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Acanthopanax sessiliflorus (Araliaceae) have been reported to exhibit many pharmacological activities. Our preliminary study suggested that A. sessiliflorus fruits include many bioactive 3,4-seco-triterpenoids. A. sessiliflorus fruits were extracted in aqueous EtOH and fractionated into EtOAc, n-BuOH, and H2O fractions. Repeated column chromatographies for the organic fractions led to the isolation of 3,4-seco-triterpenoid glycosides, including new compounds. Ultra-high-performance liquid chromatography (UPLC) mass spectrometry (MS) systems were used for quantitation and quantification. BV2 and RAW264.7 cells were induced by LPS, and the levels of pro-inflammatory cytokines and mediators and their underlying mechanisms were measured by ELISA and Western blotting. NMR, IR, and HR-MS analyses revealed the chemical structures of the nine noble 3,4-seco-triterpenoid glycosides, acanthosessilioside G–O, and two known ones. The amounts of the compounds were 0.01–2.806 mg/g, respectively. Acanthosessilioside K, L, and M were the most effective in inhibiting NO, PGE2, TNF-α, IL-1β, and IL-6 production and reducing iNOS and COX-2 expression. In addition, it had inhibitory effects on the LPS-induced p38 and ERK MAPK phosphorylation in both BV2 and RAW264.7 cells. Nine noble 3,4-seco-triterpenoid glycosides were isolated from A. sessiliflorus fruits, and acanthosessilioside K, L, and M showed high anti-inflammatory and anti-neuroinflammatory effects.

Repeated column chromatography using Sephadex LH-20, silica gel (SiO2), and octadecyl SiO2 (ODS) as well as preparative HPLC column chromatography led to isolation of a new dibenzocyclooctadiene lignan along with four known ones, gomisin L2 (1), L1 (2), M1 (3), and M2 (4). Their chemical structures were fixed based on MS, IR, and NMR data analyses. In addition, the stereochemistry of atropisomers, the absolute configuration of the axial chirality in a biphenyl structure, was confirmed by a CD experiment. The new lignan was named gomisin M3 (5).

Korean ginseng has been widely used in Eastern medicine for thousands of years. The contents of the compounds in ginseng roots change depending on the amount of steaming and drying, and the drying method used. Black ginseng (BG) is the Korean ginseng processed by repeated steaming and drying. In this study, 5-year-old fresh Korean ginseng roots were steamed and dried 3 or 5 times, and we investigated how many cycles of steaming and drying are preferable for antivirulence activities against methicillin-resistant Staphylococcus aureus (MRSA). As a result, the antivirulence activities was increased by the treatment of BG that was steamed and dried three times, and the effect was further increased by five-time processed BG. Moreover, an ELISA showed that the TNF-α production of RAW264.7 cells stimulated by MRSA supernatants was inhibited by subinhibitory concentrations of BG extract. The expression of Hla, staphylococcal enterotoxin A (SEA), and staphylococcal enterotoxin B (SEB), an important virulence factor in the pathogenicity of MRSA, was found to decrease when bacterial cells were treated with BG extract. The antivirulence activities of BG were not simply due to pathogen growth inhibition, the BG extract was shown to decrease agrA, hla, sea, and seb expression in MRSA. Therefore, BG strongly reduces the secretion of the virulence factors produced by Staphylococcus aureus, suggesting that a BG-based structure may be used for the development of drugs aimed at staphylococcal virulence-related exoproteins. This study suggests that BG could be used as a promising natural compound in the food and pharmaceutical industry.

An amendment to this paper has been published and can be accessed via the original article.

Immune checkpoint proteins including programmed cell death protein 1 (PD-1), its ligand PD-L1 and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) are involved in proliferation, angiogenesis, metastasis, chemoresistance via immune escape and immune tolerance by disturbing cytotoxic T cell activation. Though many clinical trials have been completed in several cancers by using immune checkpoint inhibitors alone or in combination with other agents to date, recently multi-target therapy is considered more attractive than monotherapy, since immune checkpoint proteins work with other components such as surrounding blood vessels, dendritic cells, fibroblasts, macrophages, platelets and extracellular matrix within tumor microenvironment. Thus, in the current review, we look back on research history of immune checkpoint proteins and discuss their associations with platelets or tumor cell induced platelet aggregation (TCIPA) and FOXP3+ regulatory T cells (Tregs) related molecules involved in immune evasion and tumor progression, clinical implications of completed trial results and signaling networks by phytochemicals for combination therapy with immune checkpoint inhibitors and suggest future research perspectives.

Non-alcoholic fatty liver disease (NAFLD) is a disease associated with metabolic syndromes such as diabetes and obesity, regardless of alcohol consumption, and refers to the accumulation of triacylglycerols in the liver. Thymol (THY) is a vegetable essential oil that is naturally contained in the Zingiberaceae and Lamiaceae families. THY was isolated from Curcuma longa L. The rhizomes of Curcuma longa L. were dried, sliced and extracted with 50% ethanol and then isolated through repeated column chromatography. This study was conducted to investigate the inhibitory effect of THY, even in non-alcoholic fatty liver disease, in relation to the inhibiting hyperlipidemia effect of THY, which was demonstrated in previous studies. Hepatocytes were treated with oleate (OA) containing THY to observe lipid accumulation by Oil Red O staining (ORO). We also tested the effect of THY on triacylglycerols (TG) and total cholesterol (TC) in HepG2 cells. Western blot and real-time RT-PCR using sterol regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), CCAAT-enhancer-binding protein (C/EBP), proliferator-activated receptor &gamma, (PPAR&gamma, ), and adenosine monophosphate (AMP)-activated protein kinase (AMPK) expressions were carried out. Consequently, inhibition of lipogenesis by THY (100 &mu, M or 200 &mu, M) in NAFLD treated with OA in HepG2 cells was confirmed. The results of TG and TC experiments confirmed a decrease in the degree of fat accumulation in the liver. Furthermore, inhibition of the SREBP-1c, FAS, ACC, C/EBP and PPAR&gamma, expressions that mediated fat accumulation and increased AMPK phosphorylation was observed. Taken together, THY is proposed as a potential natural constituent for the treatment of NAFLD.

Repeated chromatographic separations of the EtOAc fraction of Schisandra chinensis fruits on silica gel, octadecyl silica gel, and Sephadex LH-20 led to the isolation and identification of seven dibenzocyclooctadiene lignans (1–7). The NMR data reported in the literature for angeloyl gomisin H (5) were shown to be incorrect. We unambiguously identified the compounds based on detailed analysis of the 1D and 2D NMR data, especially from HMBC and NOESY experiments. In addition, MTT assays and cell viability experiments verified the cytotoxicity of the isolated dibenzocyclooctadiene lignans against the human cancer cell lines AGS, HeLa, and HT-29.

Rock bream iridovirus (RBIV) is a notorious agent that causes high mortality in aquaculture of rock bream (Oplegnathus fasciatus). Despite severity of this virus, no transcriptomic studies on RBIV-infected rock bream that can provide fundamental information on protective mechanism against the virus have been reported so far. This study aimed to investigate physiological mechanisms between host and RBIV through transcriptomic changes in the spleen based on RNA-seq. Depending on infection intensity and sampling time point, fish were divided into five groups: uninfected healthy fish at week 0 as control (0C), heavy infected fish at week 0 (0H), heavy mixed RBIV and bacterial infected fish at week 0 (0MH), uninfected healthy fish at week 3 (3C), and light infected fish at week 3 (3L). We explored clusters from 35,861 genes with Fragments Per Kilo-base of exon per Million mapped fragments (FPKM) values of 0.01 or more through signed co-expression network analysis using WGCNA package. Nine of 22 modules were highly correlated with viral infection (|gene significance (GS) vs. module membership (MM) |&gt, 0.5, p-value &lt, 0.05). Expression patterns in selected modules were divided into two: heavy infected (0H and 0MH) and control and light-infected groups (0C, 3C, and 3L). In functional analysis, genes in two positive modules (5448 unigenes) were enriched in cell cycle, DNA replication, transcription, and translation, and increased glycolysis activity. Seven negative modules (3517 unigenes) built in this study showed significant decreases in the expression of genes in lymphocyte-mediated immune system, antigen presentation, and platelet activation, whereas there was significant increased expression of endogenous apoptosis-related genes. These changes lead to RBIV proliferation and failure of host defense, and suggests the importance of blood cells such as thrombocytes and B cells in rock bream in RBIV infection. Interestingly, a hub gene, pre-mRNA processing factor 19 (PRPF19) showing high connectivity (kME), and expression of this gene using qRT-PCR was increased in rock bream blood cells shortly after RBIV was added. It might be a potential biomarker for diagnosis and vaccine studies in rock bream against RBIV. This transcriptome approach and our findings provide new insight into the understanding of global rock bream-RBIV interactions including immune and pathogenesis mechanisms.

Outbreaks of rock bream iridovirus (RBIV) infection in rock bream (Oplegnathus fasciatus) were continuously recorded, and these infections lead to vast economic losses in the aquaculture industry. However, metabolomic, proteomic, and transcriptomic studies in rock bream infected with RBIV are limited. In this study, metabolic responses in tissues of rock bream with different infection degrees of control (0C) and heavy-infected fish (0H) at 0 week, and fish with low viral titer at 3 week (3 L)-were observed by high-resolution magic-angle spinning (HR-MAS) NMR spectroscopy-based metabolomics, with multivariate, and pathway-based analyses being used to aid interpretation. In the liver, significant metabolic pathways related to energy metabolism such as glutamine and glutamate metabolism and the TCA cycle were observed only in 0H. Additionally, 0C and 3 L were clustered in the OPLS-DA (orthogonal partial least squares discriminant analysis) score plot, and 3 L showed no significant pathways compared to 0C. Thus, it is assumed that lightly infected fish (3 L) were survived from heavy-infection after 3 weeks. In the spleen, the metabolites relevant to energy production and immune response including pyruvate metabolism, the TCA cycle and branched-chain amino acid (BCAA) metabolism were changed in 0H. These results suggest that RBIV induced rapid and high energy production in the spleen by promoting both the Warburg effect, and the TCA cycle for their replication. In the kidney, elevation of the kynurenine pathway, which is related to the immunological response, and decreased osmolytes such as trimethylamine N-oxide and betaine were observed in 0H. We found that RBIV induced various disturbances in metabolism in host cells through viral replication and protective responses in the host. This study is the first metabolomics study of rock bream infected in RBIV using HR-MAS NMR spectroscopy, providing insights into how cellular metabolism is affected during RBIV infection. These results can be used as a resource to evaluate the mechanism of host-pathogen interactions.

The stress response mechanisms of Bisphenol A (BPA), an endocrine disrupting compound, remain to be elucidated. In this study, we explored the effects of BPA on the non-biting midge Chironomus riparius through basic ecotoxicity assays, DNA damage (comet assay), eco-epigenetics (global DNA and histone methylations) and non-targeted global metabolomics (NMR based) approaches. The reproduction failure, increase in DNA damage, global DNA hyper-methylation, and increased global histone modification (H3K36) status were evident due to BPA exposure at 10% lethal concentration (LC10: 1 mg/L, based on 48 h acute toxicity). Moreover, non-targeted global metabolomics followed by pathway analysis identified alterations of energy metabolism, amino acids, and methionine metabolisms etc. Most importantly, we found a potential cross-talk between altered epigenetics and metabolites, such as, increase in methionine and o-phosphocholine metabolites corresponds with the phenomena of global hyper-methylation in DNA and H3K36 mark. Overall, our results suggests that the crosstalk of global metabolomics and epigenetic modification was fundamental of the underlying mechanisms in BPA-induced stress response in C. riparius.

The present study aimed to investigate the mechanisms involved in amorphous silica nanoparticles (aSiNPs)-mediated hepatotoxicity through the evaluation of changes in global metabolomics in in vitro and in vivo systems. 1H NMR-based non-targeted global metabolomics and biochemical approaches were conducted in an aSiNPs-treated human hepatoma cell line (HepG2) and in ICR mice liver. The non-targeted NMR-based metabolomic analysis, followed by pathway analysis, revealed the perturbation of glutathione metabolism and the depletion of the glutathione pool after aSiNPs treatment in both in vitro (HepG2 cells) and in vivo systems. The total glutathione level, glutathione-S-transferase enzyme activity, and antioxidant gene expression strongly corroborated the metabolomic analysis results. The in vitro results were further supported by the in vivo data, specifically for metabolites profiling (Pearson Correlation coefficient is 0.462 (p = 0.026)). Furthermore, the depletion of glutathione, the formation of NADPH oxidase-mediated reactive oxygen species, and oxidative stress were evident in aSiNPs-treated HepG2 cells. Overall, the suppression of glutathione metabolism and oxidative stress are among the principal causes of aSiNPs-mediated hepatotoxicity.

Titanium dioxide nanoparticles (TiO2NPs) are widely used nanoparticles, whose catalytic activity is mainly due to photoactivation. In this study, the toxicity of TiO2NPs was investigated on the nematode Caenorhabditis elegans, with and without UV activation. Comparative analyses across the four treatments revealed that UV-activated TiO2NPs led to significant reproductive toxicity through oxidative stress. To understand the underlying molecular mechanism, transcriptomics and metabolomics analyses were conducted, followed by whole-genome network-based pathway analyses. Differential expression analysis from microarray data revealed only 4 DEGs by exposure to TiO2NPs alone, compared to 3,625 and 3,286 DEGs by UV alone and UV-activated TiO2NPs, respectively. Pathway analyses suggested the possible involvement of the JAK/STAT and TGF-ß pathways in the phototoxicity of TiO2NPs, which correlated with the observation of increased gene expression of those pathways. Comparative analysis of C. elegans response across UV activation and TiO2NPs exposure was performed using loss-of-function mutants of genes in these pathways. Results indicated that the JAK/STAT pathway was specific to TiO2NPs, whereas the TGF-ß pathway was specific to UV. Interestingly, crosstalk between these pathways was confirmed by further mutant analysis. We consider that these findings will contribute to understand the molecular mechanisms of toxicity of TiO2NPs in the natural environment.

Microcystins are one of the most frequently found hepatotoxic cyclic peptides in fresh- and brackish-water. Zebrafish (Danio rerio) were exposed to 20 ppb of microcystin-LR (MCLR) for 72 h in order to investigate the metabolic responses. Proton nuclear magnetic resonance (1H-NMR) spectroscopy was used to analyze the metabolic profiles. As a result, male and female zebrafish showed different patterns of metabolic perturbation in relation to their detoxification profiles. Glutathione was reduced for the MCLR excretion, and fumarate and creatine were increased for the energy demands required for detoxification in the male zebrafish. The correlation of metabolites in the metabolic pathway network was changed due to MCLR exposure. The correlations connected with myo-inositol, betaine, and NADPH considerably altered in the MCLR-exposed group.

Mesoporous carbon nanoparticles (MCNs) have been applied in a variety of drug/gene carriers. In addition to their potential benefits, many studies of their potential toxicity have been reported, showing the limitations of metabolic contextualization. In this study, we conducted 1H-nuclear magnetic resonance (NMR) profiling combined with statistical methods such as orthogonal partial least squares discriminant analysis and Pearson correlation analysis to assess metabolic alterations in the whole body of zebrafish (Danio rerio) in the presence of various concentrations of MCNs. The MCN exposure influenced numerous metabolites in energy metabolism (e.g., metabolites involved in glycolysis and tricarboxylic acid cycle) and disturbed the balance of neurotransmitters and osmoregulators. Our findings demonstrate the potential applicability of using a metabolomics approach to determine underlying metabolic disturbances caused by MCNs.

Proton nuclear magnetic resonance (1H-NMR) spectroscopy was used to study the response of zebrafish (Danio rerio) to increasing concentrations of bisphenol A (4,4′-(propane-2,2-diyl)diphenol, BPA). Orthogonal partial least squares discriminant analysis (OPLS-DA) was applied to detect aberrant metabolomic profiles after 72 h of BPA exposure at all levels tested (0.01, 0.1, and 1.0 mg/L). The OPLS-DA score plots showed that BPA exposure caused significant alterations in the metabolome. The metabolomic changes in response to BPA exposure generally exhibited nonlinear patterns, with the exception of reduced levels of several metabolites, including glutamine, inosine, lactate, and succinate. As the level of BPA exposure increased, individual metabolite patterns indicated that the zebrafish metabolome was subjected to severe oxidative stress. Interestingly, ATP levels increased significantly at all levels of BPA exposure. In the present study, we demonstrated the applicability of 1H-NMR-based metabolomi...

Alzheimer’s disease (AD) is characterized by cognitive impairment, progressive neurodegeneration, and Aβ accumulation. Aβ oligomers can lead to synaptic damage via alterations in glutamate receptors and excitotoxicity, as well as mitochondrial dysfunction. AD is associated with various biological indicators, including (1) predisposing factors such as genetic risk factors, (2) laboratory markers such as Aβ and tau protein, and (3) diagnostic markers such as MRI and PET findings. However, these markers are not confirmed, invasive, or expensive. In the present study, we employed nuclear magnetic resonance (NMR) methods that are inexpensive, time-efficient, and can be performed using samples obtained from various easily accessible sources such as cerebrospinal fluid, plasma, and peripheral tissue, thus highlighting the clinical utility of this approach. NMR analyses of blood metabolites showed that glutamine, glutamate, leucine, oxaloacetate, aspartate, isoleucine, and 3-hydroxyisovalerate are increased in patients with AD compared with control individuals. These metabolites seem to be related to mitochondrial dysfunction. Our data indicated that 3-hydroxyisovalerate, which is linked to known pathologic processes associated with mitochondrial dysfunction and accelerated neurodegeneration, was increased in the blood samples of patients with AD.

Long interspersed nuclear elements (LINEs) are widely distributed in the vertebrate genome, and can be either beneficial or detrimental to the host genome. Here we identified three members of LINE RT elements in the olive flounder genome. They showed high amino acid sequence identity (89–99 %), and the sequences of LINE reverse transcriptase (RT) in olive flounder are closely related to those of coral grouper, European seabass, and three-spined stickleback. Real-time reverse transcription-polymerase chain reaction analysis indicated that expression of the OF (Olive flounder)-LINE Chr3-1 RT increased more in spleen than in other tissues after treatment with the pathogen Streptococcus parauberis. These data may form the basis for further studies on the function of retroelements in infected olive flounder.

Various experiments using zebrafish have been highlighted recently in the scientific community. Because it is possible to conduct practical experiment from various neurological research to area of genetic study or toxicity experiment. However, gender difference effects are nearly not considered. If the gender differences of zebrafish are considered it is possible to obtain more accurate data. In this study, zebrafish which have different genders were compared each other with NMR-based metabolomics. The extracts of male and female zebrafish were measured by 600 MHz NMR spectrometer. Statistical analysis and target profiling were conducted. As a result, muscle related metabolites were observed in male zebrafish and nerve related metabolites were observed in female zebrafish.

Heavy metals are common marine and soil pollutants that are mainly the result of industrial activity, and are a threat to the environment and human health. In this study, 1H nuclear magnetic resonance (NMR)-based metabolomics was applied to adult Danio rerio to monitor the metabolic change as a response to ZnCl2 and CdCl2 exposure at different concentrations for 72 h. NMR spectroscopy was used to identify and quantify the metabolites extracted from D. rerio. The metabolite profiles of the control and heavy metal exposed group were classified by partial least squares – discriminant analysis (PLS-DA) analysis, and potential contaminant-specific biomarkers were suggested. For the ZnCl2-exposed zebrafish, the levels of ATP, aspartate and NAD+ were increased, whereas the levels of formate, inosine, hypoxanthine and succinate decreased. In addition, the CdCl2-exposed zebrafish showed an increase in the levels of ATP and formate and a decrease in the levels of glutamate, inosine and glutathione. Overall,...

Vibrio harveyi is one of the pathogens that threaten the shrimp farming industry. However, metabolic changes induced by V. harveyi infection in shrimp remain unknown. In this study, we first conducted high resolution-magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR)-based metabolomics studies on gill, hepatopancreas, and haemolymph of V. harveyi-infected white leg shrimp, Litopenaeus vannamei. Using multivariate statistical analysis, we observed a clear separation between the early (3 and 9 h post-injection (hpi)) and late phases (24, 72 and 144 hpi) of the infection in all tissues. Moreover, metabolic changes in response to V. harveyi infection were faster in the haemolymph in the early phase and significantly changed in the late phase of the infection in the gills. Extensive changes were observed in the hepatopancreas, with 24 hpi being the turning point of progression from early to late phase infection in the hepatopancreas. V. harveyi infection increased the energy demand in L. vannamei and the amino acid and carbohydrate metabolism pathways also exhibited significant changes depending on the tissue. Thus, each tissue displayed different metabolic changes, depending on the progress of the infection.

Olive flounder (Paralichthys olivaceus) is one of economically valuable fish species in the East Asia. In comparison with its economic importance, available genomic information of the olive flounder is very limited. The mass mortality caused by variety of pathogens (virus, bacteria and parasites) is main problem in aquaculture industry, including in olive flounder culture. In this study, we carried out transcriptome analysis using the olive flounder gill tissues after infection of three types of pathogens (Virus; Viral hemorrhagic septicemia virus, Bacteria; Streptococcus parauberis, and Parasite; Miamiensis avidus), respectively. As a result, we identified total 12,415 differentially expressed genes (DEG) from viral infection, 1,754 from bacterial infection, and 795 from parasite infection, respectively. To investigate the effects of pathogenic infection on immune response, we analyzed Gene ontology (GO) enrichment analysis with DEGs and sorted immune-related GO terms per three pathogen groups. Especially, we verified various GO terms, and genes in these terms showed down-regulated expression pattern. In addition, we identified 67 common genes (10 up-regulated and 57 down-regulated) present in three pathogen infection groups. Our goals are to provide plenty of genomic knowledge about olive flounder transcripts for further research and report genes, which were changed in their expression after specific pathogen infection.

Studies are underway to gather information about the mode of action (MOA) of emerging pollutants that could guide practical environmental decision making. Previously, we showed that propranolol, an active pharmaceutical ingredient, had adverse effects on Daphnia magna that were similar to its pharmaceutical action. In order to characterize the mode of action of propranolol in D. magna, which is suspected to be organ-specific pharmaceutical action or baseline toxicity, we performed time-series monitoring of behavior along with heart rate measurements and nuclear magnetic resonance (NMR) based metabolite profiling. Principle component analysis (PCA) and hierarchical clustering were used to categorize the mode of action of propranolol among 5 chemicals with different modes of action. The findings showed that the mode of action of propranolol in D. magna is organ-specific and vastly different from those of narcotics, even though metabolite regulation is similar between narcotic and non-narcotic candidates. The method applied in this study seems applicable to rapid characterization of the MOA of other cardiovascular pharmaceutical ingredients.

It is well known that fucoidan, a natural sulfated polysaccharide present in various brown algae, mediates anticancer effects through the induction of cell cycle arrest and apoptosis. Nevertheless, the role of tumor suppressor p53 in the mechanism action of fucoidan remains unclear. Here, we investigated the anticancer effect of fucoidan on two p53 isogenic HCT116 (p53+/+ and p53-/-) cell lines. Our results showed that inhibition of cell viability, induction of apoptosis and DNA damage by treatment with fucoidan were similar in two cell lines. Flow cytometric analysis revealed that fucoidan resulted in G1 arrest in the cell cycle progression, which correlated with the inhibition of phosphorylation of retinoblastoma protein (pRB) and concomitant association of pRB with the transcription factor E2Fs. Furthermore, treatment with fucoidan obviously upregulated the expression of cyclin-dependent kinase (CDK) inhibitors, such as p21WAF1/CIP1 and p27KIP1, which was paralleled by an enhanced binding with CDK2 and CDK4. These events also commonly occurred in both cell lines, suggesting that fucoidan triggered G1 arrest and apoptosis in HCT116 cells by a p53-independent mechanism. Thus, given that most tumors exhibit functional p53 inactivation, fucoidan could be a possible therapeutic option for cancer treatment regardless of the p53 status.

Polycyclic aromatic hydrocarbons (PAHs) are widespread contaminants resulting from the incomplete combustion of organic materials in the environment. The primary concern for the hazardous effect of PAHs is their ability to activate the pathway linked to the aryl hydrocarbon receptor (AhR) and lead to carcinogenesis. While previous research has demonstrated that oxidative stress plays a key role in the AhR-dependent toxic response, the effect of PAHs on the biosynthesis of glutathione (GSH), which is a powerful endogenous antioxidant, has not been extensively investigated. In the present study, we utilized a global metabolomic approach, via high resolution magic angle spinning nuclear magnetic resonance spectroscopy, and identified significant metabolome differences between non-tumorigenic liver cells (BNL CL.2; CL2) and transformed liver cells (BNL 1MEA. 7R.1; 1MEA) chronically exposed to 3-methylcholanthrene (3MC), a well‑known carcinogenic PAH. A significant change that was observed, was a lower GSH level in 1MEA cells compared with that in CL2 cells. This was contrasted by increased levels of precursor metabolites for GSH synthesis, such as S-adenosylmethionine and cysteine. These changes were accompanied by a significantly reduced expression of γ-glutamylcysteine ligase (GCL), known to be the rate‑limiting step of GSH synthesis. Furthermore, the protein level of cysteine dioxygenase was downregulated; however, the concentration of taurine was unaltered. Therefore, the present study demonstrated that cells transformed by chronic exposure to 3MC exhibited inhibition of GSH biosynthesis by suppression of GCL protein expression and reduction of cysteine availability, which may subsequently render cells vulnerable to oxidative stress.

Continuous increases in water temperature disturb homeostasis and increase oxidative stress in fish. Glutathione (GSH) is an intracellular antioxidant that helps to relieve stress in animals. In this study, we observed the effect of GSH on olive flounder exposed to high temperature using serum parameters and NMR-based metabolomics. Based on the results from the first experiment, 20 mg of GSH was chosen as an effective dose with lower infection rates and mortality. Then, fish were divided into Control, Temp (PS injection), and GSH (glutathione injection) groups, and fish in Temp and GSH groups were exposed to temperature fluctuations (20 &deg, C&rarr, 24 &deg, 27 &deg, C). In OPLS-DA score plots, Temp group was clearly distinguished from the other groups in the kidney. In the liver, the metabolic patterns of GSH group were close to the Temp group on day 4 and became similar to Control group from day 7. Serum parameters did not change significantly, but the deviation in Temp group was greater than that in GSH group. Metabolite levels that were significantly altered included GSH, lactate, O-phosphocholine, and betaine in the kidney and taurine, glucose, and several amino acids in the liver, which were related to antioxidant activity and energy system. Therefore, GSH supplements could relieve thermal stress influencing metabolic mechanisms in fish.

Department of Forensic Chemistry, National Forensic Service Southern District Office, Yangsan-si 626-742, KoreaReceived March 8, 2013, Accepted May 20, 2013Forensic and legal medicine require reliable data to indicate excessive alcohol consumption. Ethanol isoxidatively metabolized to acetate by alcohol dehydrogenase and non-oxidatively metabolized to ethylglucuronide (EtG), ethyl sulfate (EtS), phosphatidylethanol, or fatty acid ethyl esters (FAEE). Oxidativemetabolism is too rapid to provide biomarkers for the detection of ethanol ingestion. However, the non-oxidative metabolite EtG is a useful biomarker because it is stable, non-volatile, water soluble, highly sensitive,and is detected in body fluid, hair, and tissues. EtG analysis methods such as mass spectroscopy, chromato-graphy, or enzyme-linked immunosorbent assay techniques are currently in use. We suggest that nuclearmagnetic resonance (NMR) spectroscopy could be used to monitor ethanol intake. As with currentconventional methods, NMR spectroscopy doesn’t require complicated pretreatments or sample separation.This method has the advantages of short acquisition time, simple sample preparation, reproducibility, andaccuracy. In addition, all proton-containing compounds can be detected. In this study, we performed

Metabolomics is the study which detects the changes of metabolites level. Metabolomics is a terminal view of the biological system. The end products of the metabolism, metabolites, reflect the responses to external environment. Therefore metabolomics gives the additional information about understanding the metabolic pathways. These metabolites can be used as biomarkers that indicate the disease or external stresses such as exposure to toxicant. Many kinds of biological samples are used in metabolomics, for example, cell, tissue, and bio fluids. NMR spectroscopy is one of the tools of metabolomics. NMR data are analyzed by multivariate statistical analysis and target profiling technique. Recently, NMR-based metabolomics is a growing field in various studies such as disease diagnosis, forensic science, and toxicity assessment.

The putative gene cluster involved in the degradation of the raffinose family oligosaccharides (RFO) was identified in Caldicellulosiruptor bescii. Within the cluster, the gene encoding a putative α-galactosidase (CbAga36) was cloned and expressed in Escherichia coli. Size exclusion chromatography of the purified rCbAga36 indicated that the native form was a tetramer. Its primary sequence was similar to the family of glycoside hydrolase 36. The purified recombinant CbAga36 (rCbAga36) was optimally active at pH 5.0 and 70°C and had a half-life of 15 h and 10 h at 70°C and 80°C, respectively. rCbAga36 showed high activity with the artificial substrate (p-nitrophenyl α-d-galactopyranoside, pNPαGal) exhibiting lower Km and higher kcat than natural substrates such as melibiose and raffinose. Although rCbAga36 demonstrated preferential activity toward the hydrolysis of RFO such as raffinose and stachyose, it did not degrade the polymeric galactomannans. Our results imply that CbAga36 may play a role in the degradation of RFO, transported into the cytoplasm via a transporter into galactose, which is further utilized as an energy source in C. bescii. Furthermore, its ability to synthesize novel oligosaccharides by transglycosylation renders this enzyme potentially useful for the production of dietary oligosaccharides with novel function.