Jiří Kopecký

Photoadaptation of two members of the Chlorophyta (Scenedesmus and Chlorella) in laboratory and outdoor cultures: changes in chlorophyll fluorescence quenching and the xanthophyll cycle

Abstract. The role of the xanthophyll cycle in the adaptation of two chlorococcal algae Scenedesmus quadricauda and Chlorella sorokiniana to high irradiance was studied under laboratory and outdoor conditions. We wished to elucidate whether the xanthophyll cycle plays a key role in dissipating the excesses of absorbed light, as in higher plants, and to characterise the relationship between chlorophyll fluorescence parameters and the content of xanthophyll-cycle pigments. The xanthophyll cycle was found to be operative in both species; however, its contribution to overall non-photochemical quenching (NPQ) could only be distinguished in Scenedesmus (15–20% of total NPQ). The Scenedesmus cultures showed a larger pool of xanthophyll-cycle pigments than Chlorella, and lower sensitivity to photoinhibition as judged from the reduction of maximum quantum yield of photosystem II. In general, both algae had a larger xanthophyll-cycle pool when grown outdoors than in laboratory cultures. Comparing the two species, Scenedesmus exhibited a higher capacity to adapt to high irradiance, due to an effective quenching mechanism and high photosynthetic capacity; in contrast, Chlorella represents a species with a larger antennae system, less-efficient quenching and lower photosynthetic performance. Non-photochemical quenching (NPQ) induced through the xanthophyll cycle can, to a limited extent, represent a regulatory factor in diluted algal cultures grown in outdoor solar photobioreactors, as well as in natural algal phytoplankton populations exposed transiently to high irradiance. However, it does not play an appreciable role in dense, well-mixed microalgal suspensions.

Solid-phase/supercritical-fluid extraction for liquid chromatography of phenolic compounds in freshwater microalgae and selected cyanobacterial species

In the present paper a new extraction technique based on the combination of solid-phase/supercritical-fluid extraction (SPE/SFE) with subsequent reversed-phase HPLC is described. The SPE/SFE extractor was originally constructed from SPE-cartridge incorporated into the SFE extraction cell. Selected groups of benzoic acid derivatives (p-hydroxybenzoic, protocatechuic, gallic, vanillic and syringic acid), hydroxybenzaldehydes (4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde) and cinnamic acid derivatives (o-coumaric, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acid) were extracted. Cyclic addition of binary extraction solvent system based on methanol:water (1:1, v/v) and methanol/ammonia aqueous solution was used for extraction at 40MPa and 80 degrees C. The p-hydroxybenzoic, protocatechuic, vanillic, syringic, caffeic and chlorogenic acid; 4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde were identified by HPLC-electrospray mass spectrometry in SPE/SFE extracts of acid hydrolyzates of microalga (Spongiochloris spongiosa) and cyanobacterial strains (Spirulina platensis, Anabaena doliolum, Nostoc sp., and Cylindrospermum sp.). For the identification and quantification of the compounds the quasi-molecular ions [M-H](-) and specific fragments were analysed by quadrupole mass spectrometry analyzer. Our analysis showed that the microalgae and cyanobacteria usually contained phenolic acids or aldehydes at microg levels per gram of lyophilized sample. The proposed SPE/SFE extraction method would be useful for the analysis of different plant species containing trace amount of polar fraction of phenols.

High-performance liquid chromatographic determination of tramadol and its O-desmethylated metabolite in blood plasma Application to a bioequivalence study in humans

Simultaneous HPLC determination of the analgetic agent tramadol, its major pharmacodynamically active metabolite (O-desmethyltramadol) in human plasma is described. Simple methods for the preparation of the standard of the above-mentioned tramadol metabolite and N1,N1-dimethylsulfanilamide (used as the internal standard) are also presented. The analytical procedure involved a simple liquid-liquid extraction of the analytes from the plasma under the conditions described previously. HPLC analysis was performed on a 250x4 mm chromatographic column with LiChrospher 60 RP-selectB 5-microm (Merck) and consists of an analytical period where the mobile phase acetonitrile-0.01 M phosphate buffer, pH 2.8 (3:7, v/v) was used, and of a subsequent wash-out period where the plasmatic ballast compounds were eluted from the column using acetonitrile-ultra-high-quality water (8:2, v/v). The whole analysis, including the equilibration preceding the initial analytical conditions lasted 19 min. Fluorescence detection (lambda(ex) 202 nm/lambda(em) 296 nm for tramadol and its metabolite, lambda(ex) 264 nm/lambda(em) 344 nm for N1,N1-dimethylsulfanilamide) was used. The validated analytical method was applied to pharmacokinetic studies of tramadol in human volunteers.

Generic characters of the simplest cyanoprokaryotes Cyanobium, Cyanobacterium and Synechococcus

Abstract The cytomorphological features (cell morphology, type of cell division, cell structure, structure of photosynthetic apparatus) were studied in the type strains of the cyanoprokaryotic genera Cyanobium and Cyanobacterium , and in the reference strain of the traditional genus Synechococcus ( Synechococcus PCC 6301; = “ Anacystis nidulans ” sensu auct.). They were originally described by Rippka & Cohen-Bazire, based on the mean DNA-base composition (moles % G + C) and on the resistance to various cyanophages. The phenotypic diacritical characters were found to coincide well with the molecular markers, and thus, the genera Cyanobium and Cyanobacterium are acceptable also according to the traditional (botanical) taxonomic criteria. The integrated molecular, cytomorphological and ecophysiological approaches to the solution of taxonomic problems in cyanobacteria are therefore inevitable for taxonomic evaluation, from the methodological point of view. The lists of species within the revised genera Cyanobium and Cyanobacterium are reviewed.

Carbohydrate levels in rhizomes ofPhragmites australis at an oligotrophic and a eutrophic site: A preliminary study

Seasonal dynamics of total nonstructural carbohydrates (TNC) and of carbohydrate species (starch, sucrose, glucose, fructose) were followed in rhizomes ofPhragmites australis at two sites of the Třeboň Biosphere Reserve (Czech Republic): Branná sand pit and Rožmberk fishpond-western shore, which were classified (according to plant species composition and phosphorus availability) as oligotrophic and hyper-eutrophic, respectively.Phragmites stands at these sites were expanding and retreating, respectively. Rhizomes were sampled within terrestrial parts of the reed stands (at a water depth of about 10 cm).The levels of total non-structural carbohydrates were determined mainly by levels of starch and sucrose, while glucose and fructose were present at comparatively low levels. The most conspicuous differences between the sites were associated with autumnal and March levels of carbohydrates. In March, i.e. at the beginning of vegetative development, TNC and starch levels were lower at the hyper-eutrophic, as compared with the oligotrophic, site. Starch and TNC levels fell from August to September at the hyper-eutrophic, but not at the oligotrophic, site. At the low water depth investigated, the differences between stands in carbohydrate levels do not seem to be large enough to account solely for their different vigour. It is suggested that the effect of water depth needs to be evaluated in more detail.

Organic acids in the sediments of wetlands dominated by Phragmites australis: evidence of phytotoxic concentrations

Spatial and seasonal variations in concentrations of lower organic acids in the sediments of stands of Phragmites australis(Cav.) Trin. ex Steud. were studied in wetlands in the Czech Republic (Roumberk fishpond), Hungary (Lake Ferto and Kis-Balaton), and Denmark (Vejlerne Nature Reserve). Pore water concentrations of lactic, formic, acetic, propionic, butyric, oxalic, citric, tartaric, malic and fumaric acid were analysed by HPLC and shown to vary both qualitatively and quantitatively between sites. Acetic acid was prevalent at all sites, and dominated together with citric, malic, and tartaric acid at Roumberk fishpond, with lactic and oxalic acid at Lake Ferto and Kis-Balaton, and with lactic and propionic acid at Vejlerne Nature Reserve. The maximum total concentrations of organic acids recorded were 2984 and 2215mmol l 1 at a land and a deep-water site of Roumberk fishpond, 1673 and 2216 mmol l 1 at a healthy and damaged stand of Lake Ferto, 1006 and 1642mmol l 1 at a healthy and damaged stand of Kis-Balaton, and 570 and 223 mmol l 1 at a healthy reed stand and a lagoon at Vejlerne Nature Reserve, respectively. At Roumberk fishpond the concentrations of lower organic acids were considered sufficiently high to diminish the plants vigour and possibly to induce die-back. At Lake Ferto and Kis-Balaton the organic acids are unlikely to have had a toxic effect on the reeds at the sampling time because of the relatively high pH (7) in these wetlands. However, because of the great spatial and seasonal variability in organic acid speciation and concentration, organic acid phytotoxity may affect the reed also at these sites. At Vejlerne Nature Reserve, the toxic effect on the reeds is unlikely because of consistently low concentrations of organic acids. ©1999 Elsevier Science B.V. All rights reserved.

High-performance liquid chromatographic determination of tramadol in human plasma

Tramadol has been determined in human plasma samples using a sensitive high-performance liquid chromatographic method. The plasma samples were extracted with tert.-butylmethyl ether in one-step liquid-liquid extraction (recovery 86%) and analyses of the extracts were performed on reversed-phase silica gel using ion-pair chromatography (verapamil as an internal standard) and fluorescence detection. The method was applied to the determination of tramadol levels in twelve healthy volunteers after oral administration of 100 mg of tramadol in capsules of Protradon and Tramal.

Comparative biotransformation and disposition studies of nabumetone in humans and minipigs using high-performance liquid chromatography with ultraviolet, fluorescence and mass spectrometric detection

The disposition of the non-steroidal anti-inflammatory drug (NSAID) nabumetone after a single oral dose administration of nabumetone tablets to humans and minipigs was investigated. Nabumetone is a prodrug, which is metabolized in the organism to the principal pharmacodynamically active metabolite -- 6-methoxy-2-naphthylacetic acid (6-MNA), and some other minor metabolites (carbonyl group reduction products, O-desmethylation products and their conjugates with glucuronic and sulphuric acids). Standards of the above-mentioned metabolites were prepared using simple synthetic procedures and their structures were confirmed by NMR and mass spectrometry. A simple HPLC method for the simultaneous determination of nabumetone, 6-MNA and the other metabolites was developed, validated and used for xenobiochemical and pharmacokinetic studies in humans and minipigs and for distribution studies in minipigs. Naproxen was chosen as the internal standard (I.S.), both UV (for higher concentrations) and fluorescence detection (for very low concentrations) were used. The identity of the nabumetone metabolites in biological samples was confirmed using HPLC-MS experiments. Pharmacokinetics of nabumetone, 6-MNA and 6-HNA (6-hydroxy-2-naphthylacetic acid) in human and minipig plasma was evaluated and compared. The concentration levels of nabumetone metabolites in urine, bile and synovial fluid were also evaluated.

Cytotoxicity and secondary metabolites production in terrestrial Nostoc strains, originating from different climatic/geographic regions and habitats: Is their cytotoxicity environmentally dependent?

Extensive selection of cyanobacterial strains (82 isolates) belonging to the genus Nostoc, isolated from different climatic regions and habitats, were screened for both their secondary metabolite content and their cytotoxic effects to mammalian cell lines. The overall occurrence of cytotoxicity was found to be 33%, which corresponds with previously published data. However, the frequency differs significantly among strains, which originate from different climatic regions and microsites (particular localities). A large fraction of intensely cytotoxic strains were found among symbiotic strains (60%) and temperate and continental climatic isolates (45%); compared with the less significant incidences in strains originating from cold regions (36%), deserts (14%), and tropical habitats (9%). The cytotoxic strains were not randomly distributed; microsites that clearly had a higher occurrence of cytotoxicity were observed. Apparently, certain natural conditions lead to the selection of cytotoxic strains, resulting in a high cytotoxicity occurrence, and vice versa. Moreover, in strains isolated from a particular microsite, the cytotoxic effects were caused by different compounds. This result supports our hypothesis for the environmental dependence of cytotoxicity. It also contradicts the hypothesis that clonality and lateral gene transfer could be the reason for this phenomenon. Enormous variability in the secondary metabolites was detected within the studied Nostoc extracts. According to their molecular masses, only 26% of these corresponded to any known structures; thus, pointing to the high potential for the use of many terrestrial cyanobacteria in both pharmacology and biotechnology.

A Hybrid Non-Ribosomal Peptide/Polyketide Synthetase Containing Fatty-Acyl Ligase (FAAL) Synthesizes the β-Amino Fatty Acid Lipopeptides Puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum

A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.