Development of microsatellite markers in the noxious red tide‐causing dinoflagellate Heterocapsa circularisquama (Dinophyceae)

We isolated 15 polymorphic microsatellites from one of the most noxious red tide‐causing dinoflagellate species, Heterocapsa circularisquama. These loci provide one class of highly variable genetic markers, as the number of alleles ranged from two to six, and the estimate of gene diversity from 0.205 to 0.684 across the 15 microsatellites. These loci have the potential to reveal genetic structure and gene flow among H. circularisquama populations.     

THIS ARTICLE HAS BEEN RETRACTEDDevelopment of microsatellite markers in the noxious red tide‐causing dinoflagellate Heterocapsa circularisquama (Dinophyceae)

We isolated 15 polymorphic microsatellites from one of the most noxious red tide‐causing dinoflagellate species, Heterocapsa circularisquama. These loci provide one class of highly variable genetic markers, as the number of alleles ranged from two to six, and the estimate of gene diversity was from 0.205 to 0.684 across the 15 microsatellites. These loci have the potential to reveal genetic structure and gene flow among H. circularisquama populations.     

Heterocapsa circularisquama sp. nov. (Peridiniales, Dinophyceae): A new marine dinoflagellate causing mass mortality of bivalves in Japan

Heterocapsa circularisquama Horiguchi sp. nov. is described from Ago Bay, central Japan. The dinoflagellate produced large-scale red tides in the bays of central and western Japan and caused mass mortality of bivalves, notably the pearl oysters. The cell is small and is composed of a conical epitheca and a hemi-spheroidal hypothecs. The chloroplast is single and is connected to the single pyrenoid. The nucleus is elongated and is located in the left side of the cell. Thecal plate arrangement has been determined as: Po, cp, 5′, 3a, 7″, 6c, 5s, 5″′, 2″″. Heterocapsa circularisquama is morphologically very similar to Heterocapsa illdefina and it is almost impossible to distinguish these two species at light microscopical level. The characteristics which can be used to distinguish these two species are the morphology of body scales and the ultrastructure of the pyrenoid matrix. The body scales of H. circularisquama possess six radiating ridges on the circular basal plate; no such ridges can be observed on the roughly triangular basal plate of the scales of H. illdefina. Furthermore, the scales of the latter species possess substantially shorter spines compared to those of H. circularisquama. The pyrenoid matrix of H. circularisquama is hardly perforated by cytoplasmic tubules, while in H. tlldefina the pyrenoid matrix is always penetrated by many cytoplasmic tubules. Based on the arrangement of thecal plates, morphology of body scales, and ultra-structure of the pyrenoid, I am placing H. circularisquama sp nov. into the genus Heterocapsa.     

Algicidal activity of the thiazolidinedione derivative TD49 against the harmful dinoflagellate Heterocapsa circularisquama in a mesocosm enclosure

To assess the algicidal effects of the thiazolidinedione derivative TD49 on the unarmored dinoflagellate Heterocapsa circularisquama and to evaluate the response of the planktonic community and the environment to this chemical, we undertook mesocosm (1,300 L) and small-scale experiments. The reduction ratio for H. circularisquama in each experiment was dependent on the concentration of TD49. At a TD49 concentration >0.4 μM, the abundance of H. circularisquama decreased by 99 % in the small-scale experiment and by 84 % in the mesocosm during the initial 2 days. At 0.2 μM TD49, the abundance of H. circularisquama decreased by up to 85 % in the small-scale experiment, whereas the abundance in the mesocosm increased, implying the absence of an algicidal effect. The decrease in planktonic organisms, including H. circularisquama, following TD49 treatment was correlated with abrupt declines in culture pH and dissolved oxygen concentration. Following addition of TD49, there was a significant increase in the abundance of diatoms and cryptophyta species, and after 8 days, the dominant species in the TD49 treatments shifted to small pennate diatoms including Cylindrotheca and Entomoneis species. The growth of some species among the zooplankton community was promoted at low TD49 concentrations (≤0.4 μM), whereas high concentrations (≥1.0 μM) had a negative effect. This study demonstrates that TD49 is an effective agent for the control for H. circularisquama blooms and that large-scale mesocosms play a crucial role in assessing the application of algicides such as TD49 in natural environments.     

Uptake kinetics of nitrate,ammonia and phosphate by the dinoflagellate Heterocapsa circularisquama isolated from Hiroshima Bay,Japan

Heterocapsa circularisquama is a harmful dinoflagellate whose first bloom in Hiroshima Bay, Japan, appeared in 1992. As suggested by the authors’ group, in the Seto Inland Sea including Hiroshima Bay, oligotrophication particularly the reduction of phosphate starting 1980 is severe. The bloom caused serious damage to the bay's extensive oyster culture. In the present study, the uptake kinetics of nitrate, ammonia, and phosphate by this species were experimentally investigated. The maximum uptake rate (ρ_max) and the half‐saturation constant (Ks) were 0.41 pmol cell^?1 h^?1 and 4.45 μM, respectively, for nitrate, 2.02 pmol cell^?1 h^?1 and 11.1 μM for ammonium, and 0.079 pmol cell^?1 h^?1 and 1.79 μM for phosphate. The maximum specific uptake rates (V_max) for nitrate, ammonia, and phosphate were estimated to be 8.95, 44.1, and 21.3 day^?1, respectively. A comparison of V_max/Ks, which is also an index of affinity to nutrients, between this species and others suggested that H. circularisquama can utilize nitrate and ammonia efficiently, but not phosphate. Considering both reports describing that H. circularisquama has the ability to utilize dissolved organic phosphorus (DOP) and the DOP concentration is higher than phosphate in Hiroshima Bay, it was concluded that H. circularisquama became dominant due to the phosphate reduction measure.     

Antiviral activity and mode of action of a peptide isolated from Sorghum bicolor

In this paper, we describe the purification of an antiviral peptide from seeds of Sorghum bicolor L. by a procedure that included gel filtration, ion exchange, and high-performance liquid chromatography (HPLC) in a reversed-phase column. Its molecular weight, determined by chromatographic mobility on the Shim-pack DIOL-150 gel permeation column in HPLC, was found to be 2000 Da. The peptide designated 2 kD peptide strongly inhibited the replication of herpes simplex virus type 1 (HSV-1), dose-dependently, at 40–90% of the control level, after incubation with 10–50 μM of the peptide, with EC₅₀ and EC₉₀ values of 6.25 and 15.25 μM, respectively. The IC₅₀ value of the 2 kD peptide against Vero cells was 250 μM. Pre-incubation of HSV-1 with various concentrations of the 2 kD peptide showed dose-dependent cytopathic effects (CPE) reduction patterns at concentrations from 6.25 to 50 μM. The presence of the 2 kD peptide before HSV-1 infections showed moderate inhibition of virus-induced CPE as compared to during or after infections, with EC₅₀ values of 12.5, 6.25, and 6.25 μM, respectively. Similar results were observed when the 2 kD peptide was assayed against bovine herpes virus (BHV), an enveloped virus like HSV-1. On the other hand, the 2 kD peptide showed weak activity against poliovirus type 1, a non-enveloped virus. Taken together, these results indicate that the 2 kD peptide was able not only to inhibit the initiation and the spread of infection, but also had an in vitro prophylactic effect against HSV-1 infection.     

Cytotoxic activity of hirsutanone,a diarylheptanoid isolated from Alnus glutinosa leaves

BackgroundThe low efficacy of cancer therapy for the treatment of patients with advanced disease makes the development of new anticancer agents necessary. Because natural products are a significant source of anticancer drugs, it is important to explore cytotoxic activity of novel compounds from natural origin.PurposeThe aim of this work is to evaluate the cytotoxic capacity of hirsutanone, a diarylheptanoid isolated from Alnus glutinosa leaves. Hirsutanone cytotoxic way of action was also studied.Material and methodsThe cytotoxic ability of Alnus glutinosa leaves ethyl acetate extract was studied over HeLa and PC-3 cell lines, with the MTT colorimetric assay. Hirsutanone was isolated from this extract using chromatographic methods, and its structure elucidated by spectroscopic analysis. HT-29 cell viability after hirsutanone treatment was determined using SRB assay. In order to understand hirsutanone way of action, cytotoxicity was evaluated adding the diarylheptanoid and antioxidants. DNA topoisomerase II (topo II) poison activity, was also evaluated using purified topo II and a supercoiled form of DNA that bears specific topo II recognition and binding region; topo II poisons stabilize normally transient DNA-topo II cleavage complexes, and lead an increased yield of linear form as a consequence of a lack of double-strand breaks rejoining.ResultsThe diarylheptanoid hirsutanone was isolated from Alnus glutinosa (L.) Gaertn. (Betulaceae) leaves extract that showed cytotoxic activity against PC-3 and HeLa cell lines. Hirsutanone showed cytotoxic activity against HT-29 human colon carcinoma cells. Pre-treatment with the antioxidants NAC (N-acetylcysteine) and MnTMPyP (Mn(III)tetrakis-(1-methyl-4-pyridyl)porthyrin) reduced this activity, suggesting that reactive oxygen species (ROS) participate in hirsutanone-induced cancer cell death. Using human topo II and a DNA supercoiled form, hirsutanone was found to stabilize topo II-DNA cleavage complexes, acting as a topo II poison.ConclusionOur data suggest that, like curcumin, an induction of oxidative stress and topo II-mediated DNA damage may play a role in hirsutanone-induced cancer cell death. Since both compounds share similar structure and cytotoxic profile, and curcumin is in clinical trials for the treatment of cancer, our results warrant further studies to evaluate the anticancer potential of hirsutanone.     

Amphidinolide H, a novel type of actin-stabilizing agent isolated from dinoflagellate

The effect of novel cytotoxic marine macrolide, amphidinolide H (Amp-H), on actin dynamics was investigated in vitro. Amp-H attenuated actin depolymerization induced by diluting F-actin. This effect remained after washing out of unbound Amp-H by filtration. In the presence of either Amp-H or phalloidin, lag phase, which is the rate-limiting step of actin polymerization, was shortened. Phalloidin decreased the polymerization-rate whereas Amp-H did not. Meanwhile, the effects of both compounds were the same when barbed end of actin was capped by cytochalasin D. Quartz crystal microbalance system revealed interaction of Amp-H with G-actin and F-actin. Amp-H also enhanced the binding of phalloidin to F-actin. We concluded that Amp-H stabilizes actin in a different manner from that of phalloidin and serves as a novel pharmacological tool for analyzing actin-mediated cell function.     

Purification and characterization of a novel high molecular weight exotoxin produced by red tide phytoplankton,Alexandrium tamarense

Our recent studies have demonstrated that the aqueous extract prepared from Alexandrium tamarense, a harmful red tide phytoplankton, showed cytotoxicity on Vero cells. In this study, the toxic substance was purified from the culture supernatant of A. tamarense. Based on the gel‐filtration profile, the molecular mass of a purified toxin was estimated to be about 1,000 kDa. On sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis, a main band with molecular mass of 1,000 kDa was detected with periodic acid‐Schiff (PAS) staining, but no protein bands were detected by Coomassie brilliant blue (CBB) protein staining. Sugar composition analysis of the toxin suggested that the toxin contains galactose, fucose, mannose, N‐acetylglucosamine, xylose, and other minor saccharides, whereas no significant levels of amino acids were detected by amino acid analysis. These results suggest that the toxin is a polysaccharide‐based compound. The toxin showed cytotoxic effects on various cell lines in a concentration‐dependent manner. Among the cell lines tested, U937 cells were the most susceptible to the toxin. In U937 cells treated with the toxin, a typical apoptotic nuclear morphological change and DNA fragmentation were observed. This is the first report demonstrating that a polysaccharide‐based toxin isolated from red tide phytoplankton can induce apoptotic cell death. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:405–415, 2008; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20253     

Antibacterial activity and action mechanism of a novel chitosan oligosaccharide derivative against dominant spoilage bacteria isolated from shrimp Penaeus vannamei

With the aim of exploring the potential application of a novel chitosan oligosaccharide derivative (COS-All-Tio) in shrimp preservation, six dominant spoilage bacteria in the spoiled shrimp (Penaeus vannamei) were isolated and identified as Shewanella putrefaciens (RMS1), S. putrefaciens (S2), Pseudomonas weihenstephanensis (P1), P. gessardii (P2), Aeromonas bestiarum (A1) and Aeromonas molluscorum (A2). The antibacterial effect of COS-All-Tio against the six bacterial isolates were studied. Bacterial inhibition zone determination, and minimum inhibitory concentration and minimum bactericidal concentration assays indicated that the antibacterial activity of COS-All-Tio was greatly improved when compared to that of chitosan oligosaccharide (COS). The antibacterial mechanism investigation against S. putrefaciens (RMS1) revealed that COS-All-Tio could inhibit bacterial growth by influencing of membrane integrity. Such disturbance of membrane structure resulted in the leakage of intracellular substance of the bacteria. A strong synergistic antibacterial effect against S. putrefaciens (RMS1) was observed when COS-All-Tio was used in combination with food preservatives (e.g. ε-polylysine hydrochloride). Therefore, COS-All-Tio might have potential in shrimp preservation.     

Cytotoxic and apoptosis-inducing effect of ent-15-oxo-kaur-16-en-19-oic acid, a derivative of grandiflorolic acid from Espeletia schultzii

ent-Kaurenic acid and many natural derivatives of this diterpene are known to have interesting biological properties. ent-15-Oxo-kaur-16-en-19-oic acid can be easily obtained from grandiflorolic acid which was first isolated from Espeletia grandiflora. The present work describes the proapoptotic effect of ent-15-oxo-kaur-16-en-19-oic acid on the human prostate carcinoma epithelial cell line PC-3 as evidenced by the changes in the expression level of proteins associated with the execution and regulation of apoptosis. Cell viability was affected upon exposure to the compound, the IC(50) were determined as 3.7 microg/ml, which is 4 times lower than that corresponding to a primary cell culture of fibroblasts (14.8 microg/mL). Through Western blot analysis, active forms of caspace-3 associated with the specific proteolysis of Poly(ADP-ribose) polymerase (PARP) were detected. Reduced levels of the antiapoptotic protein Bcl-2, as well as the appearance of internucleosomal DNA fragmentation, were also demonstrated. Thus, ent-15-oxo-kaur-16-en-19-oic acid may be a promising lead compound for new chemopreventive strategies, alone or in combination with traditional chemotherapy agents to overcome drug resistance in tumoral cells.     

Antifungal properties and mode of action of psacotheasin, a novel knottin-type peptide derived from Psacothea hilaris

Psacotheasin is a 34-mer knottin-type peptide that is derived from Psacothea hilaris larvae. In this study, the antifungal activity and mechanism(s) by which psacotheasin affects human fungal pathogens were investigated. Psacotheasin shows remarkable antifungal properties without hemolytic activity against human erythrocytes. To understand the antifungal mechanism(s) of psacotheasin in Candida albicans, flow cytometric analysis with DiBAC₄(3) and PI was conducted. The results showed that psacotheasin depolarized and perturbed the plasma membrane of the C. albicans. Three-dimensional (3D)-flow cytometric contour-plot analysis, accompanied by decreased forward scatter (FS), which indicates cell size, confirmed that psacotheasin exerted antifungal effects via membrane permeabilization. The membrane studies, using a single GUV and FITC-dextran (FD) loaded liposomes, indicate that psacotheasin acts as a pore-forming peptide in the model membrane of C. albicans and the radius of pores were presumed to be anywhere from 2.3 to 3.3 nm. Therefore, the current study suggests that the mechanism(s) of psacotheasin’s antifungal properties function within the membrane.     

Yihiella yeosuensis gen. et sp. nov. (suessiaceae,dinophyceae), a novel dinoflagellate isolated from the coastal waters of Korea

A small (7–11 μm long) dinoflagellate with thin amphiesmal plates was isolated into culture from a water sample collected in coastal waters of Yeosu, southern Korea, and examined by LM, SEM, and TEM, and molecular analyses. The hemispheric episome was smaller than the hyposome. The nucleus was oval and situated from the central to the episomal region of the cell. A large yellowish‐brown chloroplast was located at the end of the hyposome, and some small chloroplasts extended into the periphery of the episome. The dinoflagellate had a single elongated apical vesicle (EAV) and a type E eyespot, which are key characteristics of the family Suessiaceae. Unlike other genera in this family, it had two long furrow lines, one on the episome and the other on the hyposome, and encircling the dorsal, and lateral sides of the cell body. The pyrenoid lacked starch sheaths, but tubular invaginations into the pyrenoid matrix from the cytoplasm were observed. In the TEM, the dinoflagellate was observed to have cable‐like structures (CLSs) near the eyespot but so far not observed in other dinoflagellates. The SSU rDNA sequences examined were 1.2%–5.1% different from those of other genera in the family Suessiaceae, whereas the LSU (D1‐D3) rDNA sequences of this dinoflagellate were 15.1%–31.5% different. The dinoflagellate lacked a 51‐bp fragment in domain D2 of the LSU rDNA, but it had an ~100‐bp fragment in domain D2. This feature has been found previously only in the genera Leiocephalium and Polarella, two other genera of the Suessiaceae. The molecular phylogeny and sequence divergence based on SSU, and LSU rDNA indicate that the Korean dinoflagellate holds a taxonomically distinctive position and we consider it to be a new species in a new genus in the family Suessiaceae, named Yihiella yeosuensis gen. et sp. nov.     

Alienimonas chondri sp. nov., a novel planctomycete isolated from the biofilm of the red alga Chondrus crispus

The phylum Planctomycetes comprises bacteria with peculiar and very unique characteristics among prokaryotes. In marine environments, macroalgae biofilms are well known for harboring planctomycetal diversity. Here, we describe a novel isolate obtained from the biofilm of the red alga Chondrus crispus collected at a rocky beach in Porto, Portugal. The novel strain LzC2^T is motile, rosette-forming with spherical- to ovoid-shaped cells. LzC2^T forms magenta- to pinkish-colored colonies in M13 and M14 media. Transmission and scanning electron microscopy observations showed a division by polar and lateral budding. Mother cells are connected to the daughter cells by a tubular neck-like structure. The strain requires salt for growth. Vitamins are not required for growth. Optimal growth occurs from 15 to 30 °C and within a pH range from 5.5 to 10.0. Major fatty acids are anteiso-C15:0 (54.2%) and iso-C15:0 (19.5%). Phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid represent the main lipids and menaquinone 6 (MK-6) is the only quinone present. 16S rRNA gene-based phylogenetic analysis supports the affiliation to the phylum Planctomycetes and family Planctomycetaceae, with Alienimonas as the closest relative. Strain LzC2^T shares 97% 16S rRNA gene sequence similarity with Alienimonas californiensis. LzC2^T has a genome size of 5.3 Mb and a G+C content of 68.3%. Genotypic and phenotypic comparison with the closest relatives strongly suggest that LzC2^T (=CECT 30038^T=LMG XXXT) is a new species of the genus Alienimonas, for which we propose the name Alienimonas chondri sp. nov., represented by LzC2^T as type strain.16S rRNA gene accession number: GenBank = MN757873.1.Genome accession number: GenBank = WTPX00000000.     

Induction of immunostimulatory cytokine genes expression in human PBMCs by a novel semiquinone glucoside derivative (SQGD) isolated from a Bacillus sp. INM-1

In the present study, a semiquinone glucoside derivative (SQGD) isolated from a radioresistant bacterium Bacillus sp. INM-1 was evaluated for its immunostimulatory activities. Human peripheral blood mononuclear cells (PBMCs) were stimulated by different doses (30–90 μg/ml) of SQGD for different time (3–12 h) intervals at 37 °C, and IL-12p40, IL-23p19, IL-10, RelA and c-Jun gene expression analysis was carried out by qRT-PCR method. SQGD dose dependent cytokines protein expression kinetic analysis was carried out using western blotting. As the results of SQGD (30 μg/ml) stimulation for 3 h at 37 °C, significant induction in IL-12p40, IL-23p19 and RelA gene expression was observed in PBMCs compared to unstimulated control cells. However, no such induction in IL-10 and c-Jun gene expression was observed. Time dependent protein expression study indicated significant increase in IL-12p40, IL-12p35, IL-23p19 and RelA protein expression at 3–6 h, which was found decrease at 12 h upon SQGD treatment. In contrast, IL-10 protein expression was found to enhance significantly at 12 h after SQGD treatment to the PBMCs. SQGD dose dependent study showed approximately similar level of induction in IL-12p40, IL-12p35, IL-23p19 and RelA proteins expression at all tested concentration (30–90 μg/ml) compared to control. However, no significant change in the IL-10 and c-Jun protein expression was observed at any SQGD concentration. SQGD treatment (0.25 mg/kg b wt.) was also found to enhance anti-keyhole Limpet Hemocynin (KLH) IgM antibodies significantly in the mice immunized by KLH.Thus, SQGD fraction stimulates cellular immunity by inducing immunostimulatory cytokines and humoral immunity by enhancing IgM antibodies and could be a promising immunostimulant. Further studies related to molecular mechanisms offering immunostimulation is underway, will certainly helpful to unravel its mode of action in the biological system.