Purification and in vitro cultivation of archaeocytes (stem cells) of the marine sponge <Emphasis Type="Italic">Hymeniacidon perleve</Emphasis> (Demospongiae)

Marine sponges (Porifera) are the best source of marine bioactive metabolites for drug discovery and development, although the sustainable production of most sponge-derived metabolites remains a difficult task. In vitro cultivation of sponge cells in bioreactors has been proposed as a promising technology. However, no continuous cell line has as yet been developed. Archaeocytes are considered to be toti/multipotent stem cells in sponges and, when purified, may allow the development of continuous sponge cell lines. As a prerequisite, we have developed a novel four-step protocol for the purification of archaeocytes from a marine sponge, Hymeniacidon perleve: (1) differential centrifugation to separate large sponge cells including archaeocytes; (2) selective agglomeration in low-Ca²⁺/Mg²⁺ artificial seawater in which living archaeocytes form small loose aggregates with some pinacocytes and collencytes; (3) differential adherence to remove anchorage-dependent pinacocytes, collencytes and other mesohyl cells; (4) Ficoll-Vrografin density gradient centrifugation to purify archaeocytes. The final purity of archaeocytes is greater than 80%. The proliferation potential of the archaeocytes has been demonstrated by high levels of BrdU incorporation, PCNA expression and telomerase activity. In 4-day primary cultures, the purified archaeocytes show a 2.5-fold increase in total cell number. This study opens an important avenue towards developing sponge cell cultures for the commercial exploitation of sponge-derived drugs. The authors are grateful for the financial support of the Chinese Academy of Sciences under the “100 Talent Project”, the “Innovation Fund” from the Dalian Institute of Chemical Physics, the “Hi-Tech Research and Development Program of China” (2001AA620404), and the European Commission (project: Silicon Biotechnology).     

Bioactive natural products from marine sponges and fungal endophytes

This review highlights recent findings of our group on bioactive marine natural products isolated from marine sponges and marine derived fungi. The activated chemical defence of the Mediterranean sponge Aplysina aerophoba is introduced as an example of a dynamic response of marine sponges to wounding. Following tissue disrupture preformed brominated isoxazoline alkaloids are enzymatically cleaved and thereby give rise to aeroplysinin-1 which is believed to protect sponges from invasion of pathogenic bacteria. A preliminary characterization of the membrane bound enzyme(s) involved in the cleavage reaction is presented. Bromotyrosine derived, oxime group bearing peptides, the so called bastadins, obtained from the sponge Ianthella basta and some of their synthetic derivatives were shown to exhibit pronounced antifouling activity against larvae of the barnacle Balanus improvisus. The antifouling activity could be traced to the oxime group as an important pharmacophore that was also found to be present in other sponge derived natural products exhibiting antifouling activity. Marine derived fungi that reside within invertebrates such as sponges or inside Mangrove plants are emerging as a new source of bioactive metabolites as demonstrated for Aspergillus ustus and Alternaria sp. that were isolated from the sponge Suberites domuncula or from the Mangrove plant Sonneratia alba, respectively. The former fungus yielded new moderately cytotoxic sesquiterpenoids of the drimane type whereas the latter was found to produce polyketides such as alternariol that exhibited strong and selective inhibitory activity against several protein kinases, for instance Aurora A and B which are targets for anticancer chemotherapy.     

Attachment of marine sponge cells of Hymeniacidon perleve on microcarriers

Toward the development of an in vitro cultivation of marine sponge cells for sustainable production of bioactive metabolites, the attachment characteristics of marine sponge cells of Hymeniacidon perleve on three types of microcarriers, Hillex, Cytodex 3, and glass beads, were studied. Mixed cell population and enriched cell fractions of specific cell types by Ficoll gradient centrifugation (6%/8%/15%/20%) were also assessed. Cell attachment ratio (defined as the ratio of cells attached on microcarrier to the total number of cells in the culture) on glass beads is much higher than that on Cytodex 3 and Hillex for both mixed cell population and cell fraction at Ficoll 15-20% interface. The highest attachment ratio of 41% was obtained for the cell fraction at Ficoll 15-20% interface on glass beads, which was significantly higher than that of a mixed cell population (18%). The attachment kinetics on glass beads indicated that the attachment was completed within 1 h. Cell attachment ratio decreases with increase in cell-to-microcarrier ratio (3-30 cells/bead) and pH (7.6-9.0). The addition of serum and BSA (bovine serum albumin) reduced the cell attachment on glass beads.     

Distribution of brominated compounds within the sponge<Emphasis Type="Italic"> Aplysina aerophoba</Emphasis>: coupling of X-ray microanalysis with cryofixation techniques

The major secondary metabolites of the sponge Aplysina aerophoba are brominated compounds. X-ray energy dispersive microanalysis was therefore used to locate secondary metabolites via the Br signal in energy emission spectra from sponge sections. To test the reliability of this method in the face of the loss or redistribution of metabolites during processing, we compared the results obtained by conventional aldehyde fixation with those obtained by cryofixation and cryosubstitution with and without cryoembedding. Bromine appeared to be concentrated in two sponge structures, viz. fibres and spherulous cells, when cryofixed material was examined. However, X-ray microanalysis failed to demonstrate the presence of bromine in spherulous cells in chemically fixed samples, showing the need for cryotechniques to avoid the loss of compounds. Cryofixation plus cryosubstitution methods performed best regarding structural preservation and the immobilization of metabolites. The presence of bromine in the spherulous cells suggests that this cell type is the producer of the secondary metabolites, as described for other sponge species. Nevertheless, the presence of bromine in sponge fibres indicates that they can accumulate metabolic substances, although we have been unable to assess whether the chemicals are in their original form or in a modified state within the fibres. A. aerophoba has both bacterial and cyanobacterial symbionts in its mesohyl; the absence of brominated compounds in them contrasts with previous findings in other sponges with prokaryote symbionts.     

A skeleton‐less sponge of Caribbean mangroves: invasive or undescribed?

Recent surveys of sponges occurring on Caribbean mangrove roots demonstrated the presence of a skeleton‐less sponge of the genus Halisarca, very similar in its morphology to the temperate H. dujardinii. This study evaluated the possibility that the mangrove sponge was actually H. dujardinii that had been introduced into the Caribbean mangroves. Detailed histology revealed differences between the mangrove sponge and H. dujardinii in cuticle thickness, and in characteristics of the choanocytes, spherulous, and granular cells. Also, phylogenetic reconstruction and genetic distance estimates based on cytochrome oxidase I gene sequences clearly differentiated the mangrove Halisarca sp. from H. dujardinii. Therefore, we rejected the hypothesis of the invasion of H. dujardinii, recognizing instead the mangrove Halisarca sp. as a new species and naming it H. restingaensis sp. nov. Estimated levels of genetic variation in the ribosomal internal transcribed spacers indicated that populations of H. restingaensis sp. nov. are highly differentiated between Venezuela and Panama (F_st=0.71). This level of population differentiation is consistent with the short larval competence period that is common in members of the genus Halisarca.     

Larval cells of sponge Halisarca dujardini (Demospongiae, Halisarcida). II. Some cell types marked by polyclonal antibodies

The recent morphological and experimental data concerning the involvement of flagellated cells in sponge larvae are contradictory and testify to or against the germinal layers inversion. A study of morphogenetic processes in sponges, in particular larval metamorphosis, is complicated by difficulties in identification and succession of certain cell types. It is possible to trace the destiny of flagellated and other larval cells by marking them with antibodies (AB) specified for each cell type. We separated larval and adult sponge cells of Halisarca dujardini in percoll density gradient and obtained polyclonal AB for the majority of these cell types. The protein pattern of larval flagellated cells differed significantly from that of other cell types. The major proteins of flagellated, collencyte-like and spherulous cells were used to raise the corresponding AB. Immunoblot showed all AB to be specific for certain proteins and suitable for immunofluorescence. The AB for flagellated cells reacted with the apical cytoplasm, but not with the flagellum, the AB for major protein of collencyte-like cells stained cytoplasm granules. The AB for spherulous cells of the adult sponge reacted with larval spherulous cells supposed to be of maternal origin. So, the method of cell marking with specific polyclonal AB can facilitate analysis of the layers inversion problem, as well as elucidate the degree of cell differentiation in larvae, their conformity to cells of the adult sponge or their provisional destiny.     

Localization of Two Brominated Metabolites,Aerothionin and Homoaerothionin,in Spherulous Cells of the Marine Sponge Aplysina fistularis (=Verongia thiona)

Energy dispersive X-ray microanalysis was used to localize the two brominated natural products (aerothinonin and homoaerothionin) in the tissues of a marine demosponge, Aplysina fistularis. Virtually all of these compounds were localized within the spherules of the spherulous cells in the mesohyl. This is the first localization of any secondary metabolite at the cellular or sub-cellular level in any marine invertebrate. In Aplysina fistularis, as in other species of the same genus studied by Vacelet, the spherulous cells are concentrated just beneath the exopinacoderm and just beneath the endopinacoderm of the excurrent canals. Moreover, there is electron microscopic evidence for degeneration of some spherulous cells throughout the mesohyl. Presumably, this degeneration can release some aerothionin and homoaerothionin, which are known to have antibiotic properties. After release from the spherulous cells, these brominated natural products could function (1) within the mesohyl to exclude some types of bacteria or to aggregate ingested bacteria and/or (2) within the boundary layer of the surrounding seawater for defense or offense, as considered in the discussion section.     

<Emphasis Type="Italic">Hymeniacidon perleve</Emphasis> associated bioactive bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. NJ6-3-1

Among marine bacteria isolated from the cytotoxic sponge Hymeniacidon perleve, one strain NJ6-3-1 classified as Pseudomonas sp. showed both cytotoxic and antimicrobial activities. Fatty acid analysis indicated that the bacterial strain consists mainly of C16:1, C16:0, C18:1, C18:0, C15:0, C14:0. One unusual 9,10-cyclopropane-C17:0 fatty acid and C26:0 also constitute major components, as well as the existence of squalene, the precursor of triterpenoids. The major metabolites in the culture broth were identified as alkaloids, including diketopiperazines and indole compounds, namely 3,6-diisopropylpiperazine-2,5-dione, 3-benzyl-3-isopropylpiperazine-2,5-dione, 3,6-bis-(2-methylpropyl)-piperazine-2,5-dione, indole-3-carboxaldehyde, indole-3-carboxylic acid methyl ester, indole-3-ethanol, and quinazoline-2,4-dione.From Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 35–39.Original English Text Copyright © 2005 by Li Zheng, Xiaojun Yan, Jilin Xu, Haimin Chen, Wei Lin.This article was submitted by the authors in English.     

Antimicrobial activity of marine sponge <Emphasis Type="Italic">Clathria indica</Emphasis> (Dendy, 1889)

Sponges are sessile filter feeders that have developed efficient defense mechanisms against foreign invaders such as viruses, bacteria or eukaryotic organisms. Antimicrobial peptides are known as major components of the innate immune defense system in marine invertebrates. The aim of the present work was to study the antimicrobial properties of the Indian sponge Clathria indica with special reference to the identification of antimicrobial peptides. Crude methanolic extract and its chloroform, n-butanol and aqueous fractions were tested against 16 human pathogens which include eleven bacteria with four of them being multidrug resistant and five pathogenic fungi. All fractions showed effective antibacterial activity against common and multidrug-resistant Salmonella typhi and antifungal activity against C. albicans and C. neoformans. However, they were ineffective against Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes and Staphylococcus aureus. Chloroform fraction being the most potent among the fractions tested on chemical investigation was indicative of the presence of peptides as evidenced by ninhydrin positive spots on TLC and presence of peptide bonds by NMR. Its ESI-MS showed presence of several peptides in the range of m/z 850 to 980. Structure of three peptides has been tentatively assigned by ESI-MS/MS or tandem mass analysis, on the basis of the amino acid sequence established. The results clearly show that the sponge C. indica represent an interesting source of marine invertebrates-derived antimicrobial peptides in the development of new strategies to treat various infectious diseases.     

Characterization of adrenal medullary chromaffin cells by flow cytometry.

BACKGROUND: Adrenomedullary chromaffin cells are neural crest derivatives widely used as a model system to study neurosecretory mechanisms. Morphological, immunohistochemical, and functional data indicate that chromaffin cells are heterogeneous and support the distinction between adrenaline (A)- and noradrenaline (NA)-producing and secreting cells. The aim of this study was to characterize by flow cytometry the two main chromaffin cell subtypes in suspensions of cultured bovine chromaffin cells. METHODS: An indirect immunofluorescence method was used for the specific labeling of two intracellular enzymes, dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), involved in the synthesis of NA and A, respectively. Flow cytometry analysis of fluorescence labeling was performed in two chromaffin cell fractions differentially enriched in A-containing cells by centrifugation through density gradients. PNMT and DBH-related fluorescence was also correlated with the A and NA content of the cells assayed by HPLC measurements. RESULTS: No significant differences were found in forward-side scatter plots between the two cell fractions (A-enriched cells and mixed cells); however, the degree of labeling of the enzymes and the corresponding PNMT/DBH-related fluorescence ratio was significantly greater in the A-enriched cell fraction. The existence of changes in DBH and PNMT content of chromaffin cells over time (1 week) in culture was also examined. No significant variation in enzyme related fluorescence values was detected in any of the two cell fractions, and this result correlated well with HPLC determinations of the catecholamine content (A and NA) of the cells. CONCLUSIONS: Flow cytometry appears to be a useful technique to characterize chromaffin cell subtypes and to follow their phenotypic changes in response to growth factors.     

The pterocellins,bioactive alkaloids from the marine bryozoan <Emphasis Type="Italic">Pterocella vesiculosa</Emphasis>

Although comparatively little research has been undertaken into the secondary metabolites of bryozoans as compared with those of other marine invertebrates, bryozoans have proven to be an excellent source of novel and/or biologically active compounds. The majority of bryozoan metabolites isolated to date have been alkaloids. In our continuing search for bioactive and/or novel compounds from New Zealand marine bryozoans, we undertook an investigation of an extract of Pterocella vesiculosa (order Cheilostomatida, suborder Ascophorina, family Catenicellidae) which possessed activity against P388 murine leukaemia cells. Two alkaloids, pterocellins A–B (1–2) have been isolated from the bryozoan. The biological activity of these alkaloids was examined including their activities in the in vitro 60 cell line panel and in vivo hollow fibre assays at the National Cancer Institute (NCI). The isolation and characterisation of further pterocellin analogues is currently in progress and tentative structures for two new members of this series, pterocellins C–D (3–4) are proposed, based on NMR and mass spectral data.     

海绵生物活性物质及海绵细胞离体培养

介绍了来自海绵的生物活性物质种类、分布及其潜在的应用价值。讨论了其作为抗癌、抗病毒、抗细菌等药用的生物活性物质及其相关的海绵种属 ;强调海绵生物活性物质的商业化和临床应用所面临的“供给短缺问题”。作为解决这一问题的途径之一 ,海绵细胞离体培养是最有前景的技术。讨论了海绵细胞离体培养技术的研究现状 ,存在的问题及未来的发展趋势。对我国海域的海绵生物活性物质的研究开发现状进行总结 ,强调海绵研究对开发具有我国自主知识产权的新药、新化合物的必要性及重要性 ,并提出进行研发的可能优先领域     

Streptomyces associated with a marine sponge Haliclona sp.; biosynthetic genes for secondary metabolites and products

Terrestrial actinobacteria have served as a primary source of bioactive compounds; however, a rapid decrease in the discovery of new compounds strongly necessitates new investigational approaches. One approach is the screening of actinobacteria from marine habitats, especially the members of the genus Streptomyces. Presence of this genus in a marine sponge, Haliclona sp., was investigated using culture‐dependent and ‐independent techniques. 16S rRNA gene clone library analysis showed the presence of diverse Streptomyces in the sponge sample. In addition to the dominant genus Streptomyces, members of six different genera were isolated using four different media. Five phylogenetically new strains, each representing a novel species in the genus Streptomyces were also isolated. Polyphasic study suggesting the classification of two of these strains as novel species is presented. Searching the strains for the production of novel compounds and the presence of biosynthetic genes for secondary metabolites revealed seven novel compounds and biosynthetic genes with unique sequences. In these compounds, JBIR‐43 exhibited cytotoxic activity against cancer cell lines. JBIR‐34 and ‐35 were particularly interesting because of their unique chemical skeleton. To our knowledge, this is the first comprehensive study detailing the isolation of actinobacteria from a marine sponge and novel secondary metabolites from these strains.     

New phylogenetic lineages of the <Emphasis Type="Italic">Spirochaetes</Emphasis> phylum associated with <Emphasis Type="Italic">Clathrina</Emphasis> species (Porifera)

Though spirochetes have been repeatedly found in marine sponges and other invertebrates, little attention has been paid to the specificity of this association. This study demonstrates that different genoand morphotypes of spirochetes can reside within the same sponge individual and develop in considerable numbers. Specimens of the calcareous sponge Clathrina clathrus collected from the Adriatic Sea off Rovinj (Croatia) were found to harbor spirochete-like bacteria, which were characterized by scanning electron microscopy (SEM), 16S rRNA gene analysis, and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Two novel spirochete sequence types related to the Brachyspiraceae could be retrieved. By use of specifically designed CARD-FISH probes, the C. clathrus-associated sequences could be assigned to a linear and a helical spirochete morphotype. Both were located within the sponge mesohyl and resembled the spirochete-like cells identified by SEM. In addition, from a Clathrina sp., most likely C. coriacea, that originated from Indonesian coastal waters, four different spirochete type sequences were recovered. Two of these also affiliated with the Brachyspiraceae, the other two were found associated with the Spirochaetaceae, one with the genera Borrelia and Cristispira.     

Pyrinadines B-G, new bis-pyridine alkaloids with an azoxy moiety from sponge Cribrochalina sp

Six new cytotoxic bis-3-alkylpyridine alkaloids with an azoxy moiety, pyrinadines B–G (1–6), have been isolated from an Okinawan marine sponge Cribrochalina sp., and the structures were elucidated by spectroscopic data and chemical means.     

Antiangiogenic, Antimicrobial, and Cytotoxic Potential of Sponge-Associated Bacteria

The bacteria associated with marine invertebrates are a rich source of bioactive metabolites. In the present study bacteria associated with the sponge Suberites domuncula and its primmorphs (3-dimensional aggregates containing proliferating cells) were isolated and cultured. These bacteria were extracted, and the extracts were assayed for antiangiogenic, hemolytic, antimicrobial, and cytotoxic activities. Our studies revealed that extract obtained from the bacterium (PB2) isolated from sponge primmorphs is a potent angiogenesis inhibitor. In the chick chorio-allantoic membrane (CAM) assay, it showed 50% activity at 5 μg ml⁻¹ and 100% activity at 10 and 20 μg ml⁻¹ concentrations. Extracts obtained from 5 bacterial strains isolated from sponge and its primmorphs showed hemolytic activity. The sponge-associated bacteria belonging to the α subdivision of Proteobacteria and the primmorph-associated bacterium identified as a possible novel Pseudomonas sp. displayed remarkable antimicrobial activity. It is important to note that these bacterial extracts were strongly active against multidrug-resistant clinical strains such as Staphylococcus aureus and Staphylococcus epidermidis, isolated from hospital patients. The bacterial extracts having antimicrobial activity also showed cytotoxicity against HeLa and PC12 cells. In summary, this investigation explores the importance of sponge-associated bacteria as a valuable resource for the discovery of novel bioactive molecules.     

Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller)

The tropical marine sponge Dysidea herbacea (Keller) contains the filamentous unicellular cyanobacterium Oscillatoria spongeliae (Schulze) Hauck as an endosymbiont, plus numerous bacteria, both intracellular and extracellular. Archaeocytes and choanocytes are the major sponge cell types present. Density gradient centrifugation of glutaraldehyde-fixed cells with Percoll as the support medium has been used to separate the cyanobacterial symbiont from the sponge cells on the basis of their differing densities. The protocol also has the advantage of separating broken from intact cells of O. spongeliae. The lighter cell preparations contain archaeocytes and choanocytes together with damaged cyanobacterial cells, whereas heavier cell preparations contain intact cyanobacterial cells, with less than 1% contamination by sponge cells. Gas chromatography/mass spectrometry analysis has revealed that the terpene spirodysin is concentrated in preparations containing archaeocytes and choanocytes, whereas nuclear magnetic resonance analysis of the symbiont cell preparations has shown that they usually contain the chlorinated diketopiperazines, dihydrodysamide C and didechlorodihydrodysamide C, which are the characteristic metabolites of the sponge/symbiont association. However, one symbiont preparation, partitioned by a second Percoll gradient, has been found to be devoid of chlorinated diketopiperazines. The capability to synthesize secondary metabolites may depend on the physiological state of the symbiont; alternatively, there may be two closely related cyanobacterial strains within the sponge tissue.     

Chemical Constituents of the Vietnamese Marine Sponge Gelliodes sp. and Their Cytotoxic Activities

A new decenoic acid derivative, gelliodesinic acid, and a naturally new alkaloid, together with three known furanoterpenoids and two known indole alkaloids, were isolated from the MeOH extract of the marine sponge Gelliodes sp. collected in Vietnam. The chemical structures of the isolated compounds were determined by analyses of 1D‐ and 2D‐NMR and MS data and by comparisons of the data with those reported in the literature. The cytotoxicity assay against HeLa, MCF‐7, and A549 cancer cell lines revealed that the three known furanoterpenes exhibited cytotoxic activities with IC₅₀ values ranging from 23.6 to 75.5 μM against the three cell lines, and that 1H‐indole‐3‐carboxylic acid showed cytotoxicity with an IC₅₀ value of 89.2 μM against A549 cancer cell lines.     

Aggregative properties of different cell types of the fresh-water spongeEphydatia fluviatilis isolated on ficoll gradients

Summary Cell suspensions of the fresh-watersponge Ephydatia fluviatilis have been fractionated by means ofFicoll gradient centrifugation. Three fractions were isolated. The densest contains archeocyte-like cells only; the intermediate fraction is very rich in choanocytes, and the lightest is a mixture of cell types. Earch fraction shows specificaggregative properties and potentialities to reconstitute functional sponges.It appears that the sequence of reconstitution events can be selectively altered by certain disequilibria in the cell populationThese preliminary results constitute a first approach to the analysis ofcell type specificity in sponges.     

Disruption of N-acyl-homoserine lactone-specific signalling and virulence in clinical pathogens by marine sponge bacteria

In recent years, the marine environment has been the subject of increasing attention from biotechnological and pharmaceutical industries. A combination of unique physicochemical properties and spatial niche-specific substrates, in wide-ranging and extreme habitats, underscores the potential of the marine environment to deliver on functionally novel bioactivities. One such area of ongoing research is the discovery of compounds that interfere with the cell–cell signalling process called quorum sensing (QS). Described as the next generation of antimicrobials, these compounds can target virulence and persistence of clinically relevant pathogens, independent of any growth-limiting effects. Marine sponges are a rich source of microbial diversity, with dynamic populations in a symbiotic relationship. In this study, we have harnessed the QS inhibition (QSI) potential of marine sponge microbiota and through culture-based discovery have uncovered small molecule signal mimics that neutralize virulence phenotypes in clinical pathogens. This study describes for the first time a marine sponge Psychrobacter sp. isolate B98C22 that blocks QS signalling, while also reporting dual QS/QSI activity in the Pseudoalteromonas sp. J10 and ParacoccusJM45. Isolation of novel QSI activities has significant potential for future therapeutic development, of particular relevance in the light of the pending perfect storm of antibiotic resistance meeting antibiotic drug discovery decline.