温度对厚指海绵Pachychalina sp.体内真细菌的影响

摘要：温度是影响微生物多样性的重要因素之一。【方法】本研究采用16S rDNAs扩增产物酶切片段多态性（amplified rDNA restriction analysis , ARDRA）和16S rDNAs序列分析方法,对生长在16℃和30℃条件下厚指海绵Pachychalina sp.体内真细菌（eubacteria）的多样性进行了研究,【目的】探讨温度对海绵体内细菌的影响。【结果】根据ARDRA 聚类分析,16℃条件下海绵体内100个真细菌的16S rDNAs克隆片断被分成34个类群,而30℃条件下,海绵体内100个细菌的16S rDNAs克隆片断则被分为32个类群,说明在不同温度条件下,海绵体内真细菌的组成与群落结构有所变化,但研究发现温度没有明显改变海绵体内真细菌总的群落结构。【结果】根据厚指海绵Pachychalina sp.体内真细菌的16S rDNAs序列分析结果发现：在16℃和30℃条件下,海绵体内的真细菌均属于α、γ、δ-变形菌、硫细菌、硫还原菌和烃类分解菌,此外还有少数放线菌。16℃条件下海绵体内的优势菌为γ-变形菌,而且体内的硫细菌和硫还原菌主要是耐寒细菌,而30℃条件下海绵体内的优势菌为α-变形菌。该研究还发现：同一ARDRA类型的克隆序列分析表明在同一ARDRA群内各组分间彼此关系比较密切。     

Genotoxic evaluation of extracts from Aplysina fulva, a Brazilian marine sponge

A range of biologically active secondary metabolites with pharmacological application has been reported to occur in marine sponges. The present study was undertaken to provide a set of data on the safety of a hydro-alcoholic extract (ALE) and an aqueous fraction (AQE) from Aplysina fulva Pallas, 1766 (Aplysinidae, Verongida, Porifera). Salmonella typhimurium strains TA97, TA98, TA100 and TA102, Escherichia coli strains PQ65, OG40, OG100, PQ35 and PQ37 and Balb/c 3T3 mouse fibroblasts were used to detect induction of DNA lesions by ALE and AQE. Assays used for these analyses were a bacterial (reverse) mutation assay (Ames test), the SOS-chromotest and the comet assay. Both extracts presented identical infrared 2-oxazolidone spectra. ALE treatment induced a higher frequency of type-4 comets, indicative of increasing DNA migration, in the alkaline comet assay. ALE also induced a weak genotoxic effect, as expressed by the induction factor (IF) values in the test with E. coli strain PQ35 (IF=1.5) and by cytotoxic effects in strains PQ35, PQ65 and PQ37. Positive SOS induction (IF=1.7) was detected in strain PQ37 treated with diluted AQE. No genotoxic effects were observed in strains PQ35, PQ65, OG40 and OG 100 after treatment with AQE dilutions. Using the bacterial (reverse) mutation test and survival assays with or without S9 mix, after 60min of pre-incubation, we observed for strain TA97 treated with ALE a weak mutagenic response (MI=2.2), while cytotoxic effects were seen for strains TA98, TA100 and TA102. AQE did not show mutagenic activity in any of the strains tested, but a weak cytotoxic effect was noted in strain TA102. Our data suggest that both ALE and AQE from A. fulva induce DNA breaks leading to cytotoxicity and mutagenicity under the conditions used.     

Genotoxic evaluation of extracts from Aplysina fulva, a Brazilian marine sponge

A range of biologically active secondary metabolites with pharmacological application has been reported to occur in marine sponges. The present study was undertaken to provide a set of data on the safety of a hydro-alcoholic extract (ALE) and an aqueous fraction (AQE) from Aplysina fulva Pallas, 1766 (Aplysinidae, Verongida, Porifera). Salmonella typhimurium strains TA97, TA98, TA100 and TA102, Escherichia coli strains PQ65, OG40, OG100, PQ35 and PQ37 and Balb/c 3T3 mouse fibroblasts were used to detect induction of DNA lesions by ALE and AQE. Assays used for these analyses were a bacterial (reverse) mutation assay (Ames test), the SOS-chromotest and the comet assay. Both extracts presented identical infrared 2-oxazolidone spectra. ALE treatment induced a higher frequency of type-4 comets, indicative of increasing DNA migration, in the alkaline comet assay. ALE also induced a weak genotoxic effect, as expressed by the induction factor (IF) values in the test with E. coli strain PQ35 (IF = 1.5) and by cytotoxic effects in strains PQ35, PQ65 and PQ37. Positive SOS induction (IF = 1.7) was detected in strain PQ37 treated with diluted AQE. No genotoxic effects were observed in strains PQ35, PQ65, OG40 and OG 100 after treatment with AQE dilutions. Using the bacterial (reverse) mutation test and survival assays with or without S9 mix, after 60 min of pre-incubation, we observed for strain TA97 treated with ALE a weak mutagenic response (MI = 2.2), while cytotoxic effects were seen for strains TA98, TA100 and TA102. AQE did not show mutagenic activity in any of the strains tested, but a weak cytotoxic effect was noted in strain TA102. Our data suggest that both ALE and AQE from A. fulva induce DNA breaks leading to cytotoxicity and mutagenicity under the conditions used.     

Novel spiciferone derivatives from the fungus Drechslera hawaiiensis isolated from the marine sponge Callyspongia aerizusa

From the marine sponge Callyspongia aerizusa collected from the Sea of Bali, Indonesia, fungal isolates of Drechslera hawaiiensis were obtained. Culture filtrates of the fungus yielded four spiciferone derivatives which include spiciferone A (1) and B (2), and two other novel derivatives including spiciferol A (3) which is an alcohol congener of spiciferone A (1) and compound 4 which is an monocyclic spiciferone congener featuring a butoxyl side chain. The structures of the novel compounds were established on the basis of NMR spectroscopic (1H, 13C, COSY) and mass spectrometric (EIMS) data.     

Antifouling properties of 10beta-formamidokalihinol-A and kalihinol A isolated from the marine sponge Acanthella cavernosa

Many soft-bodied sessile marine invertebrates such as sponges and soft corals defend themselves against fouling directly through the production of antifouling compounds, or indirectly through regulating the epibiotic microbes that affect larval settlement. In this study, 10beta-formamidokalihinol-A and kalihinol A were isolated and purified from the marine sponge Acanthella cavernosa (Dendy). The results indicated that both compounds inhibited the growth of bacteria isolated from the natural environment whereas kalihinol A suppressed larval settlement of a major fouling polychaete, Hydroides elegans with an EC50 of 0.5 microg ml(-1). Kalihinol A was incorporated in Phytagel that was exposed to the bacterial consortia in natural seawater for biofilm formation. Biofilms that developed on the Phytagel surfaces were analysed for bacterial abundance and bacterial species composition using a DNA fingerprinting technique, terminal restriction fragment length polymorphism (T-RFLP). The results showed that kalihinol A only slightly reduced bacterial abundance (t-test, p = 0.0497), but modified the bacterial species composition of the biofilms. Inhibition of H. elegans larval settlement was observed when biofilms developed under the influence of kalihinol A were exposed to larvae, suggesting that compounds like kalihinol A from the sponge A. cavernosa may change bacterial community composition on the sponge surface, which in turn, modulates larval settlement of fouling organisms.     

Cytotoxicity and genotoxicity of glycidyl methacrylate

Methacrylates are used in the polymer form as composite restorative materials in dentistry. However, the polymers can release monomers and co-monomers into the oral cavity and pulp, from where they can migrate into the bloodstream reaching virtually all organs. The local concentration of the released monomers can be in the millimolar range, high enough to induce adverse biological effects. Genotoxicity of methacrylate monomers is of a special significance due to potential serious phenotypic consequences, including cancer, and long latency period. In the present work, we investigated cytotoxicity and genotoxicity of glycidyl methacrylate (GMA) in the human peripheral blood lymphocytes and the CCR-CM human cancer cells. GMA at concentrations up to 5 mM evoked a concentration-dependent decrease in the viability of the lymphocytes up to about 80%, as assessed by flow cytometry. This agent did not induce strand breaks in the isolated plasmid DNA, but evoked concentration-dependent DNA damage in the human lymphocytes evaluated by the alkaline and neutral comet assay. This damage included oxidative modifications to the DNA bases, as checked by DNA repair enzymes Endo III and Fpg as well as single and double DNA strand breaks. The lymphocytes exposed to GMA at 2.5 μM were able to remove about 90% of damage to their DNA in 120 min. The ability of GMA to induce DNA double-strand breaks was confirmed by pulsed field gel electrophoresis. The drug evoked apoptosis and induced an increase in the G2/M cell population, accompanied by a decrease in the S cell population and an increase in G0/G1 cell population. Due to broad spectrum of GMA genotoxicity, including DNA double-strand breaks, and a potential long-lasting exposure to this compound, its use should be accompanied by precautions, reducing the chance of its release into blood stream and the possibility to induce adverse biological effects.     

Psammaplin A, a chitinase inhibitor isolated from the Fijian marine sponge Aplysinella rhax

Several brominated tyrosine derived compounds, psammaplins A (1), K (2) and L (3) as well as bisaprasin (4) were isolated from the Fijian marine sponge Aplysinella rhax during a bioassay guided isolation protocol. Their structures were determined using NMR and MS techniques. Psammaplin A was found to moderately inhibit chitinase B from Serratia marcescens, the mode of inhibition being non-competitive. Crystallographic studies suggest that a disordered psammaplin A molecule is bound near the active site. Interestingly, psammaplin A was found to be a potent antifungal agent.     

Purification and characterization of a dehalogenase from Pseudomonas stutzeri DEH130 isolated from the marine sponge Hymeniacidon perlevis

2-haloacid dehalogenases are enzymes that are capable of degrading 2-haloacid compounds. These enzymes are produced by bacteria, but so far they have only been purified and characterized from terrestrial bacteria. The present study describes the purification and characterization of 2-haloacid dehalogenase from the marine bacterium Pseudomonas stutzeri DEH130. P. Stutzeri DEH130 contained two kinds of 2-haloacid dehalogenase (designated as Dehalogenase I and Dehalogenase II) as detected in the crude cell extract after ammonium sulfate fractionation. Both enzymes appeared to exhibit stereo-specificity with respect to substrate. Dehalogenase I was a 109.9-kDa enzyme that preferentially utilized D-2-chloropropropionate and had optimum activity at pH 7.5. Dehalogenase II, which preferentially utilized L-2-chloropropionate, was further purified by ion-exchange chromatography and gel filtration. Purified Dehalogenase II appeared to be a dimeric enzyme with a subunit of 26.0-kDa. It had maximum activity at pH 10.0 and a temperature of 40 °C. Its activity was not inhibited by DTT and EDTA, but strongly inhibited by Cu²⁺, Zn²⁺, and Co²⁺. The K _m and V _max for L-2-chloropropionate were 0.3 mM and 23.8 μmol/min/mg, respectively. Its substrate specificity was limited to short chain mono-substituted 2-halocarboxylic acids, with no activity detected toward fluoropropionate and monoiodoacetate. This is the first report on the purification and characterization of 2-haloacid dehalogenase from a marine bacterium.     

Structural characterization of a galectin isolated from the marine sponge Chondrilla caribensis with leishmanicidal potential

BackgroundSolving primary structure of lectins leads to an understanding of the physiological roles within an organism and its biotechnological potential. Only eight sponge lectins have had their primary structure fully determined.MethodsThe primary structure of CCL, Chondrilla caribensis lectin, was determined by tandem mass spectrometry. The three-dimensional structure was predicted and the protein-carbohydrate interaction analysed by molecular docking. Furthermore, the anti-leishmanial activity was observed by assays with Leishmania infantum.ResultsThe amino acid sequence consists of 142 amino acids with a calculated molecular mass of 15,443 Da. The lectin has a galectin-like domain architecture. As observed in other sponge galectins, the signature sequence of a highly conserved domain was also identified in CCL with some modifications. CCL exhibits a typical galectin structure consisting of a β-sandwich. Molecular docking showed that the amino acids interacting with CCL ligands at the monosaccharide binding site are mostly the same as those conserved in this family of lectins. Through its interaction with L. infantum glycans, CCL was able to inhibit the development of this parasite. CCL also induced apoptosis after eliciting ROS production and altering the membrane integrity of Leishmania infantum promastigote.ConclusionsCCL joins the restricted group of sponge lectins with determined primary structure and very high biotechnological potential owing to its promising results against pathogens that cause Leishmaniasis.General significanceAs the determination of primary structure is important for biological studies, now CCL can become a sponge galectin with an exciting future in the field of human health.     

Concise synthesis of clarhamnoside, a novel glycosphingolipid isolated from the marine sponge Agela clathrodes

The first total synthesis of a novel alpha-galactoglycosphingolipid clarhamnoside has been achieved through a straightforward strategy. A thiogalactosyl donor with a benzylidene group at C-4 and C-6 and nonparticipating p-methoxybenzyl group at C-2 was successfully employed in the stereocontrolled syntheses of alpha-GalGSLs. The N-Phth-protected trifluoroacetimidate donor for terminal disaccharide was successfully applied in constructing the [GalNAc beta-(1-->6)-Gal] glycosidic linkage.     

Cytotoxicity and genotoxicity of butyl cyclohexyl phthalate

Butyl cyclohexyl phthalate (BCP) is frequently used in personal care products, medical and household applications. The aim of this study is therefore to evaluate possible cytotoxicity and genotoxicity of BCP using in vitro and in vivo assays. The in vitro cytotoxic effect of BCP was investigated on mouse fibroblastic cell line (L929 cells) by MTT assay. The result showed that BCP inhibits cell proliferation in a concentration-dependent manner (IC₅₀ value = 0.29 µg/mL). For genotoxicity assessment, tested concentrations of BCP demonstrated mutagenic activity in the presence of S9 mix with the Salmonella strain TA100 in the Ames test. Results showed that BCP is a secondary mutagenic substance even in low concentrations. The data obtained from 28-days repeated toxicity tests on mice revealed that BCP caused abnormalities of chromosome number, in a dose-dependent manner. Additionally, DNA damage, particularly DNA strand breaks, was assessed by Comet assay. The test result shows that BCP seemed to have genotoxic potential at a high level of exposure.     

Cytotoxicity and genotoxicity of chitooligosaccharides upon lymphocytes

Two COS mixtures and a low molecular weight chitosan (LMWC) were tested for potential cytotoxicity and genotoxicity upon human lymphocytes. Genotoxicity was evaluated in vitro by cytokinesis-blocked micronucleus and alkaline comet assays, while cytotoxicity was assessed by flow cytometry analysis. Our results suggest that COS do not exhibit any genotoxicity upon human lymphocytes, independently of MW or concentration. However, above 0.07 mg/mL COS induced strong cytotoxic effects. According to the concentration used, such cytotoxicity will induce cell death, essentially by necrosis (>0.10 mg/mL) and/or apoptosis (<0.10 mg/mL). The level of necrosis/apoptosis induced by high COS concentrations, suggests a promising use as apoptosis inducers in specific cancer situations.     

Effect of agitation on violacein production in Pseudoalteromonas luteoviolacea isolated from a marine sponge

AIMS: Experiments were designed to investigate the effect of agitation on the production of violacein by a marine bacterium Pseudoalteromonas luteoviolacea. METHODS AND RESULTS: A marine sponge-associated bacterium, P. luteoviolacea, was grown at different agitation speeds. Agitation did not have a significant effect on bacterial growth, but had a profound effect on the size of bacterial aggregate. The production of violacein was the highest under stagnant conditions and decreased with the increase of the agitation speed. CONCLUSIONS: Agitation affected the aggregation of bacterial cells, which, in turn, affected violacein production by P. luteoviolacea. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that P. luteoviolacea produced the highest amount of violacein when it was cultured under stagnant conditions.     

Polymorphic microsatellite loci isolated from the marine sponge Scopalina lophyropoda (Demospongiae: Halichondrida)

A total of seven microsatellites out of 88 isolated from a genomic library enriched for (CA)_n and (GA)_n repeats were characterized in the Mediterranean marine sponge Scopalina lophyropoda. The microsatellite motifs were large (34.81 ± 13.9 bp) and imperfect. The seven microsatellite loci were screened in 30 individuals collected from Blanes, northwestern Mediterranean. All of them were polymorphic (allele numbers and observed heterozygosities ranged from 3 to 6 and from 0.16 to 0.76, respectively). No significant linkage disequilibrium between pairs of loci and no departure from Hardy–Weinberg equilibrium were found. These markers are therefore promising for studies of the population structure of the species.     

Some pharmacological activities of novel adenine-related compounds isolated from a marine sponge Agelas mauritiana

Agelasimine A and agelasimine B, two novel compounds related to adenine, have been isolated from the orange sponge, Agelas mauritiana, and have been tested for a variety of biological activities. Both compounds inhibited proliferation of cultured L1210 leukemia cells at nanomolar concentrations with accumulation in the G1 stage of the cell cycle. However, no prolongation of life was observed in mice bearing P388 leukemia treated with these compounds. In the rat isolated aorta, micromolar concentrations of agelasimines were very effective in inhibiting contractions elicited by potassium chloride but had little or no effect on responses for prostaglandin F2 alpha and had modest effects on the responses to noradrenaline and significant effects on 5-hydroxytryptamine. Agelsamines A and B appeared to be equipotent in causing relaxation in rabbit jejunum and bovine coronary artery, and they also inhibited nucleoside transport into rabbit erythrocytes in micromolar concentrations.     

Diversity of microbes associated with the marine sponge, Haliclona simulans, isolated from Irish waters and identification of polyketide synthase genes from the sponge metagenome

Samples of the sponge Haliclona simulans were collected from Irish waters and subjected to a culture-independent analysis to determine the microbial, polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) diversity. 16S rRNA gene libraries were prepared from total sponge, bacterial enriched sponge and seawater samples. Eight phyla from the Bacteria were detected in the sponge by phylogenetic analyses of the 16S rRNA gene libraries. The most abundant phylum in the total sponge library was the Proteobacteria (86%), with the majority of these clones being from the γ- Proteobacteria (77%); two groups of clones were dominant and together made up 69% of the total. Both of these groups were related to other sponge-derived microbes and comprised novel genera. Within the other bacterial phyla groups of clones representing novel candidate genera within the phyla Verrucomicrobia and Lentisphaerae were also found. Selective enrichment of the bacterial component of the sponge prior to 16S rRNA gene analysis resulted in a 16S rRNA gene library dominated by a novel genus of δ- Proteobacteria , most closely related to the Bdellovibrio . The potential for the sponge microbiota to produce secondary metabolites was also analysed by polymerase chain reaction amplification of PKS and NRPS genes. While no NRPS sequences were isolated seven ketosynthase (KS) sequences were obtained from the sponge metagenome. Analyses of these clones revealed a diverse collection of PKS sequences which were most closely affiliated with PKS from members of the Cyanobacteria , Myxobacteria and Dinoflagellata .     

Anti-fertilization activity of a spirocyclic sesquiterpene isocyanide isolated from the marine sponge Geodia exigua and related compounds

(-)-10-epi-Axisonitrile-3, a spirocyclic sesquiterpene isocyanide obtained from the marine sponge Geodia exigua, immobilized sperm of sea urchin and starfish to block fertilization at the minimum effective concentration of 0.4 microg/ml. On the other hand, fertilized eggs developed normally to the gastrula stage in the presence of a 250-times higher concentration of the isocyanide. Analysis by (31)P NMR revealed an accumulation of phosphocreatine and a depletion of inorganic phosphate in the isocyanide-treated sperm, suggesting that (-)-10-epi-axisonitrile-3 inhibited the phosphocreatine shuttle participating in the high-energy phosphate metabolism, thereby immobilizing sperm to block fertilization. No analogs of (-)-10-epi-axisonitrile-3 containing different functionalities or isocyanides with different carbon skeleton exhibited such activity.     

A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species

A cultivation-based approach was employed to compare the culturable actinobacterial diversity associated with five marine sponge species (Craniella australiensis, Halichondria rugosa, Reniochalina sp., Sponge sp., and Stelletta tenuis). The phylogenetic affiliation of the actinobacterial isolates was assessed by 16S rDNA-RFLP analysis. A total of 181 actinobacterial strains were isolated using five different culture media (denoted as M1–M5). The type of medium exhibited significant effects on the number of actinobacteria recovered, with the highest number of isolates on M3 (63 isolates) and the lowest on M1 (12 isolates). The genera isolated were also different, with the recovery of three genera on M2 and M3, and only a single genus on M1. The number of actinobacteria isolated from the five sponge species was significantly different, with a count of 83, 36, 30, 17, and 15 isolates from S. tenuis, H. rugosa, Sponge sp., Reniochalina sp., and C. australiensis, respectively. M3 was the best isolation medium for recovery of actinobacteria from S. tenuis, H. rugosa, and Sponge sp., while no specific medium preference was observed for the recovery of actinobacteria from Reniochalina sp., and C. australiensis. The RFLP fingerprinting of 16S rDNA genes digested with HhaI revealed six different patterns, in which 16 representative 16S rDNAs were fully sequenced. Phylogenetic analysis indicated that 12 strains belong to the group Streptomyces, three strains belong to Pseudonocardia, and one strain belongs to Nocardia. Two strains C14 (from C. australiensis) and N13 (from Sponge sp.) have only 96.26% and 96.27% similarity to earlier published sequences, and are therefore potential candidates for new species. The highest diversity of three actinobacteria genera was obtained from Sponge sp., though the number of isolates was low. Two genera of actinobacteria, Streptomyces, and Pseudonocardia, were isolated from both S. tenuis and C. australiensis. Only the genus of Streptomyces was isolated from H. rugosa and Reniochalina sp. Sponge species have been demonstrated here to vary as sources of culturable actinobacterial diversity, and the methods for sampling such diversity presented may be useful for improved sampling of such diversity.