• Korean Journal of Ichthyology
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• v.36 no.2
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• pp.120-128
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• 2024

In this study, the characteristics of the early life history were investigated for the Hypomesus nipponensis in the west coast Daeho Bay. Egg's were adhesive eggs that had the property of sinking in water in a circular shape. The size of mature eggs was 0.52~0.66 (average of 0.59±0.03, n=30) mm. The hatching time took 140 hours at a water temperature of 22~23℃. Immediately after hatching, the yolk sac larvae was 4.78~5.60 (average of 5.25±0.26, n=30) mm in total length, and the mouth and anus were not completely opened. On the 7 days after hatching, the preflexion larvae was 5.91~6.64 (6.32±0.21) mm in total length, and the mouth and anus were opened, and feeding activities were started. On the 25 days after hatching, the flexion larvae was 9.70~12.3 (10.2±0.63) mm in total length, and the end of the spine at the tail end began to bend upward. On the 42 days after hatching, the postflexion larvae was 14.1~18.8 (16.9±1.44) mm in total length, and the end of the spine at the tail was completely bent at 45°. On the 56 days after hatching, it reached the integer with 10 dorsal fins, 16 anal fins, 7 ventral fins, and 19 caudal fins. According to the study, there were spot-shaped melanophore vesicles under the pectoral fins during the incubation period, the different positions of the egg yolk compared to the battlefield, the deposition of melanophore vesicles on the back and under the body of the caudal part during the postflexion larvae period, and the absence of melanophore vesicles on the torso between the head and the starting point of the dorsal fin. It was distinguished from related species in that melanophore vesicles were deposited in one row from the back of the body to the caudal part during the juvenile period.

• Korean Journal of Ichthyology
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• v.36 no.2
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• pp.109-119
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• 2024

This study was conducted to investigate the early life history by observing the egg development of Ladislavia taczanowskii in endangered fish and to use it as basic data for species conservation research. The broodstork used in the study was secured from the area of the Hongcheon River in Hongcheon-gun, Gangwon State. The broodstork, who was being raised in the laboratory, selected mature individuals in May 2021 and induced them to spawn by hormone injection. The size of the maturation egg was 1.50~1.79 (average 1.59±0.08, n=30) mm due to the circular invasive egg. The incubation time took 168 hours at 16.5℃ and 109 hours and 30 minutes at 25.5℃. Newly hatched larvae, the consonants had a total length of 5.55~6.31 mm (6.30±6.93, n=30) mm, and the mouth and anus did not open and had egg yolk. 5 days after hatching, the preflexion larvae had a total length of 9.91~10.8 (10.1±0.27, n=30) mm, and the mouth and anus opened, and feeding activities began. 8 days after hatching, the flexion larvae had a total length of 10.3~11.4 (10.8±0.38, n=30) mm, and the end of the vertebrae at the tail fin tip began to bend upward. 10 day after hatching, the postflexion larvae had a total length of 11.8~13.1 (12.3±0.43, n=30) mm, and the end of the vertebrae at the tail tip was completely bent at 45°. 18 days after hatching, the total length of the juveniles was 18.9~23.4 (20.4±1.69, n=30) mm, and the number of fins in each part was fin rays with 10 dorsal fins, 9 anal fins, 22 caudal fins, and 7 ventral fins. As a result of the study, the postflexion larvae showed differences in morphology from other Gobioninae fishes in the upper part of the tail's hypural, the shape of spots on the dorsal vertebrae, the vertical stripes developed on the head, and the irregularly deposited melanophore throughout the body.

• Journal of Marine Life Science
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• v.9 no.1
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• pp.9-21
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• 2024

Paralytic shellfish poisoning (PSP) including Saxitoxin (STX) is caused by harmful algae, and poisoning occurs when the contaminated seafood is consumed. The mouse bioassay (MBA), a standard test method for detecting PSP, is being sanctioned in many countries due to its low detection limit and the animal concerns. An alternative to the MBA is the Neuro-2a cell-based assay. This study aimed to establish various test conditions for Neuro-2a assay, including cell density, culture conditions, and STX treatment conditions, to suit the domestic laboratory environment. As a result, the initial cell density was set to 40,000 cells/well and the incubation time to 24 hours. Additionally, the concentration of Ouabain and Veratridine (O/V) was set to 500/50 μM, at which most cells died. In this study, we identified eight concentrations of STX, ranging from 368 to 47,056 fg/μl, which produced an S-shaped dose-response curve when treated with O/V. Through inter-laboratory variability comparison of the Neuro-2a assay, we established five Quality Control Criteria to verify the appropriateness of the experiments and six Data Criteria (Top and Bottom OD, EC₅₀, EC₂₀, Hill slop, and R² of graph) to determine the reliability of the experimental data. The Neuro-2a assay conducted under the established conditions showed an EC₅₀ value of approximately 1,800~3,500 fg/μl. The intra- & inter-lab variability comparison results showed that the coefficients of variation (CVs) for the Quality Control and Data values ranged from 1.98% to 29.15%, confirming the reproducibility of the experiments. This study presented Quality Control Criteria and Data Criteria to assess the appropriateness of the experiments and confirmed the excellent repeatability and reproducibility of the Neuro-2a assay. To apply the Neuro-2a assay as an alternative method for detecting PSP in domestic seafood, it is essential to establish a toxin extraction method from seafood and toxin quantification methods, and perform correlation analysis with MBA and instrumental analysis methods.

• The Korean Journal of Nuclear Medicine
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• v.37 no.6
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• pp.382-392
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• 2003

Purpose: Uptakes of Tc-99m MIBI (MIBI) and Tc-99m tetrofosmin (tetrofosmin) in human non-small cell lung cancer A549, multidrug-resistance associated protein (MRP) expressing cell, were investigated in vitro and in vivo. Materials and Methods: Western blot analysis and immunohistochemistry were used for detection of MRP in A549 cells with anti-MRPr1 antibody. Cellular uptakes of two tracers were evaluated at $100{\mu}M$ of verapamil (Vrp), $50{\mu}M$ of cyclosporin A (CsA) and $25{\mu}M$ of butoxysulfoximide (BSO) after incubation with MIBI and tetrofosmin for 30 and 50 min at $37^{\circ}C$, using single cell suspensions at $1{\times}10^6cells/ml$. Radioactivities in supernatants and pellets were measured with gamma well counter. A549 cells were inoculated in each flanks of 24 nude mice. Group 1 (Gr1) and Gr3 mice were treated with only MIBI or tetrofosmin, and Gr2 and Gr4 mice were treated with 70mg/kg of CsA i.p. for 1 hour before injection of 370KBq of MIBI or tetrofosmin. Mice were sacrificed at 10, 60 and 240 min. Radioactivities of organs and tumors were expressed as percentage injected dose per gram of tissue (%ID/gm). Results: Western blot analysis of the A549 cells detected expression of MRPr1 (190 kDa) and immunohistochemical staining of tumor tissue for MRPr1 revealed brownish staining in cell membrane but not P-gp. Upon incubating A549 cells for 60 min with MIBI and tetrofosmin, cellular uptake of MIBI was higher than that of tetrofosmin. Coincubation with modulators resulted in an increase in cellular uptakes of MIBI and tetrofosmin. Percentage increase of MIBI was higher than that of tetrofosmin with Vrp by 623% and 427%, CsA by 753% and 629% and BSO by 219% and 140%, respectively. There was no significant difference in tumoral uptakes of MIBI and tetrofosmin between Gr1 and Gr3. Percentage increases in MIBI (114% at 10 min, 257% at 60 min, 396% at 240 min) and tetrofosmin uptake (110% at 10 min, 205% at 60 min, 410% at 240 min) were progressively higher by the time up to 240 min with CsA. Conclusion: These results indicate that MIBI and tetrofosmin are suitable tracers for imaging MRP-mediated drug resistance in A549 tumors. MIBI may be a better tracer than tetrofosmin for evaluating MRP reversal effect of modulators.

• Tuberculosis and Respiratory Diseases
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• v.40 no.3
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• pp.236-249
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• 1993

Background: Among the injurious agents to which the lung airspaces are constantly exposed are reactive species of oxygen. It has been widely believed that reactive oxygen species may be implicated in the etiology of lung injuries. In order to elucidated how this oxidant causes lung cell injury, we investigated the effects of exogenous $H_2O_2$ on alveolar epithelial barrier characteristics. Methods: Rat type II alveolar epithelial cells were plated onto tissue culture-treated polycarbonate membrane filters. The resulting confluent monolayers on days 3 and 4 were mounted in a modified Ussing chamber and bathed on both sides with HEPES-buffered Ringer solution. The changes in short-circuit current (Isc) and monolayer resistance (R) in response to the exogenous hydroperoxide were measured. To determine the degree of cellular catalase participation in protection against $H_2O_2$ injury to the barrier, experiments were repeated in the presence of 20 mM aminotriazole (ATAZ, an inhibitor of catalase) in the same bathing fluid as the hydroperoxide. Results: These monolayers have a high transepithelial resistance (>2000 ohm-$cm^2$) and actively transport $Na^+$ from apical fluid. $H_2O_2$(0-100 mM) was then delivered to either apical or basolateral fluid. Resulting indicated that $H_2O_2$ decreased Isc and R gradually in dose-dependent manner. The effective concentration of apical $H_2O_2$ at which Isc (or R) was decreased by 50% at one hour ($ED_{50}$) was about 4 mM. However, basolateral $H_2O_2$ exposure led to $ED_{50}$ for Isc (and R) of about 0.04 mM. Inhibition of cellular catalase yielded $ED_{50}$ for Isc (and R) of about 0.4 mM when $H_2O_2$ was given apically, while $ED_{50}$ for basolateral exposure to $H_2O_2$ did not change in the presence of ATAZ. The rate of $H_2O_2$ consumption in apical and basolateral bathing fluids was the same, while cellualr catalase activity rose gradually with time in culture. Conclusion: Our data suggest that basolateral $H_2O_2$ may affect directly membrane component (e.g., $Na^+,\;K^+$-ATPase) located on the basolateral cell surface. Apical $H_2O_2$, on the other hand, may be largely degraded by catalase as it passes through the cells before reaching these membrane components. We conclude that alveolar epithelial barrier integrity as measured by Isc and R are compromised by $H_2O_2$ being relatively sensitive to basolateral (and insensitive to apical) $H_2O_2$.

• The Korean Journal of Nuclear Medicine
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• v.38 no.1
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• pp.85-98
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• 2004

Purpose: Cellular uptake of $^{99}mTc$-sestamibi (MIBI) and $^{99}mTc$-tetrofosmin (TF) is low in cancer cells expressing multidrug resistance(MDR) by p-glycoprotein(Pgp) or multidrug related protein(MRP). Verapamil is known to increase cellular uptake of MIBI in MDR cancer cells, but is recently reported to have different effects on tracer uptake in certain cancer cells. This study was prepared to evaluate effects of verapamil on cellular uptake of MIBI and TF in several cancer cells. Materials and Methods: Celluar uptakes of Tc-99m MIBI and TF were measured in erythroleukermia K562 cell, breast cancer MCF7 cell, and human ovarian cancer SK-OV-3 cells, and data were compared with those of doxorubicin-resistant K562(Ad) cells. RT-PCR and Western blot analysis were used for the detection of mdr1 mRNA and Pgp expression, and to observe changes in isotypes of PKC enzyme. Effects of verapamil on MIBI and TF uptake were evaluated at different concentrations upto $200{\mu}M\;at\;1{\times}10^6\;cells/ml\;at\;37^{\circ}C$. Radioactivity in supernatant and pellet was measured with gamma counter to calculate cellular uptake ratio. Toxicity of verapamil was measured with MTT assay. Results: Cellular uptakes of MIBI and TF were increased by time in four cancer cells studied. Co-incubation with verapamil resulted in an increase in uptake of MIBI and TF in K562(Adr) cell at a concentration of $100{\mu}M$ and the maximal increase at $50{\mu}M$ was 10-times to baseline. In contrast, uptakes of MIBI and TF in K562, MCF7, SK-OV3 cells were decreased with verapamil treatment at a concentration over $1{\mu}M$. With a concentration of $200{\mu}M$ verapamil, MIBI and TF uptakes un K562 cells were decreased to 1.5 % and 2.7% of those without verapamil, respectively. Cellular uptakes of MIBI and TF in MCF7 and SK-OV-3 cells were not changed with $10{\mu}M$, but were also decreased with verapamil higher than $10{\mu}M$, resulting 40% and 5% of baseline at $50{\mu}M$. MTT assay of four cells revealed that K562, MCF7, SK-OV3 were not damaged with verapamil at $200{\mu}M$. Conclusion: Although verapamil increases uptake of MIBI and TF in MDR cancer cells, cellular uptakes were further decreased with verapamil in certain cancer cells, which is not related to cytotoxicity of drug. These results suggest that cellular uptakes of both tracers might differ among different cells, and interpretation of changes in tracer uptake with verapamil in vitro should be different when different cell lines are used.

• The Korean Journal of Nuclear Medicine
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• v.38 no.4
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• pp.325-329
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• 2004

Purpose: Usefulness of mouse liver S9 fraction was evaluated for the measurement of the metabolites in the in vitro metabolism study of $^{18}F$-labeled radiotracers. Materials and Methods: Mouse liver S9 fraction was isolated at au early step in the course of microsome preparation. The in vitro metabolism studies were tarried out by incubating a mixture containing the radiotracer, S9 fraction and NADPH at $37^{\ciirc}C$, and an aliquot of the mixture was analyzed at the indicated time points by radio-TLC. Metabolic defluorination was further confirmed by the incubation with calcium phosphate, a bone mimic. Results: The radiotracer $[^{18}F]1$ underwent metabolic defluorination within 15 min, which was consistent with the results of the in vivo method and the in vitro method using microsome. Radiotracer $[^{18}F]2$ was metabolized to three metabolites including $4-[^{18}F]fluorobenzoic$ acid within 60 min. It is likely that the one of these metabolites at the origin of radio-TLC was identical with the one that obtained from the in vivo and in vitro (microsome) method. Compared with the in vitro method using microsome, the method using S9 fraction gave a similar pattern of the metabolites but with a different ratio, which can be explained by the presence of cytosol in the S9 fraction. Conclusion: These results suggest that the findings of the in vitro metabolism studies using S9 fraction can reflect the in vivo metabolism of novel radiotracers in the liver. Moreover, this method can be used as a tool to determine metabolic defluorination along with calcium phosphate absorption method.

• Journal of Chest Surgery
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• v.41 no.1
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• pp.12-24
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• 2008

Background: It is currently thought that tissue valve degeneration is related to an animal's immune response, which is mainly due to cell surface ${\alpha}$-Gal epitopes. Cell surface ${\alpha}$-Gal epitopes are known to be degraded by the enzyme called green coffee bean ${\alpha}$-Galactosidase. It is also well known that ${\alpha}$-Gal epitopes are immunologically stained by Griffonia Simplicifolia isolectin type B4. We know that many commercially available tissue valves are made of aortic valves and pericardial tissue of pig. So, we investigated whether ${\alpha}$-Gal epitopes of the aortic valve and pericardial tissue of a pig can be removed by green coffee bean ${\alpha}$-Galactosidase, and we did so by comparing immunologic staining of the tissues before and after the enzyme treatment. Material and method: After treating fresh porcine aortic valve and pericardial tissue with green coffee bean ${\alpha}$-Galactosidase at concentrations of 0.5 unit/mL, 1.0 unit/mL, 2.0 unit/mL, respectively, under the condition of pH 6.5, temperature. $4^{\circ}C$ and 24 hours of incubation, each sample was stained with Griffonia Simplicifolia isolectin type B4 immunpfluorescent labeling. We then examined whether the ${\alpha}$-Gal epitopes were reduced or abolished in each consecutive. concentration of green coffee bean ${\alpha}$-Galactosidase by comparing the degree of the Griffonia Simplicifolia isolectin B4 staining in each sample. Result: In the pig aortic valve tissue, a 1.0 unit/mL concentration of green coffee bean ${\alpha}$-Galactosidase at pH 6.5, $4^{\circ}C$ and reaction for 24 hours was enough for complete removal of ${\alpha}$-Gal epitopes from the cell sur face on the immunostaining with Griffonia Simplicifolia isolectin B4. On the other hand, more ${\alpha}$-Gal epitopes were present in the pig pericardial tissue on Griffonia Simplicifolia isolectin B4 staining before the enzyme treatment, and 1.0 unit/mL of galactosidase was not sufficient for complete removal of ${\alpha}$-Gal from the tissue. 2.0 units/mL of green coffee bean ${\alpha}$-Galactosidase was needed to completely remove the ${\alpha}$-Gal epitopes from the pericardial tissue on immunostaining. Conclusion: The ${\alpha}$-Gal epitopes of the pig's aortic valve and pericardial tissue were successfully stained with Griffonia Simplicifolia isolectin B4. We could remove nearly all the ${\alpha}$-Gal epitopes using green coffee bean ${\alpha}$-Galactosidase at the concentration of 1.0 unit/mL in the aortic valve. Of pig, and 2.0 unit/mL was need to nearly completely remove all the ${\alpha}$-Gal epitopes in the pericardial tissue of pig under the condition of pH 6.5, $4^{\circ}C$ and 24 hours of reaction time. In the near future, removal of ${\alpha}$-Gal epitapes in the pig's aortic valve and pericardial tissue will become a powerful tool for the improvement of the tissue valve durability. It needs to be determined if ${\alpha}$-galactosidase treated pig tissue is immune to human anti-Gal antibody or anit-Gal mooclonal antibodies.

• Tuberculosis and Respiratory Diseases
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• v.44 no.2
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• pp.379-390
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• 1997

Background : It has long been suggested that neutrophils and their products are implicated as the central mediators of the acute lung injuries. Contrary to the dominant role of neutrophils in ARDS, many cases of ARDS has occurred in the setting of severe neutropenia without pulmonary neutrophil infiltration. Therefore it is certain that effector cell(s) other than neutrophil play an important role in the pathogenesis of ARDS. This experiment was performed to define the mechanism of ARDS in the setting of neutropenia, 1) by comparing the severity of endotoxin-induced lung injury, 2) by measurement of hydrogen peroxide production and cytokine concentration in the bronchoalveolar lavage cells and fluids obtained from different rats with and without cyclophosphamide-pretreatment. Method : The male Sprague-Dawleys were divided into the normal control (NC)-, endotoxin (ETX)-, and cyclophosphamide (CPA)-group in which neutropenia was induced by injecting cyclophosphamide intraperitoneally. Acute lung injury was evoked by injecting lipopolysaccharide (LPS) into a tail vein. The bronchoalveolar lavage (BAL) was performed at 3 and 6 hour after administration of LPS to measure the change of cell counts and concentrations of protein and cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Hydrogen peroxide (HPO) production from BAL cells was measured at 6 hour after LPS administration by phenol red microassay with and without zymosan stimulation. Results : The results were as follows. A change of leukocyte counts in the peripheral blood after treatment with CPA : More than 95% of total leukocytes and neutrophils were reduced after CPA administration, resulting in severe neutropenia. A change of BAL cells : In the ETX-group, the number of total cells (p < 0.01) and of macrophage and neutrophil (p < 0.05) were increased at 3 and 6 hour after LPS administration compared to those of NC-group. In the CPA-group, the number of total leukocyte and macrophage were not changed after LPS administration, but neutrophil counts were significantly reduced and it took part in less than 0.1% of total BAL cells (p < 0.01 vs NC-group). BAL cells in this group were almost all macrophages (99.7%). A change of protein concentration in the BALF : In the ETX-group, protein concentration was increased at 3 hour and was more increased at 6 hour after LPS administration (p < 0.05 and < 0.01 vs NC-group, respectively). In the CPA-group, it was also significantly elevated at 3 hour after LPS administration (p < 0.05 vs NC-group), but the value was statistically not different from that of ETX-group. The value measured at 6 hour after LPS administration in the CPA-group became lower than that of ETX-group (p < 0.05), but showed still a higher value compared to that of NC-group (p < 0.05). A change of cytokine concentration in the BALF : TNF -alpha and IL-6 were elevated in the ETX - and CPA-group compared to those of NC-group at both time intervals. There was no statistical difference in the values of both cytokines between the ETX- and CPA-groups. Measurement of hydrogen peroxide production from BAL cells : There was no intergroup difference of HPO production from resting cells. HPO production after incubation with opsonized zymosan was significantly elevated in all groups. The percent increment of HPO production was highest in the ETX-group (89.0%, p < 0.0008 vs NC-group), and was 42.85 in the CPA-group (p = 0.003 vs NC-group ). Conclusion : Acute lung injury in the setting of neutropenia might be caused by functional activation of resident alveolar macrophages.