Sunghoon Kim

Antiobesity and hypolipidemic effects of lotus leaf hot water extract with taurine supplementation in rats fed a high fat diet

Lotus leaf hot water extract with taurine supplementation showed antiobesity and hypolipidemic effects in high fat diet-induced obese rats, which was more effective than lotus leaf hot water extract alone.BackgroundLotus (Nelumbo nucifera) leaf has been used to treat obesity. The purpose of this study was to investigate the antiobesity and hypolipidemic effects of lotus leaf hot water extract with taurine supplementation in high fat diet-induced obese rats.MethodsFour week-old male Sprague-Dawley rats were randomly divided into four groups with 8 rats in each group for a period of 6 weeks (normal diet, N group; high fat diet, HF group; high fat diet + lotus leaf hot water extract, HFL group; high fat diet + lotus leaf hot water extract + taurine, HFLT group). Lotus leaf hot water extract was orally administrated to HFL and HFLT groups and the same amount of distilled water was orally administered (400 mg/kg/day) to N and HF groups. Taurine was supplemented by dissolving in feed water (3% w/v).ResultsThe body weight gain and relative weights of epididymal and retroperitoneal adipose tissues were significantly lower in N, HFL and HFLT groups compared to HF group. HFL and HFLT groups showed lower concentrations of total cholesterol, triglyceride and low density lipoprotein cholesterol in serum. HFLT group showed higher the ratio of high density lipoprotein cholesterol/total cholesterol compared to HFL group. HFLT group showed better blood lipid profiles compared to HFL group.Conclusions

Antioxidant and hepatic protective effects of lotus root hot water extract with taurine supplementation in rats fed a high fat diet.

BackgroundNelumbo nucifera, known as sacred lotus, is a well-known medicinal plant and this lotus root is commonly used as food compared to different parts of this plant. This study was conducted to investigate the antioxidant and hepatic protective effects of lotus root hot water extract with taurine supplementation in high fat diet-induced obese rats.MethodsThirty-two male Sprague-Dawley rats (4-week-old) were randomly divided into four groups (n=8) for 6 weeks (normal diet, N group; high fat diet, HF group; high fat diet + lotus root hot water extract, HFR group; high fat diet + lotus root hot water extract + taurine, HFRT group). Lotus root hot water extract was orally administrated (400mg/kg/day) to HFR and HFRT groups and the same amount of distilled water was orally administered to N and HF groups. Taurine was supplemented by dissolving in feed water (3% w/v).ResultsThe activities of glutamate oxaloacetate transaminase and glutamate pyruvate transaminase in serum were lower in HFR and HFRT groups compared to HF group. Thiobarbituric acid reactive substance contents in all groups fed a high fat diet were higher compared to N group. The activities of hepatic antioxidant enzymes were higher in HFR and HFRT groups compared to HF group.ConclusionsThese results suggest that lotus root hot water extract with taurine supplementation shows antioxidant and hepatic protective effects in high fat diet-induced obese rats.

Evaluation of bioavailable arsenic and remediation performance using a whole-cell bioreporter.

The traditional method of evaluating the effects of soil contaminants on living organisms by measuring the total amount of contaminant has been largely inadequate, in part because testing contamination levels is hindered in real samples. Here we report a novel strategy for testing arsenic (As) bioavailability in soil samples by direct (in vivo) and indirect (in vitro) measurement using an Escherichia coli-based whole-cell bioreporter (WCB). The WCB was used to test As-amended Landwirtschaftliche Untersuchungs und Forschungsanstalt soils as well as field soils collected from a smelter area under remediation in order to evaluate the efficiency of bioavailable As removal. The percentage of bioavailable As in amended and field soils was 5.8% (range: 4.9%-7.6%) and 0.6% (0.08%-1.09%) of total As, respectively. In contaminated soils, total As was decreased, whereas bioavailable As was slightly increased after soil washing. These results emphasize the importance of considering ecotoxicological aspects of soil remediation; to this end, the WCB is a useful tool for evaluating the efficiency of soil remediation by assessing bioavailability along with the total amount of contaminant present.

Arsenic bioavailability in soils before and after soil washing: the use of Escherichia coli whole-cell bioreporters

We investigated the quantification of bioavailable arsenic in contaminated soils and evaluation of soil-washing processes in the aspect of bioavailability using a novel bacterial bioreporter developed in present study. The whole-cell bioreporter (WCB) was genetically engineered by fusing the promoter of nik operon from Escherichia coli and green fluorescent protein as a sensing domain and reporter domain. Among eight well-known hazardous heavy metals and metalloid, this system responded specifically to arsenic, thereby inferring association of As(III) with NikR inhibits the repression. Moreover, the response was proportional to the concentration of As(III), thereby it was capable to determine the amount of bioavailable arsenic quantitatively in contaminated soils. The bioavailable portion of arsenic was 5.9 (3.46–10.96) and 0.9 (0.27–1.74)u2009% of total from amended and site soils, respectively, suggesting the bioavailability of arsenic in soils was related to the soil properties and duration of aging. On the other hand, only 1.37 (0.21–2.97)u2009% of total arsenic was extracted into soil solutions and 19.88 (11.86–28.27)u2009% of arsenic in soil solution was bioavailable. This result showed that the soluble arsenic is not all bioavailable and most of bioavailable arsenic in soils is water non-extractable. In addition, the bioavailable arsenic was increased after soil-washing while total amount was decreased, thereby suggesting the soil-washing processes release arsenic associated with soil materials to be bioavailable. Therefore, it would be valuable to have a tool to assess bioavailability and the bioavailability should be taken into consideration for soil remediation plans.

Simultaneous detection of bioavailable arsenic and cadmium in contaminated soils using dual-sensing bioreporters

Whole-cell bioreporters (WCBs) have attracted increasing attention during the last few decades because they allow fast determination of bioavailable heavy metals in contaminated sites. Various WCBs to monitor specific heavy metals such as arsenic and cadmium in diverse environmental systems are available. However, currently, no study on simultaneous analysis of arsenic and cadmium has been reported, even though soils are contaminated by diverse heavy metals and metalloids. We demonstrated herein the development of dual-sensing WCBs to simultaneously quantify bioavailable arsenic and cadmium in contaminated sites by employing the promoter regions of the ars and znt operons as separate metal-sensing domains, and egfp and mcherry as reporter genes. The dual-sensing WCBs were generated by inserting two sets of genes into E. coli DH5α. The capability of WCBs was successfully proved to simultaneously quantify bioavailable arsenic and cadmium in amended Landwirtschaftliche Untersuchungs und Forschungsanstalt (LUFA) soils, and then, it was applied to contaminated field soils collected from a smelter area in Korea. As a result, it was noticed that the bioavailable portion of cadmium was higher than that of arsenic while the absolute amount of bioavailable arsenic and cadmium level was opposite. Since both cadmium and arsenic were assessed from the same E. coli cells, the data obtained by using dual-sensing WCBs would be more efficient and convenient than that from comparative WCB assay. In spite of advantageous aspects, to our knowledge, this is the first report on a dual-sensing WCB for rapid and concurrent quantification of bioavailable arsenic and cadmium in contaminated soils.

Enhancing the copper-sensing capability of Escherichia coli-based whole-cell bioreporters by genetic engineering

Metals are essential to all organisms; accordingly, cells employ numerous genes to maintain metal homeostasis as high levels can be toxic. In the present study, the gene operons responsive to metal(loid)s were employed to generate bacterial cell-based biosensors to detect target metal(loid)s. The cluster of genes related to copper transport known as the cop-operon is regulated by the interaction between the copA promoter region (copAp) and CueR, turning on and off gene expression upon copper ion binding. Therefore, the detection of copper ions could be achieved by inserting a plasmid harboring the fusion of copAp and reporter genes, such as enzymes and fluorescent genes. However, copAp is not as strong a promoter as other metal-inducible promoters, such as znt-, mer-, and ars-operons; thereby, its sensitivity toward copper ions was not sufficient for quantification. To overcome this problem, we engineered Escherichia coli with a deletion of copA to interfere with copper export from cells. The engineered E. coli whole-cell bioreporter was able to detect copper ions at 0 to 10xa0μM in an aqueous solution. Most importantly, it was specific to copper among several tested heavy metal(loid)s. Therefore, it will likely be useful to detect copper in diverse environmental systems. Although additional improvements are still required to optimize the E. coli-based copper-sensing whole-cell bioreporters presented in this study, our results suggest that there is huge potential to generate whole-cell bioreporters for additional targets by molecular engineering.

Assessing the toxicity and the dissolution rate of zinc oxide nanoparticles using a dual-color Escherichia coli whole-cell bioreporter

Particle toxicity and metal ions from the dissolution of metallic nanoparticles (NPs) can have environmentally toxic effects. Among the diverse metallic NPs, four types of zinc oxide NPs (ZnO-NPs)-two spherical (diameters <50xa0nm and <100xa0nm) and two wire (50xa0nmxa0×xa0300xa0nm and 90xa0nmxa0×xa01000xa0nm) shaped-were examined using dual-color whole-cell bioreporters (WCBs) to elucidate the relationships among size, shape, and toxicity. The amount of Zn(II) ions dissolved from NPs was determined by measuring mCherry expression because the presence of Zn(II) ions induced the expression of mCherry from pZnt-mCherry in dual-color WCBs. The overall toxic effects were assessed by measuring Escherichia coli cell growth. The toxic effect on cell growth was determined by measuring the expression of eGFP from the dual-color WCBs to avoid interferences in the signal acquisition caused by inseparable NPs. The novel approach demonstrated here used dual-color WCBs to simultaneously assess the toxicity of ZnONPs on E.xa0coli and the dissolution rates of ZnO-NPs. Toxicity varied depending upon the size and shape of the ZnONPs. The dissolution rate did not vary significantly according to size and shape; smaller sizes and wire shapes showed higher toxicity. Therefore, the physical properties of ZnONPs play a role in the overall toxic effect as well as dissolved Zn(II) ions.

Assessing bioavailability and genotoxicity of heavy metals and metallic nanoparticles simultaneously using dual-sensing Escherichia coli whole-cell bioreporters

Genetically engineered bacterial strains called whole-cell bioreporters (WCBs) generated by fusing the promoter region of stress-responsive genes and reporter genes have been widely used as biosensors to detect toxic materials in the environment. In this study, we report a dual-sensing WCB harboring recAp::egfp and zntAp::mcherry to measure the genotoxicity and bioavailability of heavy metals and metallic nanoparticles (NPs) simultaneously. Since the dual-sensing WCB harbored recAp::egfp and zntAp::mcherry, the genotoxicity and bioavailability of heavy metals and metallic NPs that activate ZntR would be assessed by measuring the fluorescence signal of enhanced green fluorescent protein (eGFP) and mCherry, respectively. Among the tested heavy metal(loid)s, only Cr induced both eGFP and mCherry expression, and some of them only induced mCherry, thereby suggesting that Cr is genotoxic. In case of the tested metallic NPs, Ti2O NPs, ZnO NPs, and Au NPs showed weak inhibitory effects on growth, but the eGFP was not induced. It was inferred that the tested NPs were not genotoxic and the inhibitory effects would not be related to direct DNA damage pathways. In addition, it was observed that ZnO NPs induced mCherry expression, indicating that the Zn ion was dissolved from the NPs. Although the dual-sensing WCB described here was limited to ZnO NPs, WCBs would be an alternative tool to investigate the dissolution of metallic NPs when the corresponding metal ion sensing systems were available.

Inhibitory potential of flavonoids on PtdIns(3,4,5)P3 binding with the phosphoinositide-dependent kinase 1 pleckstrin homology domain.

Many membrane-associated proteins are involved in various signaling pathways, including the phosphoinositide 3-kinase (PI3K) pathway, which has key roles in diverse cellular processes. Disruption of the activities of these proteins is involved in the development of disease in humans, making these proteins promising targets for drug development. In most cases, the catalytic domain is targeted; however, it is also possible to target membrane associations in order to regulate protein activity. In this study, we established a novel method to study protein-lipid interactions and screened for flavonoid-derived antagonists of PtdIns(3,4,5)P3 binding with the phosphoinositide-dependent kinase 1 (PDK1) pleckstrin homology (PH) domain. Using an enhanced green fluorescent protein (eGFP)-tagged PDK1 PH domain and 50% sucrose-loaded liposomes, the protein-lipid interaction could be efficiently evaluated using liposome pull-down assays coupled with fluorescence spectrophotometry, and a total of 32 flavonoids were screened as antagonists for PtdIns(3,4,5)P3 binding with the PDK1 PH domain. From this analysis, we found that two adjunct hydroxyl groups in the C ring were responsible for the inhibitory effects of the flavonoids. Because the flavonoids shared structural similarities, the results were then subjected to quantitative structure-activity relationship (QSAR) analysis. The results were then further confirmed by in silico docking experiments. Taken together, our strategy presented herein to screen antagonists targeting lipid-protein interactions could be an alternative method for identification and characterization of drug candidates.