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Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here, we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii , transfer sulfate in vitro to the luciferin substrate, vargulin. We find luciferin sulfotransferases (LSTs) of ostracods are not orthologous to known LSTs of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Reuse of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.

Trilobites are among the most iconic of fossils and formed a prominent component of marine ecosystems during most of their 270-million-year-long history from the early Cambrian period to the end Permian period 1 . More than 20,000 species have been described to date, with presumed lifestyles ranging from infaunal burrowing to a planktonic life in the water column 2 . Inferred trophic roles range from detritivores to predators, but all are based on indirect evidence such as body and gut morphology, modes of preservation and attributed feeding traces; no trilobite specimen with internal gut contents has been described 3,4 . Here we present the complete and fully itemized gut contents of an Ordovician trilobite, Bohemolichas incola, preserved three-dimensionally in a siliceous nodule and visualized by synchrotron microtomography. The tightly packed, almost continuous gut fill comprises partly fragmented calcareous shells indicating high feeding intensity. The lack of dissolution of the shells implies a neutral or alkaline environment along the entire length of the intestine supporting digestive enzymes comparable to those in modern crustaceans or chelicerates. Scavengers burrowing into the trilobite carcase targeted soft tissues below the glabella but avoided the gut, suggesting noxious conditions and possibly ongoing enzymatic activity., (© 2023. The Author(s).)

The Atlantic cod ( Gadus morhua ) and red king crab ( Paralithodes camtschaticus ) processing wastes are massive and unutilized in the Murmansk region of Russia. The samples of skin-containing waste of Atlantic cod fillets production were hydrolyzed using enzyme preparations derived from red king crab hepatopancreases, porcine pancreases, and Bacillus subtilis bacteria. The activity of enzymes from crab hepatopancreases was significantly higher than the activity of enzymes derived from other sources. The optimal conditions of the hydrolysis process have been figured out. The samples of cod processing waste hydrolysate were analyzed for amino acid composition and molecular weight distribution. The samples of hydrolysate were used as core components for bacterial culture medium samples. The efficiency of the medium samples was tested for Escherichia coli growth rate; the most efficient sample had an efficiency of 95.3% of that of a commercially available medium based on fish meal. Substitution of medium components with those derived from industrial by-products is one of the ways to decrease a cost of a culture medium in biopharmaceutical drug production.

In Cypridina (Vargula) hilgendorfii, Cypridina luciferin is converted from Cypridina luciferyl sulfate by a sulfotransferase with adenosine 3', 5'-diphosphate (PAP), and is used for the luminescence reaction of Cypridina luciferase. We found that the luminescence activity of crude extracts of C. hilgendorfii was significantly stimulated by the addition of acetic acid. This stimulation may be explained by an efficient supply of PAP from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) catalyzed by a sulfotransferase. Thus, acetic acid acts as a sulfate acceptor from PAPS, followed by forming acetyl sulfate and PAP. The structure of acetyl sulfate was identified using mass spectrometry and it spontaneously decomposed to acetic acid and free sulfate ion in aqueous solutions. This enzymatic conversion from Cypridina luciferyl sulfate to Cypridina luciferin could be coupled with acetic acid and PAPS by a sulfotransferase., Competing Interests: Declaration of competing interest The authors have declared no financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ostracod genus Heterodesmus Brady, 1866 is known thus far to contain only three species: H. adamsii Brady, 1866; H. apriculus Hiruta, 1992; and H. naviformis (Poulsen, 1962). This genus has been recorded from the Sea of Japan, and the coastal areas of Thailand and Vietnam. The main generic character is the presence of antero-dorsal and postero-dorsal tube-like processes on the rostrum on both valves. The three species mostly differ in the shell lateral projections. Their relationship and the position of Heterodesmus within family Cypridinidae are poorly understood, partly due to the lack of publication of DNA data so far. We study Heterodesmus collected from several localities in the Northwest Pacific, namely Tsushima and Iki Islands in Japan and Maemul Island in Korea. Besides morphological characters, we also use two mitochondrial markers (16S rRNA and mtCOI) and three nuclear regions (18S rRNA, 28S rRNA, and internal transcribed spacer - ITS) in the samples to detect the biodiversity of this genus. Our phylogenetic tree based on molecular data coupled with morphology reveals the presence of two species, H. adamsii and H. apriculus . We report on their morphological variability, molecular diversity, and phylogenetic position within Cypridinidae based on 16S, 28S and 18S rRNAs, and provide a taxonomic key for all living genera of this family. For the first time, we give an overview of the intrageneric and intrafamily DNA distances of the above markers for the entire subclass Myodocopa.

The heavy metal cadmium readily accumulates in organisms, causing damage. In this study, juvenile marine shrimp Marsupenaeus japonicus were exposed to cadmium (Cd 2+ ; 5, 50 and 500 μg L -1 ). Cd accumulation and antioxidant-related indices were determined, and damage to biomolecules was assessed, after 24, 48 and 96 h. Cd bioaccumulation in M. japonicus increased with exposure time and concentration, which reached the highest value at 96 h. The data showed that 5, 50 and 500 μg L -1 Cd increased glutathione (GSH) content and the activities of superoxide dismutase (SOD) and glutathione S-transferase (GST) in a Cd-dose-dependent manner, but 5 and 50 μg L -1 Cd had no effect on caspase-3 activity. The expression levels of SOD, GST, heat shock protein 70 (HSP70), metallothionein (MT), p53 and caspase-3 genes were rapidly increased after 50 and 500 μg L -1 Cd exposure, and remained at a significantly higher level than in the control after 96 h of exposure. After exposure to 5, 50 and 500 μg L -1 Cd, F-value (the ratio between double-stranded DNA and total DNA) remained high at 24 h, however, as the exposure time increased, the F-value decreased in a dose-dependent manner. An increase in malondialdehyde content was also observed following exposure to 50 and 500 μg L -1 Cd. Our data suggest that Cd induces oxidative stress, molecular damage and apoptosis in juvenile M. japonicus in a concentration- and time-dependent manner., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Chitinases and N-acetyl-β-glucosaminidase (NAG) are important in molting and growth of crustaceans. In ostracods, the genes encoding these enzymes have not been characterized. The aim of the present study was to clone the genes encoding chitinase (DsChi) and NAG (DsNAG) from the ostracod, Dolerocypris sinensis, elucidate the phylogenetic relationships between the cloned genes and known chitinolytic enzymes, and determine the expression patterns of these genes at different stages of growth in the presence of an environmental pollutant. The genes were amplified from the genomic DNA of the organism using polymerase chain reaction (PCR). The products from PCR were cloned and characterized with bioinformatics tools, and their expression patterns at different growth stages were determined using real-time quantitative PCR (qRT-PCR). Nine and five introns were identified in DsChi and DsNAG genes, respectively. When compared with protein sequences available in GenBank, chitinase from D. sinensis was most closely related to that of Macrobrachium nipponense (61 % homology). The NAG of D. sinensis was most closely related to that of Limulus polyphemus (55.6 % homology). Based on phylogenetic analysis of known chitinases from crustaceans and insects, the D. sinensis chitinase tightly clustered in the same branch with chitinases from species within the Malacostraca class. In contrast, NAG of D. sinensis was clustered with NAG from F. candida.The level of expression of DsChi mRNA was significantly higher than that of DsNAG throughout the period of growth (p < 0.05). Treatment of D. sinensis cells with fenoxycarb significantly downregulated the expressions of DsChi and DsNAG throughout the period of growth (p < 0.05). These results show that the protein products of DsChi and DsNAG possess remarkable biochemical properties characteristic of a chitinase and NAG, respectively.

In this study, an extracellular protease, but no chitinolytic enzyme-producing strain, Brevibacillus parabrevis TKU046, has been isolated and analyzed for the deproteinization testing of shrimp waste by liquid fermentation. Deproteinization assays of shrimp waste with this microbe showed 95% protein removal after 4 days fermentation. The efficiency of chitin extraction by B. parabrevis TKU046 on wastes of three shrimp species were also investigated in which the highest deproteinization was found on cooked tiger shrimp shell. Infrared spectra (IR) of the obtained chitin displayed characteristic profiles for chitin. The culture supernatant released after fermentation greatly exhibited growth enhancing effect on Lactobacillus rhamnosus. In addition, B. parabrevis TKU046 protease was isolated and determined the characteristics. The molecular mass of B. parabrevis TKU046 protease was determined as 32 kDa and 34 kDa, respectively, by SDS-PAGE and HPLC. Overall, the findings provide strong support for the potential candidacy of this enzyme as an effective and eco-friendly alternative to the conventional chemicals used for the deproteinization of shrimp heads in the chitin processing industry, as well as the production of prebiotics to be used in the nutraceutical industry., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Crustacean growth is characterized by molting, whereby the old exoskeleton is shed and replaced by a new and larger version. The cellular events that lead to molting are driven by steroid hormones (ecdysteroids) secreted by paired endocrine glands (Y-organs). Between molts, ecdysteroid production is suppressed by a polypeptide molt-inhibiting hormone (MIH) released from neurosecretory cells in the eyestalks. Although a decrease in the MIH titer precedes the upsurge in ecdysteroidogenesis, it is hypothesized that a positive regulatory signal is also required for full activation of Y-organs. Existing data point to an intracellular Ca 2+ signal. Ca 2+ signaling is dependent on a tightly regulated Ca 2+ gradient, achieved through membrane transport proteins. One such protein, the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA), pumps Ca 2+ from cytosol to the lumen of the ER. We have recently cloned from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative Cas-SERCA protein. In studies reported here, quantitative PCR (QPCR) was used to quantify Cas-SERCA transcript abundance in Y-organs during a molting cycle, and radioimmunoassay was used to quantify ecdysteroids in hemolymph. The abundance of the Cas-SERCA transcript in Y-organs increased gradually during pre-molt. Similarly, the level of ecdysteroids in hemolymph increased during pre-molt. The results are consistent with the hypothesis that Cas-SERCA functions to maintain Ca 2+ homeostasis in Y-organs. Cas-SERCA transcript abundance also changed in several non-ecdysteroidogenic tissues during a molting cycle. The pattern of change differed among tissues suggesting a functional role for SERCA in each., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mating behaviours are diverse and noteworthy, especially within species radiations where they may contribute to speciation. Studying how differences in mating behaviours arise between species can help us understand how diversity is generated at multiple biological levels. The bioluminescent courtship displays of cypridinid ostracods (or sea fireflies) are an excellent system for this because amazing variety evolves while using a conserved biochemical mechanism. We find that the evolution of one aspect in this behavioural phenotype-the duration of bioluminescent courtship pulses-is shaped by biochemical function. First, by measuring light production from induced bioluminescence in 38 species, we discovered differences between species in their biochemical reactions. Then, for 16 species for which biochemical, phylogenetic and behavioural data are all available, we used phylogenetic comparative models to show that differences in biochemical reaction are nonlinearly correlated with the duration of courtship pulses. This relationship indicates that changes to both enzyme (c-luciferase) function and usage have shaped the evolution of courtship displays, but that they differentially contribute to these phenotypic changes. This nonlinear dynamic may have consequences for the disparity of signalling phenotypes observed across species, and demonstrates how unappreciated diversity at the biochemical level can lead to inferences about behavioural evolution.

Sea-Nine (4,5-dichloro-2-n-octyl-4-isothiazoline3-one; DCOIT) antifoulant has been widely used owing to its broad spectrum of biocide activity against major fouling organisms. In this study, several physiological parameters of a marine mysid were analyzed upon exposure to sublethal environmental concentrations (1 and 100 ng L -1 ) of Sea-Nine in two exposure conditions, intermittent (weekly; once per week) and constant (daily; once per 24 h) exposure, for 4 weeks. In both experimental conditions, growth retardation, acetylcholinesterase (AChE) activity, glutathione S-transferase (GST) activity, and number of newborn juveniles as second generation, together with their survival were measured. Morphometric parameters of total body, antennal scale, exopod, endopod, and telson were significantly retarded by 22%, 14%, 13%, and 24%, respectively, by daily exposure to 100 ng L -1 Sea-Nine for 4 weeks. Significant inhibition of AChE activity was observed at week 4 in the 100 ng L -1 daily Sea-Nine-exposed groups, whereas no significant GST activity was measured at the same experimental conditions. Inhibition of AChE activity would be associated with impairment of cholinergic system and may adversely modulate growth parameters of the mysid. The number of newly hatched juveniles from females that were exposed daily to 100 ng L -1 Sea-Nine was significantly lower than that of the control. Although no significant differences were observed between survival percentages of newborn juveniles for 30 days, mortality (NOEC and LC50) increased in the surviving offspring from the 100 ng L -1 -exposed 1st generation of mysids. These findings suggested that constant exposure to Sea-Nine has detrimental effects on the growth parameters of marine mysids with inhibition of AChE activity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Waterborne metals can adversely affect an organism's innate defenses through oxidative stress. In the present study, the marine mysid Neomysis awatchensis was exposed to sublethal concentrations (1/10 and 1/5 of the median LC50s) of As, Cd, Cu, Pb, and Zn for 48 or 96 h at the juvenile and adult developmental stages, and the dose- and age-specific antioxidant defense system responses were characterized. Metal accumulation and modulation of four key antioxidant biomarkers, malondialdehyde (MDA), glutathione (GSH), superoxide dismutase, and catalase, were measured in juvenile and adult mysids. Based on LC50, level of metal toxicity was dependent on metal concentration, developmental stage, and the exposure duration. Intracellular MDA content was increased in the As-, Cu-, Pb-, and Zn-exposed juvenile mysids after exposure for 48 and/or 96 h, while increases in MDA content were observed in adult mysids following Cu and Zn exposure. Interestingly, GSH content was differentially modulated, where intracellular GSH levels decreased in juvenile mysids following Cd, Cu, Pb, and Zn exposure, but significantly increased in metal-exposed adult mysids, except for Pb exposure. The catalase and superoxide dismutase activities displayed similar stage-specific increases or decreases as also observed for the different GSH levels, suggesting that the susceptibility to and defense against metal-induced oxidative stress differed based on stage. Modulations in MDA and GSH content and enzymatic activity of the antioxidant defense system indicate that mysid antioxidant defense system factors are intimately connected during control of oxidative imbalances with different capacities at different developmental stages., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Increasing evidence of tight links among the gut microbiota, obesity, and host health has emerged, but knowledge of the ecological processes that shape the variation in microbial assemblages across growth rates remains elusive. Moreover, inadequately control for differences in factors that profoundly affect the gut microbial community, hampers evaluation of the gut microbiota roles in regulating growth rates. To address this gap, we evaluated the composition and ecological processes of the gut bacterial community in cohabitating retarded, overgrown, and normal shrimps from identically managed ponds. Gut bacterial community structures were distinct (P = 0.0006) among the shrimp categories. Using a structural equation modeling (SEM), we found that changes in the gut bacterial community were positively related to digestive activities, which subsequently affected shrimp growth rate. This association was further supported by intensified interspecies interaction and enriched lineages with high nutrient intake efficiencies in overgrown shrimps. However, the less phylogenetic clustering of gut microbiota in overgrown and retarded subjects may offer empty niches for pathogens invasion, as evidenced by higher abundances of predicted functional pathways involved in disease infection. Given no differences in biotic and abiotic factors among the cohabitating shrimps, we speculated that the distinct gut community assembly could be attributed to random colonization in larval shrimp (e.g., priority effects) and that an altered microbiota could be a causative factor in overgrowth or retardation in shrimp. To our knowledge, this is the first study to provide an integrated overview of the direct roles of gut microbiota in shaping shrimp growth rate and the underlying ecological mechanisms.

Cypridina noctiluca luciferase has been utilized for biochemical and molecular biological applications, including bioluminescent enzyme immunoassays, far-red luminescence imaging, and high-throughput reporter assays. Some of these applications require a large amount of purified luciferase. However, conventional protein expression systems are not capable of producing sufficient quantities of protein with a high quality and purity without laborious and costly purification processes. To improve the productivity and expand the breadth of possibilities for Cypridina luciferase applications, we employed a variety of secretion expression systems, including yeast, mammalian cells, and silk worms. In this study, we established a simple production procedure using plant cell cultures. The plant cell culture BY-2 efficiently secreted luciferase, which was easily purified using a simple one-step ion-exchange chromatography method. The production yield was 20-30 mg of luciferase per liter of culture medium, and its Km for the luciferin (0.45 μM) was similar to that of the native protein. Additionally, we characterized its glycosylation pattern and confirmed that the two potential N-glycosylation sites were modified with plant-type oligosaccharide chains. Interestingly, the oligosaccharide chains could be trimmed without any detectable decrease in recombinant protein activity. Therefore, the results of our study indicate that this method offers a more cost-effective production method for Cypridina luciferase than conventional methods., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo® is compatible with detection of yNluc bioluminescence in < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter (PCYC1-HSE ), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd., (© 2016 The Authors. Yeast published by John Wiley & Sons Ltd.)

The 19 kDa protein (KAZ) of Oplophorus luciferase is a catalytic component, that oxidizes coelenterazine (a luciferin) with molecular oxygen to emit light. The crystal structure of the mutated 19 kDa protein (nanoKAZ) was determined at 1.71 Å resolution. The structure consists of 11 antiparallel β-strands forming a β-barrel that is capped by 4 short α-helices. The structure of nanoKAZ is similar to those of fatty acid-binding proteins (FABPs), even though the amino acid sequence similarity was very low between them. The coelenterazine-binding site and the catalytic site for the luminescence reaction might be in a central cavity of the β-barrel structure., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A novel protease, named PSLTro01, with fibrinolytic and anticoagulant activity was isolated from Porcellio scaber Latreille and was purified by a combination of hollow fibre membrane molecular weight cut-off (MWCO), ammonium sulfate fractionation, gel filtration and ion-exchange chromatography. PSLTro01 is a single-chain protein with a molecular mass of 38,497 Da as estimated by non-reduced SDS-PAGE and MALDI-TOF MS spectrometry, and its N-terminal 15 amino acid sequence was determined as DINGGGATLPQPLYQ. PSLTro01 is stable in the range of 20-40 °C and pH 6.0-10.0, with a maximum fibrinolytic activity at 40 °C and pH 7.0. The PSLTro01-induced fibrinolytic activity was not influenced by K(+) or Na(+) but was slightly increased by Mg(2+) and completely inhibited by aprotinin and pepstatin A. Fibrin plate assays revealed that PSLTro01 could not directly degrade fibrin but was a plasminogen activator. PSLTro01 exhibited high specificity for the substrate S-2251 for plasmin, followed by S-2238 for thrombin and S-2444 for urokinase. Moreover, the fibrinogenolysis pattern of PSLTro01 was Aα-chains>Bβ-chains>γ-chain. Tail-thrombus of the enzyme treated group was significantly shorter than the physiological saline treated group and the thrombus decrement was correlated with the enzyme dose. PSLTro01 prolongs both thrombin time (TT) and activated partial thromboplastin time (APTT). These results indicate that PSLTro01 may have potential applications in the prevention and treatment of thrombosis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Detoxification is a fundamental cellular stress defense mechanism, which allows an organism to survive or even thrive in the presence of environmental toxins and/or pollutants. The glutathione S-transferase (GST) superfamily is a set of enzymes involved in the detoxification process. This highly diverse protein superfamily is characterized by multiple gene duplications, with over 40 GST genes reported in some insects. However, less is known about the GST superfamily in marine organisms, including crustaceans. The availability of two de novo transcriptomes for the copepod, Calanus finmarchicus, provided an opportunity for an in depth study of the GST superfamily in a marine crustacean. The transcriptomes were searched for putative GST-encoding transcripts using known GST proteins from three arthropods as queries. The identified transcripts were then translated into proteins, analyzed for structural domains, and annotated using reciprocal BLAST analysis. Mining the two transcriptomes yielded a total of 41 predicted GST proteins belonging to the cytosolic, mitochondrial or microsomal classes. Phylogenetic analysis of the cytosolic GSTs validated their annotation into six different subclasses. The predicted proteins are likely to represent the products of distinct genes, suggesting that the diversity of GSTs in C. finmarchicus exceeds or rivals that described for insects. Analysis of relative gene expression in different developmental stages indicated low levels of GST expression in embryos, and relatively high expression in late copepodites and adult females for several cytosolic GSTs. A diverse diet and complex life history are factors that might be driving the multiplicity of GSTs in C. finmarchicus, as this copepod is commonly exposed to a variety of natural toxins. Hence, diversity in detoxification pathway proteins may well be key to their survival.

Undesirable enzymatic browning mediated by polyphenol oxidase (E.C. 1.14.18.1) on the surface of seafood from crustaceans have been a great concern to food processors, causing quality losses of seafood products. Seafoods especially from crustaceans are worldwide consumed due to their delicacy and nutritional value. However, black spot formation (melanosis) is the major problem occurring in crustaceans during postmortem handling and refrigerated storage induce deleterious changes in organoleptic properties and, therefore, decreases commercial value. Polyphenoloxidase (PPO), the copper-containing metalloprotein involved in oxidation of phenol to quinone is the major biochemical reaction of melanosis formation. This enzymatic mechanism causes unappealing blackening in postharvest crustaceans. To alleviate the melanosis formation in crustaceans, use of phenolic compounds from plant extract can serve as antimelanotics and appears to be a good alternative to the conventional sulfites which are associated with health-related disorders. In this review, we focuses on the unique features about the structure, distribution, and properties of PPO as well as mechanism of melanosis formation and provide a comprehensive deeper insight on the factors affecting melanosis formation and its inhibition by various antimelanotics including newly discovered plant phenolic compounds.

We conducted a comparative study of NO-ergic system in the CNS of 10 species of crustaceans subclass Malacostraca, belonging to orders Stomatopoda and Decapoda, with a common habitat in Ussuri Bay (Sea of Japan). Both similar characteristics and differences in content and distribution of universal NO-synthase (uNOS) were revealed in homologous parts of the brain and ventral nerve cord of the investigated species of crustaceans. We discuss the involvement of nitric oxide in the regulation of physiological functions of decapod crustaceans and its role in the processes of adaptation to the environmental conditions.

As a tool to address selected issues of virus biology, we constructed a recombinant cDNA clone of bovine viral diarrhea virus (BVDV) expressing Gaussia luciferase (Gluc) reporter gene. A full-length genomic cDNA clone of a non-cytopathic BVDV isolate was assembled by recombination in yeast Saccharomyces cerevisiae. The Gluc gene was inserted between the N(pro) and Core protein coding regions by recombination. The cDNA transcribed in vitro was infectious upon transfection of MDBK cells, resulting in reporter gene expression and productive virus replication. The rescued viruses were stable for 15 passages in cell culture, maintaining the replication kinetics, focus size and morphology similar to those of the parental virus. Expression and correct processing of the reporter protein were also maintained, as demonstrated by Gluc activity. These results demonstrate that genes up to 555 bp are simply assembled by a single step in yeast recombination and are stably expressed by this cDNA clone., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012), J. Bioenerg. Biomembr. 44, 325-331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2'-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism.

The small shrimp Neocaridina denticulata has two types of arginine kinases (AKs): AK1 and AK2. We determined the full kinetic parameters for the forward reaction of the AKs at temperatures between 15 and 35°C. The catalytic efficiencies, determined by kcat/(Ka(ATP)·Kia(ARG)), of the two enzymes showed a marked difference in temperature dependence. The efficiency of AK2 decreased markedly with decreasing temperature, while that of AK1 did not decrease with decreasing temperature, suggesting that AK1 is a cold-adapted enzyme. This unusual characteristic of AK1 was attributable to the remarkable decrease in Kia(ARG) with decreasing temperature, which enlarges its catalytic efficiency. Criterion with the values of Δ(ΔH(o‡))p-m and Δ(TΔS(o‡))p-m also indicate that AK1 is a psychrophilic enzyme and AK2 is mesophilic., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The activity of cholinesterases (ChE) has been recognized as a useful tool for assessing the toxicity in the environmental assessment programs. Nevertheless, the prior optimization of the experimental conditions for the appropriate measuring of the ChE activity enables us to get reliable results. Thus, the main objective of this study was to adapt and optimize a microplate assay for measuring the activity of ChE in the tropical cladoceran Pseudosida ramosa. The best readings for the reaction rates were obtained with buffers of pH 8.0 and molarity of 0.02M. The measurements of the reaction rates for the different substrate concentrations showed that the maximum reaction rate (32mODmin(-1)) was achieved by the final concentration of 2mM of substrate. In relation to the enzyme concentration, reaction rates were directly proportional to the protein concentration, which confirmed the linear kinetics for a maximum reaction rate. On the basis of the results of the assays for the effect of the number of individuals and homogenate dilution on the reaction rate of substrate hydrolysis and ChE activity, we recommend using of 30 individuals (3 days-old) in 250μL of buffer, 20 individuals (7 days-old) in 250μL of buffer and 15 individuals (both 14 and 21 days-old) in 300μL of buffer. The limits of quantitation obtained were 1.419mODmin(-1) (≤72h-old), 1.670mODmin(-1) (7 days-old), 0.943mODmin(-1) (14 days-old) and 0.797mODmin(-1) (21 days-old). In conclusion, it was possible to measure the ChE activity in P. ramosa with the methodology adapted, thus contributing to the implementation of a biochemical biomarker in freshwater toxicity assessments in tropical regions., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A firefly luciferase reporter enabled us to monitor promoter activity in vivo as well as ex vivo; however, this requires a sufficient supply of the substrate luciferin and specific monitoring devices. To overcome these disadvantages, we developed transgenic rats carrying a secreted enzyme Cypridina luciferase (CLuc) reporter under the promoter of clock gene Per2 (Per2-CLuc). Per2-CLuc activity in serially sampled blood from freely moving rats exhibited robust circadian rhythms with a peak at early morning. The Per2-CLuc bioluminescence could be quantified even with approximately 100pl of plasma. Plasma Per2-CLuc rhythms were phase reversed, and the level was reduced by restricting food access for 2h during the light phase, suggesting that the plasma Per2-CLuc rhythms reflect the phase of peripheral clocks entrained to feeding cues as well as fuel metabolism. Fasting for 2days did not alter the circadian Per2-CLuc rhythms in rats, suggesting that feeding per se did not affect the circadian Per2-CLuc rhythms. Tissue-specific Per2-CLuc rhythms were observed in culture medium of peripheral tissues. The Per2-CLuc reporter is a powerful tool to access gene expression in vivo as well as ex vivo with ordinary laboratory equipment., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Pig trypsin is routinely used as a biotechnological tool, due to its high specificity and ability to be stored as an inactive stable zymogen. However, it is not an optimum enzyme for conditions found in wound debriding for medical uses and trypsinization processes for protein analysis and animal cell culturing, where low Ca(2+) dependency, high activity in mild conditions and easy inactivation are crucial. We isolated and thermodynamically characterized a highly active cold-adapted trypsin for medical and laboratory use that is four times more active than pig trypsin at 10(°) C and at least 50% more active than pig trypsin up to 50(°) C. Contrary to pig trypsin, this enzyme has a broad optimum pH between 7 and 10 and is very insensitive to Ca(2+) concentration. The enzyme is only distantly related to previously described cryophilic trypsins. We built and studied molecular structure models of this trypsin and performed molecular dynamic calculations. Key residues and structures associated with calcium dependency and cryophilicity were identified. Experiments indicated that the protein is unstable and susceptible to autoproteolysis. Correlating experimental results and structural predictions, we designed mutations to improve the resistance to autoproteolysis and conserve activity for longer periods after activation. One single mutation provided around 25 times more proteolytic stability. Due to its cryophilic nature, this trypsin is easily inactivated by mild denaturation conditions, which is ideal for controlled proteolysis processes without requiring inhibitors or dilution. We clearly show that cold adaptation, Ca(2+) dependency and autolytic stability in trypsins are related phenomena that are linked to shared structural features and evolve in a concerted fashion. Hence, both structurally and evolutionarily they cannot be interpreted and studied separately as previously done.

Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.

The ubiquitin-proteasome system controls a variety of essential intracellular processes through directed protein turnover. The invertebrate proteasome has recently gained increasing interest with respect to central physiological processes and pathways in different taxa. A pitfall in proteasome activity assays, represented by the trypsin-like, chymotrypsin-like or caspase-like site, lies in the fact that most commonly used experimental substrates are susceptible to degradation by non-proteasomal proteolytic enzymes, which can lead to erroneous interpretation of activity data obtained. Through the use of a proteasome-specific inhibitor, epoxomicin, we showed that the shares of proteasomal and non-proteasomal activities in the degradation of a model polypeptide substrate for chymotrypsin-like activity vary considerably between invertebrate taxa. Crustacean muscle tissue and hemocytes showed almost exclusively proteasomal activity. In yeast, approximately 90% of total proteolytic activity can be attributed to the proteasome. In contrast, proteasomal activity comprises only 20-60% of the total proteolytic activity in bivalve tissues. These results reveal that, without verification of the shares of proteasomal and non-proteasomal activities in crude extracts through the use of highly specific inhibitors, common proteasomal enzyme assays should be used and interpreted with caution.

Arginine kinase (AK), a crucial enzyme for the energy metabolism of invertebrates, catalyzes the reversible phosphorylation of arginine by Mg(2+)ATP to form phosphoarginine and Mg(2+)ADP. Arginine 330 (R330), not involved in the catalysis of phosphoryl transfer, is a residue highly conserved in the phosphagen kinase family. In order to investigate the role of R330 in AK, it was replaced by lysine (R330K). Non-reduced SDS-PAGE analysis suggested that wild type AK (Wt-AK) and R330K existed in two forms, the reduced form (R-AK or R-R330K) and the oxidized form (O-AK or O-R330K), whereas O-R330K was more susceptible to generate than O-AK. Subsequently, an intramolecular disulfide bond in O-R330K was demonstrated to be formed between Cys201 and Cys271 by site-directed mutagenesis. Biochemical analysis revealed that conformational changes of R330K were concomitant with the sharp decline of catalytic activity. These results were further confirmed by structure modeling of AK and R330K. Therefore, it can be concluded that R330 residue plays an important role in the structural stability and activity of AK., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Fluoranthene (FLU) is a priority polycyclic aromatic hydrocarbon (PAH) commonly detected in estuarine sediments, water and biota. Despite this, information on FLU detection, accumulation and effects on marine crustaceans is scarce. This work investigated the accumulation of FLU in Carcinus maenas and the responses of several early-warning biomarkers after a 7-day laboratory exposure to five FLU concentrations (2.56 to 100 μg L(-1)). After exposure to FLU, sub-samples of the crabs' digestive gland and muscle were collected for biomarker determinations. The remaining digestive gland and muscle, together with the rest of the whole-body soft tissues, were analysed for FLU residues by gas chromatography-mass spectrometry (GC-MS). The biomarkers assessed were: i) the quantification of FLU-type compounds by fixed wavelength fluorescence (FF); ii) the activities of glutathione S-transferases (GST) and glutathione reductase (GR), and the levels of total glutathione (GT) and lipid peroxidation (LPO) for oxidative stress; iii) the activity of acetylcholinesterase (AChE) for neurotoxicity; iv) the activities of isocitrate dehydrogenase (IDH) and lactate dehydrogenase (LDH) enzymes, and total protein, glycogen and lipids as indicators of changes in energy metabolism and storage; and v) the lysosomal membrane stability (LMS) as a measure of cell damage. The results showed strong (R(2)>0.95) concentration-dependent accumulation of FLU residues (as measured by GC-MS) in the remaining whole-body soft tissues and of FLU-type compounds (as measured by FF) in the digestive gland and muscle. A strong positive linear relationship (R(2)=0.91) between FLU residues and FLU-type compounds was also found. Comparing to controls, activities of GST and GR were significantly higher in crabs exposed to ≥16 and ≥40 μg L(-1) FLU, respectively. TG levels and IDH activity showed a significant trend to increase with FLU concentrations whereas AChE activity exhibited the opposite trend. FF measurements in the digestive gland and muscle proved to be an expeditious cost-effective method to assess the uptake and availability of FLU and its metabolites in C. maenas. The results suggest that under continuous environmental exposure, FLU may enhance detoxification and anti-oxidant defences, and cause alterations in the aerobic energy pathway, as well as neuromuscular toxic effects that may increase C. maenas risk of predation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Thymosin proteins are well known for their actin-binding activity. Thymosin beta 4 (Tβ4) has been associated with biological activities in tissue repair and cell migration via interaction with ATP-synthase in vertebrates, while the information of similar thymosin functions in invertebrates is limited. We have shown previously that ATP-synthase is present on the surface of crayfish hematopoietic tissue (HPT) cells, and that astakine 1 (Ast1, an invertebrate cytokine) was found to interact with this β-subunit of ATP synthase. Here, we identified five different β-thymosins from Pacifastacus leniusculus, designated Pl-β-thymosin1-5. The two dominant isoforms in brain, HPT and hemocytes, Pl-β-thymosin1 and 2, were chosen for functional studies. Both isoforms could bind to the β-subunit of ATP-synthase, and Pl-β-thymosin1, but not Pl-β-thymosin2, significantly increased extracellular ATP formation. Moreover, Pl-β-thymosin1 stimulated HPT cell migration in vitro and Ast1 blocked this effect. Pl-β-thymosin2 increased the circulating hemocyte number at an early stage after injection. Additionally, in vivo injection of Pl-β-thymosin1 resulted in significant reduction of reactive oxygen species (ROS) production in crayfish HPT whereas Pl-β-thymosin2 had a similar but transient effect. Both Pl-β-thymosins induced the expression of Ast1 and superoxide dismutase (SOD) transcripts, while silencing of endogenous Pl-β-thymosin 1 and 2 by RNAi resulted in significant reduction of the Ast1 and SOD transcripts. The diverse effects exhibited by Pl-β-thymosin1 and Pl-β-thymosin2 indicates that these proteins are involved in a complex interaction that regulates the hematopoietic stem cell proliferation and differentiation.

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.

Biomarker assays are widely used as proxies for contaminant-induced effects in aquatic organisms. However, in many cases, their intrinsic natural variability due to exogenous and endogenous factors makes the interpretation of biomarker data difficult. In the present study, we investigated the natural fluctuations of six hepatic biomarkers, namely ethoxyresorufin-O-deethylase (EROD) in fish and pentoxyresorufin-O-deethylase (PROD) in crustacea, catalase (CAT), carboxylesterase (CbE), glutathione-S-transferase (GST), total glutathione peroxidase (GPX) and glutathione reductase (GR) in two deep-sea fish species, namely Alepocephalus rostratus and Lepidion lepidion and the decapod crustacean Aristeus antennatus. The NW Mediterranean deep-sea environment is characterized by very stable temperature and salinity conditions, allowing the exclusion of these two factors as potential sources of interference with biomarker activities. Biomarker results exhibited a clear influence of reproductive processes on enzyme activities, in particular in A. rostratus, which presented a pronounced seasonal pattern linked to variations in the gonadosomatic index (GSI). In addition, other factors such as food availability may also have influenced the observed variability, in particular in specimens of L. lepidion, which did not exhibit variations in reproductive activity throughout the sampling period. Depth-related variability did not exhibit a clear trend and fluctuations across sampling depths were not attributable to any specific factor. Body size had also a significant influence on some biomarkers, although allometric scaling of certain enzyme activities appears to be species-specific. The present work has thus shown that despite the lack of fluctuations of abiotic parameters such as temperature and salinity, biomarker activities in deep-sea organisms still exhibit significant variability, mainly as a result of reproductive processes and food availability., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The comparative study of the cholinesterase activity in some crab species was carried out for the first time with use of a set of thiocholine substrates. The substrate specificity was studied in stellar nerve, heart, and hemolymph of three crab species. The crab hemolymph was shown to be characterized by the highest enzyme activity. The enzyme from various crab organs has different structure o substrate specificity. Properties of crab enzymes was compared with acetylcholinesterase (AChE) of human blood erythrocytes, butyrylcholinesterase (BuChE) of horse blood serum, enzyme o squids and bivalve molluscs. The obtained data allow the conclusion to be made on differences in properties of enzymes both at the interspecies and at the tissue levels.

Polioviruses (PVs) carrying a reporter gene are useful tools for studies of virus replication, particularly if the viral chimeras contain the polyprotein that provides all of the proteins necessary for a complete replication cycle. Replication in HeLa cells of a previously constructed poliovirus expressing the gene for Renilla luciferase (RLuc) fused to the N terminus of the polyprotein H(2)N-RLuc-P1-P2-P3-COOH (P1, structural domain; P2 and P3, nonstructural domains) led to the deletion of RLuc after only one passage. Here we describe a novel poliovirus chimera that expresses Gaussia luciferase (GLuc) inserted into the polyprotein between P1 and P2 (N(2)H-P1-GLuc-P2-P3-COOH). This chimera, termed PV-GLuc, replicated to 10% of wild-type yield. The reporter signal was fully retained for three passages and then gradually lost. After six passages the signal was barely detectable. On further passages, however, the GLuc signal reappeared, and after eight passages it had reached the same levels observed with the original PV-GLuc at the first passage. We demonstrated that this surprising observation was due to coevolution of defective interfering (DI) particles that had lost part or all of the capsid coding sequence (ΔP1-GLuc-P2-P3) and wild-type-like viruses that had lost the GLuc sequence (P1-P2-P3). When used at low passage, PV-GLuc is an excellent tool for studying aspects of genome replication and morphogenesis. The GLuc protein was secreted from mammalian cells but, in agreement with published data, was not secreted from PV-GLuc-infected cells due to poliovirus-induced inhibition of cellular protein secretion. Published evidence indicates that individual expression of enterovirus polypeptide 3A, 2B, or 2BC in COS-1 cells strongly inhibits host protein secretion. In HeLa cells, however, expression of none of the poliovirus polypeptides, either singly or in pairs, inhibited GLuc secretion. Thus, inhibition of GLuc secretion in PV-infected HeLa cells is likely a result of the interaction between several viral and cellular proteins that are different from those in COS-1 cells.

The use of Caridina nilotica whole-body acetylcholinesterase (AChE) activity as a potential biomarker of Roundup(®) pollution of aquatic ecosystems was investigated. Forty days post hatch (dph) shrimps were exposed to different concentrations of 0.0, 4.3, 6.7, 10.5, 16.4, 25.6 and 40.0 mg/L in a 96 h acute toxicity test; and 0.0, 2.2, 2.8, 3.4, 4.3 and 5.4 mg/L in a 21 d chronic toxicity test. Whole-body AChE activities were determined at the end of the exposure periods by spectrophotometric assay of sample extract; activities were then normalized against protein contents in the samples and expressed in nanomoles of substrate hydrolyzed. Results of both tests showed that AChE activity was concentration-dependent. Mean AChE activities and standard deviations (±SD) for 96 h acute toxicity were 3.6239 (± 0.4185), 3.4157 (± 1.1842), 2.537 (± 1.3989), 2.4253 (± 1.4202), 2.4127 (± 1.9097), 2.0017 (± 1.1080) and 2.316 (± 0.4001) nmol/min/mg protein; while activity levels for 21 d test were 3.6907(± 0.3401), 2.8473 (± 0.713), 2.9134 (± 0.9879), 2.6738 (± 0.7117), 2.3019 (± 0.4464) and 2.1478 (± 0.864) nmol/min/mg protein. Reference basal AChE activity for 40 dph C. nilotica based on the two control groups was estimated as 3.6907 (± 0.3401) nmol/min/mg proteins. The present work provides ecotoxicological basis for the possible use of AChE activity in C. nilotica as a biomarker for monitoring Roundup(®) pollution in freshwater systems.

In this study, a rational synthesis of superluminescent variants from marine luciferases with prolonged bioluminescence has been demonstrated. A putative active site of a model marine luciferase, Gaussia princeps Luciferase (GLuc), was assigned and modified by a site-directed mutagenesis. The potent variants were found to generate up to 10 times stronger bioluminescence, emitting red shifts of up to 33 nm with natural coelenterazine than native GLuc, rendering an efficient optical signature in bioassays. The advantageous properties were demonstrated with mammalian two-hybrid assays, single-chain probes, and metastases of murine B16 melanoma in BALB/c nude mice. The unique ideas for engineering GLuc are proved to be valid even for other marine luciferases.

We report a bioluminescence resonance energy transfer (BRET) system between Cypridina luciferase (Cluc) and QDs. After coupling of the biotinylated Cluc with streptavidin-QDs, a BRET emission of QDs through the resonance energy transfer from the oxidation of Vargula (Cypridina) luciferin was observed. A luciferin analogue having a naphtyl group at C6-position showed a large blue shift of the emission spectrum, which improved the ratio of this BRET system. After directly conjugating the luciferase with QDs, an efficient BRET was observed similar to the previous BRET using organic dyes. Furthermore, the symmetric emission spectra of QDs show a potential to realize multiplexed imaging based on the BRET of Cluc using the same luciferin., (This journal is © The Royal Society of Chemistry and Owner Societies 2011)

NanoDrop spectrophotometry and NanoDrop fluorospectrometry are used almost exclusively to determine the concentrations of nucleic acids and proteins. We propose that NanoDrop fluorospectrometry can also be applied for measuring enzyme activities using fluorogenic substrates such as the proteolytic activities of the 26S proteasome. Because the NanoDrop ND-3300 device requires only 2 μl of sample, the amount of sample extract, substrate, and cofactors used for an enzyme assay can be significantly reduced. In this report, we present exemplary microassays for proteasomal activities (chymotrypsin-, trypsin-, and PGPH [peptidyl-glutamyl peptide hydrolase]-like sites) in extracts of isolated hemocytes from a marine crab, Cancer pagurus (Crustaceae)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The antiepileptic drug carbamazapine (CBZ) readily persists in sewage-water treatment plant wastewaters and finds its way into receiving water bodies. Our study sought to examine the bioaccumulation and toxicity of CBZ using an experimental aquatic trophic chain composed of the green alga, Pseudokirchneriella subcapitata, the crustacean, Thamnocephalus platyurus, and the cnidarian, Hydra attenuata. Bioaccumulation of CBZ was estimated by liquid chromatography-tandem mass spectrometry and revealed bioaccumulation factors of 2.2 and 12.6, respectively, in algae and crustaceans. No significant bioaccumulation was observed in H. attenuata. In T. platyurus, a strong stimulation of global heme oxidase (HO) (76%), and glutathione-S-transferase activity (130%) but a drastic inhibition of cytochrome P450 3A-like activity was found which suggests alteration of enzyme activity by CBZ. However, in the hydranth H. attenuata, an increase in both global cytochrome and cytochrome P450 3A-like activity was found, while GST activity was inhibited. Lipid peroxidation was reduced in T. platyurus and H. attenuata suggesting that redox activity of the lipophilic CBZ was at play. This study highlighted the processes of carbamazepine toxicity transfer between trophic levels in aquatic organisms., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

A novel dual luciferase expression vector was designed and its expression characteristics were studied in vitro and in vivo. Firstly, the Gluc and Fluc genes were connected via the TaV 2A sequence by overlaping PCR, and inserted into the expression vector pAAV2neoCAG, obtaining the recombinant plasmid pAAV2neoCAG-Gluc-2A-Fluc. Then pAAV2neoCAG-Gluc-2A-Fluc was transfected into BHK21 cells and the activity of Gluc and Fluc in the supernatant and cell lysates were assayed respectively. Results showed that both Gluc and Fluc were expressed successfully. The Gluc was mainly detected in the culture media while the Fluc was mostly within cells. The activity of Gluc in the supernatant increased gradually with time while the Fluc activity in cells almost kept stable. To investigate the expression of pAAV2neoCAG-Gluc-2A-Fluc in vivo, the plasmid was hydrodynamically injected into BALB/c mice through tail vein. The Gluc activity was assayed in a small volume of blood taken by tail vein at different time points. Results showed that Gluc was expressed stably at least 7 days. Live bioluminescence imaging technology was used to compare the expression characteristics of Gluc and Fluc. Whole body imaging was seen when coelenterazine, a specific substrate for Gluc, was injected, and the imaging signals decreased rapidly within 10 minutes. Liver imaging was showed when Flue specific substrate named D-Luciferin was injected, and the imaging remained stable at least for half an hour. The dual luciferase expression vector pAAV2neoCAG-Gluc-2A-Fluc combines the advantages of the secreted report gene Gluc and the non-secreted report gene Fluc, and will provide a new tool for cell labeling and tracing.

A possibility of adhesion inhibition of Corynebacterium diphtheriae to human buccal epithelium by glycoside hydrolases of marine hydrobiontes was investigated using alpha-galactosidase from marine bacterium Pseudoalteromonas sp. KMM 701, total enzyme preparation and beta-1,3-glucanase from marine fungi Chaetomium, total enzyme preparation and beta-1,3-glucanase from marine mollusk Littorina kurila, and total enzyme preparation from crystalline style of marine mollusk Spisula sachalinensis were used. The enzymes were added to test-tubes containing buccal epithelial cells and/or the toxigenic bacterial strain C. diphtheriae No 1129, v. gravis. All the investigated enzymes were able to abort C. diphtheriae adherence, to human buccal epithelocytes. Inhibition of adhesion was more pronounced in the case of treatment of epithelocytes with highly purified enzymes of marine hydrobiontes in comparison with total enzyme preparations. The significant inhibition of C. diphtheriae adhesion was observed when the enzymes were added to the epithelocytes with the attached microorganisms. The results obtained show that glycoside hydrolases of marine hydrobiontes degrade any carbohydrates expressed on cell surface of bacterium or human buccal epithelocytes, impair unique lectin-carbohydrate interaction and prevent the adhesion.

The effects of betaine on prawn beta-N-acetyl-D-glucosaminidase (NAGase) activity for the hydrolysis of p-nitrophenyl-N-acetyl- beta-D-glucosaminide (pNP-NAG) have been studied. The results showed that appropriate concentrations of betaine could lead to reversible inhibition against NAGase, and the IC(50) value was estimated to be 15.00 +/- 0.30 mM. The inhibitory kinetics assay showed that betaine was a mixed type inhibitor with a K(I) value of 9.17 +/- 0.85 mM and a K(IS) value of 45.58 +/- 2.52 mM. The inhibitory model was set, and the microscopic rate constants were determined using the kinetic method of the substrate reaction. The time course of the hydrolysis of pNP-NAG catalyzed by NAGase in the presence of different betaine concentrations showed that at each betaine concentration, the rate decreased with an increase in time until a straight line was approached, indicating that the inhibition of NAGase by betaine is a slow, reversible reaction with fractional residual activity. The fact that k(+0) is much larger than k(+0)(') indicated that the free enzyme molecule is more fragile than the enzyme-substrate complex against betaine. It is suggested that the presence of the substrate offers marked protection of NAGase against inhibition by betaine.

Arthropoda is the largest of all animal phyla and includes about 90% of extant species. Our knowledge about regulation of apoptosis in this phylum is largely based on findings for the fruit fly Drosophila melanogaster. Recent work with crustaceans shows that apoptotic proteins, and presumably mechanisms of cell death regulation, are more diverse in arthropods than appreciated based solely on the excellent work with fruit flies. Crustacean homologs exist for many major proteins in the apoptotic networks of mammals and D. melanogaster, but integration of these proteins into the physiology and pathophysiology of crustaceans is far from complete. Whether apoptosis in crustaceans is mainly transcriptionally regulated as in D. melanogaster (e.g., RHG 'killer' proteins), or rather is controlled by pro- and anti-apoptotic Bcl-2 family proteins as in vertebrates needs to be clarified. Some phenomena like the calcium-induced opening of the mitochondrial permeability transition pore (MPTP) are apparently lacking in crustaceans and may represent a vertebrate invention. We speculate that differences in regulation of the intrinsic pathway of crustacean apoptosis might represent a prerequisite for some species to survive harsh environmental insults. Pro-apoptotic stimuli described for crustaceans include UV radiation, environmental toxins, and a diatom-produced chemical that promotes apoptosis in offspring of a copepod. Mechanisms that serve to depress apoptosis include the inhibition of caspase activity by high potassium in energetically healthy cells, alterations in nucleotide abundance during energy-limited states like diapause and anoxia, resistance to opening of the calcium-induced MPTP, and viral accommodation during persistent viral infection. Characterization of the players, pathways, and their significance in the core machinery of crustacean apoptosis is revealing new insights for the field of cell death.

We aimed to develop a far-red luminescence imaging technology for visualization of disease specific antigens on cell surfaces in a living body. First, we conjugated a far-red fluorescent indocyanine derivative to biotinylated Cypridina luciferase. This conjugate produced a bimodal spectrum that has long-wavelength bioluminescence emission in the far-red region as a result of bioluminescence resonance energy transfer. To generate a far-red luminescent probe with targeting and imaging capabilities of tumors, we then linked this conjugate to an anti-human Dlk-1 monoclonal antibody via the biotin-avidin interaction. This far-red luminescent probe enabled us to obtain high-resolution microscopic images of live, Dlk-1-expressing Huh-7 cells without an external light source, and to monitor the accumulation of this probe in tumor-bearing mice. Thus this far-red luminescent probe is a convenient analytical tool for the evaluations of monoclonal antibody localization in a living body.

Several biomarkers indicative of stress were characterised in the crustaceans Aristeus antennatus and Nephrops norvegicus sampled off the Barcelona coast (NW Mediterranean). The biomarkers selected were cholinesterase (ChE) activities in muscle; and catalase, glutathione reductase (GR), total glutathione peroxidase (t-GPX), DT-diaphorase (DT-D), glutathione S-transferases (GSTs) and carboxylesterases (CbEs) in hepatopancreas tissue. Lipid peroxidation (LP) levels and total protein yield (PY) were also determined in muscle and hepatopancreas tissues. The activities and levels are discussed in relation to species and season, and differences in these two factors were observed for most biomarkers. AChEs and pseudocholinesterases were present in the muscles of both crustaceans. Catalase and GST activities were higher in N. norvegicus, whereas GR and t-GPX activities varied according to the season. Hepatic CbE activities were similar in the two crustaceans, whereas LP levels and PY were different between species. Seasonality and species particularities are factors to consider when these crustaceans are used as sentinels.

Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence (lambda(max)=480 nm). This luciferase has been successfully applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase as a reporter is the capability of measuring intracellular events without destroying the cells or tissues and this property is well suited for development of high throughput screening technologies. However because Metridia luciferase is a Cys-rich protein, Escherichia coli expression systems produce an incorrectly folded protein, hindering its biochemical characterization and application for development of in vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system. Functionally active luciferase secreted by insect cells into the culture media has been efficiently purified with a yield of high purity protein of 2-3 mg/L. This Metridia luciferase expressed in the insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of Metridia luciferase purified from insect cells with that calculated from amino acid sequence, indicates that luciferase does not undergo post-translational modifications such as phosphorylation or glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted from sequence analysis.

Background: The L-lactate and D-lactate dehydrogenases, which are involved in the reduction of pyruvate to L(-)-lactate and D(+)-lactate, belong to evolutionarily unrelated enzyme families. The genes encoding L-LDH have been used as a model for gene duplication due to the multiple paralogs found in eubacteria, archaebacteria, and eukaryotes. Phylogenetic studies have suggested that several gene duplication events led to the main isozymes of this gene family in chordates, but little is known about the evolution of L-Ldh in invertebrates. While most invertebrates preferentially oxidize L-lactic acid, several species of mollusks, a few arthropods and polychaetes were found to have exclusively D-LDH enzymatic activity. Therefore, it has been suggested that L-LDH and D-LDH are mutually exclusive. However, recent characterization of putative mammalian D-LDH with significant similarity to yeast proteins showing D-LDH activity suggests that at least mammals have the two naturally occurring forms of LDH specific to L- and D-lactate. This study describes the phylogenetic relationships of invertebrate L-LDH and D-LDH with special emphasis on crustaceans, and discusses gene duplication events during the evolution of L-Ldh., Results: Our phylogenetic analyses of L-LDH in vertebrates are consistent with the general view that the main isozymes (LDH-A, LDH-B and LDH-C) evolved through a series of gene duplications after the vertebrates diverged from tunicates. We report several gene duplication events in the crustacean, Daphnia pulex, and the leech, Helobdella robusta. Several amino acid sequences with strong similarity to putative mammalian D-LDH and to yeast DLD1 with D-LDH activity were found in both vertebrates and invertebrates., Conclusion: The presence of both L-Ldh and D-Ldh genes in several chordates and invertebrates suggests that the two enzymatic forms are not necessarily mutually exclusive. Although, the evolution of L-Ldh has been punctuated by multiple events of gene duplication in both vertebrates and invertebrates, a shared evolutionary history of this gene in the two groups is apparent. Moreover, the high degree of sequence similarity among D-LDH amino acid sequences suggests that they share a common evolutionary history.