Il-Chan Kim

Heat shock protein (Hsp) gene responses of the intertidal copepod Tigriopus japonicus to environmental toxicants.

The induction of heat shock proteins (Hsps) is considered as an important protective, ecophysiologically adaptive, and genetically conserved response to environmental stress in all organisms. Among the Hsps, the heat shock protein 70 (Hsp70) family members are most extensively studied for their characterization and induction in response to environmental stressors in a range of species. We studied expression of ten Hsp transcripts in response to heat treatment in an intertidal marine copepod Tigriopus japonicus and observed that expression of Hsp70 was more pronounced than other Hsps. Subsequently, cDNA and genomic sequences of T. japonicus Hsp70 (TJ-Hsp70) were worked out by molecular cloning techniques and phylogenetic relationship was analyzed. The bacterial expression of TJ-Hsp70 and its expression in response to metal and endocrine-disrupting chemical (EDC) exposures were also studied. The TJ-Hsp70 transformed bacteria showed increased thermotolerance compared to bacteria with vector only. All the trace metals (i.e. copper, silver, and zinc) caused a concentration-dependent increase in the expression of Hsp70 transcripts. Effect of EDCs on Hsp70 expression was differential. While 4-nonylphenol (NP) and 4-t-octylpheno (OP) caused downregulation, bisphenol A (BPA) caused upregulation. The promoter region of the genomic Hsp70 sequence contained putative xenobiotic response elements (XREs) indicating that TJ-Hsp70 regulation not only by temperature but also by xenobiotics. These findings suggest that in T. japonicus, Hsp70 has a conserved role of thermotolerance and its expression in response to xenobiotics exposure appears to be a protective response.

Gene expression profiling of copper-induced responses in the intertidal copepod Tigriopus japonicus using a 6K oligochip microarray

The intertidal copepod Tigriopus japonicus has shown promising results in classical acute and chronic toxicity studies. Recently, a large number of genes have been identified from this species and their mRNA expression has been studied independently against exposure to marine environmental pollutants. T. japonicus is a promising organism for the study of mechanistic aspects of marine environmental pollutants using genomics. In this study, a 6K oligochip for T. japonicus that included mostly unique sets of genes from approximately 26K ESTs, was developed. A total of 5463 spots (2313 mRNAs upregulated and 3150 downregulated) were identified to be significantly expressed on microarray by hierarchical clustering of genes after exposure to copper for different time durations (10 microg/L for 6, 12 and 24h). However, mRNAs of only 138 and 375 genes were observed to be consistently upregulated and downregulated, respectively, at all time points. Most of the changes of mRNA expression were observed at the short exposure of 6h. It was observed that mRNA expression of several genes involved in growth, metabolism, reproduction and hormonal regulation was modulated in Cu-exposed T. japonicus. mRNA expression of genes involved in detoxification and antioxidant functions was also modulated. This indicates that Cu-induced gene transcription is complicated in T. japonicus similar to other crustaceans. Cu specifically upregulated mRNAs of genes of some isoforms of cytochrome P450 (CYP). On the other hand, a majority of downregulated mRNAs were of genes encoding for proteins important for growth and development. The expression profile of mRNAs of selected genes was verified by the quantitative real time RT-PCR. The mRNA expression profiles provide insight into the mechanism of action of copper in T. japonicus. These results demonstrate the suitability of a T. japonicus oligochip microarray for risk assessment of trace metals in the marine environment. As yet, major breakthroughs in invertebrate toxicogenomics have mainly been in Daphnia and Drosophila. Daphnias use is limited to freshwater ecotoxicogenomics. Here we propose an oligochip microarray-based approach for risk assessment of trace metals in a potential model marine test species.

Cloning of a river pufferfish (Takifugu obscurus) metallothionein cDNA and study of its induction profile in cadmium-exposed fish.

We report here the full-length cDNA sequence of metallothionein (MT) gene from an anadromous river pufferfish, Takifugu obscurus (order: Tetradotiformes; family: Tetradontidae). Phylogenetic relationship analysis revealed that the identified MT has high sequence similarity with many Perciformes fish species. The tissue distribution and concentration- and time-dependent expression of MT mRNA were studied in fish exposed to cadmium. Liver showed the highest level of MT gene expression followed by other tissues (brain, gill and kidney) in response to cadmium exposure. Muscle showed a weak expression response of MT gene. Time-course study revealed highest early phase (at 6h) expression in the brain and late persistence of induction in the intestine. MT mRNA expression showed a concentration-dependent expression in all the tissues. However, induction in brain and liver occurred at much lower concentrations as compared to other tissues. Our results demonstrate that MT in T. obscurus is induced by cadmium exposure which indicates that it plays a functionally conserved function of metal detoxification.

Effect of copper exposure on GST activity and on the expression of four GSTs under oxidative stress condition in the monogonont rotifer, Brachionus koreanus

Glutathione S-transferases (GSTs; EC 2.5.1.18) are major enzymes that function in Phase II detoxification reactions by catalyzing the conjugation of reduced glutathione through cysteine thiol. In this study, we cloned and sequenced four GST genes from the monogonont rotifer Brachionus koreanus. The domain regions of four Bk-GSTs showed a high similarity to those of other species. In addition, to evaluate the potential of GST genes as an early warning signal for oxidative stress, we exposed sublethal concentrations of copper (Cu) to B. koreanus and measured glutathione (GSH) contents and several antioxidant enzymes such as glutathione S-transferase (GST), glutathione peroxidase (GPx; EC 1.11.1.9), and glutathione reductase (GR; EC 1.8.1.7). The reactive oxygen species (ROS) at 12h and 24h after copper exposure increased significantly. GSH contents however did not increase significantly and even it decreased at 0.24mg/L at 12h. The activities of several antioxidant enzymes, particularly GPx and GR, showed a dramatic increase in 0.24mg/L of CuCl2. Messenger RNAs of each Bk-GST showed different patterns of modulations according to GST types, and particularly, Bk-GST-omega, Bk-GST-sigma, and Bk-GST zeta genes were highly sensitive to Cu. These results indicate that Bk-GSTs, functioning as one of the enzymatic defense mechanisms particularly in the early stage of oxidative stress response, were induced by Cu exposure. This also suggests that these genes and related enzymes have a potential as biomarkers for a more sensitive initial stress response.

Differential expression of metallothionein (MT) gene by trace metals and endocrine-disrupting chemicals in the hermaphroditic mangrove killifish, Kryptolebias marmoratus

Metallothionein (MT) gene expression was studied in different tissues, development stages and gender types of the mangrove killifish (Kryptolebias marmoratus). MT expression was also studied in a time-series experiment after exposure to trace metals and endocrine-disrupting chemicals (EDCs). The brain showed the highest level of MT transcripts. Although all the development stage showed some level of MT expression, the adult hermaphrodites showed the highest expression which was significantly higher than the secondary males. In the trace metal-exposed fish, cadmium caused the strongest induction of MT. However, other trace metals such as copper and zinc also caused MT gene induction. All the EDCs suppressed the expression of MT gene, and the effect of EDCs were not gender-specific. K. marmoratus has previously shown its suitability as a model species for toxicity studies and cancer research. This study demonstrated utility of MT as biomarker in K. marmoratus. However, confounding factors such as age, gender, and tissue types appear to influence the MT expression. Response of trace and organic pollutants such as EDCs also varied greatly. These observations suggest that MT would be a specific biomarker of trace metal exposure in K. marmoratus and expression would be influenced by intrinsic factors.

The copepod Tigriopus japonicus genomic DNA information (574 Mb) and molecular anatomy

The intertidal copepod, Tigriopus japonicus, has been recognized as a promising model species for marine environmental genomics. To obtain extensive genomic DNA sequences from this species, we sequenced genomic DNA from adult copepods using genomic sequencers GS-FLX and GS-FLX-Titanium and attained 1,914,995 reads (average read length 299.8 bp) including 574.2 Mb of genomic DNA information. After subjecting them to assembly, we acquired 193,642 contigs (total contigs length 129.7 Mb), and finally were able to obtain 10,894 unigenes (E-value>0.1; length>200 bp) containing 33,081,455 bp after a nonredundant (NR) blast search. In this paper, we summarize the genomic DNA sequences of T. japonicus and discuss its potential use in environmental genomics and ecotoxicological studies for uncovering mechanisms of environmental stresses and chemical toxicities to marine crustaceans.

Gamma rays induce DNA damage and oxidative stress associated with impaired growth and reproduction in the copepod Tigriopus japonicus

Nuclear radioisotope accidents are potentially ecologically devastating due to their impact on marine organisms. To examine the effects of exposure of a marine organism to radioisotopes, we irradiated the intertidal copepod Tigriopus japonicus with several doses of gamma radiation and analyzed the effects on mortality, fecundity, and molting by assessing antioxidant enzyme activities and gene expression patterns. No mortality was observed at 96h, even in response to exposure to a high dose (800Gy) of radiation, but mortality rate was significantly increased 120h (5 days) after exposure to 600 or 800Gy gamma ray radiation. We observed a dose-dependent reduction in fecundity of ovigerous females; even the group irradiated with 50Gy showed a significant reduction in fecundity, suggesting that gamma rays are likely to have a population level effect. In addition, we observed growth retardation, particularly at the nauplius stage, in individuals after gamma irradiation. In fact, nauplii irradiated with more than 200Gy, though able to molt to copepodite stage 1, did not develop into adults. Upon gamma radiation, T. japonicus showed a dose-dependent increase in reactive oxygen species (ROS) levels, the activities of several antioxidant enzymes, and expression of double-stranded DNA break damage genes (e.g. DNA-PK, Ku70, Ku80). At a low level (sub-lethal dose) of gamma irradiation, we found dose-dependent upregulation of p53, implying cellular damage in T. japonicus in response to sub-lethal doses of gamma irradiation, suggesting that T. japonicus is not susceptible to sub-lethal doses of gamma irradiation. Additionally, antioxidant genes, phase II enzyme (e.g. GSTs), and cellular chaperone genes (e.g. Hsps) that are involved in cellular defense mechanisms also showed the same expression patterns for sublethal doses of gamma irradiation (50-200Gy). These findings indicate that sublethal doses of gamma radiation can induce oxidative stress-mediated DNA damage and increase the expression of antioxidant enzymes and proteins with chaperone-related functions, thereby significantly affecting life history parameters such as fecundity and molting in the copepod T. japonicus.

Assessment of post-stroke extrapersonal neglect using a three-dimensional immersive virtual street crossing program

Kim DY, Ku J, Chang WH, Park TH, Lim JY, Han K, Kim IY, Kim SI. Assessment of post‐stroke extrapersonal neglect using a three‐dimensional immersive virtual street crossing program
Acta Neurol Scand: 2010: 121: 171–177.
© 2009 The Authors Journal compilation

The complete mitochondrial genome of the rayfish Raja porosa (Chondrichthyes, Rajidae).

We isolated mitochondrial DNA from the rayfish Raja porosa by long-polymerase chain reaction (Long-PCR) with conserved primers, and sequenced it by primer walking method using flanking sequences as sequencing primers. R. porosa mitochondrial DNA consists of 16,972 bp and its structural organization is conserved in comparison with other fishes and mammals. Based on the mitochondrial cytochrome b (cyt b) sequence, the phylogenetic position of R. porosa among cartilaginous fishes was inferred using different phylogenetic methods (ML-based quartet puzzling, Neighbor-joining (NJ) and Bayesian approaches). In this paper, we report the characteristics of the R. porosa mitochondrial genome including structural organization, base composition of rRNAs, tRNAs and protein-encoding genes and characteristics of mitochondrial tRNAs. These findings are applicable to comparative mitogenomics of R. porosa with other related taxa.

Gonadotropin-releasing hormone receptor (GnRHR) gene expression is differently modulated in gender types of the hermaphroditic fish Kryptolebias marmoratus by endocrine disrupting chemicals

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in the regulation of reproduction in vertebrates through interaction with a specific receptor. The GnRH-stimulated gonadotropin synthesis and release are regulated by the GnRH receptors (GnRHRs). In this study, we have identified a GnRH receptor (GnRHR) gene from the hermaphroditic fish Kryptolebias marmoratus. K. marmoratus GnRHR showed typical vertebrate GnRHR domains and motifs, and its cDNA contained 1634 bp including an open reading frame (ORF) of 1263 bp encoding a putative protein of 420 amino acids. To analyze expression patterns of GnRHR gene in various tissues and developmental stages of K. marmoratus, we carried out quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). The K. marmoratus GnRHR gene expression was detected in all the tissues of adult fish with highest level in brain and gonad. The expression of K. marmoratus GnRHR mRNA increased from stage 1 (2 day post fertilization, dpf) to stage 4 (12 dpf) but steeply decreased at hatching stage (stage 5). Expression of K. marmoratus GnRHR after exposure to endocrine-disrupting chemicals such bisphenol A (BPA, 600 microg/L for 96 h) and 4-tert-octylphenol (OP, 300 microg/L for 96 h) in hermaphrodites as well as secondary males was highly up-regulated in almost all the tissues. Another EDC, 4-nonylphenol (NP, 300 microg/L for 96 h) showed no consistent response. 17beta-estrodiol (E2, 100 ng/L for 96 h), a known natural estrogen, suppressed expression of GnRHR in most of the tissues from hermaphrodites as well as secondary males. Tamoxifen (TMX, 10 microg/L), an estrogen antagonist, on the other hand, caused upregulation of GnRHR expression in the liver of hermaphrodites and the gonad and liver of secondary males. This is the first report of a GnRHR gene from K. marmoratus and modulation of its expression by EDCs. This study provides an insight into the molecular mechanism of endocrinological functions of this unique fish.