Hidden in plain sight: hyperspectral documentation of complex biofluorescence produced by the green sea urchin (Strongylocentrotus droebachiensis).

Biofluorescence in echinoderms is largely unexplored, and even though the green sea urchinStrongylocentrotus droebachiensisis a well-studied species, the presence and/or function of fluorescence remains very poorly understood. Hyperspectral imaging was conducted on adult sea urchins (N = 380) while fluorospectrometric analysis was conducted on sea urchin coelomic fluid (N = 30). Fluorescence was documented in both the spines and coelomic fluid ofS. droebachiensis. Intact spines exhibited a low intensity green emission (∼550-600 nm), while broken spines averaged a high emission peak in the green spectrum (∼580 nm). Sea urchins produce a red exudate with a pronounced emission peak (∼680 nm) with a shoulder peak (∼730 nm). The sampled coelomic fluid exhibited high variability, with a majority exhibiting a low-level green fluorescence while pronounced emission peaks (N = 5) were found in the red spectrum (∼680 nm). The complex fluorescence produced byS. droebachiensiswarrants further investigation on its applicability for monitoring welfare of sea urchins in aquaculture facilities.

Functional properties of gonad protein isolates from three species of sea urchin: a comparative study.

Sea urchin Mesocentrotus nudus, Glyptocidaris crenularis, and Strongylocentrotus intermedius gonad protein isolates (mnGPIs, gcGPIs, and siGPIs) were extracted by isoelectric solubilization/precipitation (ISP) from the defatted gonads, and their functional properties were compared. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed the similar protein pattern between each protein isolate and defatted gonad, indicating the high efficiency of ISP processing for protein recovery. Amino acid profileconfirmed that the mnGPIs and siGPIs could be potential sources of essential amino acid in nature. As regard to functional properties, mnGPIs showed higher water- and oil- holding capacities followed bysiGPIs and gcGPIs and all protein isolates presented great foaming property. As for emulsifying activity index (EAI), mnGPIs, gcGPIs, and siGPIs showed the minimum solubility and EAI at pH 5, 3, and 4, respectively, and behaved a pH-dependent manner. The gcGPIs revealed the highest EAI from pH 6 to 8 among the samples. In addition, circular dichroism showed increased content of ß-sheet at the expense of α-helix and ß-turn, suggesting the structure denaturation of the protein isolates. Indeed, no statistical difference was observed between secondary structure of mnGPIs and siGPIs. Moreover, ISP processing increased free sulfhydryl content of sea urchin protein isolates, but no difference was observed among the samples. Furthermore, siGPIs revealed the highest amount of total sulfhydryl and disulfide bonds, whereas both defatted gonads and protein isolates from G. crenularis presented the maximum surface hydrophobicity. These results suggest that gonad protein isolates from three species of sea urchin possess various functionalities and therefore can be potentially applied in food system. PRACTICAL APPLICATION: Sea urchin M. nudus, G. crenularis, and S. intermedius gonads are edible, whereas the functional properties of protein isolates from sea urchin gonad remain unknown. In this case, the extraction and comparison of three species of sea urchin gonad protein isolates will not only confirm functional properties but also screen food ingredients with suitable functions. In this study, functionalities of protein isolates derived from M. nudus, G. crenularis, and S. intermedius gonads would provide potential application in bakery food and meat products or as emulsifier candidates in food system.

Green sea urchins (Strongylocentrotus droebachiensis) as potential biomonitors of metal pollution near a former lead-zinc mine in West Greenland.

In this study, metal accumulation in green sea urchins (Strongylocentrotus droebachiensis) was investigated near the former Black Angel lead-zinc mine in Maarmorilik, West Greenland. Sea urchins (n = 9-11; 31-59 mm in diameter) were collected from three stations located at < 1 km, 5 km, and 12 km (reference site) away from the former mine site, respectively. After collection, tissue of the sea urchins was divided into gonads and remaining soft parts (viscera) before subjected to chemical analyses. Focus was on eight elements found in elevated concentrations in the mine waste (iron, copper, zinc, arsenic, silver, cadmium, mercury and lead). Sea urchins at the mine site contained significantly more copper, mercury and lead compared with the reference site for both the gonads and viscera, while the latter also contained significantly more iron, zinc and silver. Arsenic and cadmium were not significantly elevated in sea urchins at the mine site. Most elements were found in higher concentrations in the viscera compared with the gonads. For comprehensive monitoring of metal pollution at mine sites, a diverse selection of monitoring organisms is necessary. The study shows that green sea urchins accumulate selected metals and can be used as a monitoring organism for mining pollution, at least for iron, copper, zinc, silver, mercury and lead. However, the results also show that green sea urchins are less likely to reflect small environmental changes in loading of most metals (except iron, copper and silver) and for arsenic compared to suspension feeders such as blue mussels.

In silico assessment and structural characterization of antioxidant peptides from major yolk protein of sea urchin Strongylocentrotus nudus.

Sea urchin gonads have been demonstrated to contain major yolk protein (MYP), which can be hydrolyzed by enzymes to release biologically active peptides. The in silico analysis of the MYP sequence in the BIOPEP database showed the presence of fragments with antioxidant activity. The sequence was hydrolyzed by 21 kinds of proteases and 23 antioxidant peptides were obtained. Eight peptides, including Leu-Trp (LW), Arg-Trp (RW), Ala-Trp (AW), Thr-Trp (TW), Ala-Asp-Phe (ADF), Leu-Trp-Lys (LWK), Ser-Asp-Phe (SDF) and Leu-Tyr (LY), were screened and a score over 0.5 was obtained using PeptideRanker. The peptides LW, TW and LWK showed a stronger antioxidant capacity with IC50 values of 8.85, 9.59 and 9.62 mmol L-1, respectively, compared to that of glutathione (10.81 mmol L-1). Furthermore, AW, LW and LY showed Trolox equivalent antioxidant capacity (TEAC) values of 3.07, 1.87 and 1.52 mmol TE per mmol peptide, respectively. These results suggest that the MYP from sea urchin (S. nudus) gonads is a good source of antioxidant peptides with abundant tryptophan.

Chemical Profiling and Bioactivity of Body Wall Lipids from Strongylocentrotus droebachiensis.

The lipids from gonads and polyhydroxynaphthoquinone pigments from body walls of sea urchins are intensively studied. However, little is known about the body wall (BW) lipids. Ethanol extract (55 °C) contained about equal amounts of saturated (SaFA) and monounsaturated fatty acids (MUFA) representing 60% of total fatty acids, with myristic, palmitic and eicosenoic acids as major SaFAs and MUFAs, respectively. Non-methylene-interrupted dienes (13%) were composed of eicosadienoic and docosadienoic acids. Long-chain polyunsaturated fatty acids (LC-PUFA) included two main components, n6 arachidonic and n3 eicosapentaenoic acids, even with equal concentrations (15 µg/mg) and a balanced n6/n3 PUFA ratio (0.86). The UPLC-ELSD analysis showed that a great majority of the lipids (80%) in the ethanolic extract were phosphatidylcholine (60 µg/mg) and phosphatidylethanolamine (40 µg/mg), while the proportion of neutral lipids remained lower than 20%. In addition, alkoxyglycerol derivatives-chimyl, selachyl, and batyl alcohols-were quantified. We have assumed that the mechanism of action of body wall lipids in the present study is via the inhibition of MAPK p38, COX-1, and COX-2. Our findings open the prospective to utilize this lipid fraction as a source for the development of drugs with anti-inflammatory activity.

Enhanced antitumor activity of gemcitabine by polysaccharide-induced NK cell activation and immune cytotoxicity reduction in vitro/vivo.

The polysaccharide SEP has been reported to activate NK and T cells via TLR2/4. Here, the combination of gemcitabine (GEM) and SEP against HepG-2 was investigated. SEP apparently enhanced antitumor activity of gemcitabine against liver cancer through stimulating NKG2D and DAP10/Akt pathway to activate NK cells. The NKG2D upregulation could improve the sensitivity of NK-92 cells targeting to its ligand MICA expressed on HepG-2 cells. Meanwhile, GEM up-regulated MICA expression and attenuated soluble MICA secretion through inhibiting ADAM10 expression, which in turn enhanced the cytotoxicity of NK-92 cells against cancer cells. SEP remarkably enhanced GEM antitumor activity with an inhibitory rate of 79.1% in an H22-bearing mouse model. Moreover, SEP reversed atrophy and apoptosis caused by GEM in both spleen and bone marrow through suppressing ROS secretion in vivo. The data indicated that the combination of SEP and GEM is a potential chemo-immunotherapy strategy for liver cancer treatment clinically.

Diversity of Polyhydroxynaphthoquinone Pigments in North Pacific Sea Urchins.

Using high-performance liquid chromatography with diode-array detection and mass spectrometry (HPLC-DAD/MS) we investigated the composition of polyhydroxynaphthoquinone (PHNQ) pigments from sea urchins Strongylocentrotus pallidus, St. polyacanthus, St. droebachiensis, Brisaster latifrons and Echinarachnius parma, collected in the Sea of Okhotsk and the Bering Sea. Identification of PHNQ pigments from sea urchins St. polyacanthus, B. latifrons, and E. parma was performed for the first time. Among the usual PHNQ pigments, mono- and dimethoxy derivatives of spinochrome E, not previously found in other sea urchins, were discovered in St. polyacanthus and St. droebachiensis. In St. droebachiensis, two monomethoxy derivatives of echinochrome A were detected, isolated previously from only tropical sea urchins. It was found that the composition and total content of pigments of St. droebachiensis depends on the collection area of the sea urchins and its depth and varies from 88 to 331 µg/g of dry shells. Sea urchins St. pallidus, B. latifrons and E. parma had average values for PHNQ pigment content, approximately 30 µg/g, and St. polyacanthus had a low PHNQ content, 13 µg/g.

Comparative stability of dimeric and monomeric pigments extracted from sea urchin Strongylocentrotus droebachiensis.

Ultraviolet-visible spectroscopy and UPLC-DAD-MS were used for analysis of stability of ethanol solutions of ethylidene-6,6'-bis(2,3,7-trihydroxynaphthazarin) (ENZ), spinochrome dimer (SDM) and spinochrome D (SD) that were isolated from Strongylocentrotus droebachiensis. In the freshly prepared solution, the concentration of ENZ at pH 6.0 was at 6 fold less comparing to pH 1.6. The increase of pH up to 4.0 resulted to increase of SD concentration and to decrease of SDM concentration. After 48 h storage, both dimers showed the highest stability at pH 1.6, while the elevation of the pH solution up to 6.0 activates degradation of SDM and ENZ at 1.3 and 3.6 fold correspondingly. The concentration of SD after 48 h storage at the pH 1.6 was at two-fold less comparing to the initial concentration, and at the pH 6.0 - at 4 fold less. This study contributes to increasing the knowledge on the stability of the spinochrome pigments.

New Aminonaphthoquinone from the Sea Urchins Strongylocentrotus pallidus and Mesocentrotus nudus.

The aminonaphthoquinone, spinamine E (5), was isolated for the first time from the sea urchins Strongylocentrotus pallidus (Sars G.O., 1872) and Mesocentrotus nudus (A. Agassiz, 1864). The structure of 5 was elucidated as 2-amino-3,5,6,7,8-pentahydroxy-1,4-naphthoquinone using ID 1H-, 13C- and 2D NMR procedures, and HR-ESI mass-spectrometric data of 5 and its trimethyl ether. Spinamine E, as well as two other aminonaphthoquinones of sea urchins, echinamines A (2) and B (3), along with their hydroxylated analogues, spinochrome E (4) and echinochrome A (1), were tested for their ability to scavenge the stable DPPH radical and to inhibit lipid peroxidation. All investigated naphthoquinones obtained from the sea urchins showed a high antiradical activity, which was up to 1.5 times higher than that of α-tocopherol. Echinamine B showed the highest scavenging effect (EC50 = 6.5-10(-6) M); this effect decreases in the series 3>5>1>2>4>α-tocopherol. In a lipid peroxidation inhibition testing model, echinamine B and spinamine E showed the highest inhibitory effect. The stability of compounds 1-5 in weakly alkaline solutions was evaluated.

SEP enhanced the antitumor activity of 5-fluorouracil by up-regulating NKG2D/MICA and reversed immune suppression via inhibiting ROS and caspase-3 in mice.

Chemotherapy and immunotherapy are the main remedies used in cancer treatment. Because immunotherapy can not only reduce the toxicity of chemotherapeutics but also enhance antitumor effects in vivo, combining these two therapies is a trend that continues to gain more attention in clinic. SEP, a polysaccharide isolated from Strongylocentrotus nudus egg, has been reported to display antitumor activity by stimulating immune cells, including NK and T cells, via TLR2 and TLR4. In the present study, the synergistic effect between SEP and 5-fluorouracil (5-FU), a traditional cytotoxic drug, in vitro and in vivo was investigated. The results obtained indicated that SEP alone stimulated NK-92 cytotoxicity and coordinated with 5-FU to augment the cytotoxicity of NK-92 cells against HepG-2 or A549 cells in vitro. SEP promoted NK-92 activity by stimulating NKG2D and its downstream DAP10/PI3K/Erk signaling pathway. Additionally, 5-FU could increase MICA expression on HepG-2 or A549 cells and prevent membrane MICA from shedding as soluble MICA, which were abrogated in the tumor cells transfected with ADAM 10 overexpression plasmid. Moreover, in H22- or Lewis lung cancer (LLC)-bearing mouse models, SEP reversed 5-FU-induced atrophy and apoptosis in both the spleen and bone marrow in vivo by suppressing ROS generation and caspase-3 activation. All of these results highlight the potential for the combination of SEP and 5-FU in cancer therapy in the future.

Extraction, preliminary characterization and immunostimulatory activity in vitro of a polysaccharide isolated from Strongylocentrotus nudus eggs.

A new water-soluble polysaccharide (SEP-2), with a molecular weight of 6.78 × 10(5)Da, was isolated from Strongylocentrotus nudus eggs under the extraction conditions optimized by response surface methodology (RSM). Preliminary characterization of SEP-2 was performed by HPSEC and GC-MS, indicating that SEP-2 could be a D-glucan containing a (1 → 4)-linked D-Glcp backbone with (1 → 3)-linked D-Glcp side chains. In subsequent immunostimulatory studies, significantly enhanced ROS level, NO production and inflammatory cytokines secretion (IL-1ß, IL-6, and TNF-α) were observed in SEP-2 treated murine macrophage cell line RAW264.7. These results suggest that SEP-2 might have the capability to enhance the microbicidal activity and killing function of macrophages through the polarization toward the pro-inflammatory M1 state.

The anti-lung cancer activity of SEP is mediated by the activation and cytotoxicity of NK cells via TLR2/4 in vivo.

Strongylocentrotus nudus egg polysaccharide (SEP) has been reported to display antitumor activity. However, the effects of SEP and its underlying mechanism in the treatment of lung cancer remain unclear, particularly with an immunodeficient mouse model of human non-small cell lung cancer (NSCLC). In the present study, we investigated the anti-lung cancer effects of SEP and its underlying mechanism of action in both Lewis lung cancer (LLC)-bearing C57/BL6J mice and human NSCLC H460-bearing nude mice. Although SEP showed no inhibitory effects on tumor cells in vitro, it markedly stimulated the percentage of CD3-NK1.1(+) cells and natural killer (NK) cell cytotoxicity in the spleens of nude mice and C57/BL6J mice. In LLC-bearing mice, SEP not only inhibited tumor growth but also promoted NK-mediated cytotoxicity, the NK1.1(+) cell population, and IL-2 and IFN-γ secretion. SEP significantly suppressed H460 growth in nude mice, which was abrogated by the selective depletion of NK cells via the intraperitoneal injection of anti-asialo GM-1 antibodies. Furthermore, anti-TLR2/4 antibodies blocked both SEP and NK cell binding and SEP-induced perforin secretion. SEP-induced proliferation and IFN-γ secretion by NK cells in wild type mice were partially impaired in TLR2 or TLR4 knockout mice. These results suggest that SEP-promoted NK cytotoxicity, which was partially mediated via TLR2 and TLR4, was the main contributing factor to lung cancer inhibition in vivo and that SEP may be a potential immunotherapy candidate for the treatment of lung cancer.

Antiallergic effects of pigments isolated from green sea urchin (Strongylocentrotus droebachiensis) shells.

This study was undertaken to evaluate possible antiallergic effects of an extract of pigments from green sea urchin (Strongylocentrotus droebachiensis) shells. Effects were studied on animal models - guinea pig ileum contraction, rabbit eyes allergic conjunctivitis, and rabbit local skin irritation. The extract significantly reduced, in a dose-dependent manner, the histamine-induced contractions of the isolated guinea pig ileum with ID50 =1.2 µg/mL (in equivalents of spinochrome B), had an inhibitory effect on the model of ocular allergic inflammation surpassing the reference drug olopatadine, and did not show any irritating effect in rabbits. The extract predominantly contained polyhydroxy-1,4-naphthoquinone which would be responsible for the pharmacological activity. The active compounds of the extract were evaluated in silico with molecular docking. Molecular docking into H1R receptor structures obtained from molecular dynamic simulations showed that all spinochrome derivatives bind to the receptor active site, but spinochrome monomers fit better to it. The results of the present study suggest possibilities for the development of new agents for treating allergic diseases on the base of pigments from sea urchins shells.

The offline combination of thin-layer chromatography and high-performance liquid chromatography with diode array detection and micrOTOF-Q mass spectrometry for the separation and identification of spinochromes from sea urchin (Strongylocentrotus droebachiensis) shells.

Thin-layer chromatography (TLC) with off-line high-performance liquid chromatography coupled to diode array detection and micrOTOF-Q mass spectrometry (HPLC-DAD-MS) resulted in the successful fractionation, separation and identification of spinochrome pigments from sea urchin (Strongylocentrotus droebachiensis) shells. Two fractions of pigments were separated by TLC and eluted with methanol using a TLC-MS interface. HPLC-DAD-MS analysis of the fractions indicated the presence of six sea urchin pigments: spinochrome monomers B and D, three spinochrome dimers (anhydroethylidene-6,6'-bis(2,3,7-trihydroxynaphthazarin) and its isomer and ethylidene-6,6'-bis(2,3,7-trihydroxynaphthazarin)), and one pigment that was preliminary identified as a spinochrome dimer with the structural formula C(22)H(16)O(16).

Strongylocins, novel antimicrobial peptides from the green sea urchin, Strongylocentrotus droebachiensis.

Sea urchins possess an innate immune system and are regarded as a potential source for the discovery of new antimicrobial peptides (AMPs). Here we report the purification and characterization of two novel antibacterial peptides (5.6 and 5.8kDa) from coelomocyte extracts of the green sea urchin, Strongylocentrotus droebachiensis. These are the first reported AMPs isolated from sea urchins. The cDNA encoding the peptides and genomic sequences was isolated and sequenced. The two peptides (named strongylocins 1 and 2) have putative isoforms (1b and 2b), similar to two putative proteins from the purple sea urchin S. purpuratus. The native strongylocins are cationic, defensin-like peptides (cysteine-rich), but show no similarity to other known AMPs concerning the cysteine distribution pattern. Strongylocin 1 consists of 83 amino acids that include a preprosequence of 35 amino acids, whereas strongylocins 2a and 2b are composed of 89 and 90 amino acids, respectively, where 38 amino acids represent a preprosequence. No introns were found in the cloned gene of strongylocin 1b, whereas three introns and four exons were found in strongylocins 1a and 2a/b. The latter gene organization was also found in genes coding for putative strongylocins in S. purpuratus. The molecular mass difference between the native peptide and the deduced strongylocin 2 suggests that the first amino acid is bromotryptophan. The native peptides display potent activities against Gram-negative and Gram-positive bacteria.