Purification of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen

The diatom Haslea ostrearia that lives in oyster ponds has the distinctive feature of synthesizing “marennine”, a blue-green pigment of which the chemical nature still remains unknown. This pigment is responsible for the greening of oyster gills. Here, we report a new method for extraction and purification of intracellular (accumulated in the apex of the cell) and extracellular (released into the external medium) forms of the pigment. Intracellular marennine is obtained by extraction from blue algal pellets with a carbonate buffer. The extract is then centrifuged and filtered. Extracellular marennine is obtained by clarification of blue-coloured culture medium. Both extracts are then purified by a semi-preparative process, using ultrafiltration through membranes and anion-exchange chromatography. This procedure allows us to produce native pigment displaying the degree of purity required to enter upon the molecular characterisation of marennine. By this process, about 35% of the initial amount of pigment can be recovered. If necessary, this method could be easily scaled up to a larger production system to accommodate potential industrial applications.     

Extraction and quantitative analysis of the blue-green pigment “marennine” synthesized by the diatom Haslea ostrearia

This study reports further information on mareninne, awater-soluble blue-green pigment synthesized by the diatom Hasleaostrearia, which is essential to the greening of maturing oysters inFrench production areas. The extraction process is reported, as well aspreliminary characterization of a partially purified marennine extract,including quantitative spectrophotometric analysis of intracellular pigment. Amean specific extinction coefficient, E ^1% _1cm = 17.2 at 669 nm, is proposed. Results forquantitative determination of marennine accumulated in cells during batchcultures are presented, and their importance for pigment production isconsidered in relation to potential industrial applications.     

Method for the quantification of the blue-green pigment “marennine” synthesized by the marine diatom <Emphasis Type="Italic">Haslea ostrearia</Emphasis> (Gaillon/Bory) Simonsen using HPLC gel-filtration and photodiode-array detection

This paper describes a new approach for quantifying marennine, a blue-green pigment synthesized by the marine diatom Haslea ostrearia, which is known to be responsible for the greening of cultured oysters in French coastal areas. The method uses gel-filtration HPLC interfaced with a photodiode-array detector (PDA). Under the chromatographic conditions applied, the peak of marennine is identified on the dextran pattern at 674 nm by its elution time (ET = 21–22 min) and its UV-Visible absorption spectrum. After calibration with pure marennine sample as external standard, consisting in plotting peak area as a function of marennine concentration, one can back-calculate concentrations of unknown samples. Results for quantitative determination of marennine using this procedure are validated and discussed in relation to those obtained by the spectrophotometric methods, the most often employed until now.     

Cloning,characterization and anion inhibition study of the δ-class carbonic anhydrase (TweCA) from the marine diatom Thalassiosira weissflogii

We investigated the catalytic activity and inhibition of the δ-class carbonic anhydrase (CA, EC 4.2.1.1) from the marine diatom Thalassiosira weissflogii, TweCA. The enzyme, obtained by cloning the synthetic gene, was an efficient catalyst for the CO₂ hydration, its physiological reaction, with a k_cat of 1.3 × 10⁵ s⁻¹ and a k_cat/K_M of 3.3 × 10⁷ M⁻¹ s⁻¹. A range of inorganic anions and small molecules were investigated as inhibitors of TweCA. Chloride and sulfate did not inhibit the enzyme (K_Is >200 mM) whereas other halides and pseudohalides were submillimolar–millimolar inhibitors (K_Is in the range of 0.93–8.3 mM). The best TweCA inhibitors were hydrogen sulfide, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_Is in the range of 9–90 μM, whereas acetazolamide inhibited the enzyme with a K_I of 83 nM. This is the first kinetic and inhibition study of a δ-class CA. However, these enzymes are widespread in the marine phytoplankton, being present in haptophytes, dinoflagellates, diatoms, and chlorophytic prasinophytes, contributing to the CO₂ fixation by sea organisms. A phylogenetic analysis with all five genetic families of CAs showed that α- and δ-CAs are evolutionarily more related to each other with respect to the γ-CAs, although these three families clustered all together. On the contrary, the β- and ζ-CAs are also related to each other but phylogenetically much more distant from the α-, γ and δ-CA cluster. Thus, the study of δ-CAs is essential for better understanding this superfamily of metalloenzymes and their potential biotechnological applications in biomimetic CO₂ capture processes, as these enzymes are part of the carbon concentrating mechanism used by many photosynthetic organisms.     

Changes in pigment content of the diatom Navicula pelliculosa (Bréb.) Hilse in silicon-starvation synchrony

Summary Exponentially grown cells of the freshwater diatom Navicula pelliculosa (Bréb) Hilse, contained chlorophyll a, chlorophyll c, fucoxanthin, diadinoxanthin, diatoxanthin, neofucoxanthin, -carotene, and an unknown pigment, the absorption spectrum of which is reported. Changes in amounts of chlorophyll a, fucoxanthin and diadinoxanthin were determined during the course of silicon-starvation synchrony carried out in the light or dark. Changes in the rate of chlorophyll a and fucoxanthin syntheses were similar. Synthesis ceased after 5–7 hr of silicon starvation, but recommenced in cultures kept in the light, once silicon was re-introduced. In cultures kept in the dark no significant synthesis was observed after re-introduction of silicon. Diadinoxanthin synthesis continued in the light at all times, although at a lower rate during the silicon-starvation period. In the dark, synthesis of this pigment ceased when cell division stopped, and the amount per unit volume of culture decreased. These results are discussed in relation both to the effect of silicon on the metabolism of the diatom and to the possible function of the carotenoids.Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday.     

Biochemical characterisation and fatty acid profiles of 25 benthic marine diatoms isolated from the Solthörn tidal flat (southern North Sea)

Twenty-five intertidal diatom species were isolated from the Solthörn tidal flat (Lower Saxony, southern North Sea) and grown in semi-continuous cultures under standardised conditions, in order to observe differences in their biochemical gross compositions (e.g. protein, lipid, carbohydrate and ash contents). Composition, expressed as % dry weight, indicated that the majority of species (52 %) contained only <15 % protein but had nearly twice the total amount of carbohydrate and two to three times higher ash content. In addition, most species contained a relatively constant percentage of lipids (19.4 to 25.6 %), whereas extraordinary high lipid contents (>30 %) were found for Amphora exigua, Gyrosigma spenceri, Pleurosigma angulatum and Gyrosigma littorale. Glucose, galactose, mannose and ribose constituted the majority of the sugars detected, although the levels of these varied between species. Lipid class composition showed high concentrations of phospholipids and galactolipids as major constituents (19–22 % and 40–43 % of total lipids). The major fatty acids in most species were 14:0, 16:0, 16:1(n-7) and 20:5(n-3). Significant differences in biochemical gross compositions were found in the temperature (10, 30 °C) and salinity tests (20, 35 PSU), suggesting special intracellular acclimatisation processes that provide possible explanations for the adaptability of the species to environmental variations and the distinct differences in the diatom assemblages.     

Adhesion of the marine fouling diatom amphora coffeaeformis to non‐solid gel surfaces

Diatom adhesion to different gel surfaces was tested under different shear conditions, using the fouling marine diatom Amphora coffeaeformis as test organism. Four polymers were selected to obtain a test matrix containing gels with different surface charge as well as different surface energies, viz. agarose, alginate, chitosan and chemically modified polyvinylalcohol (PVA‐SbQ). Three experimental systems were applied to obtain different shear rates. Experimental system 1 consisted of gels cast in a cell culturing well plate for comparing initial adhesion as well as long term biofilm development in the absence of shear. In experimental system 2, microscope slide based test surfaces were tested in aquaria under low shear conditions. A rotating annular biofilm reactor was used to obtain high and controlled shear rates. At high shear rates A. coffeaeformis cells adhered better to the charged polymer gels (alginate and chitosan) than to the low charged polymer gels (agarose and PVA‐SbQ). In the system where shear was absent A. coffeaeformis cells developed a biofilm on agarose equivalent to the charged polymer gels, while adhesion to PVA‐SbQ remained low at all shear rates. It is concluded that non‐solid surfaces did not represent an obstacle to settling and growth of this organism. As observed for solid surfaces, low charge density led to reduced attachment, particularly at high shear.     

Sulfonamide inhibition studies of the δ-carbonic anhydrase from the diatom Thalassiosira weissflogii

The δ-carbonic anhydrase (CA, EC 4.2.1.1) TweCA from the marine diatom Thalassiosira weissflogii has recently been cloned, purified and its activity/inhibition with anions investigated. Here we report the first sulfonamide/sulfamate inhibition study of a δ-class CA. Among the 40 such compounds investigated so far, 3-bromosulfanilamide, acetazolamide, ethoxzolamide, dorzolamide and brinzolamide were the most effective TweCA inhibitors detected, with K_Is of 49.6–118 nM. Many simple aromatic sulfonamides as well as dichlorophenamide, benzolamide, topiramate, zonisamide, indisulam and valdecoxib were medium potency inhibitors, (K_Is of 375–897 nM). Saccharin and hydrochlorothiazide were ineffective inhibitors of the δ-class enzyme, with K_Is of 4.27–9.20 μM. The inhibition profile of the δ-CA is very different from that of α-, β- and γ-CAs from different organisms. Although no X-ray crystal structure of this enzyme is available, we hypothesize that as for other CA classes, the sulfonamides inhibit the enzymatic activity by binding to the Zn(II) ion from the δ-CA active site.     

Isolation and functional characterisation of the genes encoding Δ(8)-Sphingolipid desaturase from Brassica rapa

△~8-Sphingohpid desaturase is the key enzyme that catalyses desaturation at the C8 position of the long-chain base of sphingolipids in higher plants.There have been no previous studies on the genes encoding△~8-sphingolipid desaturases in Brassica rapa.In this study,four genes encoding A -sphingohpid desaturases from B.rapa were isolated and characterised.Phylogenetic analyses indicated that these genes could be divided into two groups:BrD8A,BrD8C and BrD8D in groupⅠ,and BrD8B in groupⅡ.The two groups of genes diverged before the separation of Arabidopsis and Brassica.Though the four genes shared a high sequence similarity,and their coding desaturases all located in endoplasmic reticulum,they exhibited distinct expression patterns.Heterologous expression in Saccharomyces cerevisiae revealed that BrD8A/B/C/D were functionally diverse A -sphingohpid desaturases that catalyse different ratios of the two products 8(Z)- and 8(E)-C18-phytosphingenine.The aluminium tolerance of transgenic yeasts expressing BrD8A/B/C/D was enhanced compared with that of control cells.Expression of BrD8A in Arabidopsis changed the ratio of 8(Z):8(E)-C18-phytosphingenine in transgenic plants. The information reported here provides new insights into the biochemical functional diversity and evolutionary relationship of△~8-sphingolipid desaturase in plants and lays a foundation for further investigation of the mechanism of 8(Z)- and 8(E)-C18-phytosphingenine biosynthesis.     

Purification and characterisation of glutathione S-transferases from the freshwater clam Corbicula fluminea (Müller)

Glutathione S-transferases (GSTs) are involved in the phase II detoxification metabolism. To provide a molecular basis for their use as biomarkers of pollution, cytosolic GSTs from the freshwater clam Corbicula fluminea have been purified by glutathione-Sepharose affinity chromatography, anion-exchange chromatography (AEC) and reversed-phase (RP) HPLC. SDS-PAGE of visceral mass (VM) affinity-purified extracts revealed four subunits with apparent molecular masses (MW) of 30.2, 29.2, 28.5 and 27.2 kDa. Analysis by non-denaturing PAGE revealed three acidic dimeric proteins with apparent MW of 64, 55 and 45 kDa, named GSTc1, GSTc2 and GSTc3, respectively, based on their elution order by AEC. Only GSTc2 and GSTc3 exhibited GST activity towards 1-chloro-2,4-dinitrobenzene. A tissue-specific subunit pattern was obtained by RP-HPLC of affinity-purified extracts from VM and gills (GI): three major peaks were resolved, one of which was common to both tissues. MW of each VM subunit was determined by electrospray ionisation-mass spectrometry: 23602+/-1 Da for the major subunit and 23289+/-1 Da for the minor ones. Immunoblot analysis revealed all subunits from both tissues were related to the Pi-class GSTs. In addition, minor VM subunits were slightly related to the Mu-class ones. The interest of such molecular studies in biomonitoring programs is discussed.     

The metabolism of α-naphthoflavone (7,8-benzoflavone) by hepatic microsomes from the marine fish Stenotomus versicolor

Incubation of α-naphthoflavone with fish (scup; Stenotomus versicolor) liver microsomes and NADPH resulted in the production of a major component and several minor components analyzed by high pressure liquid chromatography. The appearance of these components was dependent on time, native protein, NADPH, and O₂ and was strongly inhibited by carbon monoxide. The appearance of the major component was abolished by addition of the epoxide hydrase inhibitor trichloropropene oxide. Mass spectral analysis of the major component yielded a molecular weight of 306. The results strongly indicate that α-naphthoflavone is metabolized by scup hepatic microsomal mixed-function oxygenases and epoxide hydrase and that the major product is a dihydrodiol.     

Controlling the adhesion of the diatom Navicula perminuta using poly(N-isopropylacrylamide-co–N-(1-phenylethyl) acrylamide) films

Poly(N-isopropylacrylamide)-co–N-(1-phenylethyl) acrylamide [P(NIPAAm-co-PEAAm)] thermo-responsive thin films with a lower critical solution temperature (LCST) adjusted to fit marine applications were used to investigate the effect of changes in the wetting properties of a surface on the adhesion of the diatom Navicula perminuta, an organism which forms slime films on surfaces immersed in an aquatic environment. Although the strength of attachment of cells was affected by whether the film was collapsed or expanded, no significant decrease in adhesion strength occurred upon temperature decrease. The effects were attributed to possible strong interactions between the hydrophobic segments of the responsive film when collapsed with components in the adhesive complex.     

Palynological and physico‐chemical characterisation of honeys from the north‐west of Santa Cruz (Argentinean Patagonia)

Very close similarities between the fossil genera Callimothallus Dilcher, 1965 and Microthallites Dilcher, 1965 and recent representatives of the green algae Ulvella P. L. &; H. M. Crouan, 1859, seem to rule out the assumption that fossil, disciform and radiate palynomorph microfossils are representatives of microthyrioaceous fungi. On the basis of the morphology of fossil Ulvella, a model of the general morpohlogy of encrusting, palynomorph algae is constructed. The model includes 5 morphological characters that may only be applied to encrusting life-forms and in particular not to planktonic algae. These characters, therefore, in future palynological research may serve to distinguish benthic algae from planktonic algae. A new fossil alga, Ulvella nannae sp. nov. is described.     

Characterization of a beta-D-(1-->3)-glucan from the marine diatom Chaetoceros mülleri by high-resolution magic-angle spinning NMR spectroscopy on whole algal cells

High-resolution magic-angle spinning (hr-MAS) NMR spectroscopy was used to record NMR spectra of a cell paste from the marine diatom Chaetoceros mülleri. This gave information on a cellular storage polysaccharide identified as a beta-D-(1-->3)-linked glucan, using hr-MAS one-dimensional 1H and 13C, two-dimensional 1H,1H-COSY and 13C,1H-correlation spectroscopy. The same structural information was deduced from the liquid state NMR data on the glucan extracted from C. mülleri. The extracted glucan proved to be a beta-D-(1-->3)-linked glucan with a degree of polymerization of 19 and a degree of beta-D-(1-->6) branching of 0.005. The hr-MAS spectrum of the diatom showed several nonglucan resonances in the carbohydrate region of the NMR spectrum (60-103 ppm) that were shown to be noncarbohydrate resonances by means of two-dimensional 13C,1H- and 1H,1H-correlated NMR data.     

Preliminary studies into the inhibition of the cholesterol α-glucosyltransferase from Helicobacter pylori using azasugars

Various known inhibitors of glycosidases were assessed for their ability to inhibit, both independently as well as with UDP, the cholesterol α-glucosyltransferase from Helicobacter pylori. The sub-cloning, expression and purification of the glucosyltransferase is also discussed.     

Microbial characterisation of fermented meat products from the Sicilian swine breed “Suino Nero Dei Nebrodi”

Two traditional sausage products (“salsiccia” and “salame”) processed from the raw meat of the Black Sicilian swine “Suino Nero dei Nebrodi” were microbiologically investigated during the manufacturing and ripening stages. Both products were dominated by lactic acid bacteria (LAB), especially rod-shaped types. The concentration of enterococci was consistent in salame. Coagulase-negative cocci increased slower than LAB. Yeasts showed an increasing trend during the ripening of both products. Enterobacteriaceae were counted at a constant level of about 10⁵ CFU/g in both products, while pseudomonads diminished during ripening. Coagulase-positive staphylococci, Listeria monocytogenes and Salmonella spp. were not detected at the end of the ripening process. Characterisation of LAB at the strain and species level revealed that Lactococcus lactis was found only in the meat mixture, while Lactobacillus sakei and various enterococci persisted during the monitoring period. Some LAB strains isolated from sausages were also identified on the surface of the factory equipment. Two strains (Lactobacillus sakei SS106A and Enterococcus faecalis SS91) were characterised by their anti-Listeria properties due to bacteriocin-like inhibitory substance production. A multiple strain starter composed of Lactobacillus sakei and enterococci has been proposed to maintain the typical characteristics of the two fermented meat products microbiologically investigated in this study.     

Wood-inhabiting marine fungi from the coast of Shandong, China Ⅲ

Five species of higher marine fungi were observed on the incubated drift and intertidal woods collected from the coasts of Yellow Sea and Bohai Sea. Among them, Halosphaeriopsis was a genus newly recorded for China. Taxonomy and morphology of these species were discussed in this paper. The specimens were deposited in Mycology Herbarium at Qingdao Agricultural University （MHQAU）.