ of photosystem II and subsequently PQ pool. The analysis of functional pool size of Spirulina suggests a substantial decrease in the functional pool size after 2 h UV-B exposure. These results indicate that in Spirulina low intensity of ultraviolet-B initially damages the reaction centre of photosystem II.

Copper(II)-peroxide Adduct, Modification of Protein, Copper(II)-peptide Complex We have found that copper(II) compounds containing a peptide group in the chelate exhibit high activity for modification or degradation of albumin in the presence of hydrogen peroxide, whereas no activity was detected for the copper(II) compounds without an amide-group. It is suggested that presence of the amide-group in the ligand may play an important role in the formation of a peroxide adduct and in activation of the peroxide ion, leading to cleavage of the peptide bond of a neighboring protein. It is implied that conversion of normal cellular prion protein PrP C into a disease-causing isoform, PrP Sc is attributed to the activated peroxide ion coordinated to a copper(II) captured in the NH 2 -terminal domain of the PrP C .

The individual chains in the triple helix of collagen occur in a conformation related to polyproline II because of the presence of large number of imino peptide bonds. However, these residues are not evenly distributed in the collagen molecule which also contains many non-imino residues. These non-imino regions of collagen may be expected to show preference for other than triple helical conformations. The appearance of several Raman bands in solution phase at 65 °C raises the possibility of non-uniform triple helical structure in collagen. Raman spectroscopic studies on collagen in the solid state and in solution at a temperature greater than its denaturation temperature, reported here suggest that denatured collagen may exhibit an ensemble of conformational states with yet unknown implications to the biochemical interactions of this important protein component of connective tissues.

The effect of a group of model lysosomotropic compounds on the process of Ca2+ ion desorption from lecithin liposome membranes was studied. The compounds studied were: hydrochlorides of fatty acids 2-dimethylaminoethyl esters (DM-n) for n = 9, 11, 13 and 15 carbon atoms in the fatty acid alkyl chain and methochloride of 2-dimethylaminoethyl laurate (DM S -11). It was found that all the compounds studied caused increased desorption with increasing concentration of the compound. Most effective was the quaternary ammonium salt, DMS-11. Moreover, it was found that the process of Ca 2+ desorption from the membrane depended on pH of the medium. Compound DM-11 was more active at pH 8 than at pH 5. The action of DM-n compounds depended on the alkyl chain length, DM-11 and DM-13 being the most active. Apparently free amines penetrate the phospholipid membranes and incorporate into its hydrophobic core causing structural deformations. Hydrochlorides of fatty acids and the quaternary ammonium salt induce desorption of calcium ions mostly as a result of competitive electrostatic interactions. By quantum chemistry, PM3 method, and methods of molecular modelling we established the higher hydrophilicity of the polar head of DM-n series with respect to the polar head of the DM S -n compounds. DM-n compounds possess both acceptor and donor properties for hydrogen bonding while DM S -n are instrumental as acceptors only. It should be noted, that the results obtained in this paper for model membranes are in accordance with those for biological ones.

A detailed analysis of the chemical constituents of a Caribbean specimen of Aiolochroia crassa was performed. Five brominated products (1 -5) were isolated and one of these was a new bromotyrosine metabolite. The structure of the new compound 1 has been established from spectral studies. Compounds 1 and 2, which are the major brominated metabolites and have not been previously identified in any Aiolochroia species, could be usefully employed as chemotaxonomic markers.

Three samples of Brazilian geopropolis, collected by three indigenous bee species, were investigated by GC/MS. More than 50 compounds were identified, mainly terpenoids and phenolics. The chemical composition of propolis gathered by different bee species was different.

Acetylcholine Biosynthesis, Choline Acetyltransferase, Electrophorus electricus (L.), Electrocytes It is well known that the regulation of choline acetyltransferase (ChAT) activity, under physiological conditions, is important for the development and neuronal activities of cholinergic systems. The purification of ChAT has been obtained from many sources such as electric organs of fishes, Drosophila melonogaster, and mammals. We have prepared choline acetyl-transferase from a pool of supernatants obtained by differential centrifugation of electric organ homogenates from Electrophorus electricus (L.) in Tris-phosphate buffer, 0.05 m , pH 7.6. The first step of the enzyme purification was performed by ammonium sulfate precipitation at 40% and 80% . The precipitate at 80% was solubilized with sodium-phosphate buffer 0.05м , pH 7.6, dialyzed, chromatographed on DEAE-52 column and the active fraction submitted to FPLC system columns (Mono-Q: ion exchange -Superose-12: gel filtration). ChAT activity from the eluates was estimated by Fonnun’s method [Fonnun, 1975], with Acetyl-Coenzyme A tritium labelled ([ 3 H ]AcCoA) as substrate, and the synthesis of 3 HACh formed was measured. The peak from gel filtration showed a relative molecular mass of 80 kDa with highest activity in the order of 77,42 nmoles ACh/min/mg protein. This fraction was analyzed by SDS-PAGE and a band of 42 kDa was detected with Coomassie blue stain, indicating that the enzyme is formed by two subunits. Employing an antibody, the presence of ChAT was confirmed with the Western blotting technique. Isoelectrofocusing analysis demonstrated two isoforms with pI of 6,49 and 6,56, respectively.

The glyceraldehyde-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was purified to homogeneity from electric organ of Electrophorus electricus (L.) by a hydrophobic chroma­ tography method on deacetylcolchicine-Sepharose. The purification resulted in a 162 fold increase in specific activity of the GAPDH and final yield was approximately 37%. The purified enzyme showed a single band in SDS-PAGE, with an apparent molecular mass of 36 kDa. The purity of the colchicine-Sepharose isolated material was analysed by isoelectro-phocusing and immunoblotting using a heterologous rabbit serum anti-GAPDH. Sequence analysis of the 40-N-terminal amino acids, determined by Edman degradation, revealed its identity to other GAPDHs proteins being the largest number of identical amino acids to lobster (92.5%), rabbit muscle (85%) and human liver (80%) GAPDH.

We have investigated the influence of Prostaglandin E 1 on the in vitro transcription of chromatin, isolated from spermatozoa of patients suffering from different pathologies, leading to infertility, namely, azoospermia, teratospermia and chronic prostatitis. Our studies indicate that prostaglandin E, has a stimulatory effect both on in vitro transcription, on the number of RNA polymerase molecules and the polyribonucleotide elongation rates as compared to sperm chromatin from healthy patients. Tlie results on the incorporation of α- 32 P-ATP in to RNA in the presence and absence of Prostaglandin E 1 correlate well with the data on the number of actively transcribing RNA polymerase molecules and the rate of RNA elongation, which might be due to low levels of prostaglandin E 1 in human semen.

The antioxidant activity of five new compounds from a group of quaternary ammonium salts with antioxidant function has been investigated. The effect of the compounds on the degree of lipid oxidation in the erythrocyte membrane subjected to UV radiation was studied. It was found that all the salts used decreased oxidation of the lipids in the erythrocyte membrane. The antioxidant activity of the ammonium salts studied increased with their alkyl chain length. Three compounds with the longest alkyl chains were the most active antioxidants, and their antioxidant properties were comparable to those of a flavonoid extracted from haw­ thorn. The antioxidant effectiveness of the ten-carbon alkyl chain compound was comparable with that of the known antioxidant 3,5-di-t-butyl-4-hydroxytoluene (BHT). The least effective antioxidant studied proved to be the eight-carbon alkyl chain compound. The effect of these compounds on fluidity of the erythrocyte membrane has been studied, and for all an increase of fluidity of the membrane was observed. The changes in erythrocyte ghosts fluidity depended both on concentration and type of compound. A fluorimetric study also indicated that rigidity of the erythrocyte membrane increased with degree of its oxidation, but with antioxidants present membrane rigidity increased less.

Homogenization of mammalian tissue - exemplified by porcine kidney - causes enzymatically induced lipid peroxidation (LPO) processes proven by measuring the amounts of the typical lipid peroxidation products 9- and 13-hydroxy-octadecadienoic acid (HODE) either after homogenization in aqueous (activation of enzymes) or an organic (inactivated enzymes) solvent. A kinetic study revealed that the level of the 9- and 13-isomer reached maximum values 6 hours after tissue injury. Within one day the amount of these primary oxidation products was reduced fast, indicating that they undergo degradation in their biological environment. In contrast, the level of 10-hydroxy-octadecanoic acid - obviously derived from LPO of oleic acid - increased continuously even after one day. These observations reflect that the generation and degradation of hydroperoxides occurs at different rates which might be of interest in pathological processes connected with tissue injury, e.g. myocardial infarction.