• Ocean and Polar Research
• /
• v.31 no.4
• /
• pp.349-357
• /
• 2009

Ballast water is widely recognized as a serious environmental problem due to the risk of introducing non-indigenous aquatic species. In this study we aimed to investigate measures which can minimize the transfer of aquatic organisms from ballast water. Securing more reliable technologies to determine the viability of aquatic organisms is an important initiative in ballast water management systems. To evaluate the viability of marine phytoplankton, we designed the staining methods of fluorescein diacetate (FDA) and Calcein-AM assay on each target species belonging to different groups, such as bacillariphyceae, dinophyceae, raphidophyceae, chrysophyceae, haptophyceae and chlorophyceae. The FDA method, which is based on measurements of cell esterase activity using a fluorimetric stain, was the best dye for determining live cells of almost all phytoplankton species, except several diatoms tested in this study. On the other hand, although fluorescence of Calcein-AM was very clear for a comparatively longer time, green fluorescence per cell volume was lacking in most of the tested species. According to the Flow CAM method, which is a continuous imaging technique designed to characterize particles, green fluorescence values of stained cells by FDA were significantly higher than those of Calcein-AM treatments and control, implying that the Flow CAM using FDA assay could be adapted as an important tool for distinguishing living cells from dead cells. Our results suggest that the FDA and Calcein-AM methods can be adapted for use on phytoplankton, though species-specific characters are greatly different from one organism to another.

• Journal of Pharmaceutical Investigation
• /
• v.36 no.5
• /
• pp.309-314
• /
• 2006

The multicellular layers(MCL) of human cancer cells is a three dimensional(3D) in vitro model for human solid tumors which has been used primarily for the assessment of avascular penetration of anti-cancer drugs. For anti-cancer drugs with penetration problem, MCL represents a good experimental model that can provide clinically relevant data. Calcein-AM is a fluorescent dye that demonstrates the cellular vitality in a graded manner in cancer cell culture system. In the present study, we evaluated the use of calcein-AM for determination of anti-proliferative activity of anti-cancer agents in MCL model of DLD-1 human colorectal cancer cells. Optical sectioning of confocal imaging was compromised with photonic attenuation and penetration barrier in the deep layers of MCL. By contrast, fluorescent measurement on the cryo-sections provided a feasible alternative. Cold pre-incubation did not enhance the calcein-AM distribution to a significant degree in MCL of DLD-1 cells. However, the simultaneous determination of drug penetration and cellular vitality appeared to be possible in drug treated MCL. In conclusion, these data suggest that calcein-AM can be used for the simultaneous determination of drug-induced anti-proliferative effect and drug penetration in MCL model.

• Journal of Photoscience
• /
• v.9 no.2
• /
• pp.518-520
• /
• 2002

Photodynamic therapy (PDT) is a treatment modality based on photochemical reaction and the resultant cytotoxic reactive oxygen species. The platelet thrombus formation leading to stasis observed in vivo during PDT is called vascular shut down (VSD) effect. To investigate the mechanism of the VSD effect, we observed Human Umblical Vein Endothelial Cell (HUVEC) injury induced by photochemical reaction. We observed cell retraction and blebbing after PDT. It seems that the injury was not fetal and only morphological change. Then, the cytoplasm was stained by Calcein-AM and subendothelial area was evaluated from fluorescence microscopy. The rate of subendothelial area after PDT increased significantly. Second, we investigated interaction between neutrophils and HUVEC. Human promyelocytic leukemia cells (HL-60) were differentiated into neutrophil by incubation with all-trans retinoic acid. Calcein-AM labeled neutrophil adhesion to HUVEC was evaluated from fluorescence microscopy. PDT-induced neutrophil adhesion to HUVEC depended more on the exposure of subendothlial area than on neutrophil activation. This result suggests that there is a certain interaction between neutrophil and HUVEC during PDT.

• Archives of Pharmacal Research
• /
• v.18 no.2
• /
• pp.84-89
• /
• 1995

The complexation and physical characteristics of egg phosphatidylcholine (PC) liposome containing amphotericin B(AmB) were investigated through circular dichrosim(CD) spectra, the size distribution, the turbidity change, and the calcein release. CD spectra of AmB-containing egg PC mxture exhibited a positive peak around 330 nm indicative of complexation of AmB and four negative peaks. The positive peak increased up to $2.2{\;}millidegree/{\mu}g$ AmB as AmB contents increased up to 12% (w/w), suggesting that AmB-phospholipid complexation was promoted by the antibiotics. The effective diameter of liposomesa by dynamic light scattering decreased from 450 nm to 220 nm as the amount of AmB in liposomes increased from o to 30% (w/w). The complexation may be responsible for the reduction in size. On the other hand, at around 1 mN deoxycholate (DOC), the reltive turbidities of 5 and 10% (w/w) AmB-containing liposome suspension were less than 1 probably due to the soblubilization of the complex, while those of pure PC liposome suspension were larger than 1 at the same concentration. Deoxycholate-induced release of liposomes, indicating the intercalation of the drug into the bilayers. Therefore, it is concluded that in AmB/eggPC/water system, AmB-phospholipid complexcoexists with AmB-containing liposomes.

• Journal of Life Science
• /
• v.18 no.9
• /
• pp.1244-1248
• /
• 2008

P-glycoprotein (P-gp) is a very important drug transporter, which plays an important role in drug disposition and represents an additional mechanism for the development of multidrug resistance. Flavonoids, a major class of natural compounds widely present in foods and herbal products, have been shown to be P-gp inhibitors. The objective of the present study was to identify new chemosensitizer candidates through the screening of various herbal extracts. The inhibitory effects of herbal extracts on P-gp activity were assessed by measuring accumulation of calcein AM using P-gp overexpressed L-MDR1 cells. Curcuma longa showed the most potent inhibition on P-gp function. The inhibitory potential of P-gp was in the order: Curcuma longa > Curcuma aromatica > Ageratum conizoids > Zanthoxylum planispinum > Zedoariae rhizome > Rakta chandan > Dalbergia odorifera > Caesalpinia Sappan > Aloe ferox. To identify individual constituents with inhibitory activity, the herbal extracts were analyzed by LC/MS/MS. Several flavonoids such as curcumin, a well-known P-gp inhibitor, were identified through mass spectral library search. These in vitro data indicate that herbal extracts contain constituents that can potently inhibit the activities of P-gp and suggest that these herbal extracts should be examined for potential chemosensitizer in vivo.

• Journal of Marine Bioscience and Biotechnology
• /
• v.1 no.3
• /
• pp.213-217
• /
• 2006

We evaluated the potential of major components of Phellodendri cortex to inhibit the activities of CYP2D6 and p-glycoprotein. The abilities of berberine, palmatine, limonin, and rutaecarpine to inhibit CYP2D6-mediated dextromethorphan O-demethylation and calcein AM accumulation were tested using human liver microsomes and L-MDR1 cell, respectively. Berberine strongly inhibited CYP2D6 isoform activity, whereas limonin and reuaecarpine did not. The $IC_{50}$ value of berberine was reduced after preincubation with microsomes in the presence of NADPH generating system, suggesting that berberine is a mechanism based inhibitor. In addition, all chemicals tested, didn't show inhibitory effect on p-glycoprotein activity. These results suggest that berberine has potential to inhibit CYP2D6 activity in vitro. Therefore, in vivo studies investigating the interactions between berberine and CYP2D6 substrates are necessary to determine whether inhibition of CYP2D6 activity by berberine is clinically relevant.

• Journal of Pharmaceutical Investigation
• /
• v.40 no.5
• /
• pp.269-275
• /
• 2010

P-gp plays a critical role in drug disposition and represents a mechanism for the development of multidrug resistance. Flavonoids, a major class of natural compounds widely present in foods and herbal products, have been shown to inhibit P-gp. Therefore, the aim of this study was to identify new candidate chemosensitizers by screening various plant extracts. The ability of natural plant extracts to inhibit P-gp activity was assessed by measuring cellular accumulation of calcein AM, daunorubicin and vincristine in P-gp overexpressing MDCKII-MDR1 cells. Among more than 800 plant extracts, eight were found to inhibit P-gp activity. Curcuma aromatica extract produced greatest inhibition, followed by Curcuma longa and Dalbergia odorifera extracts. Extracts of Aloe ferox, Curcuma zedoariae rhizome, Zanthoxylum planispinum, and Ageratum conyzoides showed moderate inhibitory effects. Curcumin and quercetin exhibited similar inhibition of P-gpmediated efflux of daunorubicin and vincristine, and flavones had a lesser effect. When chemosensitizing effect was evaluated by measuring daunorubicin sensitivity to MDCKII-MDR1 cells in the presence of natural plant extracts, Curcuma aromatica showed the most potent chemosensitizing effect based on daunorubicin cytotoxicity. In conclusion, natural plant extracts such as Curcuma aromatica can potently inhibit P-gp activity and may have potential as a novel chemosensitizers.

• Journal of Embryo Transfer
• /
• v.32 no.3
• /
• pp.221-226
• /
• 2017

Spermatozoa viability can be assessed by microscopy, flow cytometry, and other methods using fluorescent stain. Flow cytometry can be used to examine the morphological and functional characteristics of spermatozoa in a short time. The purpose of this study was to compare the viability of cryopreserved spermatozoa in Jeju black cattle by two dual fluorescent stain methods. Semen of Jeju black cattle raised in Subtropical Livestock Research Institute, National Institute of Animal Science, RDA were collected with artificial vaginal technique. Sperm was diluted with $Triladyl^{(R)}$-egg yolk diluent and then was performed cryopreservation. There was no significant difference in viability of spermatozoa according to the two dual fluorescent stain methods. However, when the distribution of spermatozoa according to the staining method was compared, the spermatozoa group stained with 6-CFDA/PI was more clearly distinguished than the spermatozoa group stained with calcein AM/PI.

• Herbal Formula Science
• /
• v.32 no.1
• /
• pp.99-109
• /
• 2024

Objectives : In this study, we investigated the cytoprotective effect of Eriobotrya japonica L. (EJ) extract against Arachidonic acid (AA)+iron-induced oxidative stress. Methods : To confirm the cytoprotective effect of EJ against AA+iron-induced oxidative stress in HepG2 cells, it was evaluated by MTT assay, immunoblot anaylsis, and Calcein-AM/propidium iodide (PI) staining. Additionally, the mechanism of action of the cytoprotective effect was evaluated through molecular mechanisms. Results : EJ (100 ㎍/mL) inhibited Arachidonic acid (AA)+iron-induced cell death in a concentration-dependent manner. It also inhibited AA+iron-induced mitochondrial dysfunction and ROS production. EJ activated the LKB1-AMPK signaling pathway. Conclusions : In conclusion, EJ has the ability to protect liver cells from oxidative stress, indicating that it is related to AMPK-LKB1 signaling pathways.

• Clinical and Experimental Pediatrics
• /
• v.46 no.7
• /
• pp.702-709
• /
• 2003

Purpose : $IFN{\gamma}$ sentitizes many tumor cells to $TNF{\alpha}$ and FASL-mediated apoptosis by enhancing the expression of TNF or FAS/CD95 receptor and modulating the activation of caspase and Bcl-2 family. It has been reported that $IFN{\gamma}$ and $TNF{\alpha}$ synergistically caused differentiation and growth inhibition of neuroblastoma cells. Even though some neuroblastoma cell express FASR/FASL on the cell surface, they could not induce apoptosis by ligation of the FAS/CD95 receptor. But the treatment of $IFN{\gamma}$ is reported to induce apoptosis in some neuroblastoma cell lines through the CD95/CD95L autocrine circuit. In this study, we examined whether $IFN{\gamma}$ could affect $TNF{\alpha}$ and agonistic FAS/CD95 antibody(CH-11)-induced apoptosis against neuroblastoma cell lines that had shown diverse drug sensitivity and resistance. Methods : CHLA-15, CHLA-90 and LA-N-2 neuroblastoma cells were cultured in IMDM and treated with recombinant $IFN{\gamma}$, $TNF{\alpha}$ and CH-11 antibody. Cell viability was measured by DIMSCAN with a fluorescent calcein-AM. Apoptosis was analyzed through flow cytometry using Annexin V-PE and 7-ADD staining and confirmed by pancaspase and caspase-8 blocking experiments. The expression of TNF RI and FAS/CD95 receptor was evaluated by flow cytometry using the corresponding antibody and PE-conjugated secondary antibody. Results : $IFN{\gamma}$ or $TNF{\alpha}$ alone had no demonstrable cytotoxic effects, whereas both cytokines in combination induced apoptosis synergistically in CHLA-15 and CHLA-90 cells. Although there was no cytotoxicity with the ligation of CH-11 alone in CHLA-90 cells, pretreatment of $IFN{\gamma}$ increased the sensitivity of CH-11-mediated apoptosis. The expression of TNFRI and FAS/CD95R were non-specifically enhanced after treatment of $IFN{\gamma}$ without relation to sensitivity to $TNF{\alpha}$ and CH-11. This finding suggest up-regulation of both receptors may contribute to sensitization of $TNF{\alpha}$ and CH-11-mediated apoptosis by $IFN{\gamma}$ in only sensitive cell lines. Conclusion : $IFN{\gamma}$ induced sensitization of $TNF{\alpha}$ and agonistic FAS/CD95 antibody-mediated apoptosis on some neuroblastoma cells through up-regulation of TNFRI and FAS/CD95 receptor.