Evaluation of the chronic toxicity of bisphenol A and bisphenol AF to sea cucumber Apostichopus japonicus after long-term single and combined exposure at environmental relevant concentration.

Bisphenols are emerging endocrine disrupting pollutant, and several studies have reported that they are already ubiquitous in various environmental matrices and intend to deposit in sediment. The primary sources of bisphenols are river and sewage discharge. Sea cucumber (Apostichopus japonicus), a typical deposit feeder, is one of the most important commercial marine species in Aisa. However, the effects of the bisphenol A (BPA) and its analogues bisphenol AF (BPAF) on sea cucumber was unclear. In this study, we carried out field survey in major sea cucumber farming areas in northern China, with the aim of determining which bisphenol analogue is the major bisphenol contamination in this aquaculture area. The results showed that the presence of BPAF was detected in four sampling sites (Dalian, Tangshan, Laizhou, and Longpan). The mean level of BPAF in Laizhou sediment samples was the highest which reached to 9.007 ± 4.702 µ g/kg. Among the seawater samples, the BPAF only have been detected in the samples collected at Longpan. (0.011 ± 0.003 µ g/L). Furthermore, we conducted an experiment to evaluate the single and combined toxicity of BPA and BPAF on sea cucumbers. The concentrations were informed by the findings based on the results of field research. (0.1, 1.0, and 10 µ g/L). After exposure, the body weight gain, and specific growth rate showed no significant changes (P > 0.05). We observed the histological alterations in respiratory tree of treated sea cucumbers including the fusion and detachment of lining epithelial tissue, and increase of lumen space. However, the catalase (CAT), malondialdehyde (MDA), and glutathione (GSH) activity was not significantly changed (P > 0.05). We evaluated the effects of BPA and BPAF through calculating the integrated biomarker response index (IBR), and the results indicated that the toxicity of combined treatment was higher than single treatment. Additionally, BPAF exposure to A. japonicus was more toxic than BPA.

Effects of chronic prometryn exposure on antioxidative status, intestinal morphology, and microbiota in sea cucumber (Apostichopus japonicus).

Prometryn is an occasional triazine herbicide used in aquaculture to kill algae. However, deposition of prometryn at the bottom of the pond poses a potential threat to aquatic animals, especially benthos, such as the sea cucumber. This study investigated the toxic effects of prometryn oral exposure on antioxidants, and the intestinal histomorphology and microbiome of sea cucumbers. Results showed that the accumulation of prometryn in the intestine, respiratory tree, and body wall decreased sequentially under the same level. Severe pathological damages were observed in the intestines of sea cucumbers fed with 0.080 and 1.595 g/kg prometryn (measured concentration). Moreover, hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations were significantly increased in prometryn treatment groups compared to the control group (P < 0.05), while the catalase (CAT) activity was significantly decreased (P < 0.05) in the coelomic fluid of treatment groups. At the phylum level, the abundance of Proteobacteria was significantly higher in the 0.080 g/kg treatment group than in the control group. In addition, prometryn exposure reduced the diversity of intestinal microflora in sea cucumbers. In conclusion, these results suggest that prometryn has potential toxicity to sea cucumber. Therefore, the harm of prometryn deposited in the sediment to aquatic animals must be a concern in aquaculture.

Novel lncRNA lncRNA001074 participates in the low salinity-induced response in the sea cucumber Apostichopus japonicus by targeting the let-7/NKAα axis.

Salinity fluctuations have severe impacts on sea cucumbers and therefore important consequences in sea cucumber farming. The responses of sea cucumbers to salinity changes are reflected in the expression profiles of multiple genes and non-coding RNAs (ncRNAs). The microRNA (let-7) which is a developmental regulator, the ion transporter gene sodium potassium ATPase gene (NKAα), and the long ncRNA lncRNA001074 were previously shown to be involved in responses to salinity changes in various marine species. To better understand the relationship between ncRNAs and target genes, the let-7/NKAα/lncRNA001074 predicted interaction was investigated in this study using luciferase reporter assays and gene knockdowns in the sea cucumber Apostichopus japonicus. The results showed that NKAα was the target gene of let-7 and NKAα expression levels were inversely correlated with let-7 expression based on the luciferase reporter assays and western blots. The let-7 abundance was negatively regulated by lncRNA001074 and NKAα both in vitro and in vivo. Knockdown of lncRNA001074 led to let-7 overexpression. These results demonstrated that lncRNA001074 binds to the 3'-UTR binding site of let-7 in a regulatory manner. Furthermore, the expression profiles of let-7, NKAα, and lncRNA001074 were analyzed in sea cucumbers after the knockdown of each of these genes. The results found that lncRNA001074 competitively bound let-7 to suppress NKAα expression under low salinity conditions. The downregulation of let-7, in conjunction with the upregulation of lncRNA001074 and NKAα, may be essential for the response to low salinity change in sea cucumbers. Therefore, the dynamic balance of the lncRNA001074, NKAα, and let-7 network might be a potential response mechanism to salinity change in sea cucumbers.

Anti-Infective and Antiviral Activity of Valinomycin and Its Analogues from a Sea Cucumber-Associated Bacterium, Streptomyces sp. SV 21.

The manuscript investigated the isolation, characterization and anti-infective potential of valinomycin (3), streptodepsipeptide P11A (2), streptodepsipeptide P11B (1), and one novel valinomycin analogue, streptodepsipeptide SV21 (4), which were all produced by the Gram-positive strain Streptomycescavourensis SV 21. Although the exact molecular weight and major molecular fragments were recently reported for compound 4, its structure elucidation was not based on compound isolation and spectroscopic techniques. We successfully isolated and elucidated the structure based on the MS2 fragmentation pathways as well as 1H and 13C NMR spectra and found that the previously reported structure of compound 4 differs from our analysis. Our findings showed the importance of isolation and structure elucidation of bacterial compounds in the era of fast omics technologies. The here performed anti-infective assays showed moderate to potent activity against fungi, multi drug resistant (MDR) bacteria and infectivity of the Hepatitis C Virus (HCV). While compounds 2, 3 and 4 revealed potent antiviral activity, the observed minor cytotoxicity needs further investigation. Furthermore, the here performed anti-infective assays disclosed that the symmetry of the valinomycin molecule is most important for its bioactivity, a fact that has not been reported so far.

Sex steroids and steroidogenesis-related genes in the sea cucumber, Holothuria scabra and their potential role in gonad maturation.

The sea cucumber Holothuria scabra is an economically valuable marine species which is distributed throughout the Asia-Pacific region. With the natural population declining due to over fishing, aquaculture of this species is deemed necessary. Hence, it is essential to understand the mechanisms regulating the reproduction in order to increase their populations. Sex steroids, including estrogens, androgens and progestogens, play an important role in reproduction in most vertebrates and several invertebrates. It has been proposed that sea cucumbers have the same sex steroids as vertebrates but the steroidogenic pathway in the sea cucumbers is still unclear. In this study, we demonstrated by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) that sex steroids (estradiol, progesterone, and testosterone) were present in H. scabra neural and gonadal tissues. In silico searches of available sea cucumber transcriptome data identified 26 steroidogenesis-related genes. Comparative analysis of encoded proteins for the steroidogenic acute regulatory protein (HscStAR), CYP P450 10, 17 and 3A (HscCYP10, HscCYP17, HscCYP3A) and hydroxysteroid dehydrogenases (Hsc3ß-HSD, Hsc17ß-HSD) with other species was performed to confirm their evolutionary conservation. Gene expression analyses revealed widespread tissue expression. Real-time PCR analysis revealed that HscStAR, HscCYP10, Hsc3ß-HSD, and Hsc17ß-HSD gene expressions were similar to those in ovaries and testes, which increased during the gonad maturation. HscCYP17 mRNA was increased during ovarian development and its expression declined at late stages in females but continued high level in males. The expression of the HscCYP3A was high at the early stages of ovarian development, but not at other later stages in ovaries, however it remained low in testes. Moreover, a role for steroids in reproduction was confirmed following the effect of sex steroids on vitellogenin (Vtg) expression in ovary explant culture, showing upregulation of Vtg level. Collectively, this study has confirmed the existence of steroids in an echinoderm, as well as characterizing key genes associated with the steroidogenic pathway. We propose that sex steroids might also be associated with the reproduction of H. scabra, and the identification of biosynthetic genes enables future functional studies to be performed.

Combined Effects of Elevated Temperature and Crude Oil Pollution on Oxidative Stress and Apoptosis in Sea Cucumber (Apostichopus japonicus, Selenka).

Currently, global climate change and oil pollution are two main environmental concerns for sea cucumber (Apostichopus japonicus) aquaculture. However, no study has been conducted on the combined effects of elevated temperature and oil pollution on sea cucumber. Therefore, in the present study, we treated sea cucumber with elevated temperature (26 °C) alone, water-accommodated fractions (WAF) of Oman crude oil at an optimal temperature of 16 °C, and Oman crude oil WAF at an elevated temperature of 26 °C for 24 h. Results showed that reactive oxygen species (ROS) level and total antioxidant capacity in WAF at 26 °C treatment were higher than that in WAF at 16 °C treatment, as evidenced by 6.03- and 1.31-fold-higher values, respectively. Oxidative damage assessments manifested that WAF at 26 °C treatment caused much severer oxidative damage of the biomacromolecules (including DNA, proteins, and lipids) than 26 °C or WAF at 16 °C treatments did. Moreover, compared to 26 °C or WAF at 16 °C treatments, WAF at 26 °C treatment induced a significant increase in cellular apoptosis by detecting the caspase-3 activity. Our results revealed that co-exposure to elevated temperature and crude oil could simulate higher ROS levels and subsequently cause much severer oxidative damage and cellular apoptosis than crude oil alone on sea cucumber.

Immunomodulatory effects of chicken egg yolk antibodies (IgY) against experimental Shewanella marisflavi AP629 infections in sea cucumbers (Apostichopus japonicus).

Skin ulceration syndrome in sea cucumbers is an infectious bacterial disease with fast and high mortality. This study investigated the protection of chicken egg yolk antibodies (IgY) on skin ulcer syndrome in sea cucumbers induced by intraperitoneally injecting Shewanella marisflavi AP629. Inactivated whole S. marisflavi AP629 cells were used as an immunogen to immunize laying hens. The highest titer of the obtained specific IgY by ELISA was 1:90000. Specific IgY significantly inhibited the growth of S. marisflavi AP629 in a liquid medium, dose-dependent manner at concentrations ranging from 0.5 to 2 mg/mL. Results obtained from scanning electron microscopy and confocal laser scanning microscopy showed that specific IgY could make bacteria agglutinate and damage the cell membrane of S. marisflavi AP629, resulting in a decrease of bacterial viability. Sea cucumbers treated with 25, 5, and 1 mg/mL anti-S. marisflavi AP629 IgY could achieve survival rates of 77.5%, 50%, and 22.5% at day 12 when the infection and injection therapy were carried out at the same time, respectively. However, survival rates of sea cucumbers treated with 25 mg/mL of nonspecific IgY were only 7.5% at day 12. All sea cucumbers in the positive control group died within twelve days after bacterial inoculation. Levels of the five humoral immune factors (LYZ, ACP, NOS, SOD, CAT) released by coelomocytes were significantly increased in the specific IgY group compared to the nonspecific IgY and positive control groups within 12 h. However, the activities of LYZ, ACP, and SOD decreased rapidly at the 48 h time point in the specific IgY group, indicating that specific IgY treatment could shorten the time needed to restore balance in sea cucumber immune systems. Oral prophylaxis with egg yolk powders was that all sea cucumbers were challenged with 4.2 × 106 CFU S. marisflavi AP629 by intraperitoneal injection after 60 days of feeding. Survival rates of diets containing 10%, 5%, and 1% specific egg yolk powder were 57.5%, 52.5%, and 30% by day 12, respectively, and the survival rate was 27.5% for the nonspecific group and 22.5% for the positive control group. After feeding for 60 days, enzyme activities of LZY, NOS, and SOD were all significantly enhanced in sea cucumbers fed with specific egg yolk powder when compared to the control group (p < 0.05). This study demonstrated that the phagocytic activities of coelomocytes were significantly stimulated after specific IgY treatment over that of nonspecific IgY or without IgY treatments in sea cucumbers (p < 0.05). Overall, our results revealed that anti-S. marisflavi AP629 IgY has a positive immunomodulatory effect on sea cucumbers infected with S. marisflavi AP629.

Ultraviolet-Ray-Induced Sea Cucumber (Stichopus japonicus) Melting Is Mediated by the Caspase-Dependent Mitochondrial Apoptotic Pathway.

Sea cucumber body-wall melting occurs under certain circumstances. We have shown that apoptosis but not autolysis plays a critical role in the initial stage. However, it is still unclear how apoptosis is triggered in this process. In this study, we examined the levels of reactive oxygen species (ROS), the levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax) proteins, the depolarization of mitochondrial transmembrane potentials, and cytochrome c (Cyt c) release during sea cucumber melting induced by ultraviolet (UV) exposure. We also investigated the contribution of caspase in this process by injecting a pan-caspase inhibitor. Our data showed that UV exposure stimulates ROS production, dysfunction of mitochondria, and the release of Cyt c in sea cucumber coelomic fluid cells and body walls. We found a decrease of Bcl-2 and increase of Bax in the mitochondria after UV exposure. We also demonstrated that these changes are associated with elevated caspase-9 and -3 activity. Finally, our data showed that the inhibition of caspases-9 and -3 using an inhibitor suppresses UV-induced sea cucumber melting. These results suggest that apoptosis during sea cucumber melting is mediated by mitochondrial dysfunction and follows the activation of the caspase-signaling pathway. This study presents a novel insight into the mechanism of sea cucumber melting.

Molecular ecological network analysis reveals the effects of probiotics and florfenicol on intestinal microbiota homeostasis: An example of sea cucumber.

Animal gut harbors diverse microbes that play crucial roles in the nutrition uptake, metabolism, and the regulation of host immune responses. The intestinal microbiota homeostasis is critical for health but poorly understood. Probiotics Paracoccus marcusii DB11 and Bacillus cereus G19, and antibiotics florfenicol did not significantly impact species richness and the diversity of intestinal microbiota of sea cucumber, in comparison with those in the control group by high-throughput sequencing. Molecular ecological network analysis indicated that P. marcusii DB11 supplementation may lead to sub-module integration and the formation of a large, new sub-module, and enhance species-species interactions and connecter and module hub numbers. B. cereus G19 supplementation decreased sub-module numbers, and increased the number of species-species interactions and module hubs. Sea cucumber treated with florfenicol were shown to have only one connecter and the lowest number of operational taxonomic units (OTUs) and species-species interactions within the ecological network. These results suggested that P. marcusii DB11 or B. cereus G19 may promote intestinal microbiota homeostasis by improving modularity, enhancing species-species interactions and increasing the number of connecters and/or module hubs within the network. In contrast, the use of florfenicol can lead to homeostatic collapse through the deterioration of the ecological network.

Effects of Acute and Chronic Heavy Metal (Cu, Cd, and Zn) Exposure on Sea Cucumbers (Apostichopus japonicus).

Acute and chronic toxicity tests were conducted with sea cucumber (Apostichopus japonicus) exposed to heavy metals. Acute toxicity values (96 h LC50) were 2.697, 0.133, and 1.574 mg L(-1) for Zn, Cu, and Cd, respectively, and were ranked in order of toxicity: Cu > Cd > Zn. Under chronic metal exposure the specific growth rates of sea cucumbers decreased with the increase of metal concentration for all the three metals. After acute metal exposure, the oxygen consumption rate (OCR) decreased. The OCRs in all groups were significantly different than control (P < 0.05) except in the group treated with 1.00 mg L(-1) Zn (P < 0.05), where the increase of OCR was observed. The OCRs in groups chronically exposed to metals were significantly lower than that in the control group (P < 0.05). The activity of both pyruvate kinase (PK) and hexokinase (HK) in sea cucumbers followed: respiratory tree > muscle > intestine in natural sea water. After chronic Zn, Cu, and Cd exposure, the change pattern of HK and PK in respiratory tree, muscle, and intestine varied slightly. However, the activity of the enzyme showed a general trend of increase and then decrease and the higher the exposure concentration was, the earlier the highest point of enzyme activity was obtained.

Enrichment, Distribution of Vanadium-Containing Protein in Vanadium-Enriched Sea Cucumber Apostichopus japonicus and the Ameliorative Effect on Insulin Resistance.

Sea cucumbers are a potential source of natural organic vanadium that may improve insulin resistance. In this work, vanadium was accumulated rapidly in blood, body wall, and intestine by sea cucumber Apostichopus japonicus. Furthermore, water-soluble vanadium-containing proteins, the main form of the organic vanadium, were tentatively accumulated and isolated by a bioaccumulation experiment. It was also designed to evaluate the beneficial effect of vanadium-containing proteins (VCPs) from sea cucumber rich in vanadium on the development of hyperglycemia and insulin resistance in C57BL/6J mice fed with a high-fat high-sucrose diet (HFSD). HFSD mice treated with VCPs significantly decreased fasting blood glucose, serum insulin, and HOMA-IR values as compared to HFSD mice, respectively. Serum adiponectin, resistin, TNF-α, and leptin levels in insulin-resistant mice were dramatically reduced by a VCP supplement. These results show an ameliorative effect on insulin resistance by treatment with VCPs. Such compound seems to be a valuable therapy to achieve and/or maintain glycemic control and therapeutic agents in the treatment arsenal for insulin resistance and type 2 diabetes.

The effects of dietary lead on growth, bioaccumulation and antioxidant capacity in sea cucumber, Apostichopus japonicus.

Three different diets amended with lead nitrate [Pb(NO3)2] (100, 500 and 1000mg Pb/kg dry weight) and a Pb-free control diet (1.03mg Pb/kg dry weight) were fed to sea cucumber (Apostichopus japonicus) for 30 days. The patterns of Pb accumulation over time were determined in various tissues (body wall, intestine and respiratory tree), as well as growth performance and antioxidant enzymes activities. Pb accumulation in body wall and intestine increased with time in all dietary Pb treatments. When fed the highest Pb diet, the body wall exhibited the greatest Pb burden (16.37mg Pb/kg tissue wet weight), while Pb content in the intestine (2.68mg Pb/kg tissue wet weight) and the respiratory tree (1.78mg Pb/kg tissue wet weight) were lower than Pb content in the body wall by day 30. The body weight gain (BWG), specific growth rate (SGR) and survival rate (SR) had not been affected by 30 days oral administration of Pb supplemented diet. However, the antioxidant enzymes activities [superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px)] of test groups were lower than control group in body wall and malondialdehyde (MDA) concentration in the body wall was opposite after 30 days in sea cucumbers. In summary, this work reports toxic effects in sea cucumber, A. japonicus, after dietary exposure to Pb.

Beneficial Effects of Rhodotorula sp. C11 on Growth and Disease Resistance of Juvenile Japanese Spiky Sea Cucumber Apostichopus japonicus.

The purpose of this study was to evaluate the effects of dietary administration of the live yeast, Rhodotorula sp. C11, on growth and disease resistance against Vibrio splendidus infection in juvenile Japanese spiky sea cucumber Apostichopus japonicus. Sea cucumbers were fed diets containing Rhodotorula sp. C11 at 0 (control), 104, 105, and 106 CFU/g of feed for 45 d. There were three replicate tanks per dietary treatment. The specific growth rates were higher in all sea cucumbers treated with Rhodotorula sp. C11 than in the controls. Following a challenge with V. splendidus NB13, the cumulative prevalence and mortality of sea cucumbers fed diets supplemented with Rhodotorula sp. C11 were lower than in animals fed the basal diet. In sea cucumbers fed diets supplemented with Rhodotorula sp. C11 for 42 d, the only viable yeast found in the intestine was Rhodotorula sp. C11, which had counts of 1.58-1.98 × 104CFU/g. No yeast was isolated from the intestine of animals fed the basal diet. For the colonization study, 20 sea cucumbers from each dietary treatment were removed to separate tanks and fed the control diet from day 16 to day 46. The viable yeast (Rhodotorula sp. C11) counts in the intestine decreased to 60-80 CFU/g by day 37. Moreover, as demonstrated by denaturing gradient gel electrophoresis, Rhodotorula sp. C11 colonization of the intestine could be detected until day 46. The differences in culture and PCR-denaturing gradient gel electrophoresis may be due to differences in the sensitivity of both methods. The present result showed that Rhodotorula sp. C11 was able to successfully colonize the intestine of juvenile Japanese spiky sea cucumbers by dietary supplementation, which improved its growth and disease resistance.

Transcriptome sequencing of sea cucumber (Apostichopus japonicus) and the identification of gene-associated markers.

Sea cucumber (Apostichopus japonicus) is an ecologically and economically important species in East and South-East Asia. This project aimed to identify large numbers of gene-associated markers and differentially expressed genes (DEGs) after lipopolysaccharides (LPS) challenge in A. japonicus using high-throughput transcriptome sequencing. A total of 162 million high-quality reads of 174 million raw reads were obtained by deep sequencing using Illumina HiSeq™ 2000 platform. Assembly of these reads generated 94 704 unigenes, with read length ranging from 200 to 16 153 bp (average length of 810 bp). A total of 36 005 were identified as coding sequences (CDSs), 32 479 of which were successfully annotated. Based on the assembly transcriptome, we identified 142 511 high-quality single nucleotide polymorphisms (SNPs). Among them, 33 775, 63 120 and 45 616 were located in sequences without predicted CDS (non-CDSs), CDSs and untranslated regions (UTRs), respectively. These putative SNPs included 82 664 transitions and 59 847 transversions. Totally, 89 375 (59.1%) were distributed in 15 473 known genes. A total of 6417 microsatellites were detected in 5970 unigenes, 3216 of which were annotated and 2481 were successfully subjected for primer design. The numbers of simple sequence repeats (SSRs) identified in non-CDSs, CDSs and UTRs were 2367, 2316 and 1734. These potential SNPs and SSRs are expected to provide abundant resources for genetic, evolutionary and ecological studies in sea cucumber. Transcriptome comparison revealed 1330, 1347 and 1291 DEGs in the coelomocytes of A. japonicus at 4 h, 24 h and 72 h after LPS challenge, respectively. Approximately 58.4% (1802) of total DEGs have been successfully annotated.

Sewage-exposed marine invertebrates: survival rates and microbiological accumulation.

A large number of bacteria, including agents responsible for diseases, characterise sewage-polluted seawaters. Apart from standards for bathing waters and bivalve aquaculture waters, there are no general microbiological standards applicable to seawaters to help decide if bacterial pollution is within acceptable ranges. This study represents an attempt towards the issue of comparing the susceptibility of different marine invertebrates subjected to polluted seawater with a high microbial contamination. We explored the survival rates and the microbiological accumulation of mollusc bivalves, echinoderms and crustaceans species exposed to sewage-polluted seawaters. Microbiological analyses were performed on the polluted seawater and on the homogenates of exposed and unexposed specimens. Culturable bacteria (22 °C and 37 °C) and microbial pollution indicators (total coliforms, Escherichia coli and intestinal enterococci) were measured. When exposed to the sewage-polluted seawater, the examined invertebrates showed different survival rates. In the filter feeders, bacterial densities at 22 °C and 37 °C rose after 96 h of exposure to sewage. The highest concentrations of total coliforms and intestinal enterococci were found in exposed bivalve Mytilus galloprovincialis. The concentrations of bacteria growing at 37 °C were lower in the exposed deposit feeders compared to the polluted seawater. Some yeasts were absent in several exposed species although these yeasts were present in the polluted seawater. Our data suggest that the examined filter feeders, given their capability to survive and accumulate bacteria, may counteract the effects of sewage and restore seawater quality.

Polysaccharides from Astragalus membranaceus promote phagocytosis and superoxide anion (O2-) production by coelomocytes from sea cucumber Apostichopus japonicus in vitro.

The potential immunostimulatory effects of Astralagus membranaceus polysaccharides (APS) on sea cucumber, Apostichopus japonicus (Selenka), were investigated in vitro. Phagocytosis and superoxide anion (O(2)(-)) production by phagocytic amoebocytes (PA) from A. japonicus coelomic fluid were measured during incubation at 18 degrees C, 22 degrees C, or 25 degrees C with APS at 0, 10, 20, or 40 microg mL(-1) (n=3). Phagocytic activity against yeast cells was quantified by direct visualization, and O(2)(-) production by nitroblue tetrazolium (NBT) reduction assay. Compared with controls, including APS at 20 microg mL(-1) significantly increased (P<0.05) the percentage of phagocytic capacity (PC) and phagocytic index (PI) at 18 degrees C and 22 degrees C, but no significant enhancement was observed at 25 degrees C. In contrast, the coelmocytes of A. japonicus can have an obvious generation of O(2)(-) after the stimulation. The concentration of 20 microg mL(-1) APS resulted in a significant increase in nitroblue tetrazolium (NBT) positive cells (P<0.05) at different temperature and even 10 microg mL(-1) APS could increase O(2)(-) generation significantly at 18 degrees C and 22 degrees C. Both phagocytosing and O(2)(-) production increased with the increase of APS concentration from 0 to 20 microg mL(-1) at different temperature, and when APS at 40 microg mL(-1), they were decreased. It suggested that immunocytes activity in A. japonicus decreased with the temperature increasing from 18 degrees C to 25 degrees C, and APS could be an effective immunostimulant to enhance phagocytic activity and O(2)(-) production.

RGD-containing peptides inhibit intestinal regeneration in the sea cucumber Holothuria glaberrima.

The sea cucumber Holothuria glaberrima is an echinoderm capable of regenerating its viscera. Previous studies from our group have shown a striking remodeling of the extracellular matrix (ECM) during intestinal regeneration. To study the role of the ECM during regeneration, we have focused on the RGD sequences present in many ECM molecules. Regenerating animals were treated with an RGDS (Arg-Gly-Asp-Ser) peptide that competes with the interaction between RGD sequence and cellular integrins. Saline and RGES (Arg-Gly-Glu-Ser) peptide injections were done as controls. The size of the regenerating intestine was determined, and the regenerating structures were analyzed by immunohistochemistry for the presence of collagen and fibronectin, as well as for muscle and other cells. The results show a delay in intestinal regeneration in animals injected with the RGDS peptide, suggesting that the ECM-integrin interaction plays an important function in the regenerative process.

Carbohydrate-dependent hemolytic activity of the conjugate composed of a C-type lectin, CEL-I, and an amphiphilic alpha-helical peptide, 4(3)-beta Ala2.

A lectin-cationic peptide conjugate, 4(3)-CEL-I, was prepared from an invertebrate C-type lectin, CEL-I, and an amphiphilic alpha-helical peptide, 4(3)-beta Ala2 [Ac-(Leu-Ala-Arg-Leu)3-beta Ala2]. When 4(3)-CEL-I was incubated with rabbit erythrocytes, hemolysis was observed, especially at basic pH. Inhibition experiment using some carbohydrates suggested that hemolytic activity of 4(3)-CEL-I was caused by the interaction between 4(3)-beta Ala2 portion in the conjugate and the lipid bilayer after binding to the carbohydrate chains on the cell surface by the lectin activity of CEL-I.