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In this work we analyzed the effects of Sulfosato Touchdown®, a glyphosate-based herbicide, on the ontogenic development and biochemical markers of the freshwater copepod Notodiaptomus carteri. A 30-days life-cycle experiment was carried out with three different glyphosate concentrations (0, 0.38, and 0.81 mg L -1 ) to analyze the developmental time from nauplii to adult copepods and their individual growth. An additional 10-days experiment with the same glyphosate concentrations was designed to evaluate the energy reserves (glycogen, proteins and lipids) and the activity of three antioxidant enzymes, superoxide dismutase (SOD), catalase, and glutathione-S-transferase (GST) in adult copepods, separately for females and males. We found that the lowest glyphosate concentration increased the nauplii and total development time. The highest glyphosate concentration prevented copepods from reaching the adult stage, inhibited the growth of the first copepodite stage and increased the GST and SOD activity in adult females. According to our results, the presence of this herbicide in freshwater systems could impose a risk in the ecological role of copepods in nature. This study will contribute to propose the Notodiaptomus genus as model specie for monitoring purposes in the Neotropical aquatic systems., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Listeria innocua DNA binding protein from starved cells (LiDps) belongs to the ferritin family and provides a promising self-assembling spherical 12-mer protein scaffold for the generation of functional nanomaterials. We report the creation of a Gaussia princeps luciferase (Gluc)-LiDps fusion protein, with chemical conjugation of Zinc (II)-protoporphyrin IX (ZnPP) to lysine residues on the fusion protein (giving Gluc-LiDps-ZnPP). The Gluc-LiDps-ZnPP conjugate is shown to generate reactive oxygen species (ROS) via Bioluminescence Resonance Energy Transfer (BRET) between the Gluc (470-490 nm) and ZnPP. In vitro, Gluc-LiDps-ZnPP is efficiently taken up by tumorigenic cells (SKBR3 and MDA-MB-231 breast cancer cells). In the presence of coelenterazine, this construct inhibits the proliferation of SKBR3 due to elevated ROS levels. Following exposure to Gluc-LiDps-ZnPP, migration of surviving SKBR3 cells is significantly suppressed. These results demonstrate the potential of the Gluc-LiDps-ZnPP conjugate as a platform for future development of an anticancer photodynamic therapy agent., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

To validate the use of recombinant aequorin (reAequorin) as a light emission standard, the protein concentrations of highly purified reAequorin were determined by amino acid composition analysis, and the presence of active reAequorin was confirmed by the ratio of absorbance peak at 460 nm to that at 280 nm. The high correlation of the luminescence intensity with the protein concentration showed that reAequorin could be used for a light emission standard to study the luminescence properties of luciferases and to evaluate the detection sensitivity of luminometers. The specific activity of Gaussia luciferase with I max was 7.5-fold higher than that of reAequorin and was calculated to be 3.8 × 10 16 quanta/mg protein., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase from the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The bright bioluminescence of copepod Metridia longa is conditioned by a small secreted coelenterazine-dependent luciferase (MLuc). To date, three isoforms of MLuc differing in length, sequences, and some properties were cloned and successfully applied as high sensitive bioluminescent reporters. In this work, we report cloning of a novel group of genes from M. longa encoding extremely psychrophilic isoforms of MLuc (MLuc2-type). The novel isoforms share only ∼54-64% of protein sequence identity with the previously cloned isoforms and, consequently, are the product of a separate group of paralogous genes. The MLuc2 isoform with consensus sequence was produced as a natively folded protein using baculovirus/insect cell expression system, purified, and characterized. The MLuc2 displays a very high bioluminescent activity and high thermostability similar to those of the previously characterized M. longa luciferase isoform MLuc7. However, in contrast to MLuc7 revealing the highest activity at 12-17 °C and 0.5 M NaCl, the bioluminescence optima of MLuc2 isoforms are at ∼5 °C and 1 M NaCl. The MLuc2 adaptation to cold is also accompanied by decrease of melting temperature and affinity to substrate suggesting a more conformational flexibility of a protein structure. The luciferase isoforms with different temperature optima may provide adaptability of the M. longa bioluminescence to the changes of water temperature during diurnal vertical migrations., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 SS bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of ∼3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aquatic organisms are constantly exposed to both physical (e.g. temperature and salinity variations) and chemical (e.g. endocrine disruptor chemicals, heavy metals, hydrocarbons, diatom toxins, and other toxicants) stressors which they react to by activating a series of defense mechanisms. This paper reviews the literature on the defense systems, including detoxification enzymes and proteins (e.g. glutathione S-transferases, heat shock proteins, superoxide dismutase and catalase), studied in copepods at the molecular level. The data indicate high inter- and intra-species variability in copepod response, depending on the type of stressor tested, the concentration and exposure time, and the enzyme isoform studied. Ongoing -omics approaches will allow the identification of new genes which will give a more comprehensive overview of how copepods respond to specific stressors in laboratory and/or field conditions and the effects of these responses on higher trophic levels., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The technology of real-time imaging in living cells is crucial for understanding of intracellular events. For this purpose, bioluminescent reporters have been introduced as sensitive and convenient tools. Metridia luciferase (MLuc) from the copepod Metridia longa is a coelenterazine-dependent luciferase containing a natural signal peptide for secretion. We report the high-active MLuc mutants with deletion of the N-terminal variable part of amino acid sequence. The MLuc variants were produced in Escherichia coli cells, converted to an active protein, and characterized. We demonstrate that the truncated MLucs have significantly increased bioluminescent activity as against the wild type enzyme but substantially retain other properties. One of the truncated variants of MLuc was transiently expressed in HEK 293 cells. The results clearly suggest that the truncated Metridia luciferase is well suited as a secreted reporter ensuring higher detection sensitivity in comparison with a wild type enzyme., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Hybridoma formation is an indispensable step in the production of monoclonal antibodies. Obtaining highly efficient fusion of an antibody-producing cell to the myeloma cell to form the hybridoma is an important step in this process. The electrofusion method is superior to chemical fusion methods such as the polyethylene glycol (PEG) method due to its high fusion efficiency. However, this method requires cell activation prior to electrofusion, a process that is time-consuming and tends to cause cell death. In this study, we achieved much higher fusion efficiency by stimulating B cells with CpG oligodeoxynucleotide (CpG ODN) over shorter periods. Splenocytes were isolated from immunized mice and cultured in the presence of a CpG ODN for 1 or 2 days. This CpG ODN stimulation evokes about one order of magnitude higher fusion efficiency than other stimulators. CpG ODN stimulation not only increases the fusion efficiency but also the number of antibody-producing cells. This leads to a substantial increase in the number of positive clones obtained. This highly efficient fusion method was used to produce a functional antibody against Gaussia luciferase. This method was found to produce greater numbers of hybridomas and to enable direct screening for antibodies with functional characteristics such as inhibition of the luminescence activity of an antigen. We were able to establish a functional antibody against Gaussia luciferase after a single fusion experiment using our electrofusion method., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We have developed a method of video rate bioluminescence imaging to investigate protein secretion from a single mammalian cell and analyzed the localization, secretory frequency, and quantification of secreted protein. By detecting the luminescence signals from the Gaussia luciferase (GLase) reaction using a high-speed electron-multiplying charge-coupled device (EM-CCD) camera, video rate imaging was performed with a time resolution within 500 ms/image over 30 min in living cells. As a model study, we applied the method to visualize the glucose-stimulated insulin secretion from clustered pancreatic MIN6 β cells using the fused protein of GLase with preproinsulin. High-quality video images clearly showed that the glucose-stimulated insulin secretion from the clustered MIN6 β cells oscillated within a period of a few minutes over 10 min. In addition, the glibenclamide-induced insulin secretion from the clustered MIN6 β cells was visualized, suggesting that bioluminescence video rate imaging is a useful method for validating drug action in living cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The mutated recombinant Gaussia luciferase (hgGLase) having the hinge sequence with a reactive cysteine residue at the carboxyl terminal region was purified from Escherichia coli cells by nickel-chelate affinity chromatography and hydrophobic chromatography. The biotinylated hgGLase (Biotin-hgGLase) was prepared by chemical conjugation with a maleimide activated biotin and apply to bioluminescent immunoassay. In the streptavidin and biotin complex system using Biotin-hgGLase, the measurable range of α-fetoprotein as a model analyte was 0.02-100ng/ml with the coefficient of variation between 2.5% and 5.2%. The sensitivity of Biotin-hgGLase was similar to that by using the detection system of aequorin, alkaline phosophatase and horseradish peroxidase as a label enzyme., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc-scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide-alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA-tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc-scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker.

Gaussia luciferase (GLuc) from the copepod Gaussia princeps is both the smallest and brightest known luciferase. GLuc catalyzes the oxidation of coelenterazine to produce an intense blue light but with a very short emission half-life. We report mutated GLucs with much longer luminescence half-lives that retain the same initial intensity as the wild-type enzyme. The GLuc variants were produced using cell-free protein synthesis to provide high yields and rapid production of fully active product as well as simple non-natural amino acid substitution. By incorporating homopropargylglycine and attaching PEG using azide-alkyne click reactions, we also show that the four methionines in GLuc are surface accessible. The mutants provide a significantly improved reporter protein for both in vivo and in vitro studies, and the successful non-natural amino acid incorporation and PEG attachment indicate the feasibility of producing useful bioconjugates using click attachment reactions.

Gaussia luciferase secreted by the copepod Gaussia princeps catalyzes the oxidation of coelenterazine to produce blue light. The primary structure of Gaussia luciferase deduced from the cDNA sequence shows two repeat sequences of 71 amino acid residues, suggesting the luciferase consists of two structural domains. Two domains in Gaussia luciferase were expressed independently in Escherichia coli cells, purified and characterized. We found that both domains have luminescence activity with coelenterazine, and the catalytic properties including luminescence spectrum, optimal pH, substrate specificity and luminescence stimulation by halogen ions (Cl-, Br- and I-) are identical to intact Gaussia luciferase. Thus, Gaussia luciferase has two catalytic domains for the luminescence reaction.

To investigate the impacts of marine pollution on aquatic organisms, we tested the intertidal copepod Tigriopus japonicus as a model species. To analyze the copepods' responses to endocrine-disrupting chemicals (EDCs), we exposed them to two different chemicals: 4,4'-octylphenol (4,4'-OP, 12.5-100 microg/L for 2 h) and polychlorinated biphenyl (PCB, 6.25-25 microg/L for two days). 4,4'-OP was toxic, although exposure time was limited to 2h. After extracting total RNA from the exposed T. japonicus, we performed reverse transcriptase-polymerase chain reaction (RT-PCR) to determine gene expression patterns following chemical exposure. To analyze the gene expression of T. japonicus, we used glutathione S-transferase with GAPDH as an internal control. Of the genes tested using EDC-exposed samples, 4,4'-OP induced upregulation of the glutathione S-transferase (GST) gene, while PCB caused downregulation of the GST gene. These results suggest that the two EDCs act in different manners in T. japonicus.

Secreted alkaline phosphatase (SEAP) and Metridia luciferase (MLuc) are useful reporter molecules in vitro, but little is understood about their usefulness in vivo. In this study, we investigated in vivo activity of recombinant SEAP and MLuc in blood and urine. When SEAP-transfected cells or recombinant SEAP were injected into rats, substantial increase in the level of serum SEAP was observed. In contrast, activity of SEAP was not detected in urine of rats injected with either the SEAP-transfected cells or recombinant SEAP. SEAP activity was also undetectable in urine of SEAP-injected Nagase analbuminemic rats in which glomerular permeability to macromolecules is enhanced. When MLuc-transfected cells were implanted into rats, activity of MLuc was undetectable not only in urine but also in serum. Even immediately after intravenous injection of recombinant MLuc, activity of MLuc was not detected in serum. Subsequent experiments revealed that, in contrast to SEAP, MLuc was rapidly inactivated either by rat serum, fetal bovine serum, or human serum. Albumin was identified as the molecule responsible for the inhibition of MLuc activity. These data elucidated advantages and limitations of secreted reporter molecules SEAP and MLuc under in vivo situations.

The salmon louse Lepeophtheirus salmonis, a marine ectoparasitic copepod that feeds on salmonids, cause huge financial losses in the aquaculture industry through fish mortality, reduced growth and therapeutic costs. Detailed knowledge about the salmon louse life cycle, at the functional molecular level, is of fundamental importance to evaluate alternative therapeutic or prophylactic strategies. In quantitative real-time PCR, a powerful technique in biological studies of differentially expressed genes, the transcription level of a regulated gene can be measured relative to an unregulated reference "housekeeping" gene. In the present study we validate candidate reference genes for transcription profiling throughout the life cycle of the salmon louse. Our results show that the structural ribosomal protein S20 (RPS20) and the translation elongation factor 1alpha (eEF1alpha) are valid as reference genes showing less than two fold variation in transcript levels. The frequently used reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), however, is up to six fold regulated during the L. salmonis life cycle. Furthermore our results indicated that 18S RNA, although constitutively expressed, is not a convenient reference gene for relative quantification of most transcripts.