New Ceramides and Cerebrosides from the Deep-Sea Far Eastern Starfish Ceramaster patagonicus.

Three new ceramides (1−3) and three new cerebrosides (4, 8, and 9), along with three previously known cerebrosides (ophidiocerebrosides C (5), D (6), and CE-3-2 (7)), were isolated from a deep-sea starfish species, the orange cookie starfish Ceramaster patagonicus. The structures of 1−4, 8, and 9 were determined by the NMR and ESIMS techniques and also through chemical transformations. Ceramides 1−3 contain iso-C21 or C23 Δ9-phytosphingosine as a long-chain base and have C16 or C17 (2R)-2-hydroxy-fatty acids of the normal type. Cerebroside 4 contains C22 Δ9-sphingosine anteiso-type as a long-chain base and (2R)-2-hydroxyheptadecanoic acid of the normal type, while compounds 8 and 9 contain saturated C-17 phytosphingosine anteiso-type as a long-chain base and differ from each other in the length of the polymethylene chain of (2R)-2-hydroxy-fatty acids of the normal type: C23 in 8 and C24 in 9. All the new cerebrosides (4, 8, and 9) have ß-D-glucopyranose as a monosaccharide residue. The composition of neutral sphingolipids from C. patagonicus was described for the first time. The investigated compounds 1−3, 5−7, and 9 exhibit slight to moderate cytotoxic activity against human cancer cells (HT-29, SK-MEL-28, and MDA-MB-231) and normal embryonic kidney cells HEK293. Compounds 2, 5, and 6 at a concentration of 20 µM inhibit colony formation of MDA-MB-231 cells by 68%, 54%, and 68%, respectively. The colony-inhibiting activity of compounds 2, 5, and 6 is comparable to the effect of doxorubicin, which reduces the number of colonies by 70% at the same concentration.

1-O-Alkylglycerol Ethers from the Marine Sponge Guitarra abbotti and Their Cytotoxic Activity.

The cytotoxicity-bioassay-guided fractionation of the ethanol extract from the marine sponge Guitarra abbotti, whose 1-O-alkyl-sn-glycerol ethers (AGEs) have not been investigated so far, led to the isolation of a complex lipid fraction containing, along with previously known compounds, six new lipids of the AGE type. The composition of the AGE fraction as well as the structures of 6 new and 22 previously known compounds were established using 1H and 13C NMR, GC/MS, and chemical conversion methods. The new AGEs were identified as: 1-O-(Z-docos-15-enyl)-sn-glycerol (1), 1-O-(Z-docos-17-enyl)-sn-glycerol (2), 1-O-(Z-tricos-15-enyl)-sn-glycerol (3), 1-O-(Z-tricos-16-enyl)-sn-glycerol (4), 1-O-(Z-tricos-17-enyl)-sn-glycerol (5), and 1-O-(Z-tetracos-15-enyl)-sn-glycerol (6). The isolated AGEs show weak cytotoxic activity in THP-1, HL-60, HeLa, DLD-1, SNU C4, SK-MEL-28, and MDA-MB-231 human cancer cells. A further cytotoxicity analysis in JB6 P+ Cl41 cells bearing mutated MAP kinase genes revealed that ERK2 and JNK1 play a cytoprotective role in the cellular response to the AGE-induced cytotoxic effects.

Edible Far Eastern Ferns as a Dietary Source of Long-Chain Polyunsaturated Fatty Acids.

Young fronds of ferns are consumed as a vegetable in many countries. The aim of this study was to analyze three fern species that are available for sale in the Russian Far East as dietary sources in terms of fatty acids that are important for human physiology: arachidonic acid (20:4n-6, ARA), eicosapentaenoic acid (20:5n-3, EPA) and other valuable long-chain polyunsaturated fatty acids. The content of ARA and EPA was 5.5 and 0.5 mg/g dry weight, respectively, in Pteridium aquilinum, 4.1 and 1.1 in Matteuccia struthiopteris, and 2.2 and 0.8 in Osmundastrum asiaticum. Salted fronds of P. aquilinum contained less these fatty acids than the raw fronds, with a decrease of up to 49% for ARA and 65% for EPA. These losses were less pronounced or even insignificant in dried fronds. Cooked ferns preserved significant portions of the long-chain polyunsaturated fatty acids: cooked P. aquilinum contained 4.4 mg/g dry weight ARA and 0.3 mg/g dry weight EPA. The ferns may provide a supplemental dietary source of these valuable long-chain polyunsaturated fatty acids, especially for vegetarian diets.

Investigation of Deep-Sea Ecosystems Using Marker Fatty Acids: Sources of Essential Polyunsaturated Fatty Acids in Abyssal Megafauna.

Abyssal seafloor ecosystems cover more than 50% of the Earth's surface. Being formed by mainly heterotrophic organisms, they depend on the flux of particulate organic matter (POM) photosynthetically produced in the surface layer of the ocean. As dead phytoplankton sinks from the euphotic to the abyssal zone, the trophic value of POM and the concentration of essential polyunsaturated fatty acids (PUFA) decrease. This results in pronounced food periodicity and limitations for bottom dwellers. Deep-sea invertebrate seston eaters and surface deposit feeders consume the sinking POM. Other invertebrates utilize different food items that have undergone a trophic upgrade, with PUFA synthesized from saturated and monounsaturated FA. Foraminifera and nematodes can synthesize arachidonic acid (AA), eicosapentaenoic acid (EPA), while some barophylic bacteria produce EPA and/or docosahexaenoic acid. FA analysis of deep-sea invertebrates has shown high levels of PUFA including, in particular, arachidonic acid, bacterial FA, and a vast number of new and uncommon fatty acids such as 21:4(n-7), 22:4(n-8), 23:4(n-9), and 22:5(n-5) characteristic of foraminifera. We suppose that bacteria growing on detritus having a low trophic value provide the first trophic upgrading of organic matter for foraminifera and nematodes. In turn, these metazoans perform the second-stage upgrading for megafauna invertebrates. Deep-sea megafauna, including major members of Echinodermata, Mollusca, and Polychaeta display FA markers characteristic of bacteria, foraminifera, and nematodes and reveal new markers in the food chain.

New Conjugates of Polyhydroxysteroids with Long-Chain Fatty Acids from the Deep-Water Far Eastern Starfish Ceramaster patagonicus and Their Anticancer Activity.

Four new conjugates, esters of polyhydroxysteroids with long-chain fatty acids (1-4), were isolated from the deep-water Far Eastern starfish Ceramaster patagonicus. The structures of 1-4 were established by NMR and ESIMS techniques as well as chemical transformations. Unusual compounds 1-4 contain the same 5α-cholestane-3ß,6ß,15α,16ß,26-pentahydroxysteroidal moiety and differ from each other in the fatty acid units: 5'Z,11'Z-octadecadienoic (1), 11'Z-octadecenoic (2), 5'Z,11'Z-eicosadienoic (3), and 7'Z-eicosenoic (4) acids. Previously, only one such steroid conjugate with a fatty acid was known from starfish. After 72 h of cell incubation, using MTS assay it was found that the concentrations of compounds 1, 2, and 3 that caused 50% inhibition of growth (IC50) of JB6 Cl41 cells were 81, 40, and 79 µM, respectively; for MDA-MB-231 cells, IC50 of compounds 1, 2, and 3 were 74, 33, and 73 µM, respectively; for HCT 116 cells, IC50 of compounds 1, 2, and 3 were 73, 31, and 71 µM, respectively. Compound 4 was non-toxic against tested cell lines even in three days of treatment. Compound 2 (20 µM) suppressed colony formation and migration of MDA-MB-231 and HCT 116 cells.

Fatty Acids of Abyssal Echinodermata, the Sea Star Eremicaster vicinus and the Sea Urchin Kamptosoma abyssale: A New Polyunsaturated Fatty Acid Detected, 22:6(n-2).

Fatty acids (FA) of two species of abyssal Echinodermata-the sea star (Asteroidea) Eremicaster vicinus and the sea urchin (Echinoidea) Kamptosoma abyssale-from the Kuril-Kamchatka Trench, collected at depths of 5210 and 6183 m, were analyzed. Lipids of these deposit-feeding animals showed similar FA compositions: 20.05-16.08% saturated, 42.20-39.50% monoenoic, and 37.75-44.42% polyunsaturated FA, respectively. The contents of odd- and branched-chain FA were 17.35% and 8.80%, respectively. A significant part of FA was represented by uncommon FA such as 21:4(n-7), 22:4(n-8), 22:5(n-5), and 23:4(n-9), earlier discovered in deep-sea foraminifera. Also, the newly found acid, related to the ω2 family, Δ5,8,11,14,17,20-22:6(n-2), amounted 1.60% and 0.33% of total FA, respectively, for the two species. Such unusual FA composition of these abyssal species can be explained by the transfer and modification of FA from consumed foraminifera, which in turn, feed on bacteria.

Variability of fatty acid profiles in ferns: Relation to fern taxonomy and seasonal development.

Ferns are known to contain long chain polyunsaturated fatty acids which may provide health benefits. The objective of this study was to investigate ferns of Pacific temperate regions (Far East of Russia and New Zealand) as sources of valuable fatty acids: arachidonic (20:4n-6) and eicosapentaenoic (20:5n-3). Fatty acids were analyzed in fronds of 23 fern species from 12 families. Major fatty acids include: 18:3n-3 (6-68% of total fatty acids), 16:0 (6-33%), 18:2n-6 (5-46%), 18:1n-9 (1-60%), 20:4n-6 (1-16%). Polyunsaturated fatty acids of fern fronds belong to the omega-6 (16:2n-6, 18:2n-6, 18:3n-6, 20:2n-6, 20:3n-6, 20:4n-6) and omega-3 (16:3n-3, 18:3n-3, 18:4n-3, 20:3n-3, 20:4n-3, 20:5n-3) families. For the first time, Δ5-unsaturated polymethylene-interrupted fatty acids were reported for ferns: sciadonic (5,11,14-20:3) and juniperonic (5,11,14,17-20:4) acids (up to 1.9% and 0.4%, respectively). Fatty acid profiles in fern fronds were unrelated to fern taxonomy, but affected by spore presence: fronds with sporangia/spores contained more 18:1n-9 and/or 18:2n-6. The absolute content of 20:4n-6 was found to be relatively constant for a species in different seasons. 20:5n-3 was a minor fatty acid (traces-5%) which accumulates during the vegetation period. Young fronds of the New Zealand ferns Phymatosorus pustulatus and Pteridium esculentum were enriched in 20:4n-6, while aged fronds of Cyathea dealbata had the highest level of 20:5n-3. The mature fronds of the Far Eastern ferns Phegopteris connectilis, Dryopteris expansa, and Athyrium sinense were also enriched in 20:5n-3.

New and Uncommon Fatty Acids in Lipids of Deep-Sea Foraminifera.

Fatty acids (FA) of agglutinating foraminifera of two genera, Bathysiphon and Rhabdammina, collected from a depth of 3307-3377 m in the Kuril Basin, Sea of Okhotsk, and adjacent deep-sea waters of the Pacific Ocean, were analyzed. In lipids of two Bathysiphon species, in addition to common FA typical for marine organisms, the uncommon ∆4 and ∆7 monoenoic acids and ∆4,7-dienoic acid were found in noticeable amounts. FA of two Rhabdammina species included also such uncommon acids as ∆5,8,11,14-21:4, ∆5,8,11,14-22:4, and ∆5,8,11,14-23:4, which are homologues to arachidonic acid. High levels of cis-vaccenic and arachidonic acids were typical for lipids of all the studied species. At the same time, we observed a substantial difference in FA composition between members of these two genera. Using GC and GC-MS of FAME, pyrrolidide and DMOX derivatives, the structures of 10 new FA were confirmed: ∆4-i-16:1 (1.1-0.6%), ∆7-i-21:1 (0.4%), ∆7-ai-21:1 (0.3-0.8%), ∆4,11-18:2 (0.3-0.9%), ∆4,7-20:2 (8.8%), ∆7,12-20:2 (0.4-0.6%), ∆4,7-21:2 (0.6%), ∆7,12-22:2 (6.4-2.1%); ∆5,8,11,14-22:4 (1.2-2.0%), and ∆5,8,11,14-23:4 (1.7-2.3%). The origin of the new FA and the role of foraminifera FA in deep-sea communities are discussed.

Fatty acid, lipid class, and phospholipid molecular species composition of the soft coral Xenia sp. (Nha Trang Bay, the South China Sea, Vietnam).

The soft corals of the genus Xenia are common for Indo-Pacific reef ecosystems. Lipid class, fatty acid (FA), phospho- and phosphonolipid molecular species compositions were identified for the first time in the soft coral Xenia sp. from Vietnam. Total lipids consisted predominantly of waxes, monoalkyl diacylglycerols, triacylglycerols, sterols, and polar lipids (21.4, 7.7, 14.2, 10.5, and 36.7 %, respectively). Sesquiterpene alcohol, valerenenol, was found. Acids 16:0, 18:3n-6, 20:4n-6, and 20:5n-3 dominated in total FA. The markers of zooxanthellae (18:4n-3 and 18:5n-3) and octocorals (24:5n-6 and 24:6n-3) were detected. Acids 18:5n-3, 20:4n-6, 22:4n-6, and 24:5n-6 concentrated in FA of polar lipids, whereas 14:0, 16:0, 16:1n-7, 18:2n-6, and 18:3n-6 were the major FA of neutral lipids. ChoGpl, EtnGpl, SerGpl, CAEP, PtdIns, and lyso ChoGpl constituted 39.5, 20.8, 20.5, 9.7, 4.3, and 5.3 %, respectively, of the sum of phospho- and phosphonolipids. Thirty-two molecular species of phospholipids and ceramide aminoethylphosphonate (CAEP) were determined by high resolution tandem mass spectrometry. Lyso 18:0e PakCho (4.1 %), 18:0e/20:4 PakCho (20.5 %), 18:1e/20:4 PlsEtn (18.0 %), 18:0e/24:5 PakSer (14.0 %), and 16:0 CAEP (9.6 %) were the major molecular species. EtnGpl and PtdIns mainly consisted of alkenyl acyl and diacyl forms, respectively. Alkyl acyl forms predominated in ChoGpl and SerGpl. Acid 24:5n-6 was a principal FA in SerGpl, whereas 20:4n-6 was more abundant in ChoGpl and EtnGpl. PtdIns contained various C20-24 PUFA. In the context of chemotaxonomy of corals, Xenia sp. has the lipid composition typical for soft corals and the FA profile similar to that of alcyonarians with the high level of 18:3n-6.

Isomerization of octadecapentaenoic acid (18:5n-3) in algal lipid samples under derivatization for GC and GC-MS analysis.

During gas chromatography (GC) analysis of fatty acid (FA) composition of the dinoflagellate Gymnodinium kowalevskii, we found unex-pectedly low and irreproducible content of all-cis-3,6,9,12,15-octadecapentaenoic acid (18:5n-3), which is an important chemotaxonomic marker of several classes of microalgae. We compared chromatographic behavior of 18:5n-3 methyl ester and other GC derivatives obtained using different conventional methods of derivatization. The use of methods based on saponification or base-catalyzed transesterification resulted in a mixture of double-bond positional isomers of 18:5. On a SUPELCOWAX 10 column, the equivalent chain length (ECL) value for authentic 18:5n-3 methyl ester was 20.22, whereas the main component after base-catalyzed methylation had ECL 20.88. Attempts to prepare N-acyl pyrrolidides or 4,4-dimethyloxazoline (DMOX) derivatives of 18:5n-3 also gave inadequate results. These derivatives also showed a main peak corresponding to isomerized 18:5. Mass spectra for both DMOX and pyrrolidide derivatives of this compound showed the base peak at m/z 139, probably corresponding to 2,6,9,12,15-18:5 acid. Of all methods tested for methylation, only derivatization with 5% HCl or 1% sulphuric acid in methanol gave satisfactory results. Therefore, GC or GC-mass spectrometry analyses of algal lipids containing 18:5n-3 may be inaccurate when base-catalyzed methods of FA derivatization are applied. The best and simplest way to avoid incorrect GC results is to use standard acid-catalyzed methylation.

Poseidonocella pacifica gen. nov., sp. nov. and Poseidonocella sedimentorum sp. nov., novel alphaproteobacteria from the shallow sandy sediments of the Sea of Japan.

The taxonomic study of two Gram-negative, aerobic, non-pigmented bacteria KMM 9010(T) and KMM 9023(T) isolated from a sandy sediment sample collected from the Sea of Japan seashore was performed. On the basis of the nearly complete 16S rRNA gene sequences, strains KMM 9010(T) and KMM 9023(T) clustered with the Roseobacter lineage (class Alphaproteobacteria) forming a distinct phylogenetic line adjacent to the genus Donghicola. Novel strains shared the highest sequence similarity of 96.4% to each other and lower than 96.1% similarities to other validly named genera of the class Alphaproteobacteria. In both strains, ubiquinone Q-10 was found to be the major respiratory quinone; phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidic acid, and an unknown aminolipid were the major polar lipids and C(18:1)ω7c and 11-methyl C(18:1)ω7c were predominant fatty acids. The DNA G+C content was 60.5 mol% (KMM 9010(T)) and 65.4 mol% (KMM 9023(T)). Based on phenotypic properties and phylogenetic evidence, strains KMM 9010(T) and KMM 9023(T) should be classified as two novel species in a new genus, Poseidonocella gen. nov., with Poseidonocella pacifica sp. nov., the type species with the type strain KMM 9010(T) (= NRIC 0794(T) = JCM 17310(T)), and Poseidonocella sedimentorum sp. nov. as the second species with the type strain KMM 9023(T) (= NRIC 0796(T) = JCM 17311(T)).

Mild method for preparation of 4,4-dimethyloxazoline derivatives of polyunsaturated fatty acids for GC-MS.

A mild and convenient method has been developed for preparing 4,4-dimethyloxazoline (DMOX) derivatives of fatty acids for GC-MS analysis. First, fatty acid methyl esters are converted to corresponding amides by incubation overnight at room temperature with 2-amino-2-methyl-1-propanol and a catalytic amount of sodium methoxide. The resulting 2-(methylpropanol) amides were isolated by partition between hexane-diethyl ether and water, and then converted to 4,4-dimethyloxazoline derivatives by treatment with trifluoroacetic anhydride under mild conditions (50 °C for 45 min). Structures of 2-methylpropanol amide and a DMOX derivative of oleic acid were confirmed by GC-MS. This method was applied to different FAME prepared from animal, plant or microbial lipids. The suggested method is most suitable for structure analysis of polyunsaturated fatty acids (PUFA) and for acids with double bonds in close to terminal positions. Application of the method is illustrated with spectra of the DMOX derivatives of 16:1(n-13), 24:5(n-6) and 24:6(n-3) acids.

Vadicella arenosi gen. nov., sp. nov., a novel member of the class Alphaproteobacteria isolated from sandy sediments from the Sea of Japan seashore.

A taxonomic study of three aerobic, Gram-negative, non-pigmented, non-motile rod-shaped bacterial strains, designated KMM 9008, KMM 9017, and KMM 9024(T), which were isolated from a sandy sediment sample collected from the Sea of Japan seashore, was undertaken. The DNA-DNA hybridization values of 88-96% obtained between novel strains confirm their assignment to the same species. An analysis of the nearly complete 16S rRNA gene sequences showed that the novel isolates were closely related to each other (99.6-100% sequence similarity) and shared highest sequence similarities to the described genera Celeribacter (96.2-95.9%), Pseudoruegeria (95.6-94.3%), and Thalassobacter (95.2-93.1%) within the class Alphaproteobacteria. The major isoprenoid quinone was Q-10, polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, an unknown aminolipid and an unknown lipid as prevalent, and phosphatidylethanolamine was a minor component, and major fatty acids were C(18:1) ω7c , followed by 11-Methyl C(18:1) ω7c, C(12:1) and C(10:0) 3-OH in all strains. The DNA G+C content of strains KMM 9008, KMM 9017, and KMM 9024(T) was in the range of 56.7-60 mol%. Based on distinctive phenotypic characteristics and phylogenetic distance, strain KMM 9024(T) (=NRIC 0787(T) = JCM 17190(T)) represents the type strain of a novel species in a novel genus, for which the name Vadicella arenosi gen. nov., sp. nov. is proposed.

Primorskyibacter sedentarius gen. nov., sp. nov., a novel member of the class Alphaproteobacteria from shallow marine sediments.

Two gram-negative, aerobic, non-pigmented, non-motile, rod-shaped bacteria, strains KMM 9015 and KMM 9018(T), were isolated from a sample of shallow sediment collected from the Sea of Japan. An analysis of the nearly complete 16S rRNA gene sequences showed that the isolates were very close to each other phylogenetically (99.9 % sequence similarity) and their close relatives were Marinovum algicola FF3(T) (95.8 and 95.9 %, respectively) and members of the genera Leisingera (95.7-95.1 and 95.8-95.2 %), Phaeobacter (95.0-94.2 and 95.1-94.2 %) and Thalassobius (96.3-94.8 and 96.2-94.7 %) of the class Alphaproteobacteria. In phylogenetic trees based on 16S rRNA gene sequences, strains KMM 9015 and KMM 9018(T) were positioned as a distinct phylogenetic line adjacent to Marinovum algicola. The major isoprenoid quinone was Q-10, the polar lipids consisted of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unknown lipid and the major fatty acid was C(18 : 1)ω7c, followed by 11-methyl C(18 : 1)ω7c, in both strains. The DNA G+C contents of strains KMM 9015 and KMM 9018(T) were 60.2 and 61.9 mol%, respectively. Based on distinctive phenotypic characteristics and phylogenetic analysis, strains KMM 9015 and KMM 9018(T) represent a novel species in a novel genus, for which the name Primorskyibacter sedentarius gen. nov., sp. nov. is proposed. The type strain of Primorskyibacter sedentarius is strain KMM 9018(T) ( = NRIC 0784(T)  = JCM 16874(T)).

Pacificibacter maritimus gen. nov., sp. nov., isolated from shallow marine sediment.

An aerobic, Gram-stain-negative, non-pigmented, non-motile bacterium, strain KMM 9031(T), was isolated from a sandy sediment sample collected from the shore of the Sea of Japan and subjected to phenotypic and phylogenetic analysis. Based on comparative 16S rRNA gene sequence analysis, strain KMM 9031(T) constituted a separate phylogenetic line within the Roseobacter clade of the class Alphaproteobacteria, sharing highest sequence similarities with members of the genera Roseovarius (92.7-95.3 %), Pseudoruegeria (94.5 %), Sulfitobacter (92.7-94.4 %) and Thalassobacter (94.2-94.3 %). The predominant fatty acid of strain KMM 9031(T) was C(18 : 1)ω7c, with C(16 : 0), C(10 : 0) 3-OH and C(12 : 1) 3-OH present in lesser amounts. The DNA G+C content of the isolate was 52.6 mol%. The major isoprenoid quinone was Q-10 and polar lipids comprised phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol and two unknown lipids. On the basis of phylogenetic analysis and physiological and biochemical characterization, strain KMM 9031(T) represents a novel species in a new genus, for which the name Pacificibacter maritimus gen. nov., sp. nov. is proposed; the type strain is KMM 9031(T) ( = NRIC 0785(T)  = JCM 17096(T)).