• 한국진공학회:학술대회논문집
• /
• 한국진공학회 2013년도 제44회 동계 정기학술대회 초록집
• /
• 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.

• 약학회지
• /
• 제32권1호
• /
• pp.70-79
• /
• 1988

The effect of Ginseng on global myocardial ischemia and reperfusion was examined in isolated perfused rat hearts. The Ginseng ethanol extract (100mg/kg/day) was administered orally for 10 days. The rat hearts were removed and perfused at 75cm $H_{2}O$ by the Langendorff method. After 25 min. of global ischemia, the hearts were reperfused. The myocardial contents of adenosine 5'-triphosphate, creatine phosphate, and calcium were assayed. There no differences in ATP levels in all group of normal and Ginseng-treated hearts. Both in non-ischemic and ischemic heart, Ginseng increased significantly tissue creatine phosphate levels compared with control. Whereas, in ischemic-reperfused heart, there was no significant difference. In the control groups, myocardial calcium contents in the ischemic hearts were decreased compared with the non-ischemic hearts. But, in the Ginseng-treated groups, the calcium contents in the ischemic herts were not changed with the nonischemic hearts. Therefore, Ginseng appears to exert its protective effect against ischemic heart condition, not against ischemic-reperfused heart condition, by regulating energy metabolism and maintaing cellular function.

• Applied Microscopy
• /
• 제28권4호
• /
• pp.563-575
• /
• 1998

To investigate the changes during the differentiation of the cerebral neurons of chick embryo of tne embryogenic day (ED) 7 and 8, the ultrastructural changes in the cerebral neurons, the activity of dehydronases (LDH, MDH and SDH), protein expression profile and adenosine triphosphate concentration were analyzed. In ED 7 chick embryos, relatively large nucleus, centrally located nucleolus, evenly spread chromatin over nucleoplasm, and prominent nuclear envelope were observed. Oval-shaped mitochondria with well-developed cristae were present over entire cytoplasm. In ED 8 chick embryos, evenly spread chromatin over nucleoplasm, and prominent nuclear envelope were observed. In the cytoplasm, well-developed rough endoplasmic reticulum and Golgi complex were observed. In ED 7 chick embryos and ED 8 chick embryos, 31 polypeptide bands and 34 polypeptide bands were observed, respectively. The activities of dehydrogenases were lower in ED 7 chick embryos than in ED 8 chick embryos. LDH activity was 8.16 (ED 7) and 9.28 (ED 8), MDH activity was 7.98 (ED 7) and 10.10 (ED 8), and SDH activity was 5.49 (ED 7) and 7.14 (ED 8) respectively. The ATP concentration remained unchanged over ED 7 and 8.

• Journal of Gastric Cancer
• /
• 제15권4호
• /
• pp.223-230
• /
• 2015

Purpose: The purpose of this pilot study was to evaluate the association between adenosine triphosphate-based chemotherapy response assays (ATP-CRAs) and subsets of tumor infiltrating lymphocytes (TILs) in gastric cancer. Materials and Methods: In total, 15 gastric cancer tissue samples were obtained from gastrectomies performed between February 2007 and January 2011. Chemotherapy response assays were performed on tumor cells from these samples using 11 chemotherapeutic agents, including etoposide, doxorubicin, epirubicin, mitomycin, 5-fluorouracil (5-FU), oxaliplatin, irinotecan, docetaxel, paclitaxel, methotrexate, and cisplatin. TILs in the tissue samples were evaluated using antibodies specific for CD3, CD4, CD8, Foxp3, and Granzyme B. Results: The highest cancer cell death rates were induced by etoposide (44.8%), 5-FU (43.1%), and mitomycin (39.9%). Samples from 10 patients who were treated with 5-FU were divided into 5-FU-sensitive and -insensitive groups according to median cell death rate. No difference was observed in survival between the two groups (P=0.216). Only two patients were treated with a chemotherapeutic agent determined by an ATP-CRA and there was no significant difference in overall survival compared with that of patients treated with their physician's choice of chemotherapeutic agent (P=0.105). However, a high number of CD3 TILs was a favorable prognostic factor (P=0.008). Pearson's correlation analyses showed no association between cancer cell death rates in response to chemotherapeutic agents and subsets of TILs. Conclusions: Cancer cell death rates in response to specific chemotherapeutic agents were not significantly associated with the distribution of TIL subsets.

• The Korean Journal of Physiology
• /
• 제17권2호
• /
• pp.125-133
• /
• 1983

Red cell glycolytic intermediates, metabolites and metabolic ratios were studied. Glycolytic intermediates were measured in neutralized perchloric acid extracts of red cell suspensions after 3 hr incubation at $37^{\circ}C$ in the presence and absence of saponin. Adenosine triphosphate(ATP), adenosine diphosphate(ADP), pyruvate and lactate were measured by enzymatic procedures involving stoichiometric oxidation or reduction of a pyridine nucleotide. Glucose was determined using glucose oxidase after zinc hydroxide extraction. The redox state was calculated from the lactate dehydrogenase equilibrium. Adenosine triphosphatase activity(ATPase) was measured by determining the amount of phosphate released from ATP by washed erythrocyte membranes(ghost) during 20 min. incubation. Both total hydrolysis and the amount of hydrolysis that occured in the presence of ouabain were measured. The second measurement yields Mg-ATPase and represents nonspecific ATPase activity of the membranes. The difference between total and Mg-ATPase activity can be attributed to Na-K-ATPase. For the measurement of sodium fluxes, human erythrocytes were preincubated in $^{22}Na$ for 3 hr at $37^{\circ}C$, washed and suspended in a tracer-free medium. The amount of $^{22}Na$ transported out of cells at any time was determined by analysis of supernatant samples taken at various time after addition of the labeled cells to isotope-free medium. The cells and medium were separated and the radioactivity appearing in the medium was measured. From the total radioactivity in the suspension and the radioactivity appearing in the medium at known time, the rate constant for sodium release was computed. The results are summarized as follows: 1) ATP and ATP/ADP were found to increase at every concentration of saponin tested whereas ADP declined at every cone. of saponin. The increase in pyruvate and lactate were observed at every cone, of saponin and thus $NAD^+/NADH$ computed from pyruvate/lactate also increased. Glucose utilization was stimulated by saponin. 2) $Na^+-K^+-ATPase$ activities showed a biphasic response to saponin, first increasing in lower concentration and then decreasing in higher concentration of saponin. 3) The efflux of sodium was significantly increased by saponin in the range of 5 to 10 mg%. The stimulatory effect of saponin on the rate constants for active(ouabain-sensitive) sodium efflux was inhibited by addition of ouabain.

• The Korean Journal of Physiology and Pharmacology
• /
• 제18권4호
• /
• pp.297-305
• /
• 2014

Flavonoids have an ability to suppress various ion channels. We determined whether one of flavonoids, cyanidin-3-glucoside, affects adenosine 5'-triphosphate (ATP)-induced calcium signaling using digital imaging methods for intracellular free $Ca^{2+}$ concentration ([$Ca^{2+}$]i), reactive oxygen species (ROS) and mitochondrial membrane potential in PC12 cells. Treatment with ATP ($100{\mu}M$) for 90 sec induced [$Ca^{2+}$]i increases in PC12 cells. Pretreatment with cyanidin-3-glucoside ($1{\mu}g/ml$ to $100{\mu}g/ml$) for 30 min inhibited the ATP-induced [$Ca^{2+}$]i increases in a concentration-dependent manner ($IC_{50}=15.3{\mu}g/ml$). Pretreatment with cyanidin-3-glucoside ($15{\mu}g/ml$) for 30 min significantly inhibited the ATP-induced [$Ca^{2+}$]i responses following removal of extracellular $Ca^{2+}$ or depletion of intracellular [$Ca^{2+}$]i stores. Cyanidin-3-glucoside also significantly inhibited the relatively specific P2X2 receptor agonist 2-MeSATP-induced [$Ca^{2+}$]i responses. Cyanidin-3-glucoside significantly inhibited the thapsigargin or ATP-induced store-operated calcium entry. Cyanidin-3-glucoside significantly inhibited the ATP-induced [$Ca^{2+}$]i responses in the presence of nimodipine and ${\omega}$-conotoxin. Cyanidin-3-glucoside also significantly inhibited KCl (50 mM)-induced [$Ca^{2+}$]i increases. Cyanidin-3-glucoside significantly inhibited ATP-induced mitochondrial depolarization. The intracellular $Ca^{2+}$ chelator BAPTA-AM or the mitochondrial $Ca^{2+}$ uniporter inhibitor RU360 blocked the ATP-induced mitochondrial depolarization in the presence of cyanidin-3-glucoside. Cyanidin-3-glucoside blocked ATP-induced formation of ROS. BAPTA-AM further decreased the formation of ROS in the presence of cyanidin-3-glucoside. All these results suggest that cyanidin-3-glucoside inhibits ATP-induced calcium signaling in PC12 cells by inhibiting multiple pathways which are the influx of extracellular $Ca^{2+}$ through the nimodipine and ${\omega}$-conotoxin-sensitive and -insensitive pathways and the release of $Ca^{2+}$ from intracellular stores. In addition, cyanidin-3-glucoside inhibits ATP-induced formation of ROS by inhibiting $Ca^{2+}$-induced mitochondrial depolarization.

• 한국수산과학회지
• /
• 제50권5호
• /
• pp.500-505
• /
• 2017

The nucleotides and their related compounds, including ATP (adenosine triphosphate), ADP (adenosine diphosphate), AMP (adenosine monophosphate), IMP (inosine monophosphate), HxR (inosine) and Hx (hypoxanthine), were nearly identical in oysters Crassostrea gigas from the two culture methods. The K-value was lower than the threshold value such as 11.2-12.1. Although oysters have low amount of IMP, it was detected in this experiment. DPPH radical scavenging activity did not vary significantly with sample amounts (100, 300, and $500{\mu}g/mL$). DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging activity was 76.0-80.7% compare with the ascorbic acid standard. Superoxide anion scavenging activity reached 49.3% in the rack-and-bag culture sample at $500{\mu}g/mL$. However, the reducing power and $Fe^{2+}$ chelating activity were very low compared with their respective standard. The oyster culture methods did not affect oyster quality in terms of antioxidant activities.

• 대한수의학회지
• /
• 제29권3호
• /
• pp.393-405
• /
• 1989

This study was carried out to treatment test for bovine mastitis by the determination of adenosine triphosphate (ATP) based on firefly bioluminescence. The results obtained are followed; 1. In the susceptibility test, cephalothin which looks the most effective were sensitive to Staphylococcus sp. (72.3%), Micrococcus sp. (84.2%), Streptococcus sp. (72.7%) and Gram positive bacilli (72.7%), Gram negative bacilli were sensitive to gentamicin (92.3%) and Yeast-like-fungi was the most sensitive to clotrimazole, and nystatin in order. 2. When the number of bacteria, such as Staphylococcus aureus, Candida tropicalis isolated from the mastitis milk were counted by conventional agar plating technique, and compared with the concentration of bacterial ATP, it gave a good linear relationship. The content of ATP per Staphylococcus aureus, cell was 3.1fM and Candida tropicalis showed the high level of A TP (90fM). 3. The ATP assay was applied to the determination of minimal inhibitory concentration (MIC) of various antibiotics. When Staphylococcus aureus was incubated in the presence of different concentration of tetracycline, erythromycin, kanamycin and streptomycin sulfate and the growth was monitored by the conventional agar plating technique and ATP assay, both methods shown the same results that they were 1mcg/ml, 2mcg/ml, 6.25mcg/ml and 8mcg/ml, respectively. 4. For the determination of susceptibility of sensitive and resistant Staphylococcus au reus isolated for the milk with mastitis to tetracycline, erythromycin kanamycin and streptomycin sulfate, the minimum time required for the test was determined by the assay of ATP every 30 minutes during incubation of 3 hours at $37^{\circ}C$. ATP concentration time curve calculated on both resistant and sensitive strains incubated 3 hours as the optimum time for the determination of susceptibilities of various antibiotics exemed. The ATP concentration of each test broth (antibiotic containing), expressed as a percentage of its own control brith (antibioticfree) indicated values of 30% to be indicative of each antibiotic sensitivity. Single time point ATP assay carried out on the various sensitive and resistant of Staphylococcus aureus to antibiotics examined after 3 hours at $37^{\circ}C$ correlated exactly with disc diffusion and MIC. 5. In the cure of intramammary treatment of bovine mastitis in lactating quarters, the cure rate of Staphylococcal mastitis showed to cephalexin (80%), cloxacillin and gentamicin (70%), ampicillin and oxytetracycline (60%), and Streptococcal mastitis showed to cephalexin (85%), penicillin (80%), cloxacillin and oxytetracycline (75%), and ampicillin (70%), but intramammary antimycotic drug (clotrimazol) were only a little effect about fungal mastitis.

• Asian-Australasian Journal of Animal Sciences
• /
• 제21권8호
• /
• pp.1127-1133
• /
• 2008

This study was designed to develop a method of liquid storage of boar sperm at $4^{\circ}C$ by using the modified Beltsville F5 (BF5) diluent with bovine serum albumin (BSA) and N-acetyl-D-glucosamine. Boar sperm were stored in lactose-egg yolk and N-acetyl-D-glucosamine (LEN), BF5 and Golden-Pig liquid 4 (GPL4) diluents at $4^{\circ}C$ for 5 days and were examined for sperm viability, adenosine triphosphate (ATP) and in vitro fertilization (IVF). The percentage of sperm viability in GPL4 diluent was higher than in LEN and BF5 diluent from 1 to 5 days of storage at $4^{\circ}C$. The percentage of sperm viability steadily declined from 1 to 5 days of storage in the three different diluents. Sperm ATP in GPL4 diluent was higher than in LEN and BF5 diluents from 1 to 5 days of storage. Sperm ATP rapidly declined after 5 days of storage in the three different diluents. Porcine oocytes matured in vitro were inseminated with different sperm concentrations of liquid semen stored for 3 days in GPL4 diluent. The percentage of monospermic oocytes did not show any differences from 2.5 to $20{\times}10^5$ sperm/ml. However, the percentage of polyspermic oocytes in the sperm concentration of $2.5{\times}10^5$ sperm/ml was lower than in concentrations of 5, 10 and $20{\times}10^5$5 sperm/ml. The percentage of blastocysts from the cleaved oocytes at $2.5{\times}10^5/ml$ sperm concentration was significantly lower than at 5, 10 and $20{\times}10^5sperm/ml$ concentrations. In conclusion, GPL4 diluent can be stored at $4^{\circ}C$ for 5 days and showed higher sperm viability and sperm ATP concentration compared with LEN and BF5 diluents. Also, we found that GPL4 diluent can be used for IVF of porcine oocytes.

• Applied Microscopy
• /
• 제26권3호
• /
• pp.253-266
• /
• 1996

To investigate the effects of mercuric chloride ($HgCl_2$) on the differentiation of the cerebral neuron of chick embryo 9 days, the ultrastructural changes in nerve cells injected with a various doses of mercuric chloride were observed with transmission electron microscope. The enzyme activity of the some dehydrogenases, cerebral proteins and adenosine triphosphate (ATP) were also analyzed. The results obtained are as follows: The ultrastructural changes in 0.5 and 1.0mg-injected groups were undetectable, but in 2.0mg-injected group, the nuclear envelops were very irregular and mitochondria, were swelled and destroyed partly. The number of polypeptide bands separated by SDS-PAGE in the normal group were 37 bands. According to the in creased dose of mercuric chloride, contends of the bands were increased in 7 bands. The activities of dehydrogenases were declined by increasing the dose of mercuric chloride. Lactate dehydrogenase (LDH) activity failed to 78% in 1.0mg-injected group and greatly to 68% in 2.0 mg-injected group. Malate dehydrogenase (MDH) activity failed to 81% in 2.0 mg-injected group. On the other hand, succinate dehydrogenase (SDH) activity decreased to 80% in 1.0 mg-injected group and greatly to 63% in 2.0 mg-injected group. ATP content in 1.0 mg-injected group was increased slightly and in 2.0 mg-injected group was increased greatly.