Volume 54, Issue 5-6

Echimidine was isolated from Echium rauwolfii and Echium horridum and identified by MS, 1 H-and 13 C NMR as a major alkaloid. In addition, structures of 12 minor alkaloids were inferred from GLC and GLC-MS analyses: 7-angeloylretronecine, 7-tigloylretronecine, lycopsamine, 7-acetyllycopsamine, uplandicine, 7-angeloyllycopsamine, 7-tigloyllycopsamine, tigloyl isomer of echimidine, 7-angeloyl-9-(2-methylbutyryl)retronecine, 7-tigloyl-9-(2-methylbutyryl)retronecine, 7-angeloyl-9-(2,3-dihydroxybutyryl)retronecine, and 7-tigloyl-9-(2,3-dihydroxybutyryl)retronecine. Both species had similar alkaloid profiles. Alkaloid extracts exhibited antibacterial effects with a MIC of 1.7 mg/ml in E. coli. Microscopic examination of E. coli treated with different subtoxic alkaloid concentrations (13-52 μg/ml) revealed extensive filamentation.

The structure of the pyoverdin siderophore of Pseudomonas fluorescens 1.3 was elucidated by spectroscopic methods and chemical degradation. It shows structural similarities with the pyoverdins of several other strains. Whether mutual recognition occurs was investigated by growth tests.

The lipid/resin components of Laretia compacta were characterized by GC-MS and CSIA. The lipid components consist of n-alkanes, n-alkanoic acids and n-alkanols all with low carbon number maxima and high CPI values. The resin components are comprised of major amounts of tetracyclic diterpenoids with a minor content of mono-and sesquiterpenoids. The kaurene skeleton predominates with some phyllocladenes. Phytosterols are trace components. The δ 13 C data of the individual alkanes in the hydrocarbon fraction place this species in the C 4 plant group.

The systematic affinities of Ganoderma have largely been resolved in the extensive publications of Moncalvo and coworkers (Moncalvo et al., 1995a, b; Hseu et al., 1996). The present communication adds further sequences of the ITS1 region of Ganoderma isolates from Po­ land and corrects some of the classifications of Ganoderma species. The sequence data indicate that G. australe and G. adspersum are different species. Both morphological and molecular data are in accord with an interspecific separation of G. pfeifferi and G. resinaceum. The ITS1 region is particularly suited for the taxonomic segregation of Ganoderma by molecular methods.

The antioxidative activity of three anthocyanin pigments, extracted from the fruits of chokeberry, honeysuckle and sloe, were studied. Lipid oxidation in the liposome membrane, induced by UV radiation, was evaluated with a thiobarbituric acid-reactive substances assay. The antioxidant efficiency of the studied compounds follows this sequence: chokeberry > sloe > honeysuckle. The extract concentrations at which a 50% reduction of phosphatidylcho­ line oxidation was observed, were respectively: 48, 54 and 60 mg/1. The end products of lipid membrane oxidation were evaluated using HPLC. It was found that the antioxidative potency of anthocyanin extracts is concentration-dependent. As shown by E P R technique the efficiency of the extracts to eliminate free radicals from the solution follows the order of the antioxidant activity.

The polyether antibiotic maduramicin and its methyl ester derivative inhibited photophos­ phorylation and proton uptake in isolated spinach chloroplasts. Both compounds also en­ hanced basal and phosphorylating electron transport and stimulated Mg 2+ -dependent AT­ Pase activity, therefore, they behave as uncouplers of photophosphorylation being the methyl ester derivative more potent than the parent compound. On the other hand, maduramicin inhibited germination and radicle elongation of several crop and weed species. In addition, the antibiotic caused phytotoxic injury and fresh weight reduction to 4-to-6 week old seed­ lings of two weed and two crop species when applied at 10 -4 ᴍ by foliar application in the greenhouse.

Potato microtubers turn green within a few days when kept in the light. The initial phases in this process were observed as early as 12 hours after the onset of illumination. The changes included a pronounced increase in chlorophyll and carotenoid concentrations, accompanied by changes in the protein pattern and in the transformation of amyloplasts and leucoplasts to chloroplasts. The bleaching herbicides amitrole and norflurazon inhibited the synthesis of carotenoids in the illuminated potato microtubers. However, amitrole only delayed greening and an increase in chlorophyll and carotenoid levels became visible as late as four days after the onset of illumination, and the LHC II protein of the photosynthetic membrane was not detected before the seventh day of light exposure. Norflurazon, in contrast, acted as a stronger inhibitor, and microtuber tissues stayed yellowish throughout the experiment. The concentrations of both carotenoids and chlorophylls were very low in tissues treated with this herbicide. The LHC II protein could not be detected after a seven-day light exposure and the plastids were damaged, small in size, without normal thylakoids and with numerous plastoglobules.

Barley (Hordeum vulgare) was grown for eight days in the presence of a range of salt concentrations or subjected to repeated cycles of wilting and rehydration. Changes in apoplastic protein content, protein pattern, enzymic activities and ion composition were in­ vestigated under salinity and drought. The protein content of intercellular washing fluid (IWF) increased 2.5-to 3.0-fold when the NaCl concentration in the growth medium was increased from 0 to 100 mᴍ. The elevated protein content was the result of a general increase in most polypeptides and a pronounced increase in the abundance of specific polypeptides of apparent molecular masses of 15, 21, 22, 26, 36, 40 and 62 kDa. Conversely, the IWF protein content decreased during wilting similar as after application of colchicin, cytochalasin B or cycloheximide suggesting that inihibition of protein synthesis or vesicle transport may be the cause for the decrease in apoplastic protein content and enzyme activities in dehydrating plant tissue. The changes in apoplastic protein content were accompanied by stress-specific alterations in activities of apoplastic enzymes. The greater apoplastic protein content was the consequence of stimulated protein synthesis in the presence of NaCl, as evidenced by increased incorporation of [ 35 S]-methionine into IWF protein. The results demonstrate that the leaf apoplast is a compartment which sensitively and differentially responds to drought and salinity with consequences for plant growth.

Chlorella vulgaris, Chlorophyll Fluorescence Quenching, Green Algae, Molecular Oxygen Molecular oxygen can act as a collisional quencher of the singlet excited state of chlorophyll a. This effect is well described for chlorophyll a in various solvents but not for chlorophyll a in the antenna complexes of photosynthetic organisms. We studied the chlorophyll fluorescence decay of Chlorella vulgaris cells under different oxygen concentrations but did not find any evidence for quenching by oxygen.

A cDNA, A p L 1a , corresponding to a homologue of the large subunit of ADP-glucose pyrophosphorylase (AG Pase), has been isolated/characterised by screening a cDNA library prepared from leaves of Arabidopsis thaliana, followed by rapid amplification of cDNA 3′-ends (3′-RACE). Within the 1685 nucleotide-long sequence (excluding polyA tail), an open reading frame encodes a protein of 522 amino acids (aa), with a calculated molecular weight of 57.7 kDa. The derived aa sequence does not contain any discernible transit peptide cleavage site motif, similarly to two other recently sequenced full-length Arabidopsis homo-logues for AGPase, and shows ca. 58–78 % identity to homologous proteins from other plants/tissues. The corresponding gene was found (rosette and stem leaves, stems, flowers and fruits), consistent with its critical role in starch synthesis in

A DNA probe specific for biocontrol strains of Pseudomonas was produced by screening randomly amplified polymorphic DNA (RAPD) PCR fragments. Specificity of the probe was assessed by dot blot and colony hybridization. It was used to specifically determine the population of these strains on roots of Cucumis sativus cv. Delikatess. Two polymorphic RAPD fragments of 750 bp, and 550 bp showed identical specificity. The biocontrol strain Pseudomonas sp. W34 was shown to be competitive in the rhizosphere of cucumber and to maintain a stable population for at least 10 days when inoculated on the seed. The phylogenetic relationships between the biocontrol and reference strains were analyzed at the strain level by means of RAPD and repetitive sequence-based PCR genomic finger­ printing (rep-PCR), and at higher taxonomic levels by means of amplified 16S ribosomal DNA restriction analysis A RDRA . It was shown that the antagonistic strains are closely related, forming a separate cluster from other non-antagonistic and reference Pseudomonas strains, their taxonomic placement remaining uncertain.

The activities of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoA reductase; EC 1.1.1.34), rate-limiting enzyme of cholesterol biosynthesis, and cholesterol 7α-hydroxylase (EC 1.14.13.17), key enzyme of the neutral bile acid synthesis pathway, were measured in the microsomal fraction of rat liver and in rat liver cells to investigate the coordinate regulation of the two pathways. Both enzyme activities exhibited the same diurnal rhythm and responded in a coordinate fashion to fasting or bile acid-feeding (decrease) and to cholestyramine-feeding (increase). Cholesterol-feeding decreased the activity of HMGCoA reductase, increased that of cholesterol 7α-hydroxylase, and concomitantly increased free cholesterol in microsomes. In an ex vivo setting using primary hepatocytes from animals fed a high cholesterol diet the activity of HMGCoA reductase was initially low and that of cholesterol 7α-hydroxylase was elevated. Release of cholesterol into the medium with ongoing incubation caused HMGCoA reductase activity to increase, and that of cholesterol 7α-hydroxylase to decline. Incubation of hepatocytes with a cholesterol-containing lipoprotein fraction stimulated the activity of cholesterol 7α-hydroxylase, but left HMGCoA reductase activity unaffected. The results confirm the idea of a joint regulation of the two key enzymes of cholesterol metabolism in response to the levels of substrate and metabolites, and support the notion that with respect to bile acid and cholesterol levels, respectively, regulation of HMGCoA reductase activity may be secondary to that of cholesterol 7α-hydroxylase. The in vitro studies supply evidence that the effects of cholesterol and bile acid excess or deficiency are direct and do not involve accessory changes of hormone levels or mediators.

Protease and virulence of the extracellular products (ECP) of Vibrio carchariae strain EmI82KL, a causative agent of gastroenteritis in E pinephelus coioides, cultured on different media were studied. The bacteria grown on peptone agar, nutrient agar or brain heart infu­sion agar produced higher protease activities than that grown on tryptic soy agar (TSA) in terms of protein content. The addition of ethylenediamine di(o-hydroxyphenylacetic acid) or horse serum in TSA enhanced the ECP protease production while the addition of grouper serum apparently reduced the enzyme activity indicating the presence of protease inhibitor(s) in the fish serum. Furthermore, the use of grouper meat or peptone as a single nutrient source remarkably enhanced the production of ECP protease. Adding proteinaceous materials from animal sources (horse serum, grouper meat or peptone) on agar manifested higher ECP protease activity than that from plant source (TSA), indicating the intestine of carnivorous groupers might favour the existence, survival or infection of the bacterium. The protease was a metal-chelator-sensitive serine protease since it was inhibited by 3,4-dichloroisocoumarin and phenylmethanesulfonyl fluoride while about 80% of its activity inhibited by chelating agents (ethylene-diaminetetraacetic acid and ethylene glycol-bis(y3-amino-ethylether) N,N,N′,N′-tetraacetic acid). The ECP obtained from each medium was not lethal to the groupers suggesting that the bacterium is low virulent. As grouper is carnivorous, therefore, the role of the protease played in the fish intestine probably is competing for nutrients and/ or associated with the cause of edema leading to gastroenteritis.

We report identification of the sex attractant of the rosy Russian gypsy moth, Lymantria mathura Moore. Two compounds, Z,Z,Z-3,6,9-nonadecatriene 1 and its monoepoxide Z,Z-(9S,10R)-9,10-epoxy-3,6-nonadecadiene 4a, have been identified from abdominal tip ex­ tracts of female moths based on coupled gas chromatography/electroantennogram detector responses and dose response curves. Single cell recordings showed that only one of the mo­ noepoxide enantiomers (S,R) was active. In field tests, both the (S,R)-monoepoxide and the racemate were active. This type of pheromone system, unusual for a Lymantriid, is more typical of those found in the families Arctiidae, Noctuidae and Geometridae.

The underivatized hexane and acetone extracts of two propolis samples (from Brazil’s southwest) were analyzed by HT-HRGC (high temperature high resolution gas chromatogrphy) and HT-HRGC coupled to mass spectrometry (HT-HRGC-MS). Several compounds, including flavonoid aglycones, phenolic acids and high molecular weight compounds could be readily characterized in the crude extracts by HTHRGC-MS. HTHRGC and HTHRGC-MS were shown to be quick and informative tools for rapid analysis of crude extracts without need for prior derivatization and purification.

Propolis and plant secretions from three species, most frequently mentioned as botanical sources of the bee glue in Brazil (Baccharis dracunculifolia, Araucaria angustifolia and Eucalyptus citriodora) have been investigated using GC-MS. Based on chemical evidence, B. dracunculifolia was shown to be the main propolis source in São Paulo state. The antibacterial and antifungal activities of all four materials were also tested, the most active being propolis and Baccharis leaf exudate.

Propolis from Central Chile was investigated for its plant origin by microscopical analysis of pollen grains and leaf fragments found in the sample. The pollen grains that appear with significant higher frequency in the sample corresponded to four native and two introduced species, whereas leaf fragments corresponded to four native species. Seventeen phenolic com­ pounds that belong to the phenylpropane, benzaldehyde, dihydrobenzofuran, or benzopyran classes, were isolated from an organic extract that was found to have a moderate growth inhibitory activity against Mycobacterium avium , M. tuberculosis, and two strains of Staphylococcus aureus. The components responsible for activity were determined.

In order to check the structure-activity relationship and find more potent derivatives of the natural products 1 and 2 obtained from sponge Ircinia spinosula, a series of oxidation, hydrogenation, acetylation and methylation derivatives was prepared. All compounds (natural and synthetic ones) were screened for their cytotoxic and antibacterial activities. The biological studies showed a wide range of antibacterial activity even though only 2 and 2d showed a moderate cytotoxicity against the clone C98. The oxidation of the hydroquinone to quinone and the hydrogenation of the side-chain increased the antibacterial effect of the molecules.

A new series of amphiphilic compounds with incorporated antioxidant functional group has been investigated. Piperidinium bromides, differing in the alkyl chain length (8, 10, 12, 14 and 16 carbon atoms in the chain) were synthesised to protect biological and/or model membranes against peroxidation and following negative consequences. Their antioxidant activity was studied with erythrocytes subjected to UV radiation. The salts used inhibited lipid oxidation in the erythrocyte membrane. The degree of this inhibition depended on the alkyl chain length of the bromide used and increased with increasing alkyl chain length. A comparison of the results obtained for piperidinium bromides with those obtained for the widely used antioxidant 3,5-di-t-butyl-4-hydroxytoluene (BHT) revealed that only two shortest alkyl chain salts were less efficient than BHT in protecting erythrocyte membranes. A similar comparison with antioxidant efficiency of flavonoids extracted from Rosa rugosa showed that they protected the membranes studied more weakly than the least effective eight-carbon alkyl chain piperidinium bromide. The three compounds of longest alkyl chains were the most active antioxidants. Their activities did not differ significantly.

The aim of the work was to determine the synergism and antagonism of mixtures of the cationic amphiphilic compounds: N-benzyl-N,N-dimethyl-N-tetradecylammonium chloride (BD TA), N-benzyl-N,N-dimethyl-N-dodecylammonium chloride (BDDA), N-methyl-N-te-tradecyl-morpholinium bromide (MTM), N,N,N-trimethyl-N-tetradecylammonium bromide (TMTA) and those of tripropyltin chloride in a mixture with amphiphilic anionic compounds (a homologous series of sodium alkylsulfonates with n = 2, 4, 6, 8, 10, 12 and 14 alkyl carbon atoms) in the process of calcium ion desorption from liposome lecithin membranes. Radioactive labels and spin labels were used in the investigations. All compounds studied caused an increase in the kinetic rate constant of calcium ion desorption from liposome membranes with increasing concentration. In case of mixtures containing cationic and anionic compounds both synergism and antagonism were observed. It was shown that the regulatory effect depended on the properties of both cationic and anionic compounds. A mixture of the most effective cationic compound (BDTA) with each of the anionic compounds decreased the effect. Mixtures of the remaining ammonium com­ pounds with sodium alkylsulfonates produced both synergism and antagonism. Anionic com­ pounds with short chains increased, and with longer chains decreased efficiency, while com­ pounds with comparable chains caused totally blocked desorption. A mixture of an organometallic compound having three short chains with anionic compounds gave a reversed effect: short-chain compounds caused a decreased and those with long-chains -increased efficiency of tripropyltin chloride. Studies with spin labels supported the conclusion that the most important factor responsi­ ble for the regulatory effect is the molecular shape of the complexes formed in a mixture of both groups of compounds -a factor that determines membrane packing density

The effects of cortical tissue preparations (CTP) from human brain on the production of reactive oxygen species (ROS) has been investigated with several biochemical model reactions. As indicators for ROS, fragmentation of the methionine derivatives, α-keto-γ-methyl-thiobutyric acid (KMB) or 1-amino-cyclopropane-1-carboxylic acid (ACC), yielding ethene have been used. With these systems we have shown that production of OH-radical-type oxidants by the xanthine oxidase (XOD)-system is strongly stimulated by CTP. This activity is due to intrinsic iron ions since ethene formation from KMB is stimulated by EDTA , inhibited by desferrioxamine (Desferal R ) and also visible with heat-denatured CTP. CTP by themselves have no XOD activity. 3-Hydroxykynurenine (3HK) is another possible substrate for XOD but produces H 2 O 2 without XOD-catalysis, whereas allopurinol is not inhibiting. CTP contain measurable NAD(P)H oxidoreductase activity, producing OH-radical-type oxidants at the expense of NADPH and (to a lesser extent) NADH as electron donors , shown as redox-cycling of 2-methyl-5-hydroxy-1.4-naphthoquinone, plumbagin. Ethene formation from KMB is also driven by both morpholinosydnonimine (SIN) or ONOOH. The reaction driven by SIN is stimulated by CTP and inhibited by catalase, SOD and hemoglobin. Since ethene release from KMB driven by ONOOH is inhibited by CTP the mechanisms driving KMB fragmentation are different for SIN and ONOOH. Furthermore CTP contain approx. 4 U catalase activity per mg protein and very weak peroxidase (POD) activity shown as ACC fragmentation yielding ethene in the presence of both H 2 O 2 and KBr or NaCl. Since ACC binds to CTP and both compounds, ACC and KMB are natural products, present in food (ACC) or synthesized from methionine in vivo (KMB), these compounds may represent protecting agents in systems where reactive oxygen species are formed. One might even speculate that the production of ethene at these membrane receptor sites may have biological functions, since ethene is known to possess anaesthetic activities.

1. Single-photon responses (bumps) and small macroscopic photocurrents were studied in ventral photoreceptors of the horseshoe crab Limulus. Lowering the calcium concentration in the bath from 10 mᴍ to 250 μᴍ led to increased bump size. Adaptation of the cells by a moderately bright conditioning flash was not impaired. 2. Pressure-injection of 1.2 mᴍ EGTA into the dark-adapted cells resulted in reduced bump size. EGTA weakened the effect of the conditioning light flash although it did not completely abolish light adaptation. 3. The microsomal calcium-ATPase inhibitor cyclopiazonic acid strongly desensitized the cells, and bumps were suppressed below detection. When the bathing saline contained 10 mᴍ calcium, macroscopic photoresponses after extracellular application of the agent had amplitudes smaller than under control conditions but normal response kinetics: The response to a light step still consisted of a fast transient photocurrent and a much smaller plateau. However, when applied in calcium-free bathing saline, cyclopiazonic acid additionally influenced the waveform of the photoresponse. The clear distinction between transient and plateau was no longer possible, and the photocurrent appeared “square”. 4. Our results support the idea that a transient elevation of the cytosolic calcium concentration is obligatory for light adaptation in the ventral photoreceptor. It is also obligatory for the generation of the so-called C 2 component of the photocurrent which is represented by “standard” bumps and the fast transient phase of a prolonged response. However, a rise in cytosolic calcium appears not necessary for the initiation of a slow electrical photoresponse.

Dendroligotrichum dendroides and Dawsonia superba, which belong to two exclusively Gondwanalandic genera of the Polytrichales, accumulate the same type of coumarins as species of the cosmopolitan genera of that order. The structures of the isolated compounds, one of which is a new natural product, were elucidated spectroscopically.

Kinetics of triiodothyronine (T 3 ) induced changes were studied in cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH) and lactate dehydrogenase (LDH) of the liver and skeletal muscle of a catfish, Clarias batrachus. The rates of gradual inductions in the activities of all the three metabolic enzymes were faster in skeletal muscle than those of the liver. These time-dependent and tissue-specific inductions may be due to the possible differences in the rates of different enzymic syntheses. The maximum inductions in the activities of cMDH, mMDH and LDH were recorded around 19 hr after T 3 treatment. Thereafter, the activities of all the enzymes gradually declined to their half levels within the next 12 hr which reflected the physiological half-life of these metabolic enzymes in the freshwater catfish.