Molecular profiling of marine endophytic fungi from green algae: Assessment of antibacterial and anticancer activities

Bioactive natural metabolites, especially from the marine endophytic fungi, are largely unexplored. Endophytic fungi are being increasingly recognized as a group of organisms that produce novel metabolites of industrial importance. This study investigated the anticancer and antibacterial potential of the marine algal endophyte, Penicillium chrysogenum. The different organic solvent extracts of the endophytic fungi grown on different growth medium were analyzed for anticancer and antibacterial activities. The highest inhibitory activity was observed for the ethyl acetate (EA) extract of the culture filtrate grown in potato dextrose broth (PDB) for 21 days, against the tested human breast cancer cell (MCF-7) line. Similarly, the PDB-EA extract showed an appreciable activity against the human pathogens. The biochemical analysis of the Cha EA metabolites revealed terpenoids, steroids, phenolics and flavones. Gas Chromatography (GCMS) data revealed several bioactive compounds such as anthraquinone and cinnamic acid. The Cha EA extract induced membrane damage and thus, apoptosis in MCF-7cells. The secondary metabolites produced by these marine endophytic fungi have contributed to considerable anticancer and antimicrobial activities and hence, this study is an evidence of potential sources of antimicrobial and anticancer compounds from Penicillium chrysogenum.     

Crassocolides N-P, three cembranoids from the Formosan soft coral Sarcophyton crassocaule

Three new cembranoids crassocolides N-P (1-3), was isolated from the organic extract of a Formosan soft coral Sarcophyton crassocaule. These structures were elucidated on the basis of spectroscopic analyses and by comparison with those previously reported in literature. The cytotoxicity of these compounds toward various cancer cell lines has also been determined.     

Distribution of sarcophytol A in soft coral of the Sarcophyton genus

The distribution of sarcophytol-A in the Sarcophyton genus was investigated in seven samples belonging to S. glaucum (3 samples), S. infundibulifurme (2 samples), S. crassocaule (1 sample) and S. trocheliophorum (1 sample) that were collected on Ishigaki Island in Okinawa Prefecture. Sarcophytol-A was present in one sample each of S. glaucum and S. infundibulifurme. This study indicates that the composition of cembranoids in the Sarcophyton genus is not related with the respective species, but with the individual samples collected.     

Cytotoxic and anti-inflammatory cembranoids from the Dongsha Atoll soft coral Sarcophyton crassocaule

Five new cembranoids, sarcocrassocolides A–E (1–5), along with three known cembranoids 6–8, have been isolated from a Formosan soft coral Sarcophyton crassocaule. The structures of the new metabolites were elucidated by extensive spectroscopic analysis and the absolute configuration of 1 was determined by a modified Mosher’s method. Compounds 1–4 exhibited significantly cytotoxic activity against a limited panel of cancer cell lines. Compounds 1–4, 6 and 8 were shown to exert significant in vitro anti-inflammatory activity in LPS-stimulated RAW264.7 macrophage cells. Compound 6 also significantly inhibited the accumulation of pro-inflammatory COX-2 protein.     

Chemical constituents of soft coral Sarcophyton infundibuliforme from the South China Sea

Chemical investigation on soft coral Sarcophyton infundibuliforme collected from the South China Sea led to the isolation and identification of 14 secondary metabolites, including ten cembrene diterpenoids (1–10), one α-tocopheryl quinone derivative (11), one prostaglandin (12), one lipid (13) and one carotinoid (14). Their structures were determined by extensive analysis of their spectroscopic data. All of these metabolites were isolated from this species for the first time. Diterpenoids 1, 2, 7 and 10 showed potent antifouling activity against the larval settlement of barnacle Balanus amphitrite.     

Trochelioid A and B,new cembranoid diterpenes from the Red Sea soft coral Sarcophyton trocheliophorum

Chemical investigations of the soft coral Sarcophyton trocheliophorum, has led to the isolation of six cembranoids, two of which are new, Trochelioid A (1) and B (2), and one, 16-oxosarcophytonin E (3) isolated from nature for the first time. Additionally, two have been isolated from S. trocheliophorum for the first time (4 and 6). Structures were elucidated by employing extensive NMR and HR-FAB-MS experimentation.     

Phylogenetic relationships within the tropical soft coral genera Sarcophyton and Lobophytum (Anthozoa, Octocorallia)

Abstract. The alcyonacean soft coral genera Sarcophyton and Lobophytum are conspicuous, ecologically important members of shallow reef communities throughout the Indo-West Pacific. Study of their ecology is, however, hindered by incomplete knowledge of their taxonomy: most species cannot be identified in the field and the two genera cannot always be distinguished reliably. We used a 735-bp fragment of the octocoral-specific mitochondrial protein-coding gene msh1 to construct a phylogeny for 92 specimens identified to 19 species of Lobophytum and 16 species of Sarcophyton . All phylogenetic methods used recovered a tree with three strongly supported clades. One clade included only morphologically typical Sarcophyton species with a stalk distinct from the polypary, poorly formed club-shaped sclerites in the colony surface, and large spindles in the interior of the stalk. A second clade included only morphologically typical Lobophytum colonies with lobes and ridges on the colony surface, poorly formed clubs in the colony surface, and interior sclerites consisting of oval forms with regular girdles of ornamental warts. The third distinct clade included a mix of Sarcophyton and Lobophytum nominal species with intermediate morphologies. Most of the species in this mixed clade had a polypary that was not distinct from the stalk, and the sclerites in the colony surface were clubs with well-defined heads. Within the Sarcophyton clade, specimens identified as Sarcophyton glaucum belonged to six very distinct genetic sub-clades, suggesting that this morphologically heterogeneous species is actually a cryptic species complex. Our results highlight the need for a complete taxonomic revision of these genera, using molecular data to help confirm species boundaries as well as to guide higher taxonomic decisions.     

Overproduction and biological activity of prodigiosin-like pigments from recombinant fusant of endophytic marine Streptomyces species

Thirty-four endophytic marine Actinomycetes isolates were recovered from the Egyptian marine sponge Latrunculia corticata, out of them 5 isolates (14.7 %) showed red single colonies on yeast-CzAPEK plates. Isolates under the isolation code NRC50 and NRC51 were observed with the strongest red biomass. After application of protoplast fusion between NRC50 and NRC51 isolates, 26 fusants were selected and produced widely different amounts of prodigiosin-like pigments (PLPs) on different fermentation media. Among them fusant NRCF69 produced 79 and 160.4 % PLPs more than parental strains NRC50 and NRC51, respectively. According to the analysis of 16S rDNA sequence (amplified, sequenced, and submitted to GenBank under Accession no. JN232405 and JN232406, respectively), together with their morphological and biochemical characteristics, parental strains NRC50 (P1) and NRC51 (P2) were identified as Streptomyces sp. and designated as Streptomyces sp. NRC50 and Streptomyces sp. NRC51. This study describes a low cost, effective production media by using peanut seed broth, sunflower oil broth or dairy processing wastewater broth alone, or supplemented with 0.5 % mannitol that supports the production of PLPs by the Streptomyces fusant NRCF69 under study (42.03, 40.11, 36.7 and 47 g L^?1, respectively). PLPs compounds exhibited significant cytotoxic activities against three human cancer cell lines: colon cancer cell line (HCT-116), liver cancer cell line (HEPG-2) and breast cancer cell line (MCF-7) and antimycotic activity against clinical dermatophyte isolates of Trichophyton, Microsporum and Epidermophyton.     

Isolation of marine bacteria with antimicrobial activities from cultured and field-collected soft corals

Bacteria associated with eight field-collected and five cultured soft corals of Briareum sp., Sinularia sp., Sarcophyton sp., Nephtheidae sp., and Lobophytum sp. were screened for their abilities in producing antimicrobial metabolites. Field-collected coral samples were collected from Nanwan Bay in southern Taiwan. Cultured corals were collected from the cultivating tank at National Museum of Marine Biology and Aquarium. A total of 1,526 and 1,138 culturable, heterotrophic bacteria were isolated from wild and cultured corals, respectively; seawater requirement and antimicrobial activity were then assessed. There is no significant difference between the ratio of seawater-requiring bacteria on the wild and cultured corals. The ratio of antibiotic-producing bacteria within the seawater-requiring bacteria did not differ between the corals. Nineteen bacterial strains that showed high antimicrobial activity were selected for 16S rDNA sequencing. Three strains could be assigned at the family level (Rhodobacteraceae). The remaining 16 strains belong to eight genera: Marinobacterium (2 strains), Pseudoalteromonas (1), Vibrio (5), Enterovibrio (1), Tateyamaria (1), Labrenzia (2), and Pseudovibrio (4). The crude extract from bacteria strains CGH2XX was found to have high cytotoxicity against the cancer cell line HL-60 (IC₅₀?=?0.94???g/ml) and CCRF-CEM (IC₅₀?=?1.19???g/ml). Our results demonstrate that the marine bacteria from corals have great potential in the discovery of useful medical molecules.     

Marine sterols X. Minor constituents of the sterols of the soft coral Sarcophyton glaucum

The sterol mixture of the southern Japan soft coral $\text{Sarcophyton glaucum}$ was found to contain a variety of minor components overlooked in a previous study. Five 4α-methylsterols ($\text{1}$ to $\text{5}$) and three 4-demethyl-sterols ($\text{6}$ to $\text{8}$) were isolated and their structures were confirmed.     

A molecular phylogenetic analysis of reproductive trait evolution in the soft coral genus Alcyonium

The soft coral genus Alcyonium is among the most reproductively diverse invertebrate taxa known: The genus includes species that vary both in mode of reproduction (including broadcast spawners, internal brooders, and external brooders) and sexual expression (gonochores, hermaphrodites, and a unisexual parthenogen). Such diversity offers a unique opportunity to examine associations between reproductive and morphological traits in a phylogenetic context. We used an approximately 900-bp sequence of the nuclear ribosomal gene complex spanning the internal transcribed spacer (ITS) regions to construct a molecular phylogeny for 14 European and North American species of Alcyonium onto which we mapped the known distribution of reproductive and morphological traits. The phylogeny suggests that hermaphroditism or parthenogenesis has evolved independently at least twice in this genus, and always in internally brooding species. Broadcast spawning and external brooding only occur in species with large colony size, whereas all species with small colony size brood their larvae internally. Internal brooding and small size appear to be ancestral in this genus; if this is the case, an association between broadcast spawning and large colony size has evolved independently in at least two clades. This tendency of small adults to brood their larvae while large adults broadcast spawn them into the plankton has been observed in a variety of solitary invertebrate taxa, but to date has not been documented in any other colonial invertebrates. Moreoever, it has been suggested that organisms with a colonial growth form should not experience the allometric constraints on brood space that have been proposed to explain the association between adult size and mode of reproduction in solitary organisms. Unlike many other colonial groups, however, module (polyp) size is strongly correlated with colony size in Alcyonium, and constraints on brooding may be imposed by module, rather than colony, allometry. The very close genetic relationship (< 1% sequence divergence) and shared polymorphisms among A. digitatum (a large, gonochoric broadcast spawner), A. siderium, and A. sp. A (intermediate-sized and small hermaphroditic, internal brooders) suggest that evolutionary transitions between broadcast spawning and brooding and between gonochorism and hermaphroditism can occur easily and rapidly in this group.     

Antiprotozoal,anticancer and antimicrobial activities of dihydroartemisinin acetal dimers and monomers

Nine dihydroartemisinin acetal dimers (6–14) with diversely functionalized linker units were synthesized and tested for in vitro antiprotozoal, anticancer and antimicrobial activity. Compounds 6, 7 and 11 [IC₅₀: 3.0–6.7 nM (D6) and 4.2–5.9 nM (W2)] were appreciably more active than artemisinin (1) [IC₅₀: 32.9 nM (D6) and 42.5 nM (W2)] against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of the malaria parasite, Plasmodium falciparum. Compounds 10, 13 and 14 displayed enhanced anticancer activity in a number of cell lines compared to the control drug, doxorubicin. The antifungal activity of 7 and 12 against Cryptococcus neoformans (IC₅₀: 0.16 and 0.55 μM, respectively) was also higher compared to the control drug, amphotericin B. The antileishmanial and antibacterial activities were marginal. A number of dihydroartemisinin acetal monomers (15–17) and a trimer (18) were isolated as byproducts from the dimer synthesis and were also tested for biological activity.     

Optimized production of scygonadin in Pichia pastoris and analysis of its antimicrobial and antiviral activities

The crab antimicrobial peptide scygonadin is confirmed to have antimicrobial activity against bacteria and it is probably associated with the reproductive immunity in Scylla paramamosain. To obtain large quantity of scygonadin for further biological assays, a 306 bp cDNA sequence encoding the mature peptide of scygonadin was cloned into a secretion vector of pPIC9K, and a high-level of the recombinant scygonadin was achieved in Pichia pastoris. The optimal expression condition was determined as incubation with 0.5% methanol for 48 h at 28 °C under pH 6.0, and a total of 70 mg scygonadin was expressed in 1 L culture medium. The recombinant product was purified and 97% pure scygonadin was obtained using immobilized metal affinity chromatography with a yield of 46 mg/L. The recombinant scygonadin was confirmed using SDS-PAGE analysis and MS-fingerprinting. P. pastoris-derived scygonadin exhibited relatively higher antimicrobial activities against bacteria than Escherichia coli-derived scygonadin. The antimicrobial activity of the recombinant scygonadin against pathogenic Aeromonas hydrophila showed salt resistant and the killing kinetics of A. hydrophila was time dependent. Besides, the antiviral assay demonstrated that scygonadin could interfere with white spot syndrome virus (WSSV) replication in vitro-cultured crayfish haematopoietic (Hpt) cells. Taken together, this is the first report on the heterologous expression of scygonadin in P. pastoris, and P. pastoris is an effective expression system for producing large quantities of biological active scygonadin for both research and agricultural application.     

New marine steroids, yonarasterols, isolated from the okinawan soft coral, Clavularia viridis

Six new marine steroids, yonarasterols A through F, were isolated from the Okinawan soft coral, Clavularia viridis. Their structures were determined based on the results of spectroscopic analysis.     

灯台树内生放线菌多样性及抗菌活性评价

从云南西双版纳采集7份灯台树样品,经过表面消毒,用4种分离培养基分离得到105株内生放线菌。经16S rRNA基因序列分析鉴定,分布于7个科9个属。利用5种植物病原真菌指示菌对所有菌株的发酵液进行抗菌活性检测。结果显示,有12.4%、14.3%、11.4%、12.4%、8.6%的菌株分别对镰刀霉、疫霉、赤星霉、苹果炭疽、白色念珠菌有抗性。对3株具有广谱抗菌活性的菌株进行再次发酵和抗菌活性复筛,结果显示这3株的抗菌活性稳定,并可能含有生物碱类化合物。     

Cembrane diterpenoids from the soft coral Sarcophyton trocheliophorum Marenzeller as a new class of PTP1B inhibitors

A detailed investigation of the South China Sea soft coral Sarcophyton trocheliophorum Marenzeller yielded, along with six known terpenes (6?11), the new sarcophytonolides N?R (1?5), whose structures have been elucidated by detailed spectroscopic analysis. Sarcophytonolides N–R are mono- or bicyclic cembranoids characterized by the presence of three/four double bonds and oxidized methyl groups. Some of the isolated compounds showed significant inhibitory activity against human protein tyrosine phosphatase 1B (PTP1B) enzyme, a key target for the treatment of type-II diabetes and obesity, and some preliminary structure–activity relationships have been drawn. This is the first report on the anti-PTP1B activity of cembrane diterpenoids.     

Genome-based phylogenetic analysis of Streptomyces and its relatives

MotivationStreptomyces is one of the best-studied genera of the order Actinomycetales due to its great importance in medical science, ecology and the biotechnology industry. A comprehensive, detailed and robust phylogeny of Streptomyces and its relatives is needed for understanding how this group emerged and maintained such a vast diversity throughout evolution and how soil-living mycelial forms (e.g., Streptomyces s. str.) are related to parasitic, unicellular pathogens (e.g., Mycobacterium tuberculosis) or marine species (e.g., Salinispora tropica). The most important application area of such a phylogenetic analysis will be in the comparative re-annotation of genome sequences and the reconstruction of Streptomyces metabolic networks for biotechnology.MethodsClassical 16S-rRNA-based phylogenetic reconstruction does not guarantee to produce well-resolved robust trees that reflect the overall relationship between bacterial species with widespread horizontal gene transfer. In our study we therefore combine three whole genome-based phylogenies with eight different, highly informative single-gene phylogenies to determine a new robust consensus tree of 45 Actinomycetales species with completely sequenced genomes.ResultsNone of the individual methods achieved a resolved phylogeny of Streptomyces and its relatives. Single-gene approaches failed to yield a detailed phylogeny; even though the single trees are in good agreement among each other, they show very low resolution of inner branches. The three whole genome-based methods improve resolution considerably. Only by combining the phylogenies from single gene-based and genome-based approaches we finally obtained a consensus tree with well-resolved branches for the entire set of Actinomycetales species. This phylogenetic information is stable and informative enough for application to the system-wide comparative modeling of bacterial physiology.     

Phytochemical analysis and in vitro antiviral activities of the essential oils of seven Lebanon species

The chemical composition of the essential oils of Laurus nobilis, Juniperus oxycedrus ssp. oxycedrus, Thuja orientalis, Cupressus sempervirens ssp. pyramidalis, Pistacia palaestina, Salvia officinalis, and Satureja thymbra was determined by GC/MS analysis. Essential oils have been evaluated for their inhibitory activity against SARS-CoV and HSV-1 replication in vitro by visually scoring of the virus-induced cytopathogenic effect post-infection. L. nobilis oil exerted an interesting activity against SARS-CoV with an IC(50) value of 120 microg/ml and a selectivity index (SI) of 4.16. This oil was characterized by the presence of beta-ocimene, 1,8-cineole, alpha-pinene, and beta-pinene as the main constituents. J. oxycedrus ssp. oxycedrus oil, in which alpha-pinene and beta-myrcene were the major constituents, revealed antiviral activity against HSV-1 with an IC(50) value of 200 microg/ml and a SI of 5.