Effect of red seaweed sulfated galactans on initial steps of complement activation in vitro.

The research described here presents data on the effect of galactans of red algae, carrageenans (λ/µ/ν-, κ-, κ/ß-, and ι/κ-types), and agar on complement system activation in normal human serum. The experiments were based on well surfaces coated with triggering agents for binding initiating complement components -C3 and C4. The sulfated galactans inhibited C3 binding to lipopolysaccharide with direct dependence on the sulfation degree of polysaccharides. Sulfation degree was also important in carrageenans' capacity to reduce C4 binding to mannan. However, C4 binding to antibodies was considerably activated by carrageenans, especially with 3,6-anhydrogalactose. The gelling carrageenans were able to block antigen binding centers of total serum IgM and with more intensity than non-gelling. No structural characteristics mattered in ameliorating C5 cleavage by plasmin in extrinsic protease complement activation, but λ/µ/ν- and κ/ß-carrageenans almost completely inhibited C5 cleavage. Thus, galactans participated in cell surface biology by imitating surface glycans in inhibition of C3 binding and mannose binding lectin, but as to the tthe heclassical pathway these substances stimulated complement, probably due to their structure based on carrabiose.

Expression and biochemical characterization of two recombinant fucoidanases from the marine bacterium Wenyingzhuangia fucanilytica CZ1127T.

Genomic analysis of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T revealed the presence of four fucoidanase genes fwf1, fwf2, fwf3, fwf4 that belonged to the glycoside hydrolase family 107 (GH107, CAZy), which is located in one gene cluster putatively involved in fucoidan catabolism. Genes encoding two fucoidanases fwf1 and fwf2 were cloned, and the proteins FWf1 and FWf2 were produced in Escherichia coli cells. The recombinant fucoidanases were purified and the biochemical properties of these enzymes were studied. The amino acid sequences of FWf1 and FWf2 showed 41 and 51% identity respectively with a fucoidanase FcnA from the marine bacterium Mariniflexile fucanivorans, with the established 3D structure. Structures of the oligosaccharides produced during enzymatic hydrolysis of fucoidan by FWf1 and FWf2 have been determined by NMR spectroscopy. Detailed substrate specificities of FWf1 and FWf2 were studied using fucoidans and sulfated fucooligosaccharides with different structures. Both fucoidanases catalyzed hydrolysis of 1→4-glycosidic bonds between sulfated α-l-fucose residues but had different specificities regarding sulfation patterns of the fucose residues in fucoidan molecules. Specific cleavage sites recognizable by the fucoidanases in fucoidan molecules were determined. The obtained results provide new knowledge about differences between specificities of the fucoidanases belonging to the GH107 family.

Cancer-preventive activities of secondary metabolites from leaves of the bilberry Vaccinium smallii A. Gray.

Ten secondary metabolites including flavonoids (1-8), caffeic (9) and chlorogenic (10) acids were structurally identified from the extract of Sakhalin bilberry Vaccinium smallii leaves and studied in vitro as potential cancer-preventive agents. The results showed that compounds 1-10 inhibited EGF-induced neoplastic transformation of mouse JB6 Cl 41 P+ cells in soft agar with an inhibition concentration (INCC50) of 20-80 µm. Moreover, all these natural products were non-toxic against JB6 Cl 41 P+ cells up to a concentration of 200 µm.

New steroid glycosides from the starfish Asterias rathbuni.

Two novel steroidal 24-O-xylosides, designated as rathbuniosides R(1) (1) and R(2) (2), and the known amurensoside A (3) and 3-O-sulfomarthasterone (4) have been isolated from the starfish Asterias rathbuni. The structures of all the compounds were determined from their spectroscopic data, including one- and two-dimensional NMR methods. The compounds 1 and 4 inhibit the cell division of fertilized sea urchin eggs at doses of 7.0 x 10(-5) and 2.9 x 10(-5) M, respectively.

Free sterols from the holothurians Synapta maculata, Cladolabes bifurcatus and Cucumaria sp.

Free sterol fractions from the holothurians (sea cucumbers) Synapta maculata, Cladolabes bifurcatus and Cucumaria sp. have been isolated and studied by HPLC, GLC, GLC-MS and NMR methods. Forty seven sterols were identified, including several rare ones. In contrast with previously studied holothurians, the presence of a minor amount of Delta7 sterols was indicated in the sterols of S. maculata. This animal contains predominantly Delta(9(11))sterols as well as an abnormally high concentration of Delta5 sterols. In C. bifurcatus and Cucumaria sp., 14alpha-methyl and 4alpha,14alpha-dimethyl-Delta(9(11))sterols were found to be the main sterol constituents. Peculiarities of sterol distribution and the relationship between sterol compositions and taxonomic positions, ecology and toxicity of the corresponding sea cucumbers were discussed.

Triterpene glycosides from the far eastern sea cucumber Pentamera calcigera II: disulfated glycosides.

Three new triterpene glycosides, calcigerosides D(1) (1), D(2) (2), and E (3), have been isolated from the sea cucumber Pentamera calcigera. Their structures have been deduced from extensive spectral analysis (NMR and MS) and chemical evidence. All the compounds are disulfated pentaosides differing in aglycon structure and position of sulfate group, which were determined by the measurement of NT(1) values in the cases of glycosides 1 and 2. Glycoside 1 is a nonholostane derivative, that is, it lacks an 18(20)-lactone, which is very rare among the sea cucumber glycosides.

Hemolytic polar steroidal constituents of the starfish Aphelasterias japonica.

A reinvestigation of the polar steroid fraction from the starfish Aphelasterias japonica, collected near the Russian shore of the Sea of Japan, has afforded two new compounds, the disulfated quinovoside aphelasteroside C (1) and the monosulfated polyhydroxysteroid aphelaketotriol (2). Compounds 1 and 2 contain a unique 23-oxo-24-hydroxylated side chain that is unprecedented in marine steroids. The known compounds cheliferoside L1 (3), 3-O-sulfoasterone (4), forbeside E3 (5), and 3-O-sulfothornasterol A (6) were also isolated from this source. Compounds 1-3, 5, and 6 showed hemolytic activity to mouse erythrocytes.

Triterpene glycosides from the Far-Eastern sea cucumber Pentamera calcigera. 1. Monosulfated glycosides and cytotoxicity of their unsulfated derivatives.

Three new monosulfated triterpene glycosides, calcigerosides B (2), C(1) (3), and C(2) (4), along with the known cucumarioside G(2) (1), have been isolated from the sea cucumber Pentamera calcigera. Their structures have been deduced from extensive spectral analysis (NMR and MS) and chemical evidence. Compounds 2-4 present a novel pentasacharide chain never reported before in sea cucumber triterpene glycosides. The desulfated derivatives of calcigerosides B, C(1), and C(2) (5, 7, and 9, respectively) showed moderate cytotoxicity (IC(50) = 5 microg/mL) against a selection of four human and mouse tumor cell lines.

New steroid glycosides from the deep-water starfish Mediaster murrayi.

Four new 24-O-biosides of 5alpha-cholestane-3beta,6alpha,8,15beta, 24-pentaol, designated as mediasterosides M1 (1), M2 (2), M3 (3), and M4 (4), and the previously known 5alpha-cholestane-3beta,6beta,8, 15alpha,16beta,26-hexaol (5) have been isolated from the deep-water starfish Mediaster murrayi. Glycosides 1-3 contain rare carbohydrate moieties with (1-->5) bonds between the monosaccharide units. Compounds 1 and 2 showed the inhibition of cell division of fertilized sea urchin eggs and exhibited moderate hemolytic activities.

Koreoside A, a new nonholostane triterpene glycoside from the sea cucumber Cucumaria koraiensis.

The new triterpene glycoside koreoside A (1) has been isolated from the sea cucumber Cucumaria koraiensis. Koreoside A (1) is the first glycoside reported from holothurians that presents a delta(7) nonholostane aglycon without a lactone group and with a shortened side chain. Its structure has been elucidated by 13C and 1H NMR as well as FABMS studies.

Structure of eximisoside A, a novel triterpene glycoside from the Far-Eastern sea cucumber psolus eximius.

The new triterpene saponin eximisoside A (1) has been isolated from the Far-Eastern sea cucumber Psolus eximius and its structure elucidated by 1D and 2D NMR (13C, 1H, 1H-1H COSY, HMQC, and NOESY spectra), and FABMS studies. The structural features of eximisoside A (1) are the rare presence of a 23 double bond in the aglycon and the absence of quinovose, typically found in the oligosaccharide chain of holothurian glycosides.

Structure of cucumarioside G2, a novel nonholostane glycoside from the sea cucumber Eupentacta fraudatrix.

The new triterpene glycoside cucumarioside G2 [1] has been isolated from the sea cucumber Eupentacta fraudatrix. Glycoside 1 is the first triterpene glycoside with the 23,24,25,26,27-pentanorlanostane type of aglycone. Its structure has been established by chemical transformations as well as 13C- and 1H-nmr, eims, and liquid sims studies.

Sea anemone Radianthus macrodactylus--a new source of palytoxin.

A very potent non-protein toxin was isolated from the sea anemone Radianthus macrodactylus with the use of chromatography on polytetrafluoroethylene, CM-Sephadex C-25 and by cation and anion exchange HPLC. The toxin was identified as palytoxin by u.v.-, i.r.- and 500 MHz 1H NMR spectroscopy. Its LD50 was 0.74 +/- 0.29 micrograms/kg by i.v. injection into mice. So far, palytoxin has been associated with zoanthids only. The toxin caused the loss of haemoglobin from erythrocytes but only in about 2 hr after the beginning of incubation, which is characteristic for palytoxin from zoanthids. Sea anemone palytoxin was divided into major and minor components by HPLC. The latter proved to be a product of degradation of palytoxin.

Physicochemical characteristics of interaction of toxic triterpene glycosides from holothurians with rat brain Na+-K+-ATPase.

High-angle X-ray diffraction spectra showed that triterpene glycosides form crystalline complexes with membrane cholesterol. Electron microscopy demonstrated a decreased vesicle size, of the membrane preparation from rat brain which is enriched in Na+-K+-ATPase, by the triterpene glycosides. The Arrhenius plot was linear in the presence of triterpene glycosides. The half-width of the phosphatidylcholine N-methyl proton line in proton NMR spectra was not altered in the presence of marine glycosides. The excimer formation of pyrene, a hydrophobic fluorescent probe, was significantly decreased by triterpene glycosides. The increase of tryptophanyl residue fluorescence demonstrated a change of the Na+-K+-ATPase conformation after treatment with cytotoxic glycosides.

Triterpene glycosides from sea cucumber Stichopus japonicus Selenka.

Triterpenoidal oligoglycosides from the far eastern sea cucumber Stitchopus japonicus Selenka collected in the Posiet Bay, Japan Sea were investigated. The glycoside fraction was shown to consist of two main components, holotoxins A1 and B1 which have been isolated by chromatography on silica gel columns. The chemical structures of the holotoxins were elucidated on the basis of chemical and physiochemical evidence (for formula see text). The proposed structures were compared with close related but not identical structures of holotoxin A and B from other collection of the same holothurian. The probable reasons of the distinction were discussed. The previously-established difference in glycoside structures between Stichopus japonicus and other representatives of the family Stichopodidae was corroborated.

Steroids in Porifera. II. Steroid derivatives from two sponges of the family Halichondriidae. Sokotrasterol sulfate, a marine steroid with a new pattern of side chain alkylation.

A trisulfated derivative of 24,25,26,26-tetramethyl-5 alpha-cholest-23E-ene-2 alpha, 3 beta, 6 alpha-triol (sokotrasterol sulfate) has been isolated from the sponge Halichondriidae gen. sp., collected near Sokotra Island (Arabian Sea), and its structure has been elucidated. The side chain of the new steroid involves a "normal" alkylation at C-24 and the unprecedented addition of two extra methyl groups at C-26 and one extra methyl group at C-25. A free sterol fraction contained only 24-isopropyl-5-cholesten-3 beta-ol and 24-isopropyl-5, 22E-cholestadien-3 beta-ol. 24-Isopropyl-5, 22E-cholestadien-3 beta-ol as sole monohydroxy sterol and halistanol sulfate as major polyhydroxylated steroid derivative have been detected in Halichondria sp., a Madagascar sponge.

Structural studies on the O-specific polysaccharide side-chains of Yersinia pseudotuberculosis, type III, lipopolysaccharides.

Lipopolysaccharide from pathogenic Yersinia pseudotuberculosis type III, strain 209, has been isolated and characterized. Lipopolysaccharide was shown to contain lipid A (30%), protein (4.4%), 2-keto-3-deoxyoctonate (7.8%) and a polysaccharide component (45%), which consisted of paratose, L-fucose, D-mannose, D-glucose, D-galactose (trace), L- and D-glycero-D-manno-heptose, 2-deoxy-2-amino-D-glucose and 2-deoxy-2-amino-D-galactose residues. Molecular -sieve chromatography on Sephadex G-50 afforded on O-specific polysaccharide, being hydrolysed to yield equimolar amounts of paratose, L-fucose, D-mannose and 2-deoxy-2-amino-D-galactose. The chemical repeating unit of the O-specific polysaccharide has been suggested on the basis of methylation studies and periodate oxidation data.