Ascorbic acid biosynthesis in Pacific abalone Haliotis discus hannai Ino and L-gulonolactone oxidase gene loss as an independent event.

Up to now, it has been believed that invertebrates are unable to synthesize ascorbic acid (AA) in vivo. However, in the present study, the full-length CDs (Coding sequence) of L-gulonolactone oxidase (GLO) from Pacific abalone (Haliotis discus hannai Ino) were obtained through molecular cloning. The Pacific abalone GLO contained a FAD-binding domain in the N-termination, and ALO domain and conserved HWAK motif in the C-termination. The GLO gene possesses 12 exons and 11 introns. The Pacific abalone GLO was expressed in various tissues, including the kidney, digestive gland, gill, intestine, muscle and mantle. The GLO activity assay revealed that GLO activity was only detected in the kidney of Pacific abalone. After a 100-day feeding trial, dietary AA levels did not significantly affect the survival, weight gain, daily increment in shell length, and feed conversion ratio of Pacific abalone. The expression of GLO in the kidney was downregulated by dietary AA. These results implied that the ability to synthesize AA in abalone had not been lost. From the evolutionary perspective, the loss of GLO occurred independently as an independent event by matching with the genomes of various species. The positive selection analysis revealed that the GLO gene underwent purifying selective pressure during its evolution. In conclusion, the present study provided direct evidence to prove that the GLO activity and the ability to synthesize AA exist in abalone. The AA synthesis ability in vertebrates might have originated from invertebrates dating back 930.31 million years.

Characterization and crystal structure of prolyl endopeptidase from abalone (Haliotis discus hannai).

Aimed to study the characteristics of prolyl endopeptidase (PEP, EC 3.4.21.26) and its possible role in the degradation of collagen, we cloned the full-length cDNA sequence of PEP from abalone (Haliotis discus hannai) (Hdh-PEP). Recombinant Hdh-PEP (rHdh-PEP) was expressed in vitro, its enzymatic properties were detected, and its secondary structure was analyzed by Circular Dichroism (CD). We for the first time determined the 1.5 Å crystal structure of rHdh-PEP. The decomposition effect of rHdh-PEP on collagen peptides was analyzed. Our data revealed that the molecular weight of rHdh-PEP is 85 kDa, consisting of a catalytic domain and a ß-propeller domain. The optimal pH and temperature of rHdh-PEP were pH 6.0 and 20 °C, respectively. Using small collagen peptides as substrates, HPLC-ESI-MS analysis confirmed that rHdh-PEP specifically cleaved at the carboxyl side of proline residues, suggesting its role in the degradation of collagen peptides during autolysis.

Molecular characterization and spatiotemporal expression of prohormone convertase 2 in the Pacific abalone, Haliotis discus hannai.

Prohormone convertases (PCs) are subtilisin-like proteases responsible for the intracellular processing of prohormones and proneuropeptides in vertebrates and invertebrates. The full-length PC2 cDNA sequence was cloned from pleuropedal ganglion of Haliotis discus hannai, consisted of 2254-bp with an open reading frame of 1989-bp and encoded a protein of 662 amino acid residues. The architecture of Hdh PC2 displayed key features of PCs, including a signal peptide, a pro-segment domain with sites for autocatalytic activation, a catalytic domain, and a pro-protein domain (P-domain). It shares the highest homology of its amino acid sequence with the PC2 from H. asinina and to lesser extent with that of Homo sapiens and Rana catesbeiana PC2. Sequence alignment analysis indicated that Hdh PC2 was highly conserved in the catalytic domain, including a catalytic triad of serine proteinases of the subtilisin family at positions Asp-195, His-236, and Ser-412. The cloned sequence contained a canonical integrin binding sequence, and four cysteine residues involved in the formation of an intramolecular disulfide link. Phylogenetic analysis revealed that the Hdh PC2 is robustly clustered with the Has PC2. Quantitative PCR assay demonstrated that the Hdh PC2 was predominantly expressed in the pleuropedal ganglion rather than in other examined tissues. Although PC2 mRNA was expressed throughout the gametogenetic cycle of male and female abalone, the expression level was significantly higher in the ripening stage of female abalone. Also, a significantly higher expression was observed in the pleuropedal ganglion and gonadal tissues at a higher effective accumulative temperature (1000°C). In situ hybridization revealed that the PC2 mRNA expressing neurosecretory cells were distributed in the cortex region of the pleuropedal ganglion. According to the results, it can be concluded that pleuropedal ganglion is the highest site of PC2 activity, and this enzyme might be involved in the abalone reproduction process.

Effect of pathogenic bacteria on a novel C-type lectin, hemocyte and superoxide dismutase/ alkaline phosphatase activity in Onchidium reevesii.

Bacterial infection in the marine environment is a serious problem to maintain the stability of marine ecosystems. Nevertheless, there is little report so far for the biological effects of pathogenic bacteria in coastal ecosystems. Hence, we investigated the responses of shell-less Onchidium reevesii to resist against pathogenic bacterial infection. Analysis of data here could be used as fundamental information for assessment of innate immune response of O. reevesii. The full-length OrCTL cDNA was cloned and consists of 1849 base pair (bp) encoding protein of 192 amino acids. Constructing multiple alignments suggested that OrCTL has conserved carbohydrate recognition domain (CRD) of CTLs, containing an EPS (Glu-Pro-Ser) motif that may imply the function of recognition of carbohydrates like others invertebrate. OrCTL mRNAs were mainly detected in ganglion and hepatopancreas, and expression was highly up-regulated from 2 h after Vibrio harveyi challenge, rapidly decreased at 4 h, and significantly increased at 12 h. In addition, after challenge with Vibrio parahaemolytics, OrCTL gene expression was slightly up-regulated from 2 h, peaked at 12 h. Enzyme activity (in the hepatopancreas) and cell immune (in the hemolymph) were investigated along with Superoxide dismutase (SOD) activity, alkaline phosphatase (ALP) activity and cell cycle. SOD activities were significantly higher after V. harveyi and V. parahaemolytics challenge than that in the control group, respectively. By contrast, ALP activities were significantly inhibited after challenged with bacteria than that in the control group, respectively. Enzyme activities in the hepatopancreas obviously fluctuated, and ALP activity was more sensitive to bacteria. Cell responses illustrated that there were a significant higher percentage of cells in the S and G2/M phase in hemolymph after challenged with bacteria. Our results suggested that the immune response of O. reevesii could be activated by pathogenic bacteria, and the data will provide referent for the disease prevention of systematic investigation in aquatic animal.

Early embryonic exposure of freshwater gastropods to pharmaceutical 5-alpha-reductase inhibitors results in a surprising open-coiled "banana-shaped" shell.

In vertebrates, the steroidogenesis enzyme 5α-reductase converts testosterone to the more potent androgen 5α-dihydrotestosterone. Homologues of 5α-reductase genes have been identified in molluscs. However, recent findings suggest that vertebrate-type steroid androgens are not utilised in molluscan reproductive development. Genomic searches have revealed that molluscs do not possess many of the steroidogenic enzymes required to make testosterone, nor a nuclear androgen receptor. Consequently, the role of 5α-reductase in molluscs presents a mystery. Here, developmental exposures of Biomphalaria glabrata to selective pharmaceutical 5α-reductase inhibitors elicited a strong, highly reproducible phenotypic response characterised by the development of elongated "banana-shaped" shell morphology. In comparison to untreated snails, the shells are open-coiled and the whorls are unattached. Dutasteride (5α-reductase inhibitor) is approximately 10-times more potent at provoking the banana-shaped shell phenotype than finasteride, paralleling the pharmaceuticals' efficacy in humans. Other enzyme inhibitors with different modes of action were tested to investigate the specificity of the phenotype. However, only the pharmaceutical 5α-reductase inhibitors provoked the response. Dutasteride elicited the same phenotype in a second gastropod, Physella acuta. In the absence of evidence for de novo androgen steroidogenesis in molluscs, these findings suggest that novel substrates for 5α-reductase exist in gastropods, lending support to the contention that molluscan endocrinology differs from the well-characterised vertebrate endocrine system.

Effects of the probiotic Bacillus amyloliquefaciens on the growth, immunity, and disease resistance of Haliotis discus hannai.

The effects of a diet containing the probiotic Bacillus amyloliquefaciens on the survival and growth of Haliotis discus hannai were evaluated by measuring growth and hematological parameters and the expression levels of nonspecific immune genes. In addition, the abalone's response to Vibrio parahaemolyticus infection was assessed. H. discus hannai (shell length: 29.35 ±â€¯1.81 mm, body weight: 4.28 ±â€¯0.23 g) were exposed to an 8-week culture experiment in indoor aquariums and a 2-week V. parahaemolyticus artificial infection experiment. In each experiment, the control group (C) was fed daily with the basal feed; the experimental groups were fed daily with the experimental feed, prepared by spraying B. amyloliquefaciens onto the basal feed at final concentrations of 103 (group A1), 105 (A2), and 107 (A3) cfu/g. The survival rate, body weight specific growth rate, and food conversion efficiency in A2 and A3 were significantly higher than those in A1 and C (P < 0.05). The total number of blood lymphocytes, the O2- and NO levels produced from respiratory burst, the activities of acid phosphatase, superoxide dismutase, and catalase, and the expression levels of catalase and thiol peroxidase in A2 were not significantly different from those in A3, but these factors were significantly higher in A2 compared to A1 and C (P < 0.05). The total antioxidant capacity and expression levels of glutathione S-transferase in A1, A2 and A3 were significantly higher than those in C (P < 0.05). At day 9 after infection with V. parahaemolyticus, all abalone in C were dead; at the end of the experiment, the cumulative mortality of abalone in A2 was significantly lower than that in any other group (P < 0.05). Thus, the experimental feed containing 105 cfu/g B. amyloliquefaciens not only facilitated the food intake and growth of abalone, but also effectively enhanced their non-specific immunity and resistance to V. parahaemolyticus infection. In this regard, B. amyloliquefaciens may be a useful probiotic strain for abalone aquaculture.

Stability and Hydrolysis of Desomorphine-Glucuronide.

Desomorphine, the principal opioid in Krokodil, has an analgesic potency approximately ten-times that of morphine. Similar to other opioids, during phase II metabolism it undergoes conjugation with glucuronic acid to form desomorphine-glucuronide. Although hydrolysis of conjugated species is sometimes required prior to analysis, desomorphine-glucuronide has not been fully investigated. In this study, six hydrolysis procedures were optimized and evaluated. Deconjugation efficiencies using chemical and enzymatic hydrolysis were evaluated and stability in aqueous solution was assessed. Acid hydrolysis was compared with five ß-glucuronidase sources (BGTurbo™, IMCSzyme™, Escherichia coli, Helix pomatia and Patella vulgata). At optimal conditions, each hydrolysis method produced complete hydrolysis (≥96%). However, under simulated challenging conditions, P. vulgata was the most efficient ß-glucuronidase for the hydrolysis of desomorphine-glucuronide. Both BGTurbo™ and IMCSzyme™ offered fast hydrolysis with no need for sample cleanup prior to liquid chromatography-quadrupole/time of flight-mass spectrometry (LC-Q/TOF-MS) analysis. Hydrolysates using E. coli, H. pomatia and P. vulgata underwent additional sample treatment using ß-Gone™ cartridges. Additionally, the stability of free and conjugated drug was evaluated at elevated temperature (60°C) in aqueous solutions between pH 4 and 10. No degradation was observed for either desomorphine or desomorphine-glucuronide under any of the conditions tested.

Systematic Evaluation and Validation of Benzodiazepines Confirmation Assay Using LC-MS-MS.

Benzodiazepines is a large drug group used extensively to treat sleep disorders and anxiety; these drugs are frequently associated with various crimes such as murder and drug-facilitated sexual assault. With growing use and misuse of these compounds, confirmatory assays are increasingly required in clinical laboratories. In this study, 4 ß-glucuronidase enzymes were systematically evaluated for their hydrolysis efficiencies. Additionally, the matrix effects and extraction recoveries of three protein precipitation plates were systematically evaluated. The recombinant IMCSzyme ß-glucuronidase enzyme showed higher hydrolysis efficiency than did ß-glucuronidase from abalone, Patella vulgata and Helix pomatia. Lower ion suppression and more favorable overall recovery were observed in the Supelco protein precipitation plate than in the two other plates. Analytes were separated on a superficially porous particle column (ultra biphenyl) within 4.5 min and characterized through electrospray ionization-tandem mass spectrometry in the positive ion multiple-reaction monitoring mode. The accuracy, carryover, extraction recovery, detection limit, matrix effect, quantification limit and precision of the method were validated. Sixty opioid-positive urine samples were analyzed; a high incidence of benzodiazepines was detected in these samples. The proposed technique is robust and suitable for efficiently monitoring the numerous benzodiazepines that occur in urine samples.

Comparing mutagenesis and simulations as tools for identifying functionally important sequence changes for protein thermal adaptation.

Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from -1.9 °C (Antarctica) to ∼55 °C (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability, we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme's thermal responses and foster evolutionary adaptation of function.

Identification and functional characterization of thioredoxin-related protein of 14 kDa in Oncomelania hupensis, the intermediate host of Schistosoma japonicum.

Oncomelania hupensis is the unique intermediate host of the blood fluke Schistosoma japonicum, which causes schistosomiasis. In snails, highly toxic reactive oxygen species (ROS) can be continually generated by hemocytes in response to foreign particles or pathogens, and may be involved in damaging and eliminating digenean larvae. Thioredoxin-related protein of 14 kDa (TRP14) is a member of the Trx superfamily, and plays an important role in the scavenging of ROS. This study was designed to identify and characterize TRP14 from O. hupensis (OhTRP14), and investigate the involvement of OhTRP14 in the scavenging of ROS in snail host immune response to the parasite S. japonicum. Here we expressed and purified the recombinant OhTRP14 and its mutant, and rOhTRP14 displayed oxidoreductase activity dependent on the CPDC motif. OhTRP14 protein was ubiquitously present in all the tested snail tissues, and especially immunolocalized in the cytoplasm of immune cell types (hemocytes). Both the expression of OhTRP14 and ROS level increased significantly in snails following challenge with S. japonicum. The dsRNA-mediated knockdown of OhTRP14 was successfully conducted by oral feeding, and ROS production was increased by OhTRP14 knockdown, implying that OhTRP14 was involved in the scavenging of ROS in O. hupensis circulating hemocytes. Therefore, we conclude that OhTRP14 may be involved in the scavenging of ROS in snail host immune response to the parasite S. japonicum. The results expand our understanding of the interaction between this parasite and host, and lay a foundation for the establishment of Oncomelania-schistosome infection models.

Possible Mechanisms of Hatching from Egg Capsules in the Gastropods Crepipatella dilatata and Crepipatella peruviana, Species with Different Modes of Early Development.

Many invertebrates enclose their embryos within egg capsules, from which the offspring hatch. In marine gastropods that brood their egg capsules, hatching could involve radular activity by the mother or by unhatched stages, increased osmotic concentration of the intracapsular fluid, or production of hatching enzymes. The present research sought to determine whether mechanical action by the brooding female or by the encapsulated embryos was involved in the hatching for two sympatric and closely related species of calyptraeid: Crepipatella dilatata, which exhibits direct development without free-living larvae, and Crepipatella peruviana, which releases free-living veliger larvae. We also considered the role that enzymatic action or osmotic changes in the intracapsular fluid might play in hatching. Using scanning electron micrograph analyses, we found no evidence that the well-developed, pre-hatching juvenile radula of C. dilatata played any role in the hatching process and that the radula of C. peruviana did not even develop until long after hatching; so there was no evidence of radular activity involved in the hatching of either species. For C. peruviana, the intracapsular fluid osmolality was always higher than that of the surrounding seawater, suggesting that there is a strong natural water inflow during development. Moreover, when egg capsules of C. peruviana were exposed to lower ambient salinities, the substantial entry of water correlated well with high percentages of hatching, particularly for egg capsules containing advanced veligers, suggesting that an osmotic mechanism may be involved in the hatching process of this species. In contrast, hatching in C. dilatata appeared to be enzymatically mediated.

[Genotyping and polymorphism analysis of cytochrome c oxidase subunit â gene of Pomacea canaliculata from Lincang City in Yunnan Province].

OBJECTIVE: To analyze the genetic diversity of Pomacea canaliculata based on the mitochondria DNA cytochrome c oxidase subunit Ⅰ (mtDNA COⅠ) gene as a molecular marker in Lincang City of Yunnan Province, so as to provide the scientific data for monitoring Angiostrongylus cantonensis in local areas. METHODS: The genotypes and polymorphisms of 38 specimens of P. canaliculata collected from Mengding Town of Lincang City were analyzed by sequencing COⅠ gene. The phylogenetic tree and genetic distances were produced based on the haplotypes from GenBank and the present study by using the neighbourjoining method with the software MEGA version 6.06. RESULTS: Totally 31 sequences were acquired in the present study and they produced 3 unique haplotypes. Haplotype 1 showed a higher frequency compared to the others and it accounted for 83.9 % (26/31). The data showed that the least genetic distances ranged from 0 to 0.052 between P. canaliculata and 3 haplotypes, as well as the largest genetic distances ranged from 0.021 to 0.239 between Pila conica and 3 haplotypes. Otherwise, the analysis of the phylogenetic trees based on COⅠ gene sequences of P. canaliculata indicated that all of 3 haplotypes clustered into one big clade with that from Japan (GenBank accession number: AB433769), China (GenBank accession number: KT313034) and USA (GenBank accession number: EU523129), which owned the closet relationship amongst them. Their genetic relationships were distantly related to the GenBank's reference sequences of P. insularum (GenBank accession number: EF514942), P. camena (GenBank accession number: EF515059) and so on. CONCLUSIONS: There is a P. canaliculata species in Lincang City of Yunnan Province as well as a high genetic diversity amongst the acquired 3 haplotypes in this study.

Toxicokinetic and toxicodynamic studies of carbaryl alone or in binary mixtures with azinphos methyl in the freshwater gastropod Planorbarius corneus.

Carbamate insecticides such as carbaryl and organophosphates such as azinphos-methyl share the ability to inhibit the activity of B-esterases. This study aimed to (1) assess the inhibitory effects of carbaryl on B-esterase activity in soft tissues and hemolymph of Planorbarius corneus; (2) establish whether binary mixtures of carbaryl and azinphos-methyl depart or not from a model of concentration addition on the inhibition of cholinesterase activity; (3) determine the bioconcentration and elimination of the pesticides. The results showed that exposure of gastropods to increasing concentrations of carbaryl (0.1-5 mg L-1) for 48 h inhibited cholinesterase activity in a concentration-dependent manner, with an EC50 of 1.4 ±â€¯0.3 mg L-1 and 1.2 ±â€¯0.1 mg L-1 for soft tissue and hemolymph, respectively. Carboxylesterase activity, measured with the substrates p-nitrophenyl butyrate and p-nitrophenyl acetate, was between 2.3 and 25 times more sensitive to carbaryl inhibition than cholinesterase activity. Binary mixtures corresponding to 0.5 EC50 carbaryl + 0.5 EC50 azinphos-methyl and 0.75 EC50 carbaryl + 0.75 EC50 azinphos-methyl produced inhibitions of cholinesterase activity similar to those of individual pesticides, following a model of concentration addition. Bioconcentration was analyzed using a one-compartment model. The absorption kinetics (k1) for both pesticides alone (1.4 mg L-1 of carbaryl or 1.8 mg L-1 of azinphos-methyl) or mixed (1.4 mg L-1 of carbaryl + 1.8 mg L-1 of azinphos-methyl) were similar. The elimination kinetics ratio (k2) estimated for the pesticides alone or in the mixtures showed that carbaryl was eliminated 3.5 times faster than azinphos-methyl. These results suggest that exposure of Planorbarius corneus to binary mixtures of carbaryl and azinphos-methyl for 48 h follow a concentration addition model on inhibition of cholinesterase activity and that the pesticide mixtures do not change the toxicokinetic parameters of the parent compounds.

Mannitol oxidase and polyol dehydrogenases in the digestive gland of gastropods: Correlations with phylogeny and diet.

Mannitol oxidase and polyol dehydrogenases are enzymes that convert polyalcohols into sugars. Mannitol oxidase was previously investigated in terrestrial snails and slugs, being also present in a few aquatic gastropods. However, the overall distribution of this enzyme in the Gastropoda was not known. Polyol dehydrogenases are also poorly studied in gastropods and other mollusks. In this study, polyalcohol oxidase and dehydrogenase activities were assayed in the digestive gland of 26 species of gastropods, representing the clades Patellogastropoda, Neritimorpha, Vetigastropoda, Caenogastropoda and Heterobranchia. Marine, freshwater and terrestrial species, including herbivores and carnivores were analyzed. Ultrastructural observations were undertake in species possessing mannitol oxidase, in order to investigate the correlation between this enzyme and the presence of tubular structures known to be associated with it. Mannitol oxidase activity was detected in the digestive gland of herbivores from the clades Caenogastropoda and Heterobranchia, but not in any carnivores or in herbivores from the clades Patellogastropoda, Neritimorpha and Vetigastropoda. In most of the species used in this study, dehydrogenase activities were detected using both D-mannitol and D-sorbitol as substrates. Nevertheless, in some carnivores these activities were not detected with both polyalcohols. Ultrastructural observations revealed tubular structures in digestive gland cells of some species having mannitol oxidase activity, but they were not observed in others. Based on our results, we suggest that mannitol oxidase first occurred in a herbivorous or omnivorous ancestor of Apogastropoda, the clade formed by caenogastropods and heterobranchs, being subsequently lost in those species that shifted towards a carnivorous diet.

[A comparative study of phenoloxidase activity between Cipangopaludina chinensis and Oncomelania hupensis].

OBJECTIVE: To study the differences of the phenoloxidase (PO) relative activity among ribbed shelled Oncomelania hupensis, smooth shelled O. hupensis and Cipangopaludina chinensis. METHODS: The crude PO fluid was extracted from the soft tissue of O. hupensis and C. chinensis by homogenation and centrifugation. The PO activity was detected with catechol as the substrate in the reaction systems. RESULTS: The PO relative activities in the ribbed shelled O. hupensis, smooth shelled O. hupensis and C. chinensis were (25.72 ± 2.27), (14.56 ± 1.24) U / mL and (13.72 ± 1.06) U / mL. The PO relative activity in the smooth shelled O. hupensis was higher than that in the ribbed shelled O. hupensis (q = 21.46, P < 0.05) and C. chinensis (q = 12.00, P < 0.05), while the difference between the PO relative activities of the latter two was not statistically significant (q = 1.62, P > 0.05) . CONCLUSIONS: There is a difference in the relative PO activity between O. hupensis and C. chinensis, which may be related to the living environment of snails.

Involvement of clip-domain serine protease in the anti-Vibrio immune response of abalone (Haliotis discus hannai)-Molecular cloning, characterization and functional analysis.

Vibrio parahemolyticus (V. parahemolyticus) is a major pathogen for abalone, an important economical shellfish in coastal area of China. There is little known about the abalone innate immune system against pathogen infection. Clip-domain serine proteases (cSPs) are increasingly recognized to play important roles in host immune defense in invertebrates. In this study, we cloned a cSP (Hdh-cSP) from abalone (Haliotis discus hannai). We found out that Hdh-cSP was widely expressed in multiple tissues of abalone, with highest level in the immune-like organ, hepatopancreas. V. parahemolyticus infection induced significantly elevated expression of Hdh-cSP in addition to better-characterized innate immune component genes including Rel/NF-κB, allograft inflammatory factor (ALInFa), macrophage expressed protein (MEP) and caspase-8. Importantly, the silencing of Hdh-cSP reduced the expression of these genes, suggesting that Hdh-cSP was an upstream regulatory factor in V. parahemolyticus infection. Further analysis showed that apoptosis of hemocytes was inhibited when the transcription of Hdh-cSP was knocked down, suggesting that Hdh-cSP participated in cell apoptosis by regulation of caspase 8 expression in V. parahemolyticus infection. Therefore, our study established an important role of cSP in the innate immunity against V. parahemolyticus infection in abalone.

Optimization of recombinant ß-glucuronidase hydrolysis and quantification of eight urinary cannabinoids and metabolites by liquid chromatography tandem mass spectrometry.

Prolonged urinary cannabinoid excretion in chronic frequent cannabis users confounds identification of recent cannabis intake that may be important in treatment, workplace, clinical, and forensic testing programs. In addition, differentiation of synthetic Δ9-tetrahydrocannabinol (THC) intake from cannabis plant products might be an important interpretive issue. THC, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) urine concentrations were evaluated during previous controlled cannabis administration studies following tandem alkaline/E. coli ß-glucuronidase hydrolysis. We optimized recombinant ß-glucuronidase enzymatic urinary hydrolysis before simultaneous liquid chromatography tandem mass spectrometry (LC-MS/MS) quantification of THC, 11-OH-THC, THCCOOH, cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), tetrahydrocannabivarin (THCV) and 11-nor-9-carboxy-THCV (THCVCOOH) in urine. Enzyme amount, incubation time and temperature, buffer molarity and pH were optimized using pooled urine samples collected during a National Institute on Drug Abuse, Institutional Review Board-approved clinical study. Optimized cannabinoid hydrolysis with recombinant ß-glucuronidase was achieved with 2000 IU enzyme, 2 M pH 6.8 sodium phosphate buffer, and 0.2 mL urine at 37°C for 16 h. The LC-MS/MS quantification method for hydrolyzed urinary cannabinoids was validated per the Scientific Working Group on Toxicology guidelines. Linear ranges were 1-250 µg/L for THC and CBG, 2-250 µg/L for 11-OH-THC, CBD, CBN, THCV and THCVCOOH, and 1-500 µg/L for THCCOOH. Inter-batch analytical bias was 92.4-112.4%, imprecision 4.4-9.3% CV (n = 25), extraction efficiency 44.3-97.1% and matrix effect -29.6 to 1.8% (n = 10). The method was utilized to analyze urine specimens collected during our controlled smoked, vaporized, and edible cannabis administration study to improve interpretation of urine cannabinoid test results.

High-level expression and purification of a molluskan endoglucanase from Ampullaria crossean in Pichia pastoris.

EG27I is an endogenous glucanase belonging to glycoside hydrolase family (GHF) 45 from the mollusk Ampullaria crossean. In this study, the mature EG27I peptide gene fused to the HFBII secretion signal of Trichoderma reesei was expressed under the GAP promoter of Pichia pastoris in SMD1163 strain. A bioactive EG27I with a molecular weight of 27 kDa was successfully expressed and secreted into our culture medium. The respective final OD600 and hydrolytic activity were 333 and 1.28 U/mL when high-cell-density fermentation of the recombinant P. pastoris was performed in a 7.5 L fermenter through a fed-batch strategy for 132 h. The recombinant protein concentration of the fermentation supernatant was 47.7 mg/L. EG27I was consecutively purified from the fermentation supernatant through ultrafiltration, cation exchange, and hydrophobic interaction. The specific activity of the recombinant EG27I was 26.8 U/mg, and the optimal pH and temperature of the enzyme were 5 and 50 °C, respectively. The half-life of the enzyme activity at 100 °C could reach 40 min. The N-terminal amino acid sequence analysis of the purified recombinant protein confirmed that the amino terminal sequence was consistent with the natural structure. The high quantity and purity of the EG27I provide a basis for future structural and functional studies.

A cysteine protease (cathepsin Z) from disk abalone, Haliotis discus discus: Genomic characterization and transcriptional profiling during bacterial infections.

Cathepsin Z (CTSZ) is lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense via phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. Hence, CTSZ is also acknowledged as an acute-phase protein in host immunity. In this study, we sought to identify the CTSZ homolog from disk abalone (AbCTSZ) and characterize it at the molecular, genomic, and transcriptional levels. AbCTSZ encodes a protein with 318 amino acids and a molecular mass of 36kDa. The structure of AbCTSZ reveals amino acid sequences that are characteristic of the signal sequence, pro-peptide, peptidase-C1 papain family cysteine protease domain, mini-loop, HIP motif, N-linked glycosylation sites, active sites, and conserved Cys residues. A pairwise comparison revealed that AbCTSZ shared the highest amino acid homology with its molluscan counterpart from Crassostrea gigas. A multiple alignment analysis revealed the conservation of functionally crucial elements of AbCTSZ, and a phylogenetic study further confirmed a proximal evolutionary relationship with its invertebrate counterparts. Further, an analysis of AbCTSZ genomic structure revealed seven exons separated by six introns, which differs from that of its vertebrate counterparts. Quantitative real time PCR (qPCR) detected the transcripts of AbCTSZ in early developmental stages and in eight different tissues. Higher levels of AbCTSZ transcripts were found in trochophore, gill, and hemocytes, highlighting its importance in the early development and immunity of disk abalone. In addition, we found that viable bacteria (Vibrio parahaemolyticus and Listeria monocytogenes) and bacterial lipopolysaccharides significantly modulated AbCTSZ transcription. Collectively, these lines of evidences suggest that AbCTSZ plays an indispensable role in the innate immunity of disk abalone.

Evidence of metabolic microevolution of the limpet Nacella concinna to naturally high heavy metal levels in Antarctica.

The gastropod Nacella concinna is the most conspicuous macroinvertebrate of the intertidal zone of the Antarctic Peninsula and adjacent islands. Naturally high levels of copper and cadmium in coastal marine ecosystems are accumulated in N. concinna tissues. We aimed to study the effects of metal cations on N. concinna arginase in the context of possible adaptive microevolution. Gills and muscle had the highest argininolytic activity, which was concentrated in the cytosol in both tissues. Gills had the highest levels of arginase and may be involved in the systemic control of l-arginine levels. The relatively high argininolytic activity of the N. concinna muscular foot, with KM=25.3±3.4mmolL-1, may be involved in the control of l-arginine levels during phosphagen breakdown. N. concinna arginases showed the following preferences for metal cations: Ni2+>Mn2+>Co2+>Cu2+ in muscle and Mn2+>Cu2+ in gills. Cu2+ activation is a unique characteristic of N. concinna arginases, as copper is a potent arginase inhibitor. Cu2+ partly neutralized N. concinna arginase inhibition by Cd2+, worked synergistically in muscle arginase activation by Co2+ and neutralized muscle arginase activation by Ni2+. Mn2+ was able to activate muscle arginase in the presence of Fe3+ and Pb2+. The selection of arginases that are activated by Cu2+ and resistant to inhibition by Cd2+ in the presence of Cu2+ over evolutionary timescales may have favored N. concinna occupation of copper- and cadmium-rich niches.