Effects of dietary astaxanthin on growth performance, muscle composition, non-specific immunity, gene expression, and ammonia resistance of juvenile ivory shell (Babylonia areolate).

Astaxanthin is one of the important immunopotentators in aquaculture. However, little is known about the physiological changes and stress resistance effects of astaxanthin in marine gastropods. In this study, the effects of different astaxanthin concentrations (0, 25, 50, 75, and 100 mg/kg) on the growth, muscle composition, immune function, and resistance to ammonia stress in Babylonia areolata were investigated after three months of rearing. With the increase in astaxanthin content, the weight gain rate (WGR), specific growth rate (SGR), and survival rate (SR) of B. areolata showed an increasing trend. The 75-100 mg/kg group was significantly higher than the control group (0 mg/kg). There was no significant difference in the flesh shell ratio (FSR), viscerosomatic index (VSI), and soft tissue index (STI) of the experimental groups. Astaxanthin (75 mg/kg) significantly increased muscle crude protein content and increased hepatopancreas alkaline phosphatase (AKP), superoxide dismutase (SOD), and catalase (CAT) activity. Astaxanthin (75-100 mg/kg) significantly increased the total antioxidant capacity (T-AOC) and acid phosphatase (ACP) of the hepatopancreas and decreased the malondialdehyde (MDA) content of B. areolata. Astaxanthin significantly induced the expression levels of functional genes, such as SOD, Cu/ZnSOD, ferritin, ACP, and CYC in hepatopancreas and increased the survival rate of B. areolata under ammonia stress. The addition of 75-100 mg/kg astaxanthin to the feed improved the growth performance, muscle composition, immune function, and resistance to ammonia stress of B. areolata.

Metal-Specificity Divergence between Metallothioneins of Nerita peloronta (Neritimorpha, Gastropoda) Sets the Starting Point for a Novel Chemical MT Classification Proposal.

Metallothioneins' (MTs) biological function has been a matter of debate since their discovery. The importance to categorize these cysteine-rich proteins with high coordinating capacity into a specific group led to numerous classification proposals. We proposed a classification based on their metal-binding abilities, gradually sorting them from those with high selectivity towards Zn/Cd to those that are Cu-specific. However, the study of the NpeMT1 and NpeMT2isoforms of Nerita peloronta, has put a new perspective on this classification. N. peloronta has been chosen as a representative mollusk to elucidate the metal-binding abilities of Neritimorpha MTs, an order without any MTs characterized recently. Both isoforms have been recombinantly synthesized in cultures supplemented with ZnII, CdII, or CuII, and the purified metal-MT complexes have been thoroughly characterized by spectroscopic and spectrometric methods, leading to results that confirmed that Neritimorpha share Cd-selective MTs with Caenogastropoda and Heterobranchia, solving a so far unresolved question. NpeMTs show high coordinating preferences towards divalent metal ions, although one of them (NpeMT1) shares features with the so-called genuine Zn-thioneins, while the other (NpeMT2) exhibits a higher preference for Cd. The dissimilarities between the two isoforms let a window open to a new proposal of chemical MT classification.

Toxicological Impacts of Some Compounds on Massylaea vermiculata (O.F. Müller 1774), (Gastropoda: Helicidae) Under Laboratory Conditions.

<b>Background and Objective:</b> Effect of some compounds such as Plant extracts (Techno oil and Berna Star), natural origin compounds (Top-nine, Repcar and Chitosan 5%) and classical chemical pesticides (methomyl and lambda-cyhalothrin) were studied against the terrestrial snail <i>Massylaea vertmiculata</i> using the bait technique. Material and Methods: LC₅₀ of the each tested compound of natural compounds were estimated after 14 days of treatment, while LC₅₀ of pesticide were evaluated after 72 hrs of treatment. The impact of LC₅₀of each tested compound on some biochemical parameters, total protein content, alkaline phosphatase (ALP) and acid phosphatase (ACP) activity were determined 48 hrs post treatment. <b>Results:</b> The results revealed that the methomyl and lambda-cyhalothrin were the most effective compounds against test land snails, followed by Repcar, Top-nine and Techno oil, while Berna Star and Nema Ultra Chem come in the last rank. The pesticide compound methomyl was the most toxic one against the tested terrestrial snail species, while the Chitosan 5% was the least toxic one. The results showed that all tested compounds caused fluctuated effect whether increasing or decreasing on all the studies parameters such as total protein content, ALP and ACP activity as well. However, the Techno oil and the Berna Star caused sever decreasing on total protein content and ACP, followed by Top-nine, Repcar, Chitosan 5% (Nema Ultra Chem) and plant extracts. <b>Conclusion:</b> The both tested natural compounds and plant extracts recorded satisfying results compared with methomyl and lambda-cyhalothrin effect and that can be used in the pest controlling programs against terrestrial snails to reduce the environmental pollution.

Arginine Regulates TOR Signaling Pathway through SLC38A9 in Abalone Haliotis discus hannai.

Arginine plays an important role in the regulation of the target of the rapamycin (TOR) signaling pathway, and Solute Carrier Family 38 Member 9 (SLC38A9) was identified to participate in the amino acid-dependent activation of TOR in humans. However, the regulations of arginine on the TOR signaling pathway in abalone are still unclear. In this study, slc38a9 of abalone was cloned, and the slc38a9 was knocked down and overexpressed to explore its function in the regulation of the TOR signaling pathway. The results showed that knockdown of slc38a9 decreased the expression of tor, ribosomal s6 protein kinase (s6k) and eukaryotic translation initiation factor 4e (eif4e) and inhibited the activation of the TOR signaling pathway by arginine. Overexpression of slc38a9 up-regulated the expression of TOR-related genes. In addition, hemocytes of abalone were treated with 0, 0.2, 0.5, 1, 2 and 4 mmol/L of arginine, and abalones were fed diets with 1.17%, 1.68% and 3.43% of arginine, respectively, for 120 days. Supplementation of arginine (0.5-4 mmol/L) increased the expressions of slc38a9, tor, s6k and eif4e in hemocytes, and abalone fed with 1.68% of dietary arginine showed higher mRNA levels of slc38a9, tor, s6k and eif4e and phosphorylation levels of TOR, S6 and 4E-BP. In conclusion, the TOR signaling pathway of abalone can be regulated by arginine, and SLC38A9 plays an essential role in this regulation.

Wound-healing activity of glycoproteins from white jade snail (Achatina fulica) on experimentally burned mice.

Burns are a global public health problem and the treatment of burn wounds is a major medical and economic issue. White jade snails (Achatina fulica) are now widely distributed in Asia, and they have been used to treat burns in folk medicine of China. In this study, the glycoproteins from white jade snails were investigated and their effect on burn healing was evaluated by a mouse burn model. The results showed that the snail mucus was mainly composed of proteins and polysaccharides, and it had good adhesion. The main component of snail mucus was glycoprotein from the results of DEAE Sepharose FF ion exchange chromatography. The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging effect of 1 mg/mL snail mucus reached 13.77%. The wound healing rate of the snail mucus group was higher than that of the control group (p < 0.0001). Histopathological results showed that mice in the snail mucus group had a faster healing than that of the control group. The biochemical analysis was in agreement with the histopathological findings. These results suggested that glycoproteins from snail mucus showed effective wound healing activities in the skin of experimentally burned mice.

Ganoderma Lucidum Polysaccharide, an Extract from Ganoderma Lucidum, Exerts Suppressive Effect on Cervical Cancer Cell Malignancy through Mitigating Epithelial-Mesenchymal and JAK/STAT5 Signaling Pathway.

BACKGROUND/AIMS: We aimed to explore whether ganoderma lucidum polysaccharide (GLP) exhibits antitumor effect on cervical cancer cells. METHODS AND RESULTS: Different concentration of GLP was used to treat cervical cell. The data from cell counting kit-8 assay proved that the optimal working concentration and time of GLP were 200 µg/mL and treated for 48 h. The transwell assay demonstrated that GLP could attenuate the invasion and migration abilities of cervical cancer cells. Moreover, flow cytometry illustrated that GLP could promote the apoptosis of cervical cancer cells and limit the cycle of cervical cancer cells. Western blot assay discovered that the expression of proapoptosis proteins including Bax, Cleaved Caspases 3 and 9 increased and the antiapoptosis protein Bcl-2 decreased after treated with GLP. Moreover, we found that the expression of E-cadherin was increased, and N-cadherin, Vimentin, and Slug were decreased. Meanwhile, the expression of phosphorylated-JAK and phosphorylated-STAT5 was also decreased in cervical cancer cells treated by GLP, suggesting the inhibitory effect on JAK/STAT5 pathways. CONCLUSIONS: All of these data illustrated that GLP could alleviate the activity and aggressiveness, block the cell cycle, and promote the apoptosis of cervical cancer cells, which were possible via inhibiting epithelial-mesenchymal and JAK/STAT5 pathways.

The effects of selenium biofortification on mercury bioavailability and toxicity in the lettuce-slug food chain.

The effects of foliar Se biofortification (Se+) of the lettuce on the transfer and toxicity of Hg from soil contaminated with HgCl2 (H) and soil collected near the former Hg smelter in Idrija (I), to terrestrial food chain are explored, with Spanish slug as a primary consumer. Foliar application of Se significantly increased Se content in the lettuce, with no detected toxic effects. Mercury exerted toxic effects on plants, decreasing plant biomass, photochemical efficiency of the photosystem II (Fv/Fm) and the total chlorophyll content. Selenium biofortification (Se+ test group) had no effect on Hg bioaccumulation in plants. In slugs, different responses were observed in H and I groups; the I/Se+ subgroup was the most strongly affected by Hg toxicity, exhibiting lower biomass, feeding and growth rate and a higher hepatopancreas/ muscle Hg translocation, pointing to a higher Hg mobility in comparison to H group. Selenium increased Hg bioavailability for slugs, but with opposite physiological responses: alleviating stress in H/Se+ and inducing it in I/Se+ group, indicating different mechanisms of Hg-Se interactions in the food chain under HgCl2 and Idrija soil exposures that can be mainly attributed to different Hg speciation and ligand environment in the soil.

Localization, ligand environment, bioavailability and toxicity of mercury in Boletus spp. and Scutiger pes-caprae mushrooms.

This study provides information on mercury (Hg) localization, speciation and ligand environment in edible mushrooms: Boletus edulis, B. aereus and Scutiger pes-caprae collected at non-polluted and Hg polluted sites, by LA-ICP-MS, SR-µ-XRF and Hg L3-edge XANES and EXAFS. Mushrooms (especially young ones) collected at Hg polluted sites can contain more than 100 µg Hg g-1 of dry mass. Imaging of the element distribution shows that Hg accumulates mainly in the spore-forming part (hymenium) of the cap. Removal of hymenium before consumption can eliminate more than 50% of accumulated Hg. Mercury is mainly coordinated to di-thiols (43-82%), followed by di-selenols (13-35%) and tetra-thiols (12-20%). Mercury bioavailability, as determined by feeding the mushrooms to Spanish slugs (known metal bioindicators owing to accumulation of metals in their digestive gland), ranged from 4% (S. pes-caprae) to 30% (B. aereus), and decreased with increasing selenium (Se) levels in the mushrooms. Elevated Hg levels in mushrooms fed to the slugs induced toxic effects, but these effects were counteracted with increasing Se concentrations in the mushrooms, pointing to a protective role of Se against Hg toxicity through HgSe complexation. Nevertheless, consumption of the studied mushroom species from Hg polluted sites should be avoided.

Effects of acetaminophen in oxidative stress and neurotoxicity biomarkers of the gastropod Phorcus lineatus.

The growing use of pharmaceutical drugs has become a major environmental issue considering that these substances (or their metabolites) end up inevitably in sewage waters after excretion. In the wild, these chemicals may affect non-target organisms, and their potential toxicity is not sufficiently studied, a reality that is particularly true for marine organisms. Acetaminophen (also known as paracetamol) is known to be toxic in high dosages, namely, by triggering oxidative effects. These effects may be potentiated in marine organisms subjected to contamination resulting from large human settlements along coastal areas. In order to assess how different exposure regimes (acute vs. chronic) may affect aquatic wildlife, individuals of the gastropod species Phorcus lineatus were acutely (96 h) and chronically (28 days) exposed to ecologically relevant concentrations of acetaminophen. The effects were evaluated through the quantification of selected biomarkers-catalase (CAT), glutathione-S-transferase (GST), and cholinesterase (ChE) activities. The results from acute exposure showed no significant effects in all three biomarkers, but chronically exposed organisms showed significant increases in the activities of CAT and ChEs. The data show that P. lineatus triggered a defensive biological response in the presence of acetaminophen, and also show that realistically low levels of acetaminophen can exert adaptive changes with unknown consequences.

Protection of dietary selenium-enriched seaweed Gracilaria lemaneiformis against cadmium toxicity to abalone Haliotis discus hannai.

Seaweed Gracilaria lemaneiformis is the main dietary source of the abalone mariculture industry in China. In this study, we examined the protection of selenium (Se)-enriched G. lemaneiformis against cadmium (Cd) toxicity in the abalone, Haliotis discus hannai, using various indices including metal concentration (Se and Cd), growth rate, GPx enzymatic antioxidants, and metallothionein (MT) concentration over a period of 28 days of exposure. The growth rates and Se contents increased significantly in abalones fed with Se-enriched G. lemaneiformis, while the toxicity of Cd was reduced. Seven to 12 days Cd exposure to the Se-enriched G. lemaneiformis not only affected GPx activity but the MT levels fluctuated irregularly. MT concentrations increased after 3 days exposure and then gradually decreased to the control level after Day 7. There were statistically significant positive correlations between MT levels, GPx activity and Se concentrations, and negative relationships between MT levels, GPx activity and Cd levels in abalones. These findings suggest that Se-enriched Gracilaria protects abalone against Cd toxicity. The possible mechanism is the induction of MT with a concomitant increased capacity of GPx enzymatic antioxidants.

Antioxidant responses in estuarine invertebrates exposed to repeated oil spills: Effects of frequency and dosage in a field manipulative experiment.

We have experimentally investigated the effects of repeated diesel spills on the bivalve Anomalocardia brasiliana, the gastropod Neritina virginea and the polychaete Laeonereis culveri, by monitoring the responses of oxidative stress biomarkers in a subtropical estuary. Three frequencies of exposure events were compared against two dosages of oil in a factorial experiment with asymmetrical controls. Hypotheses were tested to distinguish between (i) the overall effect of oil spills, (ii) the effect of diesel dosage via different exposure regimes, and (iii) the effect of time since last spill. Antioxidant defense responses and oxidative damage in the bivalve A. brasiliana and the polychaete L. culveri were overall significantly affected by frequent oil spills compared to undisturbed controls. The main effects of diesel spills on both species were the induction of SOD and GST activities, a significant increase in LPO levels and a decrease in GSH concentration. N. virginea was particularly tolerant to oil exposure, with the exception of a significant GSH depletion. Overall, enzymatic activities and oxidative damage in A. brasiliana and L. culveri were induced by frequent low-dosage spills compared to infrequent high-dosage spills, although the opposite pattern was observed for N. virginea antioxidant responses. Antioxidant responses in A. brasiliana and L. culveri were not affected by timing of exposure events. However, our results revealed that N. virginea might have a delayed response to acute high-dosage exposure. Experimental in situ simulations of oil exposure events with varying frequencies and intensities provide a useful tool for detecting and quantifying environmental impacts. In general, antioxidant biomarkers were induced by frequent low-dosage exposures compared to infrequent high-dosage ones. The bivalve A. brasiliana and the polychaete L. culveri are more suitable sentinels due to their greater responsiveness to oil and also to their wider geographical distribution.

Fe, oxidative and nitrosative metabolism in the Antarctic limpet Nacella concinna.

The hypothesis of this work was that oxidative and nitrosative metabolism in the digestive gland (DG) of two limpet populations (intertidal and subtidal) of the Antarctic species Nacella concinna show different behavior when they were exposed to either intermittent (intertidal) or constant (subtidal) natural Fe. Total Fe content and labile Fe pool were higher in the DG of the subtidal compared to the intertidal population. However, no significant differences between populations were seen on the Fe atoms content of the isolated ferritin. Ascorbyl radical content was 2.0±0.4 and 6.5±0.8pmol/mg FW in the DG of the intertidal and subtidal animals, respectively. Lipid damage, assessed as content of thiobarbituric reactive substances, was different between the tissues of intertidal and subtidal samples, 491±102 and 1242±367pmol/mg FW, respectively. Catalase and superoxide dismutase activities showed no differences between the limpets. Nitric oxide (NO) content was 25±3 and 22±2pmol/mg FW in DG from intertidal and subtidal animals, respectively. NO synthase-like (NOS-like) activity was evaluated supplementing the samples with the enzyme co-factors, and the inhibitory effect of Nω-nitro-L-arginine methyl ester hydrochloride was tested. NO generation rate was 3.4±0.3 and 4.7±0.6pmol/minmg FW in DG from the intertidal and subtidal population, respectively. These results showed that the oxidative condition of the limpet population constantly covered by the Fe enriched water is more affected than the intertidal population. However, the nitrosative metabolism seems to be independent of the environmental high Fe content since similar NO steady state concentration and NOS-like activity were measured in both populations.

Ecotoxicological assessment of diamondback terrapin (Malaclemys terrapin) pond habitat, prey and eggs in Bermuda.

Total petroleum hydrocarbons, PAH and various trace metal residues were extracted and analyzed from fresh whole diamondback terrapin (Malaclemys terrapin) eggs, whole brackish-water gastropods (terrapin prey) and benthic sediment from anchialine pond environments in Bermuda inhabited by terrapins. Gastropods and terrapin eggs showed higher concentrations of trace metals and organic contaminants than sediments. Conversely, PAHs were mostly found within the sediment and smaller amounts detected in gastropods and terrapin eggs. Results indicated that contaminants in prey were transferred to terrapin eggs, and that concentrations of several contaminants exceeded potentially toxic concentrations for aquatic vertebrates. Necropsy of unhatched eggs from nests that had yielded viable hatchlings showed significantly compromised embryonic development. Bermudian diamondback terrapins reside and feed in brackish wetland habitats characterized by widespread, multifactorial contamination. This study suggests that environmental contamination plays a role in the recorded low hatching success in terrapin eggs in Bermuda.

Two variants of selenium-dependent glutathione peroxidase from the disk abalone Haliotis discus discus: Molecular characterization and immune responses to bacterial and viral stresses.

Glutathione peroxidase (GPx) is an essential member of the antioxidant systems of living organisms and may be involved in immune defense against pathogenic invasion. In the current study, two selenium-dependent glutathione peroxidases (AbSeGPxs) that shared 54.3% identity were identified from the disk abalone Haliotis discus discus. The open reading frames (ORFs) of AbSeGPx-a and AbSeGPx-b coded for 222 and 220 amino acids, respectively, with a characteristic selenocysteine residue encoded by an opal stop codon (TGA). The conserved selenocysteine insertion sequence (SECIS) element was predicted in the 3' untranslated region (UTR) of both isoforms, and they were found to form two stem-loop structures. Amino acid comparison and phylogenetic studies revealed that the AbSeGPxs were closely related to those in other mollusk species and were evolutionarily distinct from those of other taxonomic groups. The SYBR Green qPCR was employed in investigating the transcripts of AbSeGPxs. The expression of AbSeGPxs mRNA was examined in different embryonic developmental stages and differential expression patterns for AbSeGPx-a and AbSeGPx-b were noted. Meanwhile, the highest expression of AbSeGPxs was detected in the hepatopancreas of healthy adult animals. Next, transcriptional levels were profiled in hemocytes of adults to determine the immune responses of AbSeGPxs to microbial infections. The results revealed the significant up-regulation of AbSeGPx-a in a time-dependent manner after bacterial (Listeria monocytogenes and Vibrio parahaemolyticus) and viral (viral hemorrhagic septicemia virus) infections. Consequently, these findings indicate that AbSeGPx-a and AbSeGPx-b might be involved in the embryonic development of disk abalone and the regulation of immune defense system of adult animals.

Pharmacokinetics, tissue distribution and excretion of isoalantolactone and alantolactone in rats after oral administration of Radix Inulae extract.

Radix Inulae is endemic to China and has been used in traditional medicine to treat upper body pain, emesis and diarrhoea, and to eliminate parasites. Here, an UPLC-MS/MS method was developed and applied to study the pharmacokinetics, distribution and excretion of isoalantolactone and alantolactone, which are two main active sesquiterpene lactones in Radix Inulae, in Sprague-Dawley rats following oral administration of total Radix Inulae extract. Isoalantolactone, alantolactone and osthole (internal standard) were prepared using acetonitrile precipitation, and the separation of isoalantolactone and alantolactone was achieved by isocratic elution using water (containing 0.1% formic acid) and acetonitrile as the mobile phase using a ZORBAX Eclipse Plus C18 column. The total run time was 6.4 min. The present study showed poor absorption of isoalantolactone and alantolactone in vivo. The apparent Cmax, Tmax, T1/2 and total exposure (AUC0-12h) in rat plasma were 37.8 ng/mL, 120 min, 351.7 min and 6112.3 ng-min/mL for isoalantolactone and 25.9 ng/mL, 90 min, 321.0 min and 4918.9 ng-min/mL for alantolactone, respectively. It was shown that the highest concentration was achieved in the small intestine and feces clearance was shown to be the dominant elimination pathway of the lactones.

Dietary ascorbic acid modulates the expression profile of stress protein genes in hepatopancreas of adult Pacific abalone Haliotis discus hannai Ino.

This study was conducted to investigate the effects of dietary ascorbic acid (AA) on transcriptional expression patterns of antioxidant proteins, heat shock proteins (HSP) and nuclear factor kappa B (NF-κB) in the hepatopancreas of Pacific abalone Haliotis discus hannai Ino (initial average length: 84.36 ± 0.24 mm) using real-time quantitative PCR assays. L-ascorbyl-2-molyphosphate (LAMP) was added to the basal diet to formulate four experimental diets containing 0.0, 70.3, 829.8 and 4967.5 mg AA equivalent kg(-1) diets, respectively. Each diet was fed to triplicate groups of adult abalone in acrylic tanks (200 L) in a flow-through seawater system. Each tank was stocked with 15 abalone. Animals were fed once daily (17:00) to apparent satiation for 24 weeks. The results showed that the dietary AA (70.3 mg kg(-1)) could significantly up-regulate the expression levels of Cu/Zn superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), feritin (FT) and heat shock protein 26 (HSP26) in the hepatopancreas of abalone in this treatment compared to the controls. However, the expression levels of Mn-SOD, glutathione peroxidase (GPX), thioredoxin peroxidase (TPx), selenium-binding protein (SEBP), HSP70 and HSP90 were significantly down-regulated. Compared with those in the group with 70.3 mg kg(-1) dietary AA, the expression levels of CAT, GST and HSP26 were decreased in abalone fed with very high dietary AA (4967.5 mg kg(-1)). In addition, significant up-regulations of expression levels of Mn-SOD, GPX, TPx, SEBP, FT, HSP70, HSP90 and NF-κB were observed in abalone fed with apparently excessive dietary AA (829.8 and 4967.5 mg kg(-1)) as compared to those fed 70.3 mg kg(-1) dietary AA. These findings showed that dietary AA influenced the expression levels of antioxidant proteins, heat shock proteins and NF-κB in the hepatopancreas of abalone at transcriptional level. Levels of dietary AA that appeared adequate (70.3 mg kg(-1)) reduced the oxidative stress by influencing gene expression of antioxidant proteins, but excessive dietary AA (829.8 and 4967.5 mg kg(-1)) induced oxidative stress in Pacific abalone H. discus hannai.

Splice variants of perlucin from Haliotis laevigata modulate the crystallisation of CaCO3.

Perlucin is one of the proteins of the organic matrix of nacre (mother of pearl) playing an important role in biomineralisation. This nacreous layer can be predominately found in the mollusc lineages and is most intensively studied as a compound of the shell of the marine Australian abalone Haliotis laevigata. A more detailed analysis of Perlucin will elucidate some of the still unknown processes in the complex interplay of the organic/inorganic compounds involved in the formation of nacre as a very interesting composite material not only from a life science-based point of view. Within this study we discovered three unknown Perlucin splice variants of the Australian abalone H. laevigata. The amplified cDNAs vary from 562 to 815 base pairs and the resulting translation products differ predominantly in the absence or presence of a varying number of a 10 mer peptide C-terminal repeat. The splice variants could further be confirmed by matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF MS) analysis as endogenous Perlucin, purified from decalcified abalone shell. Interestingly, we observed that the different variants expressed as maltose-binding protein (MBP) fusion proteins in E. coli showed strong differences in their influence on precipitating CaCO3 and that these differences might be due to a splice variant-specific formation of large protein aggregates influenced by the number of the 10 mer peptide repeats. Our results are evidence for a more complex situation with respect to Perlucin functional regulation by demonstrating that Perlucin splice variants modulate the crystallisation of calcium carbonate. The identification of differentially behaving Perlucin variants may open a completely new perspective for the field of nacre biomineralisation.

GFP facilitates native purification of recombinant perlucin derivatives and delays the precipitation of calcium carbonate.

Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro. First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 µg/ml and higher, the inhibitory effect was observed predominantly for HCO(3) (-) as the first ionic interaction partner, but not necessarily for Ca(2+). The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals.

Cloning, characterization, and expression analysis of a putative 17 beta-hydroxysteroid dehydrogenase 11 in the abalone, Haliotis diversicolor supertexta.

The 17-beta-hydroxysteroid dehydrogenases (17ß-HSDs) are key enzymes for sex steroid biosynthesis. To date, relatively little is known about the presence and function of 17ß-HSDs in marine gastropods. In the present study, a cDNA sequence encoding putative 17ß-HSD type 11 (17ß-HSD-11) was identified in marine abalone (Haliotis diversicolor supertexta). The full-length cDNA contains 1058bp, including an open reading frame (ORF) of 900bp that encodes a protein of 299 amino acids. Comparative structural analysis revealed that abalone 17ß-HSD-11 shares relatively high homology with other 17b-HSD-11 hormologues, and a lesser degree of amino acid identity with other forms of 17b-HSD, especially in the functional domains, including the cofactor binding domain (TGxxxGxG) and catalytic site (YxxSK). Phylogenetic analysis showed that abalone 17ß-HSD-11 belongs to the short-chain dehydrogenase/reductase (SDR) family. Functional analysis following transient transfection of the ORF into human embryonic kidney-293 (HEK-293) cells indicated that abalone 17ß-HSD-11 has the ability to convert 5α-androstane-3α,17ß-diol (3α-diol) to androsterone (A) and testosterone (T) to androstenedione (4A). Expression analysis in vivo demonstrated that abalone 17ß-HSD-11 is differentially expressed during three stages (non-reproductive, reproductive, and post-reproductive). Taken together, these results indicate that ab-17ß-HSD-11 is an SDR family member with a potential role in steroid regulation during the reproductive stage.

Molecular characterization and gene expression of ferritin in red abalone (Haliotis rufescens).

Ferritin is the principal iron storage protein in the majority of living organisms. Its capacity to capture the toxic cellular iron in excess in a compact and safe manner, gives to this protein a key role in detoxification and iron storage. It has a main role in cellular homeostasis and in cellular defense against oxidative stress produced by the reactive oxygen species (ROS). In this research, the cDNA coding sequence of ferritin for Red abalone (Haliotis rufescens) was obtained, which had an open reading frame (ORF) of 516 bp. The deduced amino acid sequence was consisted of 171 residues with a calculated molecular weight of 19.77 kDa. In addition, tissue expression profiles of ferritin in Red abalone were induced by thermal stress showed an expression peak from 16 °C to 22 °C. The transcriptional level of ferritin was mainly achieved in muscle, digestive gland, gills, foot, mantle and gonad respectively. This research providing more information to better understands the structural and functional properties of this protein in Haliotis.