• Applied Microscopy
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• v.27 no.2
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• pp.197-215
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• 1997

A morphologic study on the effect of butylated hydroxytoluene (BHT) on experimental hepatocarcinogenesis induced by 3'-methyl-4-dimethyl aminoazobenzene (3'-MeDAB) was investigated. A total of 110 Sprague-Dowley male rats weighting about 200 g each were used for the experiment, and divided into 4 groups; the 3'-MeDAB, BHT, 3'-MeDAB/BHT treated group, and the control group. Four to eight rats of each group were sacrified on the 4th, 8th, 14th and 16th experimental weeks, with continuous pelletized feeding containing 0.09% 3'-MeDAB and 0.5% BHT. The liver was examined by transmission and scanning electron microscopy. The results were as follows; Electron microscopically, the fine structure of the hepatocytes remained consistently abnormal up to 16 weeks after the 3'-MeDAB treatment. There was no significant difference in the groups observed earlier than in the ones observed later. Many subcellular changes were observed : nuclear change, decreased glycogen, mitochondrial abnormalities, disaggregated rough endoplasmic reticulum, marked proliferation of smooth endoplasmic reticulum, dilatation and distortion of bile canaliculi, increased lysosomes, apoptotic bodies, migration of bile ductule cell. In the BHT treated group, the ultrastructural changes of hepatocytes were not significant, except for the lipid droplets and proliferated smooth endoplasmic reticulum among hepatocytes depending on the experimental duration. The various subcellular changes of 3'-MeDAB/BHT treated groups were simillar to those of the 3'-MeDAB treated group, but the degree of changes in the 3'-MeDAB/ BHT treated group decreased compared with those of the 3'-MeDAB treated group. These results suggest that dietary butylated hydroxytoluene has a protective/inhibitory effect on the hepatocarcinogenesis induced by 3'-methyl-4-dimethyl -aminoazobenzene.

• Applied Microscopy
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• v.33 no.1
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• pp.79-92
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• 2003

Ultrastructural changes of the pericarp in Citrus reticulata has been investigated during hesperidium abscission. The pericarp was composed of compactly arranged parenchyma cell layers during early stages of fruit development. The outermost exocarp was green and active in photosynthesis. However, cells in the exocarp soon changed into collenchyma cells by developing unevenly thickened walls within a short time frame. As the fruit approached maturation, the chlorophyll gradually disappeared and chloroplasts were transformed into carotenoid-rich chromoplasts. In the mature fruit the exocarp consisted of large, lobed collenchyma cells with primary pit fields and numerous plasmodesmata. The immature mesocarp was a relatively hard and thick layer, located directly under the exocarp. With development, the deeper layers of the exocarp merged into the white, spongy mesocarp. Before separation of the hesperidium from the plant, some unusual features were detected in the plasma membrane of the exocarp cells. The number of small vacuoles and dark, irregular osmiophilic lipid bodies also increased enormously in the exocarp collenchyma after the abscission. They occurred between the plasma membrane and the wall, and invaginated pockets of the plasma membrane containing double-membraned vesicles were also frequently noticed. The lipid bodies in the cytoplasm were often associated with other organelles, especially with plastids and mitochondria. The plastids, which were irregular or amoeboid in shape, contained numerous large lipid droplets, and occasional clusters of phytoferritin, as well as few loosely -oriented peripheral lamellae. Myelin-like configurations of membrane were frequently observed in the vacuoles, as was the association of lipid bodies with the vacuolar membrane. Most vacuoles had an irregular outline, and lipid bodies were often connected to the tonoplast of the vacuoles. The structural changes underlying developmental, particularly to senescence, processes in various hesperidium will be reported in the separate paper.

• Parasites, Hosts and Diseases
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• v.26 no.3
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• pp.179-188
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• 1988

The life cycle of Fibricola seoulensis was studied in the laboratory and in the field, with special interests in the larval developments within the eggs and in the intermediate hosts. The first emergence of miracidia after incubation of eggs in 26C water began on the ninth day. The miracidia, elongate and cylindrical shape, had epidermal plates in the formula of 6, 9, 4 and 3, with two pairs of flame cells and lateral processes. A kind of fresh water snail, Hippeutis (H.) cantori, was found to shed furcocercous cercariae from the 13th day after experimental challenge with miracidia while Physa acute failed to shed. The same kind of snail collected from the field also shed the same cercariae. The cercariae were equipped with 2 pairs of penetration glands and 5 pairs of fame cells. The tadpoles of Rana nigromaculata were found susceptible to experimental infection with the cercariae. The same kind of tadpoles collected from various areas were also found naturally infected. The metacercariae in the tadpoles which were infected experimentally became infective to the definitive host in 21 days. The metacercariae were located free in the body cavity of tadpoles, and attained sexual maturity in rats in 7 days. The present study successfully followed the complete life cycle of F. seoulensis and found that it is possible to maintain the life cycle in the laboratory.

• Natural Product Sciences
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• v.5 no.3
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• pp.113-120
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• 1999

Nitric oxide (NO) is a free radical synthesized from L-arginine by nitric oxide synthase (NOS). It is believed that NO is an important mediator in numerous physiological and inflammatory responses. Particularly, a large amount of NO released from the inducible nitric oxide synthase (iNOS) is mostly associated with inflammatory processes. Overproduction of NO in these processes including sepsis and autoimmune diseases can have deleterious consequences and pathophysiologic relevance. Therefore, for the discovery of new inhibitory agents against iNOS activity, we have evaluated about 100 kinds of natural products after partition into three layers (n-hexane, ethyl acetate and aqueous) from 100% methanol extracts to study inhibitory effects on iNOS activity induced by lipopolysaccharide (LPS) in RAW264.7 cells culture system. As a positive control, curcumin, which is known as an anti-tumor promoter, anti-inflammatory agent as an iNOS inhibitor, was used and showed the dose-dependent inhibitory effect $(IC_{50},\;2.5\;{\mu}g/ml)$. Among tested fractions, the n-hexane fraction of Cimicifuga heracleifolia $(IC_{50}:\;9.65\;{\mu}g/ml)$, Forsythiae fructus $(IC_{50}:\;6.36\;{\mu}g/ml)$, Saposhnikovia divaricata $(IC_{50}:\;5.92\;{\mu}g/ml)$, and the ethyl acetate fraction of Chrysanthemum sibiricum $(IC_{50}:\;2.56\;{\mu}g/ml)$, Gastrodia elata $(IC_{50}:\;3.46\;{\mu}g/ml)$, and the aqueous fraction of Dianthus chinensis $(IC_{50}:\;6.73\;{\mu}g/ml)$, Euonymus alatus $(IC_{50}:\;6.78\;{\mu}g/ml)$, Mechania urticifoloria $(IC_{50}:\;8.01\;{\mu}g/ml)$ showed strong inhibitory activity against LPS-stimulated iNOS. Especially, the ethyl acetate fraction of Chrysanthemum sibiricum $(IC_{50}:\;2.56\;{\mu}g/ml)$, which exhibited the strongest inhibition against iNOS, was fractionated with silica-gel column chromatography. These subfractions exhibited dose-dependent inhibition against iNOS activity in the range of $2.59-5.6\;{\mu}g/ml$ except for fraction No. 3, 4, 5, 6, 8, 9, and 16. Our study shows that Chrysanthemum sibiricum has the strongest inhibitory effect against iNOS activity and has similar effect to curcumin. Therefore, further studies for the identification of active principles from Chrysanthemum sibiricum and investigation for the mechanism of the inhibition of iNOS by active principles will be performed.

• Journal of Chest Surgery
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• v.36 no.4
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• pp.219-228
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• 2003

Background:Liquid nitrogen freezing techniques have already met with widespread success in biology and medicine as a means of long-term storage for cells and tissues. The use of cryoprotectants such as glycerol and dimethylsulphoxide to prevent ice crystal formation, with carefully controlled rates of freezing and thawing, allows both structure and viability to be retained almost indefinitely. Cryopreservation of various tissues has various con-trolled rates of freezing. Material and Method: To find the optimal freezing curve and the chamber temperature, we approached the thermodynamic calculation of tissues in two ways. One is the direct calculation method. We should know the thermophysical characteristics of all components, latent heat of fusion, area, density and volume, etc. This kind of calculation is so sophisticated and some variables may not be determined. The other is the indirect calculation method. We performed the tissue freezing with already used freezing curve and we observed the actual freezing curve of that tissue. And we modified the freezing curve with several steps of calculation, polynomial regression analysis, time constant calculation, thermal response calculation and inverse calculation of chamber temperature. Result: We applied that freezing program on mesenchymal stem cell, chondrocyte, and osteoblast. The tissue temperature decreased according to the ideal freezing curve without temperature rising. We did not find any differences in survival. The reason is postulated to be that freezing material is too small and contains cellular components. We expect the significant difference in cellular viability if the freezing curve is applied on a large scale of tissues. Conclusion: This program would be helpful in finding the chamber temperature for the ideal freezing curie easily.

• Korean Journal of Veterinary Research
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• v.30 no.4
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• pp.373-381
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• 1990

Endocrine cells in the cardiac and fundic regions of the Korean hedgehog were studied ultrastructurally. Five types of endocrine cells classified as EC, ECL, $D_1$. G and A-like cells were identified in these regions. EC cells contained pleomorphic granules, 170~500nm in diameter, with high electron density and highly dense bodies in a dense matrix. ECL cells were characterized by the presence of round or oval granules, 220~450nm in diameter, with high electron density. Some granules of ECL cells showed a small amounts of contents or empty. $D_1$ cells contained round and relatively small granules, 140~400nm in diameter, with low to moderate electron density. G cells were characterized by the presence of round or oval granules, 200~400nm in diameter, with moderate electron density. Some granules of these cells showed a narrow halo between the limiting membrane and the granular matrix. A-like cells contained round granules, 170~260nm in diameter, With high electron density. The granules of these cells showed homogeneous matrix surrounded by the tight membrane.

• Molecules and Cells
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• v.39 no.2
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• pp.103-110
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• 2016

As a degenerative joint disease, osteoarthritis (OA) constitutes a major cause of disability that seriously affects the quality of life of a large population of people worldwide. However, effective treatment that can successfully reverse OA progression is lacking until now. The present study aimed to determine whether two small non-coding RNAs miR-29a and miR-140, which are significantly down-regulated in OA, can be applied together as potential therapeutic targets for OA treatment. MiRNA synergy score was used to screen the miRNA pairs that potentially synergistically regulate OA. An in vitro model of OA was established by treating murine chondrocytes with IL-$1{\beta}$. Transfection of miR-29a and miR-140 via plasmids was investigated on chondrocyte proliferation and expression of nine genes such as ADAMTS4, ADAMTS5, ACAN, COL2A1, COL10A1, MMP1, MMP3, MMP13 and TIMP metallopeptidase inhibitor 1 (TIMP1). Western blotting was used to determine the protein expression level of MMP13 and TIMP1, and ELISA was used to detect the content of type II collagen. Combined use of miR-29a and miR-140 successfully reversed the destructive effect of IL-$1{\beta}$ on chondrocyte proliferation, and notably affected the MMP13 and TIMP1 gene expression that regulates extracellular matrix. Although co-transfection of miR-29a and miR-140 did not show a synergistic effect on MMP13 protein expression and type II collagen release, but both of them can significantly suppress the protein abundance of MMP13 and restore the type II collagen release in IL-$1{\beta}$ treated chondrocytes. Compared with single miRNA transfection, cotransfection of both miRNAs exceedingly abrogated the suppressed the protein production of TIMP1 caused by IL-$1{\beta}$, thereby suggesting potent synergistic action. These results provided1novel insights into the important function of miRNAs' collaboration in OA pathological development. The reduced MMP13, and enhanced TIMP1 protein production and type II collagen release also implies that miR-29a and miR-140 combination treatment may be a possible treatment for OA.

• The Journal of Korean Society of Virology
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• v.27 no.1
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• pp.19-27
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• 1997

To develop the polymerase chain reaction (PCR) for the detection of type D simian retrovirus (SRV) infection, an oligonucleotide primer pair was designed to hybridize to the sequences within env gene of SRV subtype 1 (SRV-1). The 3' proximal env sequences annealing to the primers had been rather conserved among three different subtypes of SRV, SRV-1, SRV-2, and SRV-3 (Mason-Pfizer Monkey Virus: MPMV). The PCR using the primer pair targeting an env region successfully detected and amplified all three subtypes of SRV with excellent specificity after single round of reaction. The tests with peripheral blood mononuclear cells infected either with simian immunodeficiency virus or simian T-Iymphotropic virus type 1, major immunosuppressive viral agents together with SRV in simian, verified the specificity of the PCR by excluding any cross reactivity. Semiquantitative titration PCR, amplifying serially diluted plasmid DNA of each subtype, was performed to evaluate sensitivity limits of the reaction. Based on molecular weight of each cloned SRV genome, the PCR should be able to detect one SRV-infected cell per more than $5-7{\times}10^4$ uninfected cells after simple ethidium bromide staining of resulting products. The PCR must be very efficient screening system with its quickness, certainty, and sensitivity for SRV-infected animals used in human AIDS research model. Second round amplification of the reaction products from the first PCR, or Southern hybridization by radiolabeled probes shall render to compete its efficacy to ELISA which has been the most sensitive technique to screen SRV infection but with frequent ambiguity problem.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.153-159
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• 2007

In this study, using FIV-based lentivirus vector system, we tried to express hG-CSF in tetracycline-controllable manner. hG-CSF influences the proliferation, differentiation, and survival of cells in the neutrophil lineage. To enhance stability and translation of hG-CSF transcript, WPRE sequence was also introduced into FIV-Tet-On vector at downstream region of either the hG-CSF gene or the sequence encoding rtTA. Primary culture cells (CEF, chicken embryonic fibroblast; PFF, procine fetal fibroblast) infected with the recombinant FIV were cultured in the medium supplemented with or without doxycycline for 48 hours, and induction efficiency was measured by comparing the hG-CSF gene expression level using quantitative real-time PCR, Western blot and ELISA. Higher hG-CSF expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hG-CSF (in CEF) or rtTA (in PEE) gene. This FIV-Tet-On vector system may be helpful in solving serious physiological disturbance problems which has continuously hampered successful production of transgenic animals and gene therapy.

• Development and Reproduction
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• v.19 no.4
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• pp.243-252
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• 2015

The pregnancy and abortion process involves a complex mechanism with various immune cells present in the implantation sites and several hormones associated with pregnancy, such as leptin, ghrelin and nesfatin-1. However, the mechanism underlying spontaneous abortion by maternal T helper 17 (Th17) present in the implantation sites and nesfatin-1, which is of anorexigenic hormones, is not fully understood so far. Therefore, the purpose of this study was to examine the possible roles of Th17 cells present in the implantation sites and nesfatin-1 expressed in the uterus on spontaneous abortion using the $CBA/j{\times}DBA/2$ mouse model. Th17 transcription factor, ROR-${\gamma}t$ mRNA expression was significantly increased in the abortion sites compared with the implantation sites of abortion model mice on day 14.5 and 19.5 of pregnancy. In addition, the expression levels of IL-17A mRNA were significantly higher in abortion sites than in implantation sites on day 14.5 and 19.5. Moreover, the nesfatin-1/NUCB2 protein and mRNA levels were increased in abortion sites compared with levels in implantation sites of both normal pregnant and abortion model mice on day 14.5 of pregnancy. Interestingly, nesfatin-1/NUCB2 serum levels were not changed throughout the whole pregnancy in abortion model mice, but its serum level was dramatically increased on day 14.5, and then rapidly decreased on day 19.5 in normal pregnant mice. In this study, we showed for the first time the expression of nesfatin-1/NUCB2 mRNA and protein in implantation sites during pregnancy. The present results suggest that Th17 cells in the uterus may play an important role in the period of implantation and for maintenance of pregnancy. Furthermore, the present results suggest that Th17 cells in implantation sites may be a key regulator for maintenance of pregnancy and provides evidence that activation of these cells may be regulated by nesfatin-1/NUCB2. Further study is needed to elucidate the role of nesfatin-1 expressed in the uterus during pregnancy.