Isolation of (Z)-17-tetracosenal from the marine sponge Halichondria panicea

The isolation and characterization from Halichondria panicea of the long chain aldehyde (Z)-17-tetracosenal (1), one of the longest ever isolated from a marine sponge, is described. Structural assignment of the compound was confirmed by spectroscopy (1H-NMR and MS) as well as by selective ozonolysis.     

Fatty acids from the sponge Halichondria panicea from the Sea of Japan

The fatty acid (FA) composition of total lipids isolated from the marine sponge Halichondria panicea inhabiting Peter the Great Bay of Sea of Japan was studied. GC and GC-MS techniques helped identify 63 FAs, with the main attention being paid to FAs with 14-22 carbon atoms. 4, 8, 12-Trimethyl-13:0 FA was for the first time identified as the main saturated FA along with the branched FAs br-25:1, br-27:1, and br-27:2. The contents of arachidonic, eicosapentaenoic, docosapentaenoic, and the major demospongic acids [26:3(5, 9, 19), 26:3(5, 9, 17), 27:3(5, 9, 20), and 28:3(5, 9, 21)] considerably differed from those previously found for H. panicea, which may be due to seasonal changes in the species composition of organisms consumed by the sponge.     

Preliminary study on the occurrence of brominated organic compounds in Dutch marine organisms

The extracts of three marine organisms; the ascidian Ciona intestinalis, the brown seaweed Sargassum muticum and the sponge Halichondria panicea, all elicited a number of brominated compounds, some of which were tentatively identified. Tribromophenol was observed in all species. This compound, also industrially produced as flame retardant and fungicide, was likely due to endogenous production.     

Bacterial diversity in the breadcrumb sponge Halichondria panicea (Pallas)

The aim of this study was to investigate the diversity and variability of bacterial communities associated with the marine sponge Halichondria panicea with respect to tissue compartmentalization as well as seasonal and small-scale geographic variation. Diversity of microorganisms in sponges was investigated recently, but work on the variability and succession of associated bacterial communities is rare. Despite some information on Pacific and Mediterranean sponges, it is still uncertain whether bacteria and sponges are specifically associated. In this study, H. panicea specimens were sampled throughout the year at different stations around the island of Helgoland (North Sea) and investigated using molecular tools. The bacterial community associated with H. panicea was diverse, consisting of one denaturing gradient gel electrophoresis (DGGE) band occurring in most 'tissue' samples and additional variable bands. Variability was observed between different sponge fractions (i.e. the aquiferous system and the 'tissue'), sampling locations, and sampling dates. A PCR-DGGE specific for the Roseobacter group of marine Alphaproteobacteria displayed low diversity and a marked similarity between all samples. Phylogenetic analysis also pointed to specific Alphaproteobacteria of the Roseobacter group, which was predominant in most sponge 'tissue' samples. We conclude that H. panicea harbour a specific Roseobacter population with varying bacterial co-populations occurring seasonally or on a small-scale geographically, sometimes even dominating the bacterial community.     

Morphogenetic processes and seasonal changes in the anatomic and histological organization of the Barents Sea sponge Halichondria panicea (Pallas)

As a result of histological investigations of seasonal dynamics in the anatomical-tissue organization of the litoral sponge Halichondria panicea (Pallas), morphogenetical processes during different periods of its life cycle are described in detail. Correlations between different phases of sexual reproduction and conditions of anatomical and tissue organization in the maternal organism are stated. It is noted that tissue reduction of the definitive sponge Halichondria panicea during autumn--winter season is a natural phenomenon, regularly repeating in the life of the litoral sponge investigated, as a result of the maternal organism's "exhaustion" in the period of sexual embryogenesis.     

Isolation of 2-methyl branched unsaturated very long fatty acids from marine sponge Halichondria panicea and identification of them by GC-MS and NMR

In order to study the biomarker fatty acids of symbionts in the marine sponge Halichondria panicea, purification and structural identification of two new 2-methyl branched monoenoic very long fatty acids (2-Me-24:1 n-7 and 2-Me-26:1 n-9) were performed for the first time. These acids amounted to 7.1% of total sponge FAs, but our attempts to determine their structures by one-step GC-MS analysis were unsuccessful because of low yields of the correspondent N-acyl pyrrolidide derivatives. Silver-ion thin-layer chromatography isolated enriched fractions of monoenoic fatty acids extracted from the sponge. Further purification of unknown fatty acid methyl esters was carried out by reversed-phase high-performance liquid chromatography. Determination of the chain length, degree and position of unsaturations was achieved by gas chromatography-mass spectrometry on methyl esters and dimethyldisulfide adducts. Structures, position of methyl substitution, and double bonds cis isomery were confirmed by 1H nuclear magnetic resonance.     

Animals associated with sponges at North Hayling, Hampshire

Sponges were collected over a period of two and a half years from the shore at North Hayling, Hampshire and their associated faunas studied.
Species of nematode, annelids, crustaceans, pyenogonids, echinoderms and fish associate with sponges Halichondria panicea, Hymeniacidon perleve and Mycale macilenta; other sponge species attract noticably very few. No animals were sponge specific.     

Cross-species induction of antibacterial activity produced by epibiotic bacteria isolated from Indian marine sponge Pseudoceratina purpurea

Four antibiotic producing bacteria were isolated from the surface of the marine sponge Pseudoceratina purpurea and exposed to living cells of two human pathogenic bacteria as well as some marine fouling bacteria to induce the production of antimicrobial activity. Experimental results showed that these four marine epibiotic bacteria enhanced their antibacterial production, when exposed to these test strains. The highest induction was exhibited by the sponge isolate PS79 against fouling bacterium FB-9 (from 3 mm to 7 mm inhibition zone). All the four strains were induced and showed increased activity specifically against the challenged pathogenic or fouling bacteria tested. Specific induction by these species suggests that the induction might be attributed to the response to the chemical signals received from potential challenger strains.     

In situ manifestations of nonself recognition between incrusting sponges

The present study investigates the morphological aspects of nonself recognition between three incrusting sponge species living in contact. Two types of association have been observed: nonmerging fronts and epizoism. In two of the three combinations studied, Hymeniacidon sanguinea/Halichondria panicea and Halichondria panicea/Ophlitaspongia seriata, nonself contacts consisted of either epizoism or of nonmerging fronts. In contrast, only nonmerging fronts were observed between Hymeniacidon sanguinea and Ophlitaspongia seriata. In the two types of associations, the main phenomenon consists of the isolation of the foreign sponges by a spongin barrier secreted by the partners in the zone of contact. The microscpic structure and the thickness of the barrier vary according to the type of association and the species paired, but they are clearly defined for a given combination. The conditions required for the establishment of epizoism and the existence of a natural xenogeneic tolerance between sponges are discussed.     

Axenic Cultivation of Entamoeba dispar Brumpt 1925, Entamoeba insolita Geiman and Wichterman 1937 and Entamoeba ranarum Grassi 1879

ABSTRACT. Three species of Entamoeba have been grown in axenic culture for the first time. In two cases, novel methods for adapting the organisms to growth without bacteria were employed. While E. ranarum was axenized by the classic technique of Diamond, from a monoxenic culture with Trypanosoma cruzi as the associate, both E. dispar and E. insolita were first grown in axenic culture medium supplemented with lethally irradiated bacteria. From there, E. insolita was axenized directly, but E. dispar initially required the presence of fixed bacteria. After prolonged culture under this technically axenic but unwieldy culture system, E. dispar was eventually adapted to growth in the absence of added bacteria.     

CvL, a lectin from the marine sponge Cliona varians: Isolation, characterization and its effects on pathogenic bacteria and Leishmania promastigotes

CvL, a lectin from the marine sponge Cliona varians was purified by acetone fractionation followed by Sepharose CL 4B affinity chromatography. CvL agglutinated papainized treated human erythrocytes with preference for type A erythrocytes. The lectin was strongly inhibited by monosaccharide d-galactose and disaccharide sucrose. CvL is a tetrameric glycoprotein of 28 kDa subunits linked by disulphide bridges with a molecular mass of 106 kDa by SDS-PAGE and 114 kDa by Sephacryl S300 gel filtration. The lectin was Ca2+ dependent, stable up to 60 degrees C for 60 min, with optimum pH of 7.5. CvL displays a cytotoxic effect on gram positive bacteria, such as Bacillus subtilis and Staphylococcus aureus. However, CvL did not affect gram negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa. Leishmania chagasi promastigotes were agglutinated by CvL up to 2(8) titer. These findings are indicative of the physiological defense roles of CvL and its possible use in the antibiosis of bacteria and protozoa pathogenic.     

Construction of a metagenomic library for the marine sponge Halichondria okadai

Symbionts of the marine sponge Halichondria okadai are promising as a source of natural products. Metagenomic technology is a powerful tool for accessing the genetic and biochemical potential of bacteria. Hence, we established a method of recovering bacterial-enriched metagenomic DNA by stepwise centrifugation. The metagenomic DNA was analyzed by ultrafast 454-pyrosequencing technology, and the results suggested that more than three types of bacterial DNA, Alphaproteobacteria, Actinobacteria, and Cyanobacteria, had been recovered, and that eukaryotic genes comprised only 0.02% of the metagenomic DNA. These results indicate that stepwise centrifugation and real-time quantitative PCR were effective for separating sponge cells and symbiotic bacteria, and that we constructed a bacteria-enriched metagenomic library from a marine sponge, H. okadai, selectively for the first time.     

Bacterial antigenic polysaccharides. 12. Structure and 13C NMR spectrum of the polysaccharide chain of Shigella boydii type 8 lipopolysaccharide

A specific acidic polysaccharide was isolated from Sh. boydii type 8 antigenic lipopolysaccharide after mild hydrolysis followed by chromatography on Sephadex G-50. The polysaccharide consists of D-glucuronic acid, D-galacturonic acid, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose and 2-amino-1,3-propanediol residues in 1:1:1:1:1 ratio. From the results of methylation analysis, partial acid hydrolysis and Smith degradation, the structure of the repeating unit of the specific polysaccharide was deduced as: (Formula: see text). The 13C NMR spectra of native, O-deacetylated and carboxyl-reduced polysaccharides, as well as the spectrum of oligosaccharide produced by Smith degradation were interpreted. The 13C NMR data fully confirmed the structure of the polysaccharide repeating unit.     

Structure and anticomplementary activity of an acidic polysaccharide from the leaves of Malva sylvestris var. mauritiana

A main acidic polysaccharide preparation was isolated from the leaves of Malva sylvestris L. var mauritiana Mill. It is composed of L-rhamnose, D-galactose, D-galacturonic acid, and D-glucuronic acid in the molar ratio of 22:6:22:11, and it contains 7.7% peptide. It was homogeneous by electrophoresis and gel chromatography, which gave a value of 11,000 as molecular weight. The structure of the polysaccharide component was elucidated by methylation analysis and partial hydrolysis. The substance showed considerable anticomplementary activity.     

Aggregation of sponge cells: 22. Species-specific reactivity of a lectin from the sponge Geodia cydonium

Single cells of the marine sponge Geodia cydonium aggregate species-specifically in the presence of a soluble aggregation factor to form large cell clumps. A lectin isolated from the same sponge species does not cause agglutination of Geodia cells but agglutinates only cells from heterologous species (e.g. Tethya lyncurium, Hemimycale columella, Pellina semitubulosa, Cacospongia scalaris, Verongia aerophoba). The process of agglutination is independent of divalent cations (they do not affect the agglutination process at concentrations up to 50 mM), occurs at 2°C, causes a reduction in the viability of the cells and results in an inhibition of programmed syntheses. The observed differences between the properties of cell agglutination (effect of a lectin in a heterologous system) and cell aggregation (effect of an aggregation factor in the homologous system) is discussed. Cell aggregation is dependent upon the presence of an aggregation factor, the presence of cations and an incubation temperature 2̃0°C; cell aggregation results in a stimulation of programmed syntheses. Cell agglutination requires a heterologous macromolecule (e.g. lectin), it is independent of divalent cations and causes inhibition of programmed syntheses in the cells.     

Evidence for rapid uptake of D-galacturonic acid in the yeast Saccharomyces cerevisiae by a channel-type transport system

D-Galacturonic acid is a major component of pectins but cannot be metabolized by Saccharomyces cerevisiae. It is assumed not to be taken up. We show that yeast displays surprisingly rapid low-affinity uptake of D-galacturonic acid, strongly increasing with decreasing extracellular pH and without saturation up to 1.5 M. There was no intracellular concentration above the extracellular level and transport was reversible. Among more than 160 single and multiple deletion mutants in channels and transporters, no strain was affected in D-galacturonic acid uptake. The uptake was not inhibited by any compound tested as candidate competitive inhibitor, including D-glucuronic acid, which was also transported. The characteristics of D-galacturonic acid uptake are consistent with involvement of a channel-type system, probably encoded by multiple genes.     

Isolation and characterization of okadaic acid binding proteins from the marine sponge Halichondria okadai

Okadaic acid, first isolated from the marine sponge Halichondria okadai, is a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A, respectively). Photoaffinity labeling experiments previously performed with biotinylated photoreactive okadaic acid revealed the presence of okadaic acid binding protein (OABP) in the crude extract of H. okadai. In this article, OABP1 and OABP2 were purified from H. okadai as guided by the binding affinity of [27-3H]okadaic acid. OABP1 has an approximate molecular mass of 37 kDa in SDS-PAGE analysis. Edman degradation followed by molecular cloning and sequencing identified OABP1 as being 88% identical to the rabbit PP2Abeta catalytic subunit. On the other hand, HPLC analysis revealed that OABP2 consists of three 22 kDa proteins (OABP2.1, OABP2.2, and OABP2.3). Electrospray ionization mass spectrometry indicated that OABP2.1 and OABP2.2 form a complex with okadaic acid. The complete amino acid sequence of OABP2, determined by Edman degradation and molecular cloning, showed that OABP2.1 is 96% identical to OABP2.2 and 66% identical to OABP2.3, while being very slightly homologous to any protein phosphatases known to date. OABP2 did not exhibit phosphatase activity, though it bound to okadaic acid with a Kd of 0.97 nM. Furthermore, OABP2 was not detected in the sponge Halichondria japonica or the dinoflagellate Prorocentrum lima. We thus speculated that OABP2 might be involved in detoxifying okadaic acid.     

Tauropine dehydrogenase from the marine sponge Halichondria japonica is a homolog of ornithine cyclodeaminase/mu-crystallin

The partial amino acid sequence including the N- and C-terminal portions of tauropine dehydrogenase (EC 1.5.1.23) from the marine sponge Halichondria japonica was determined by enzymatic cleavages followed by peptide sequencing. This information was used to design degenerate primers for amplification of cDNA encoding the tauropine dehydrogenase. The cDNA included 1231 nucleotides with an open reading frame of 1002 nucleotides that encodes a protein of 334 amino acid residues. From the peptide and nucleotide sequencing, the mature tauropine dehydrogenase was estimated to consist of 333 amino acid residues with an acetylated N-terminal serine residue and no intrachain disulfide bonds. The primary structure of the H. japonica enzyme showed apparent similarity with a homolog of ornithine cyclodeaminase from Rhizobium meliloti and other proteins of the ornithine cyclodeaminase/mu-crystallin family, but it showed no significant similarity with the known sequences of octopine dehydrogenases and tauropine dehydrogenases from marine invertebrates. These findings indicate that opine dehydrogenases in marine invertebrates are not all homologous.     

基于皱皮软海绵宏基因组的PKS基因筛选的研究

提取皱皮软海绵及其共附生微生物的宏基因组总DNA,使用聚酮合酶(PKS)基因的酮酰合酶(KS)域引物PCR扩增PKS基因片段获得一条671bp的片段,以pUCm-T vector为载体将该基因片段克隆到大肠杆菌中,从阳性克隆中分离出PKS基因片段,测序推导出氨基酸序列。通过BLAST比对发现此氨基酸序列与红细菌目的Rhodobacterales bacterium PKS基因KS域的氨基酸序列有96%的同源性。通过基于氨基酸序列的系统发育分析,推测此筛选得到的PKS基因属于trans-AT型。本文首次证实了皱皮软海绵中存在细菌来源的PKS基因。