X-ray structure of a sodium salt of digeneaside isolated from red alga Ceramium botryocarpum

X-ray diffraction analysis has been recently used to determine the crystal structure of the floridoside (2-O-α-d-galactopyranosylglycerol) isolated from red alga Palmaria palmata and Dilsea carnosa, respectively [Simon-Colin, C.; Michaud, F.; Léger, J.-M.; Deslandes, E. Carbohydr. Res.2003, 338, 2413-2416; Vonthron-Senechau, C.; Sopkova-de Oliveira Santos. J.; Mussio, I.; Rusig, A. M. Carbohydr. Res. 2008, 343, 2697-2698]. In this present study, a similar analysis was performed on another compound belonging to the glycopyranosyl-glycerols family present in red algae, digeneaside. The crystal structure of a hydrated sodium salt of digeneaside (sodium 2-O-α-d-mannopyranosyl-d-glycerate monohydrate) was determined by single-crystal X-ray diffraction analysis at 110 ± 3 K. The space group is C2 with Z = 4, a = 17.9315(12), b = 6.2693(4), c = 10.7805(7) Å, beta = 90.746(7)°.     

The production of antimicrobial compounds by British marine algae I. Antibiotic-producing marine algae

Using five species of bacteria as the test organisms, 151 species of British marine algae have been screened for the production of antibiotics. Of these, Asparagopsis armata, Bonnemaisonia asparagoides, Bonnemaisonia hamifera, Chondrus crispus, Dilsea carnosa, Gloiosiphonia capillaris, Sphondylothamnion multifidum, Desmarestia aculeata, Desmarestia ligulata, Laminaria digitata, Dictyopteris membranacea, Dictyota dichotoma, Halidrys siliquosa and most members of the family Rhodomelaceae appear to possess outstanding antibacterial properties. Although the production of antibiotics would appear to be a characteristic of several families, it has not been possible to establish any major correlation between taxonomy and antibiotic production. In the case of two closely related and morphologically similar species, Chondrus crispus and Gigartina stellata, the former possesses considerable degrees of antimicrobial activity whilst the latter exhibits no such activity. The results also indicate that the production of antibiotics by the algae is affected by the season of the year.     

Floridoside suppresses pro-inflammatory responses by blocking MAPK signaling in activated microglia

Inflammatory conditions mediated by activated microglia lead to chronic neuro-degenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. This study was conducted to determine the effect of floridoside isolated from marine red algae Laurencia undulata on LPS (100 ng/ml) activated inflammatory responses in BV-2 microglia cells. The results show that floridoside has the ability to suppress pro-inflammatory responses in microglia by markedly inhibiting the production of nitric oxide (NO) and reactive oxygen species (ROS). Moreover, floridoside down-regulated the protein and gene expression levels of iNOS and COX-2 by significantly blocking the phosphorylation of p38 and ERK in BV-2 cells. Collectively, these results indicate that floridoside has the potential to be developed as an active agent for the treatment of neuro-inflammation. [BMB Reports 2013; 46(8): 398-403]     

Impact of the salt stress on the photosynthetic carbon flux and C-label distribution within floridoside and digeneaside in Solieria chordalis

The flux of photosynthetic carbon used in the synthesis of low-molecular weight carbohydrates (digeneaside and floridoside) was investigated by ¹³C and ¹H NMR spectroscopy in samples of the red seaweed, Solieria chordalis, incubated at different salinities (22, 34 and 50 psu). Carbohydrates were labelled, by pulse-chase, with the stable isotope ¹³C from NaH¹³CO₃. In vivo NMR analyses carried out with a cryogenic probe optimised for ¹³C detection were performed directly on the living algal tissues to evidence the labelling of the carbohydrates with neither preliminary extraction nor purification step(s). The isotopic enrichment of each compound was determined by high-resolution ¹H and ¹³C NMR spectroscopy. These analyses evidenced different orientations of the flux of the photosynthetic carbon in the algae according to the salt stress. At normal and low salinities, the photosynthetic carbon flux was responsible of 70% and 67% of the floridoside synthetized during the pulse period, respectively, whereas it was only of 30% in the thalli exposed to the high salinity, meaning a biosynthesis of high floridoside amount from endogen source leading to the osmotic regulation. Under normal and hyper-osmotic conditions, the stock of floridoside was used for cellular needs during the chase period, whereas it was not under hypo-osmotic conditions.The characterization of isotopomers composition of floridoside and digeneaside and the analysis of adjacent ¹³C-labelling gives much more details on the effects of salinity on the metabolic pathways leading to the synthesis or the degradation of these molecules. High turnover of floridoside was evidenced at normal salinity during the chase period and products issued from the degradation of floridoside would not be used for the novo biosynthesis. That suggests that synthesis and degradation of floridoside may be realized in two different cellular compartments. The presence of more numerous ¹³C-¹³C blocks in the carbon skeleton of the molecules from the up salt stressed thalli than in those from no salt stressed algae, concomitant with a slower degree of isotopic enrichment of the molecule, provided evidence that the two metabolic pathways (endogen and photosynthetic) may not share the precursor molecules involved in the floridoside synthesis and that these two routes may be totally separate until the constitution of floridoside molecule.     

Crystal structure and chirality of natural floridoside

The crystal structure and absolute configuration of natural floridoside (2-O-alpha-D-galactopyranosylglycerol) were determined by single-crystal X-ray diffraction analysis. The space group is orthorhombic P2(1)2(1)2(1) with Z=4, a=4.885(1), b=9.734(1), c=23.886(2) A at 296 +/- 2 K. The structure was solved by a direct method and refined to R=0.0351 from 1914 reflections of Cu Kalpha radiation.     

Evolutionary relationships of Mycaureola dilseae (Agaricales), a basidiomycete pathogen of a subtidal rhodophyte

Mushroom-forming fungi (homobasidiomycetes) are major examples of morphological and ecological diversification in terrestrial habitats. Homobasidiomycetes includes only nine described species that are known from marine environments. Morphological traits that have concealed the ancestry of these fungi include reduced fruiting bodies with hairy surfaces and extremely modified spores, both of which may function as floating devices to aid successful dispersal and adhesion to various substrates such as driftwood. Our previous results suggested that all marine forms as yet investigated are placed in the Nia clade (euagarics) and that they have primarily evolved from cypelloid forms (minute, cup-shaped, terrestrial saprotrophs) via transitions through mangroves to fully marine habitats. We show here that Mycaureola dilseae, which parasitizes the red alga Dilsea carnosa, is a second independent lineage of marine fungi in the euagarics clade that is not related to cyphelloid forms. Phylogenetic reconstructions were based on two data sets: a partial four-region rDNA data set (nuc-ssu, nuc-lsu, mt-ssu, and mt-lsu) with inclusive sampling of 249 taxa and a densely sampled ITS data set including 32 taxa, which formed a clade with Mycaureola in the four-region rDNA analyses. Inferences using constrained and unconstrained six-parameter weighted parsimony, Bayesian Markov chain Monte Carlo methods, and maximum likelihood approaches place M. dilseae in the morphologically diverse /physalacriaceae clade next to Gloiocephala spp., a group of highly reduced stipitate-pileate saprotrophs.     

Comparative genomics of the white-rot fungi, Phanerochaete carnosa and P. chrysosporium, to elucidate the genetic basis of the distinct wood types they colonize

ABSTRACT: BACKGROUND: Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus, Phanerochaete carnosa, has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by a white-rot fungus, the present study reports the P. carnosa genome sequence and its comparative analysis with the previously reported P. chrysosporium genome. RESULTS: P. carnosa encodes a complete set of lignocellulose-active enzymes. Comparative genomic analysis revealed that P. carnosa is enriched with genes encoding manganese peroxidase, and that the most divergent glycoside hydrolase families were predicted to encode hemicellulases and glycoprotein degrading enzymes. Most remarkably, P. carnosa possesses one of the largest P450 contingents (266 P450s) among the sequenced and annotated wood-rotting basidiomycetes, nearly double that of P. chrysosporium. Along with metabolic pathway modeling, comparative growth studies on model compounds and chemical analyses of decomposed wood components showed greater tolerance of P. carnosa to various substrates including coniferous heartwood. CONCLUSIONS: The P. carnosa genome is enriched with genes that encode P450 monooxygenases that can participate in extractives degradation, and manganese peroxidases involved in lignin degradation. The significant expansion of P450s in P. carnosa, along with differences in carbohydrate- and lignin-degrading enzymes, could be correlated to the utilization of heartwood and sapwood preparations from both coniferous and hardwood species.     

VARIATIONS IN FLORIDOSIDE CONTENT AND FLORIDOSIDE PHOSPHATE SYNTHASE ACTIVITY IN PORPHYRA PERFORATA (RHODOPHYTA)1

The diurnal and seasonal variations in floridoside content and floridoside phosphate synthase (FPS) activity were measured in samples of Porphyra perforata J. Ag. growing in the field. Floridoside content generally increased about fourfold from early morning to the middle of the day and then dropped gradually in the afternoon and the evening. On a monthly basis, there was a steady increase in floridoside content from February to May. A similar trend in monthly increase in FPS activity was also observed from February to April. The level of FPS activity varied with the time of day. The highest activity was observed early in the morning shortly after dawn; subsequently, it decreased, reaching about 50% of the peak value late in the afternoon/evening. On a daily as well as seasonal basis, a change in FPS activity correlated with a change in floridoside content. Among environmental factors, floridoside content and FPS activity showed a positive correlation with daylength and temperature on a seasonal basis. However, on a daily basis, salinity and water temperature did not seem to affect the floridoside content or the FPS activity.     

Mode of coniferous wood decay by the white rot fungus Phanerochaete carnosa as elucidated by FTIR and ToF-SIMS

The softwood degrading white-rot fungus, Phanerochaete carnosa, was investigated for its ability to degrade two coniferous woods: balsam fir and lodgepole pine. P. carnosa grew similarly on these wood species, and like the hardwood-degrading white-rot fungus Ceriporiopsis subvermispora, P. carnosa demonstrated selective degradation of lignin, as observed by Fourier transform infrared spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Lignin degradation across cell walls of decayed pine samples was also evaluated by ToF-SIMS and was shown to be uniform. This study illustrates softwood lignin utilization by a white-rot fungus and reveals the industrial potential of the lignocellulolytic activity elicited by this fungus.     

Smith degradation of the O-antigenic polysaccharide of Salmonella Dakar: structural studies of the products

Two different oligosaccharides were obtained from the Smith degradation of the O-polysaccharide isolated from the lipopolysaccharide of Salmonella Dakar. The structures of these oligosaccharides were investigated by chemical analysis, NMR spectroscopy and MALDI-TOF mass spectrometry. The following structures of these products were determined: alpha-D-GalpNAc-(1-->4)-alpha-D-Quip3NAc-(1-->3)-alpha-L-Rhap-(1-->2)-threitol and [FORMULA: SEE TEXT] where Quip3NAc is 3-acetamido-3,6-dideoxyglucose. The reaction products confirmed the structure of the repeating unit of the Salmonella Dakar O-polysaccharide reported previously [Kumirska, J.; Szafranek, J.; Czerwicka, M.; Paszkiewicz, M.; Dziadziuszko, H.; Kunikowska, D.; Stepnowski, P. Carbohydr. Res. 2007,342, 2138-2143].     

红藻糖苷对超低温冻存微藻的保护作用

为研究红藻糖苷对超低温冻存微藻细胞的保护作用,研究将3种不同的微藻置于含10% DMSO和不同浓度红藻糖苷的冻存液中,冻存并解冻后,以流式细胞仪检测细胞存活率,测定复养后藻株的生长曲线及相关生理参数。结果显示,由于冷冻损伤,冻存后各种藻细胞的生长速率、细胞密度及生理指标都显著性下降,而红藻糖苷协同DMSO能够显著增加细胞的存活率,尤其15%红藻糖苷能将紫球藻存活率提升20%（P0.05）;生长曲线得到明显改善;且对PSII最大光能转化效率也有显著性提高（P0.05）。总体结果来看,红藻糖苷对超低温冻存微藻,特别是紫球藻具有明显的保护作用,且效果强于蔗糖。     

LOW MOLECULAR WEIGHT CARBOHYDRATES OF THE BANGIOPHYCIDAE (RHODOPHYTA)1

In the order Porphyridiales there are three clades based on molecular evidence. These show parallels with the low molecular weight carbohydrate (LMWCs) in different genera. Clade Porphyridiales 1 includes Dixoniella, Glaucosphaera, Rhodella, and one undescribed genus (3987) that all contain mannitol. Clade Porphyridiales 2 comprises taxa of the Stylonematales Rhodosorus and Stylonema species and contains digeneaside and sorbitol, whereas Chroodactylon has only sorbitol. In clade Porphyridiales 3 Flintiella, Porphyridium, and the undescribed genus (3797) all possess only floridoside. In the Erythropeltidales Rhodochaete contains floridoside and digeneaside, Erythrotrichia species contain only floridoside, Sahlingia subintegra has floridoside and traces of D‐floridoside, and Smithora has L‐isofloridoside plus floridoside. In the Compsopogonales Boldia and Compsopogon have only floridoside. Within these genera as presently circumscribed, the LMWCs appear to be a reliable character to supplement the usual cytological characters.     

Separation of floridoside and isofloridosides by HPLC and complete (1)H and (13)C NMR spectral assignments for D-isofloridoside

Isofloridosides (1-O-alpha-D-galactopyranosylglycerol) and floridoside (2-O-alpha-D-galactopyranosylglycerol) were extracted from the red alga Porphyra umbilicalis (Linné) Kützing (Bangiales, Rhodophyta). Their separation was achieved by HPLC (NH(2) P50 column) after successive purification of the crude extract by ion-exchange chromatography and HPLC (Sugar-Pak TM1 column). 1D and 2D NMR spectroscopy experiments allowed to completely assign the (1)H and (13)C spectra of D-isofloridoside.     

The structure of Cryptococcus neoformans galactoxylomannan contains β-d-glucuronic acid

The structure of galactoxylomannan, a capsular polysaccharide from the opportunistic yeast Cryptococcus neoformans, was re-examined by NMR spectroscopy and GC-MS. The residue that is 3-linked to the side chain galactose and was previously assigned as β-d-xylose [Vaishnav, V. V.; Bacon, B. E.; O’Neill, M.; Cherniak, R. Carbohydr. Res.1998, 306, 315-330] was determined to be β-d-glucuronic acid. A revised structure for this polymer is presented, along with a proposal that this compound be termed glucuronoxylomannogalactan (GXMGal).     

Structural analysis of lipopolysaccharide oligosaccharide epitopes expressed by non-typeable Haemophilus influenzae strain 176

The structure of the lipopolysaccharide (LPS) from non-typeable Haemophilus influenzae strain 176 has been investigated. Electrospray ionization-mass spectrometry (ESIMS) on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) samples obtained after mild-acid hydrolysis of LPS provided information on the composition and relative abundance of the glycoforms. ESIMS tandem-mass spectrometry on LPS-OH confirmed the presence of minor sialylated and disialylated glycoforms. Oligosaccharide samples were studied in detail using high-field NMR techniques. It was found that the LPS contains the common inner-core element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A having glycosyl substitution at the O-3 position of the terminal heptose as recently observed for non-typeable H. influenzae strain 486 [M?nsson, M.; Bauer, S. H. J.; Hood, D. W.; Richards, J. C.; Moxon, E. R.; Schweda, E. K. H., Eur. J. Biochem. 2001, 268, 2148--2159]. The following LPS structures were identified as the major glycoforms, the most significant being indicated with an asterisk (*) (glycoforms are partly substituted with Gly at the terminal Hep):     

Structure of the O-polysaccharide of Cronobacter sakazakii O2 with a randomly O-acetylated l-rhamnose residue

Studies by sugar analysis and partial acid hydrolysis along with one- and two-dimensional ¹H and ¹³C NMR spectroscopy and high-resolution ESI MS showed that the O-polysaccharide (O-antigen) Cronobacter sakazakii ATCC 29004 (serotype O2) possesses a branched hexasaccharide O-unit with a randomly mono-O-acetylated terminal rhamnose residue in the side chain and the following structure:A similar structure has been reported for the O-polysaccharide of C. sakazakii 767, which differs in the presence of an additional lateral α-d-Glcp residue on GlcNAc and the pattern of O-acetylation (Czerwicka, M., Forsythe, S. J.; Bychowska, A.; Dziadziuszko, H.; Kunikowska, D.; Stepnowski, P.; Kaczynski, Z. Carbohydr. Res.2010, 345, 908-913).     

EFFECT OF NUTRIENT DEPRIVATION AND RESUPPLY ON METABOLITES AND ENZYMES RELATED TO CARBON ALLOCATION IN GRACILARIA TENUISTIPITATA (RHODOPHYTA)1

The starch content of red algae normally increases during nitrogen limitation. Based on this we hypothesized that nutrient deprivation would result in an increased activity of starch‐synthesizing enzymes and a decrease in the activity of starch‐degrading enzymes, with the opposite scenario when nutrients were sufficient. We therefore examined the effect of the nutrient status of Gracilaria tenuistipitata Chang et Xia on the content of starch and floridoside and on the activity of enzymes involved in the allocation of carbon into starch, floridoside, and agar; floridoside phosphate synthase and α‐galactosidase involved in synthesis and degradation of floridoside; starch synthase and starch phosphorylase involved in the metabolism of starch; uridine 5′‐diphosphate (UDP)‐glucose pyrophosphorylase; adenosine 5′‐diphosphate‐glucose pyrophosphorylase; UDP‐glucose 4‐epimerase; and phosphoglucomutase. During the period of nutrient limitation the starch and floridoside content increased, as did dry weight and C/N ratio, whereas growth rate and protein content decreased. A general decrease in the enzyme activities during nutrient limitation was also observed, indicating a decrease in overall cellular metabolism. The addition of nutrients caused an increase in enzyme activities and a decrease in the contents of starch and floridoside. Of the enzymes examined, only the activity of UDP‐glucose pyrophosphorylase increased during nutrient limitation and decreased abruptly after nutrient addition. This implies a regulatory role for this enzyme in the supply of UDP‐glucose for starch synthesis. It also supports our suggestion that UDP‐glucose is the substrate for starch synthesis in red algae. This assertion is further strengthened by the observation that of the potential starch synthases only the UDP‐glucose starch synthase could support the observed rate of starch synthesis.     

Reinvestigation of the iodocyclization of 4,5,7-tri-O-benzyl-3-(N-benzylacetamido)-1,2,3-trideoxy-D-gluco-hept-1-enitol: unexpected formation of a 1,3-imino-heptitol derivative

The NIS-mediated iodocyclization of 4,5,7-tri-O-benzyl-3-(N-benzylacetamido)-1,2,3-trideoxy-D-gluco-hept-1-enitol gave unexpectedly a 1,3-imino-heptitol derivative, namely 2-O-acetyl-N-benzyl-4,5,7-tri-O-benzyl-1,3-dideoxy-1,3-imino-D-glycero-D-ido-heptitol. This compound is a new example of a rare class of azetidine imino alditol derivatives which have interesting properties such as glycosidase inhibitors. The physical and spectral data for this imino heptitol were essentially identical to those reported for 2,6-anhydro-4,5,7-tri-O-benzyl-3-(N-benzylacetamido)-3-deoxy-D-glycero-D-ido-heptitol, a derivative of C-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)methanol obtained from the same precursor [Lay, L.; Nicotra, F.; Panza, L.; Verani, A. Gazz. Chim. Ital. 1992, 122, 345-348]; these findings cast doubts on the structure reported for the latter product.     

FIXED CARBON PARTITIONING IN THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)

The main products of carbon fixation in the red algae are sulfated cell-wall polysaccharides, floridean starch, and low molecular weight (LMW) carbohydrates, mainly floridoside. In the red microalga Porphyridium sp., sulfated polysaccharide—cell bound and soluble—comprises up to 70% of the algal biomass. The purpose of this study was to elucidate the partitioning of fixed carbon in Porphyridium sp. toward the different products of carbon fixation. Using pulse-chase technique with [¹⁴C]bicarbonate, we followed ¹⁴C flow into the major compounds, namely, cell-wall polysaccharide, floridoside, starch, and protein, under various environmental conditions (i.e. carbon dioxide enrichment and nitrate starvation). ¹³C-NMR and gas chromatography analysis showed the main LMW product in Porphyridium sp. to be floridoside. After the short [¹⁴C]bicarbonate pulse (20 min), 42%–53% of total ¹⁴C uptake was initially found in floridoside. The appearance of ¹⁴C in the soluble polysaccharide was evident immediately at the end of the 20-min [¹⁴C]bicarbonate pulse. The specific radioactivity in the floridoside fraction declined by 80% after the 48-h chase, this decline being accompanied by increased labeling of starch and the soluble polysaccharide. In cells exposed to high CO₂ concentration, larger amounts of ¹⁴C (about twice as much) were channeled into starch and soluble polysaccharide than in cells under low CO₂ concentration. The most significant increase (1500%) in labeling during chase was found in the soluble polysaccharide of the nitrate-deprived cultures. It therefore seems likely that the large amounts of carbon incorporated by Porphyridium sp. cells into floridoside were subsequently used for the synthesis of macromolecular components. The data thus support the premise that floridoside serves as a dynamic carbon pool, which channels the fixed carbon toward polysaccharides and other end products according to the ambient conditions.