• Proceedings of the Korean Vacuum Society Conference
• /
• 2013.02a
• /
• pp.108-109
• /
• 2013

Mitochondria play key roles in the production of cell's energy. Their dominant function is the synthesis of adenosine 5'-triphosphate (ATP) from adenosine diphosphate (ADP) and phosphate (Pi) through the oxidative phosphorylation. Evaluation of drug-induced mitochondrial toxicity has become increasingly important since mitochondrial dysfunction has recently been implicated in numerous diseases including cancer and diabetes mellitus. Mitochondrial functions have been monitored via oxygen consumption, mitochondrial membrane potential, and more importantly via ATP synthesis since ATP synthesis is the most essential function of mitochondria. Various analytical methods have been employed to investigate ATP synthesis in mitochondria, including high performance liquid chromatography (HPLC), bioluminescence technique, and pH measurement. However, most of these methods are based on destructive analysis or indirect monitoring through the enzymatic reaction. Infrared absorption spectroscopy (IRAS) is one of the useful techniques for real-time, label-free, and direct monitoring of biological reactions [1,2]. However, the strong water absorption requires very short path length in the order of several micrometers. Transmission measurements with thin path length are not suitable for mitochondrial assays because solution handlings necessary for evaluating mitochondrial toxicity, such as rapid mixing of drugs and oxygen supply, are difficult in such a narrow space. On the other hand, IRAS in the multiple internal reflection (MIR) geometry provides an ideal optical configuration to combine solution handling and aqueous-phase measurement. We have recently reportedon a real-time monitoring of drug-induced necrotic and apoptotic cell death using MIR-IRAS [3,4]. Clear discrimination between viable and damaged cells has been demonstrated, showing a promise as a label-free and real-time detection for cell-based assays. In the present study, we have applied our MIR-IRAS system to mitochondria-based assays by monitoring ATP synthesis in isolated mitochondria from rat livers. Mitochondrial ATP synthesis and hydrolysis were in situ monitored with MIR-IRAS, while dissolved oxygen level and solution pH were simultaneously monitored with O2 and pH electrodes, respectively. It is demonstrated that ATP synthesis and hydrolysis can be monitored by the IR spectral changes in phosphate groups in adenine nucleotides and MIR-IRAS is useful for evaluating time-dependent drug effects of mitochondrial toxicants.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.24 no.5
• /
• pp.713-719
• /
• 1995

These studies were carried out to investigate the effects of Puerariae radix catechins(PRC) administration on the biochemical parameters of liver function in liver of carbon tetrachloride(CCl4)-treated rats. Thirty six healthy Sprague-Dawley rats weighing about 120g were used for this experiment and divided intot he following 3 groups : normal control group(NCON), $CCl_4$ control group(CCON), PRC treated group(PRC). Fifty percent $CCl_4$ in oil was administered(I.P.) by 2ml per kg body weight two times a week for 3 weeks. PRC treated groups were administered orally at the leaves of 1% per day in distilled water for 8 weeks. Lipid hydroperoxides were analyzed by using chemiluminescence-high performance liquid chromatography(CL-HPLC) method as a phosphatidylcholine hydroperoxide value(PCOOH) in liver tissues. $CCl_4$ treatment significantly(p<0.05) resulted in an increase in GPT & GOT activities and liver hydroperoxide values comparing with those of the untreated control, while administration of PRC to the $CCl_4-treated$ rats significantly(p<0.001) decreased GPT & GOT activities and liver hydroperoxide value. Their ultrastructual changes of hepatocellular organelles were shown to clarify the morphologic nature of protective effects of PRC on hepatocytic injuries. $CCl_4$ treatment observed to change the ultrastructual nature of outer membrane of hepatocytes. However, the hepatic changes on PRC treatment to $CCl_4$ group was not found. PRC administration may inhibit the formatiion of liver lipid hydroperoxides in vivo and were very effective in recovering the liver function in $CCl_4-treated$ rats.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.2
• /
• pp.225-236
• /
• 2013

Among several bacteria examined, an antibacterial-producing Lactobacillus strain with probiotic characteristics was selected and identified based on 16S rRNA gene sequencing. Subsequent purification and mode of action of the antibacterial compounds on target cells including E. coli were investigated. Maximum production of the antibacterial compound was recorded at 18 h incubation at $30^{\circ}C$. Interestingly, antibacterial activity remained unchanged after heating at $121^{\circ}C$ for 45 min, 24 h storage in temperature range of $70^{\circ}C$ to room temperature, and 15 min exposure to UV light, and it was stable in the pH of range 2-10. The active compounds were inactivated by proteolytic enzymes, indicating their proteinaceous nature, and, therefore, referred to as bacteriocin-like inhibitory substances. Isolation and partial purification of the effective agent was done by performing ammonium sulfate precipitation and gel filtration chromatography. The molecular mass of the GFC-purified active compound (~3 kDa) was determined by Tris-Tricine SDS-PAGE. To predict the mechanisms of action, transmission electron microscopy (TEM) analysis of ultrathin sections of E. coli before and after antibacterial treatment was carried out. TEM analysis of antibacterial compounds-treated E. coli demonstrated that the completely altered bacteria appear much darker compared with the less altered bacteria, suggesting a change in the cytoplasmic composition. There were also some membrane-bound convoluted structures visible within the completely altered bacteria, which could be attributed to the response of the E. coli to the treatment with the antibacterial compound. According to the in vivo experiments oral administration of L. plantarum HKN01 resulted in recovery of infected BALB/c mice with Salmonella enterica ser. Typhimurium.

• Korean Journal of Food Science and Technology
• /
• v.29 no.3
• /
• pp.417-426
• /
• 1997

Rice bran protein hydrolysates were prepared and some of their physicochemical properties were investigated to utilize rice bran as starting material for functional food ingredient. Rice bran proteins (RBP) were prepared from defatted rice bran by alkaline extraction and isoelectric precipitation. The enzyme for hydrolysis of RBP was selected through measuring relative activity by pH-drop method and comparing the degree of hydrolysis (DH) of hydrolysates. The enzymatic hydrolysates prepared by $Esperase^{\circledR}$ treatment were partitioned into two fractions by ultrafiltration(UF) with a 10 kDa molecular weight cut-off membrane. Each fraction was applied to a cholic acid-conjugated ${\omega}-aminohexyl$ Sepharose 4B column and the bile acid-binding components were obtained by eluting with deoxycholate. Gel permeation chromatography on a Sephadex G-50 column revealed that molecular weight of the bile acid-binding fraction of UF permeate was distributed in ranges of $2\;kDa{\sim}10\;kDa$ and $0.2\;kDa{\sim}0.6\;kDa$. Three peaks (R-1, R-2 and R-3) were obtained by prep-HPLC of bile acid-binding fraction of UF retentate and analyzed for total and free amino acid composition. The results showed that proline content of the bile-acid binding polypeptides and peptides was four times as much as that of rice bran protein and that the peak corresponding to higher average hydrophobicity had a higher free amino acid content. Average hydrophobicity slightly increased with enzymatic hydrolysis.

• Bulletin of the Korean Chemical Society
• /
• v.30 no.12
• /
• pp.2999-3005
• /
• 2009

Among the more than 120 different types of human papillomavirus (HPV), types 16 and 18 have been known to be high risk agents that cause cervical cancer. We examined, in an immuno-chromatographic analysis, the potential of using the early gene product, E7 protein, as a diagnostic marker of cervical cancer caused by HPV. We developed monoclonal antibodies specific to HPV-16 and 18 E7 proteins that were produced from bacterial cells using gene recombinant technology. For each E7 protein, the optimal antibody pair was selected using the immuno-chromatographic sandwichtype binding system based on the lateral flow through membrane pores. Under these conditions, this rapid testing assay had a detection capability as low as 2 ng/mL of E7 protein. Furthermore, since viral analysis required the host cell to be lysed using chemicals such as detergents, it was possible that the E7 protein was structurally damaged during this process, which would result in a decrease in detection sensitivity. Therefore, we examined the detrimental effects caused by different detergents on the E7 protein using HeLa cells as the host. In these experiments, we found that the damage caused by the detergent, nonylphenylpolyethylene glycol (NP-40), was minimal relative to Triton X-100 commonly used for the cell lysis. Temperature also affected the stability of the E7 protein, and we found that the E7 protein was stabilized at 4$^{\circ}C$ for about 2 h, which was 4 times longer than at room temperature. Finally, a HPV-infected cervical cancer cell line, which was used as a real sample model, was treated using the optimized conditions and the presence of E7 proteins were analyzed by immuno-chromatography. The results of this experiment demonstrated that this rapid test could specifically detect HPV-infected samples.

• KSBB Journal
• /
• v.25 no.5
• /
• pp.429-436
• /
• 2010

Keratinase is widely used in certain industrial applications. The present study sought to improve the culture conditions of Bacillus subtilis SMMJ-2 to facilitate mass production of keratinase. Strain SMMJ-2 was irradiated by ultraviolet light and the resulting isolates were tested for keratinase activity. Isolates displaying elevated keratinase activity were selected and used to determine the optimum temperature (24, 30, 37, 45, $55^{\circ}C$) for bacterial keratinase production during a 4 day incubation period. The highest enzyme activity (55 units/mL/min), from a Bacillus subtilis SMMJ-2 mutant (mutant No. 2) was demonstrated following incubation at $30^{\circ}C$. The effects of carbon and nitrogen sources on keratinase production were confirmed by measuring the enzyme activity from the culture broth of the mutant strain cultured in various media containing different carbon source and nitrogen sources during a 4 day period. The optimal medium composition for producing keratinase consisted of 1% glucose, 0.7% $K_2HPO_4$, 0.2% $K_2HPO_4$, and 1.2% soybean meal. Optimal initial pH and temperature for producing keratinase were 7.0 and $30^{\circ}C$, respectively. Keratinases produced by B. subtilis SMMJ-2 and the mutant No. 2 were purified from the culture broth which used soybean meal as a nitrogen source. Membrane ultrafiltration, DEAE-sephacel ion exchange and Sephadex G-100 gel chromatography were used to purify the enzymes. The purified keratinases from both B. subtilis SMMJ-2 and the mutant No. 2 showed single bands and their molecular weights were estimated as 28 kDa and 42 kDa, respectively on SDS-polyacrylamide gel electrophoresis.

• Microbiology and Biotechnology Letters
• /
• v.40 no.3
• /
• pp.274-277
• /
• 2012

Cytokinesis is the final stage of cell division, dividing one mother cell into two daughter cells. For the cutting of a plasma membrane during bacterial cytokinesis, a tubulin homolog FtsZ protein is recruited from the cytoplasm to the division site. FtsZ protein polymerizes in a GTP-dependent manner and its N-terminal domain has a GTPase activity. In this study, we have begun to characterize FtsZ from Staphylococcus aureus (SA). Full-length SA FtsZ was cloned into pRSFDuet-1 vector and the clone was transformed into a BL21 (DE3) star cell. The recombinant SA FtsZ protein was purified using Ni-NTA affinity chromatography and dialysis. Using a spectrofluorometer, we showed that SA FtsZ undergoes a GTP-dependant polymerization in vitro. The polymer of the SA FtsZ protein disappeared after a few minutes, suggesting that the polymer is degraded as the GTP is consumed. This assay system may well be applied for inhibitor screening targeting S. aureus FtsZ.

• Analytical Science and Technology
• /
• v.24 no.1
• /
• pp.10-14
• /
• 2011

This study was performed to compare international analytical methods of bromide and applied to determine bromide in bottled mineral water and raw ground water in Korea. Bromide in water was eluted by 10~50 mM potassium hydroxide (KOH) and determined by ion chromatography-suppressed conductivity detector (IC-CD). Sample was purified with Ba- and H-on-guard cartridge and $0.2\;{\mu}m$ membrane filter. The method detection limit (MDL) and the limit of quantitation (LOQ) of bromide were 0.2 and $0.5\;{\mu}g/L$, respectively. The calibration curve showed good lineality above 0.9993 in the ranges of the $0.5\sim80\;{\mu}g/L$. A correlation coefficient of bromide in ground water and sum of bromide and bromate in bottled mineral water is 0.808.

• Korean Journal of Microbiology
• /
• v.32 no.2
• /
• pp.147-154
• /
• 1994

Nuclease was secreted to the environmental media from the Escherichia coli JM107 tranformant harboring the extracellular nuclease gene of Serratia marcescens in the plasmid of pNUC4. Under the growth conditions, the amount of secreted enzyme was increased in parallel with bacterial growth conditions, the amount of secreted enzyme was increased in parallel with bacterial growth. The enzyme was purified using chromatofraphic procedures of Matrex green gel and heparin agarose affinity gel, resulted in 50-fold purification with 15% recovery of the enzyme. The apparent molecular weight of the enzyme was estimated to be 29Kda by sodium dodecylsulfate denaturing gel electrophoresis. Using the purified enzyme, polyclonal antibody was obtained from the rabbit. The specificity of the antibody was confirmed by immunoblotting and immunoprecipitaion. For the investigation of cellular distribution of the enzyme, cells were fractionated into three fractions; cytoplasm, periplasm and extracellular fluid. While more than 80% of the enzymatic activity was detected in the extracellular fluid and periplasm, a little was found in the cytoplasm, indicating that the enzyme was likely to be immediately exported to the membrane for excretion after biosynthesis. These results were confirmed again by immunocytochemistry technique using the antibody.

• The Korean Journal of Nuclear Medicine
• /
• v.35 no.3
• /
• pp.185-191
• /
• 2001

Objectives: Recently, $[methyl-^{11}C]-({\beta}$-Hydroxyethyl)trimethylammonium ($[^{11}C]$choline) Has been discovered to be a very effective tracer in imaging various human tumors using positron omission tomography. Because of the short half-life of C-11, it is very difficult to use in a routine imaging procedure and needs a frequent synthesis of $[^{11}C]$choline. This can be supplemented by the substitution of $[^{11}C]$choline with $[methyl-^{18}F]$fluorocholine. Here, we would like to report ceil uptake and biodistribution of $[^{11}C]$choline and $[^{18}F]$fluorocholine as a basic study. Methods: $[^{11}C]$Choline was prepared by the treatment of $[^{11}C]CH_3I$ with N,N-dimethylaminoethanol and $[^{18}F]$fluorocholine was synthesized from reaction of $CH_2Br[^{18}F]F$ with N,N-dimethylaminoethanol. The radiochemical purity was checked by high performance liquid chromatography (HPLC). The blodistribution of $[^{11}C]$choline and $[^{18}F]$fluorocholine was determined in balb/c mouse at 5 min, 20 min, 40 min and 80 min. The cell uptake was measured using glioma (9L) and colon adenocarcinoma (SW620). Results: The radiochemical purity was more than 98% after purification. In the liver, uptake did not change over time; the uptake was 20%ID/g for $[^{11}C]$choline and 13%ID/g for $[^{18}F]$fluorocholine. In the kidney, radioactivity decreased over time; the uptake was 15%ID/g for $[^{11}C]$choline and 20%ID/g for $[^{18}F]$fluorocholine, 80 min post-injection. The cell uptake of $[^{11}C]$choline was 4.93% for glioma (9L) and 18.69% for colon adenocarcinoma (SW620). For $[^{18}F]$fluorocholine, 1.77% for glioma (9L) and 2.77% for colon adenocarcinoma (SW620). Conclusion: $[^{11}C]$Choline and $[^{18}F]$fluorocholine showed a different cell uptake tendency, depending on cancer cell line.