Title: Exposure to metals premixed with microplastics increases toxicity through bioconcentration and impairs antioxidant defense and cholinergic response in a marine mysid Eom HJ, Haque MN, Lee S, Rhee JS Ref: Comparative Biochemistry & Physiology C Toxicol Pharmacol, :109142, 2021 : PubMed
Coexistence of metals and microplastics (MPs) in aquatic environments represents a growing concern; however, little is known regarding the risks associated with their combined effects. Here, the effects of five metals (As, Cd, Cu, Pb, and Zn), alone or combined with MPs for various premixing durations (30 and 60 days), on the juvenile and adult stages of the marine mysid Neomysis awatschensis were evaluated. The toxicity (50% lethal concentration for 96 h) and bioconcentration of metals premixed with MPs were measured, and their effects on the antioxidant defense and cholinergic systems were examined. Metal toxicity increased with increasing premixing period with MPs, and juveniles were more sensitive to exposure to metals premixed with MPs than adults. Metal bioconcentration in the mysid body increased following co-exposure with MPs. Metals premixed with MPs significantly increased intracellular malondialdehyde content at both stages but decreased glutathione content in juveniles. At both stages, catalase and superoxide dismutase activity was suppressed following co-exposure to metals and MPs, except under the Cu treatment. Moreover, co-exposure inhibited acetylcholinesterase activity at both stages, suggesting cholinergic impairment. Taken together, metals and MPs produce synergistic detrimental effects on marine mysids in a stage-specific manner. Further studies are warranted to elucidate the role of MPs as a vector for contaminants and stimulator of toxicity in aquatic organisms.
Title: Exposure to sublethal concentrations of zinc pyrithione inhibits growth and survival of marine polychaete through induction of oxidative stress and DNA damage Haque MN, Nam SE, Eom HJ, Kim SK, Rhee JS Ref: Mar Pollut Bull, 156:111276, 2020 : PubMed
Effects of zinc pyrithione (ZnPT) and inorganic Zn (ZnCl2) were evaluated on a marine polychaete at sublethal concentrations for 14 days. ZnPT decreased the burrowing activity and AChE activity with higher acute toxicities, implying its cholinergic effect. Both ZnPT and ZnCl2 increased MDA levels at higher concentrations, suggesting lipid peroxidation and oxidative stress. In the ZnPT-treated polychaete, enzymatic activities of CAT and SOD were elevated with an increase in DNA damage, whereas the levels of GSH, GPx, GR, and GST were decreased. However, in the ZnCl2-treated polychaete, the level of GSH and enzymatic activities of CAT, SOD, GPx, GR, and GST were significantly elevated to resist cellular damage. During 97 days depuration experiment, significant mortality and growth retardation were observed in the ZnPT-exposed polychaete. Overall, ZnPT was found to be more toxic than ZnCl2 with the harmful impact on antioxidant defense system and DNA stability in marine polychaete.
Title: Effects of sublethal concentrations of the antifouling biocide Sea-Nine on biochemical parameters of the marine polychaete Perinereis aibuhitensis Eom HJ, Haque MN, Nam SE, Lee DH, Rhee JS Ref: Comparative Biochemistry & Physiology C Toxicol Pharmacol, 222:125, 2019 : PubMed
Sea-Nine 211 is an emerging biocide that has an adverse impact on aquatic environments. In this study, the marine polychaete Perinereis aibuhitensis was exposed to Sea-Nine (0.1, 1, and 10mugL(-1)), and acute toxicity and biochemical responses such as changes in the intracellular contents of malondialdehyde (MDA) and glutathione (GSH) and enzymatic activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), and acetylcholinesterase (AChE) were evaluated over a period of 14 d. Determined median lethal doses, LC50 were 268mugL(-1), 142mugL(-1), and 55mugL(-1) at 24h, 96h, and 14 d, respectively. The MDA content increased significantly in a dose- and time-dependent manner, indicative of lipid peroxidation-related oxidative damage. Significantly higher intracellular GSH levels and antioxidant defense-related enzyme (CAT, SOD, GPx, GR, and GST) activities were observed after exposure to 10mugL(-1) Sea-Nine. In contrast, Sea-Nine treatment significantly reduced AChE activity at the highest concentration of Sea-Nine used (10mugL(-1)). Taken together, these results indicate that sublethal concentrations of Sea-Nine are toxic to marine polychaetes through potential lipid peroxidation, induction of oxidative stress, and modulation of the cholinergic system. Our results can contribute to biomonitoring of aquatic environments and ecotoxicological research through the measurements of polychaete cellular defenses against waterborne biocides.
Title: Chlorothalonil induces oxidative stress and reduces enzymatic activities of Na+/K+-ATPase and acetylcholinesterase in gill tissues of marine bivalves Haque MN, Eom HJ, Nam SE, Shin YK, Rhee JS Ref: PLoS ONE, 14:e0214236, 2019 : PubMed
Chlorothalonil is a thiol-reactive antifoulant that disperses widely and has been found in the marine environment. However, there is limited information on the deleterious effects of chlorothalonil in marine mollusks. In this study, we evaluated the effects of chlorothalonil on the gill tissues of the Pacific oyster, Crassostrea gigas and the blue mussel, Mytilus edulis after exposure to different concentrations of chlorothalonil (0.1, 1, and 10 mug L-1) for 96 h. Following exposure to 1 and/or 10 mug L-1 of chlorothalonil, malondialdehyde (MDA) levels significantly increased in the gill tissues of C. gigas and M. edulis compared to that in the control group at 96 h. Similarly, glutathione (GSH) levels were significantly affected in both bivalves after chlorothalonil exposure. The chlorothalonil treatment caused a significant time- and concentration-dependent increase in the activity of enzymes, such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR), in the antioxidant defense system. Furthermore, 10 mug L-1 of chlorothalonil resulted in significant inhibitions in the enzymatic activity of Na+/K+-ATPase and acetylcholinesterase (AChE). These results suggest that chlorothalonil induces potential oxidative stress and changes in osmoregulation and the cholinergic system in bivalve gill tissues. This information will be a useful reference for the potential toxicity of chlorothalonil in marine bivalves.
Title: Constant exposure to environmental concentrations of the antifouling biocide Sea-Nine retards growth and reduces acetylcholinesterase activity in a marine mysid Do JW, Haque MN, Lim HJ, Min BH, Lee DH, Kang JH, Kim M, Jung JH, Rhee JS Ref: Aquat Toxicol, 205:165, 2018 : PubMed
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.
Title: Effects of Antifouling Biocides on Molecular and Biochemical Defense System in the Gill of the Pacific Oyster Crassostrea gigas Park MS, Kim YD, Kim BM, Kim YJ, Kim JK, Rhee JS Ref: PLoS ONE, 11:e0168978, 2016 : PubMed
Antifouling biocides such as organotin compounds and their alternatives are potent toxicants in marine ecosystems. In this study, we employed several molecular and biochemical response systems of the Pacific oyster Crassostrea gigas to understand a potential mode of action of antifouling biocides (i.e. tributyltin (TBT), diuron and irgarol) after exposure to different concentrations (0.01, 0.1, and 1 mug L-1) for 96 h. As a result, all the three antifouling biocides strongly induced the antioxidant defense system. TBT reduced both enzymatic activity and mRNA expression of Na+/K+-ATPase and acetylcholinesterase (AChE). Lower levels of both Na+/K+-ATPase activity and AChE mRNA expression were observed in the diuron-exposed oysters compared to the control, while the irgarol treatment reduced only the transcriptional expression of AChE gene. We also analyzed transcript profile of heat shock protein (Hsp) superfamily in same experimental conditions. All antifouling biocides tested in this study significantly modulated mRNA expression of Hsp superfamily with strong induction of Hsp70 family. Taken together, overall results indicate that representative organotin TBT and alternatives have potential hazardous effects on the gill of C. gigas within relatively short time period. Our results also suggest that analyzing a series of molecular and biochemical parameters can be a way of understanding and uncovering the mode of action of emerging antifouling biocides. In particular, it was revealed that Pacific oysters have different sensitivities depend on the antifouling biocides.
Loss-of-function mutations in the synaptic adhesion protein Neuroligin-4 are among the most common genetic abnormalities associated with autism spectrum disorders, but little is known about the function of Neuroligin-4 and the consequences of its loss. We assessed synaptic and network characteristics in Neuroligin-4 knockout mice, focusing on the hippocampus as a model brain region with a critical role in cognition and memory, and found that Neuroligin-4 deletion causes subtle defects of the protein composition and function of GABAergic synapses in the hippocampal CA3 region. Interestingly, these subtle synaptic changes are accompanied by pronounced perturbations of gamma-oscillatory network activity, which has been implicated in cognitive function and is altered in multiple psychiatric and neurodevelopmental disorders. Our data provide important insights into the mechanisms by which Neuroligin-4-dependent GABAergic synapses may contribute to autism phenotypes and indicate new strategies for therapeutic approaches.
Title: Inhibitory effects of biocides on transcription and protein activity of acetylcholinesterase in the intertidal copepod Tigriopus japonicus Lee JW, Kim BM, Jeong CB, Won EJ, Rhee JS, Lee JS Ref: Comparative Biochemistry & Physiology C Pharmacol Toxicol, 167C:147, 2014 : PubMed
Acetlycholinesterase (AChE) is a serine esterase that plays an important role in the hydrolytic degradation of acetylcholine. We investigated the modulatory potential of T. japonicus-AChE (TJ-AChE) for biocide response by cloning, sequencing, and characterizing the full-length genomic DNA of the TJ-AChE1 and TJ-AChE2 genes. The deduced TJ-AChE proteins were highly conserved across species and were distinctively separated into two subtypes, AChE1 and AChE2. Each TJ-AChE protein was closely phylogenetically clustered with invertebrate AChE1 and AChE2 proteins. Transcriptional level of TJ-AChE1 was higher than TJ-AChE2 in all developmental stages. TJ-AChE1 mRNA decreased in response to five biocides (alachlor, chlorpyrifos, dimethoate, endosulfan, lindane,) but not in the molinate-exposed group. TJ-AChE2 decreased significantly only in response to chlorpyrifos and lindane. TJ-AChE enzymatic activity was significantly inhibited when exposed to alachlor, chlorpyrifos, endosulfan, or lindane for 24h. This study elucidates potential endogenous mechanisms of biocide-induced neurotoxicity in T. japonicus.
Short-term synaptic plasticity, the dynamic alteration of synaptic strength during high-frequency activity, is a fundamental characteristic of all synapses. At the calyx of Held, repetitive activity eventually results in short-term synaptic depression, which is in part due to the gradual exhaustion of releasable synaptic vesicles. This is counterbalanced by Ca(2+)-dependent vesicle replenishment, but the molecular mechanisms of this replenishment are largely unknown. We studied calyces of Held in knockin mice that express a Ca(2+)-Calmodulin insensitive Munc13-1(W464R) variant of the synaptic vesicle priming protein Munc13-1. Calyces of these mice exhibit a slower rate of synaptic vesicle replenishment, aberrant short-term depression and reduced recovery from synaptic depression after high-frequency stimulation. Our data establish Munc13-1 as a major presynaptic target of Ca(2+)-Calmodulin signaling and show that the Ca(2+)-Calmodulin-Munc13-1 complex is a pivotal component of the molecular machinery that determines short-term synaptic plasticity characteristics.
In many brain regions, gephyrin and GABAA receptor clustering at developing inhibitory synapses depends on the guanine nucleotide exchange factor collybistin (Cb). The vast majority of Cb splice variants contain an autoinhibitory src homology 3 domain, and several synaptic proteins are known to bind to this SH3 domain and to thereby activate gephyrin clustering. However, many functional GABAergic synapses form independently of the known Cb-activating proteins, indicating that additional Cb activators must exist. Here we show that the small Rho-like GTPase TC10 stimulates Cb-dependent gephyrin clustering by binding in its active, GTP-bound state to the pleckstrin homology domain of Cb. Overexpression of a constitutively active TC10 variant in neurons causes an increase in the density of synaptic gephyrin clusters and mean miniature inhibitory postsynaptic current amplitudes, whereas a dominant negative TC10 variant has opposite effects. The enhancement of Cb-induced gephyrin clustering by GTP-TC10 does not depend on the guanine nucleotide exchange activity of Cb but involves an interaction that resembles reported interactions of other small GTPases with their effectors. Our data indicate that GTP-TC10 activates the major src homology 3 domain-containing Cb variants by relieving autoinhibition and thus define an alternative GTPase-driven signaling pathway in the genesis of inhibitory synapses.
Title: Effect of pharmaceuticals exposure on acetylcholinesterase (AchE) activity and on the expression of AchE gene in the monogonont rotifer, Brachionus koreanus Rhee JS, Kim BM, Jeong CB, Park HG, Leung KM, Lee YM, Lee JS Ref: Comparative Biochemistry & Physiology C Pharmacol Toxicol, 158:216, 2013 : PubMed
Pharmaceuticals are widely used in human and veterinary medicine. However, they are emerging as a significant contaminant in aquatic environments through wastewater. Due to the persistent and accumulated properties of pharmaceuticals via the food web, their potential harmful effects on aquatic animals are a great concern. In this study, we investigated the effects of six pharmaceuticals: acetaminophen, ATP; atenolol, ATN; carbamazepine, CBZ; oxytetracycline, OTC; sulfamethoxazole, SMX; and trimethoprim, TMP on acetylcholinesterase (AChE; EC 3.1.1.7) activity and its transcript expression with chlorpyrifos (as a positive control) in the monogonont rotifer, Brachionus koreanus. ATP, CBZ, and TMP exposure also remarkably inhibited Bk-AChE activity at 100mug/L (24h) and 1000mug/L (12h and 24h). ATP, CBZ, and TMP exposure showed a significant decrease in the Bk-AChE mRNA level in a concentration-dependent manner. However, in the case of OTC and SMX, a slight decrease in Bk-AChE mRNA expression was found but only at the highest concentration. The time-course experiments showed that ATP positively induced Bk-AChE mRNA 12h after exposure at both 100 and 1000mug/L, while the Bk-AChE mRNA expression was significantly downregulated over 6 to 24h after exposure to 1000mug/L of CBZ, OTC, SMX, and TMP. Our findings suggest that Bk-AChE would be a useful biomarker for risk assessment of pharmaceutical compounds as an early signal of their toxicity in aquatic environments. Particularly, ATP, CBZ, and TMP may have a toxic cholinergic effect on rotifer B. koreanus by inhibiting AChE activity.
Title: Functional display of Pseudomonas and Burkholderia lipases using a translocator domain of EstA autotransporter on the cell surface of Escherichia coli Yang TH, Kwon MA, Song JK, Pan JG, Rhee JS Ref: J Biotechnol, 146:126, 2010 : PubMed
Functional expression of the industrially important Pseudomonas and Burkholderia lipases, such as those from P. aeruginosa, B. cepacia and P. fluorescens, was achieved on the cell surface of Escherichia coli using the C-terminal translocator moiety (EstATu) of autotransporter protein (EstA) from P. putida. In particular, lipases which required a lipase-specific foldase (Lif) for their proper folding were for the first time displayed in the active form by coexpression of the Lif protein.
Title: Enhancement of the efficiency of secretion of heterologous lipase in Escherichia coli by directed evolution of the ABC transporter system Eom GT, Song JK, Ahn JH, Seo YS, Rhee JS Ref: Applied Environmental Microbiology, 71:3468, 2005 : PubMed
The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.
Title: Identification of the tliDEF ABC transporter specific for lipase in Pseudomonas fluorescens SIK W1 Ahn JH, Pan JG, Rhee JS Ref: Journal of Bacteriology, 181:1847, 1999 : PubMed
Pseudomonas fluorescens, a gram-negative psychrotrophic bacterium, secretes a thermostable lipase into the extracellular medium. In our previous study, the lipase of P. fluorescens SIK W1 was cloned and expressed in Escherichia coli, but it accumulated as inactive inclusion bodies. Amino acid sequence analysis of the lipase revealed a potential C-terminal targeting sequence recognized by the ATP-binding cassette (ABC) transporter. The genetic loci around the lipase gene were searched, and a secretory gene was identified. Nucleotide sequencing of an 8.5-kb DNA fragment revealed three components of the ABC transporter, tliD, tliE, and tliF, upstream of the lipase gene, tliA. In addition, genes encoding a protease and a protease inhibitor were located upstream of tliDEF. tliDEF showed high similarity to ABC transporters of Pseudomonas aeruginosa alkaline protease, Erwinia chrysanthemi protease, Serratia marcescens lipase, and Pseudomonas fluorescens CY091 protease. tliDEF and the lipase structural gene in a single operon were sufficient for E. coli cells to secrete the lipase. In addition, E. coli harboring the lipase gene secreted the lipase by complementation of tliDEF in a different plasmid. The ABC transporter of P. fluorescens was optimally functional at 20 and 25 degrees C, while the ABC transporter, aprD, aprE, and aprF, of P. aeruginosa secreted the lipase irrespective of temperature between 20 and 37 degrees C. These results demonstrated that the lipase is secreted by the P. fluorescens SIK W1 ABC transporter, which is organized as an operon with tliA, and that its secretory function is temperature dependent.
Title: Cloning and expression of the gene encoding phospholipase A1 from Serratia sp. MK1 in Escherichia coli Song JK, Kim MK, Rhee JS Ref: J Biotechnol, 72:103, 1999 : PubMed
The gene encoding extracellular phospholipase A1 of Serratia sp. MK1 was cloned from a genomic DNA library. Formation of transparent halos on the PCY agar plates was used to identify E. coli carrying the phospholipase A1 gene. A 4.2 kb EcoRI fragment was isolated and sequenced. From nucleotide sequences and expression of various plasmids, two open reading frames (plaA and plaS) involved in efficient expression of phospholipase A1 in natural and recombinant host were identified. Extracellular phospholipase A1 activity was identified as the gene product of plaA encoding 321 amino acids with a predicted MW of 33,400. Analysis of the amino acid sequence revealed significant homology (around 70%) to phospholipase A1 of Serratia liquefaciens and Yersinia enterocolitica. The sequence, -Gly-X1-Ser-X2-Gly-, known as a lipase-specific consensus sequence was also found in the bacterial phospholipase A1. PlaS encoding a protein of 224 amino acids showed no enzymatic activity, but might be necessary for the efficient expression of phospholipase A1 in E. coli. To further improve the production of phospholipase A1 as a soluble and active form in E. coli, the effect of some parameters was examined. Surprisingly, a higher yield of soluble and active phospholipase A1 could be obtained under the combined conditions of a lower temperature, an enriched medium, and a lower-strength promoter.
Title: Cloning and nucleotide sequence of thermostable lipase gene from Pseudomonas fluorescens SIK W1 Chung GH, Lee YP, Jeohn GH, Yoo OJ, Rhee JS Ref: Agric and Biological Chemistry, 55:2359, 1991 : PubMed
A gene coding for a thermostable lipase of Pseudomonas fluorescens SIK W1 was cloned into Escherichia coli JM83 by inserting Sau3AI-generated DNA fragments into the BamHI site of pUC19. Twenty colonies with esterase activity on the tributyrin agar plate were isolated by screening the constructed Pseudomonas fluorescens genomic library. Only one out of the esterase positive 20 colonies had lipase activity on the agar plate containing olive oil and Rhodamine-B. The complete nucleotide sequence of the lipase gene was identified. The lipase gene consists of an open reading frame, 1347bp long, commencing with an ATG start codon encoding a polypeptide of 449 amino acid residues and a TGA stop codon. Comparison of this lipase amino acid sequence with those from another organisms sequenced to data showed the presence of the short homologous region Gly-X-Ser-X-Gly.
Title: Cloning and nucleotide sequence of an esterase gene from Pseudomonas fluorescens and expression of the gene in Escherichia coli Choi KD, Jeohn GH, Rhee JS, Yoo OJ Ref: Agric and Biological Chemistry, 54:2039, 1990 : PubMed
A gene coding for a novel esterase of Pseudomonas fluorescens was cloned in this study. DNA sequencing showed that the open reading frame is comprised of 708 nucleotides. The coding sequence of the gene is preceded by a potential Shine-Dalgarno sequence and by a promoter-like structure. Following the stop codon a structure reminiscent of the E. coli rho-independent terminator is present. The enzyme expressed in an E. coli clone was mostly in the periplasmic space, released to the outside of the cell by osmotic shock and purified to homogeneity by QAE-Sephadex A-50 and DEAE-Sepharose columns. The native form of the enzyme consisted of two identical subunits, each with a molecular weight of 27,000. By studying the properties and substrate specificity, the enzyme was classified as an arylesterase (EC 3.1.1.2).
