• Journal of Korean Academy of Oral and Maxillofacial Radiology
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• 제23권1호
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• pp.27-44
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• 1993

This study was performed to investigate the changes of mandibular condyle by low calcium diet and the effects of irradiation on the bone in osteoporotic state. In order to carry out this experiment, 80 seven-week old Sprague-Dawley strain rats weighing about 150 gm were selected and equally divided into one experimental group of 40 rats and one control group with the remainder. The experimental group and the control group were then subdivided into two group and exposed to irradiation. The two irradiation groups received a single dose of 20 Gy on the jaw area only and irradiated with a cobalt-60 teletherapy unit. The rats in the control and experimental groups were serially terminated by fours on the 3rd, the 7th, the 14th, and the 21st day after irradiation. After termination, both sides of the dead rats mandibular condyle were removed and fixed with 10% neutral formalin. The bone mineral density of mandibular condyle was measured by use of dual energy X-ray bone densitometer(model DDX-alpha, Lunn Corp., U. S. A.). The mandibular condyle was radiographed with Hitex HA-80(Hitex Co., Japan). Thereafter, the obtained radiographs were observed, and the mandibular condyle was further decalcified and embedded in paraffin as the general method. The specimen sectioned and stained with hematoxylin-eosin, PAS and Rabbit Anti-Human Tumor Necrosis Factor-a observed by a light microscope. The obtained results were as follows: 1. In the non-irradiated group with the low calcium diet, the bone mineral density of the condyle was markedly decreased after 14 days, and decrease the number of trabeculae of the condyle and resorption of the calcified cartilaginous zone were observed after 3 days. On microscopic observations, the number nd size of trabeculae were decreased after 7 days of experiment. 2. In the irradiated group with the low calcium diet, the bone mineral density of the condyle was markedly decreased after 14 days and resorption of the calcified cartilaginous zone and decrease the number and coarse of the trabeculae of the condyle were observed. These findings were extended rather than in non-irradiated group with low calcium diet. On microscopic observations, many osteoclasts were detected and the number and size of trabeculae were somewhat decreased after 7 days. Also there was degenerative changes of tissues of bone marrow on the 14th day but that condition was restored on the 21st day of experiment. 3. In the irradiated group with normal diet, the bone mineral density of the condyle was somewhat decreased with times and degree of decrease of the number of trabeculae was somewhat larger than in the non-irradiated group with normal diet. On microscopic observations, the tissues of bone marrow were atrophic and degenerative changes but that condition was restored on the 21st day of experiment. 4. In immunocytochemical findings, in the irradiated and non-irradiated groups with low calcium diet, negative or partial positive response to TNF was observed, but positive response in the normal diet groups.

• Development and Reproduction
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• 제15권1호
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• pp.41-52
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• 2011

In the present study, embryoid bodies (EBs) obtained from induced pluripotent stem cells (iPSCs) were induced to differentiate into germ lineage cells by treatment with bone morphogenetic protein 4 (BMP4) and retinoic acid (RA). The results were compared to the results for embryonic stem cells (ESCs) and multipotent spermatogonial stem cells (mSSCs) and quantified using immunocytochemical analysis of germ cell-specific markers (integrin-${\alpha}6$, GFR-${\alpha}1$, CD90/Thy1), fluorescence activating cell sorting (FACS), and real time-RT-PCR. We show that the highest levels of germ cell marker-expressing cells were obtained from groups treated with 10 ng/$m{\ell}$ BMP4 or 0.01 ${\mu}M$ RA. In the BMP4-treated group, GFR-${\alpha}1$ and CD90/Thy-1 were highly expressed in the EBs of iPSCs and ESCs compared to EBs of mSSCs. The expression of Nanog was much lower in iPSCs compared to ESCs and mSSCs. In the RA treated group, the level of GFR-${\alpha}1$ and CD90/Thy-1 expression in the EBs of mSSCs Induced pluripotent stem cells, Mouse embryonic stem cells, Multipotent spermatogonial stem cells, Germ cell lineage, Differentiation potential. was much higher than the levels found in the EBs of iPSCs and similar to the levels found in the EBs of ESCs. FACS analysis using integrin-${\alpha}6$, GFR-${\alpha}1$, CD90/Thy1 and immunocytochemistry using GFR-${\alpha}1$ antibody showed similar gene expression results. Therefore our results show that iPSC has the potential to differentiate into germ cells and suggest that a protocol optimizing germ cell induction from iPSC should be developed because of their potential usefulness in clinical applications requiring patient-specific cells.

• Journal of Embryo Transfer
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• 제25권2호
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• pp.103-110
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• 2010

The first morphogenetic event of preimplantation development, compaction, was required efficient production of porcine embryos in vitro. Compaction of the porcine embryo, which takes place at post 4-cell stage, is dependent upon the adhesion molecule E-cadherin. The E-cadherin through ${\beta}$-catenin contributes to stable cell-cell adhesion. Rho-associated kinase (ROCK) signaling was found to support the integrity of E-cadherin based cell contacts. In this study, we traced the effects of ROCK-1 on early embryonic development and structural integrity of blastocysts in pigs. Then, in order to gain new insights into the process of compaction, we also examined whether ROCK-1 signaling is involved in the regulation of the compaction mediated by E-cadherin of cellular adhesion molecules. As a result, real-time RT-PCR analysis showed that the expression of ROCK-1 mRNA was presented throughout porcine preimplantation stages, but not expressed as consistent levels. Thus, we investigated the blastocyst formation of porcine embryos treated with LPA and Y27632. Blastocysts formation and their qualities in LPA treated group increased significantly compared to those in the Y27632-treated group (p < 0.05). Then, to determine whether ROCK-1 associates embryonic compaction, we explored the effect of activator and/or inhibitor of ROCK-1 on compaction of embryos in pigs. The rate of compacted morula in LPA treated group was increased compared to that in the Y27632-treated group (39.7 vs 12.0%). Furthermore, we investigated the localization and expression pattern of E-cadherin at 4-cell stage porcine embryos in both LPA- and Y27632-treated groups by immunocytochemical analysis and Western blot analysis. The expression of E-cadherin was increased in LPA-treated group compared to that in the Y27632-treated group. The localization of E-cadherin in LPA-treated group was enriched in part of blastomere contacts compared to that Y27632-treated group. ROCK-1 as a crucial mediator of embryo compaction may plays an important role in regulating compaction through E-cadherin of the cell adhesion during the porcine preimplantation embryo. We concluded that ROCK-1 gene may affect the developmental potential of porcine blastocysts through regulating embryonic compaction.

• Journal of Pharmacopuncture
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• 제4권1호
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• pp.123-124
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• 2001

Purpose. Free radicals are implicated in the pathophysiology of aging, ischemic injury and neurodegenerative disorders. To deform]no whether Hominis Placenta extract prevents $H_2O_2$-induced apoptosis, we have performed morphological and biochemical analyses for the detection of apoptotic phenomena in the pineal tumor cell line $PGT-{\beta}$ We have also peformed cytochemical and immunocytochemical analyses for the detection of changes in nitric oxide synthase (NOS) activity and estimated the expression . of apoptotic genes using reverse transcription-polymerase chain reaction (RT-PCR) Methods. $PGT-{\beta}\;cells$ were pretreated with Hominis Placenta extracts $(0,\;10^{-2}\;{\mu}g/ml)$ for 2 hours and then exposed to $H_2O_2\;(0,\;50\;{\mu}M)$ for 3 hours. Appearance of apoptotic characteristics were monitored using 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) staining assay, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay and flow cytometric analysis. NOS activity was measured by NADPH-diaphorase cytochemistry. Expression of inducible NOS (iNOS) and nuclear factor kappa B (NF k B) was assessed via immunocytochemistry. The expression of apoptotic genes was examined by RT-PCR. Results. After 3 flours of exposure to $H_2O_2$, it was shown that $PGT-{\beta}\;cells$ treated with $H_2O_2(50\;{\mu}M)$ exhibit classical apoptotic features and increases in NOS activity and caspase-3 expression. Treatment with Hominis Placenta extract resulted in a reduced occurrence of apoptotic features. DAPI staining, TUNEL and flow cytometric assays revealed decreases in the occurrence of nuclear fragmentation and in the sub-Gl fraction in the $PGT-{\beta}\;cells$ treated with Hominis Placenta extract. Cells treated with Hominis Placenta extract also showed lower activity of NADPH-diaphorase and immunoreactivities of both iNOS and NF k B than those of $H_2O_2$-treated cells which were not treated with Hominis Placenta extract. By RT-PCR, it was shown that the level of caspase-3 mRNA was derreased In the cells treated with Hominis Placenta . extract. Conclusions. This study shows that Hominis Placenta extract prevents $H_2O_2$-induced apoptosis in $PGT-{\beta}\;cells$; inhibitions of iNOS and caspnse-3 are possible mechanisms of the protection against apoptosis.

• The Korean Journal of Zoology
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• 제36권4호
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• pp.439-451
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• 1993

Protein kinase C isozymes in a human prostate adenocarcinoma PC-3 cell line were characterized. Immunoreactive bands and immunocytochemical stains were obsenred in PC-3 cells with antibodies raised against protein kinase C ${\alpha}$, ${\beta}$, ${\gamma}$, $\delta$, $\varepsilon$, and ζ types, respectively. Protein kinase C ${\alpha}$ corresponded to a immunoreactive band at a molecular weight of 80,000-dalton, whereas molecular weights of other immunoreactive isozvmes of protein kinase C were detected at 68,000-dalton. Protein kinHse C $\delta$ and ζ antibodies detected additional bands at 55,000-dalton and 80,000-dalton, respectively Immunocvtochemical study confirmed the results of the immunoblotting experiments qualitatively: all six protein kinase C isozymes were detected in the cytoplasm of PC-3 cells. Translocation of protein kinase C in PC-3 cells were also examined with phorbol 12-myristate 13-acetate (PMA), bryostatin 2, diolein, and 1-oleoyl-2-acetyl glycerol (OAG). Differential reactions of protein kinase C isozvmes to these activators were obsenred. When PC-3 cells were treated with 10mM bryostatin 2, protein kinase C isozyme u was translocated into the nucleus, whereas s type was translocated into the plasma membrane and the nucleus. Protein kinase C ${\alpha}$ and ζ types were translocated into the nucleus following the treatment with 101M diolein, whereas protein kinase C ${\alpha}$, ${\beta}$, ${\gamma}$, and $\varepsilon$ types were translocated into the nucleus by the treatment with 10mM OAG. Protein kinase C ${\alpha}$ and $\varepsilon$ types were translocated into the nucleus in the presence of 100nM PMA. Protein kinase C $\delta$ type was translocated to the nuclear membrane by these activators, however, only PMA-induced translocation was inhibited by protein kinase C inhibitor, 1-(5-isoquinolinesulfonyll-2-methvlpiperazine dihvdrochloride (H7) . H7 inhibited translocation of protein kinase C ${\alpha}$ type induced by PMA, ${\beta}$ type by OAG and s type by PMA and OAG, whereas it did not affect translocations induced by bryostatin and diolein, respectively. These results suggest that there exist six isoformes of protein kinase C (${\alpha}$, ${\beta}$, ${\gamma}$, $\delta$, $\varepsilon$ and ζ types) in PC-3 cells and that each of these isozvmes distinctivelv reacts to bryostatin, diolein, OAG and PMA, in part due to an altered molecular size and conceivably discrete binding site(s).

• Journal of Life Science
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• 제15권4호
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• pp.607-612
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• 2005

Heat shock protein 70 (HSP70) is a multigene family composed of constitutively expressed members(Hsc70) and stress-inducible members (Hsp70). In the mammalian nervous system, a considerable amount of HSPs is also synthesized under normal conditions suggesting that they play an important role in the metabolism of unstressed cells. In this study we examined the expression of Hsp70 in the synapses of rat cerebellar neurons. Immunohistochemistry using specific antibodies revealed that both Hsp70 and Hsc70 are expressed in the cerebellar tissue, with strongest expression in Purkinje cells followed by granule cells. Neurons in deep cerebellar nuclei were also intensely stained by Hsp70 antibody. Immunocytochemical stainings of cultured cerebellar cells showed that Hsp70 is expressed in both Purkinje and granule cells. The expression was punctate in the soma and along dendritic trees, and the punctae were colocalized with those of PSD95, a postsynaptic marker. Immunoblotting also indicates that Hsp70 is associated with the postsynaptic density fraction. Taken together, our results indicate that the Hsp70 is expressed in cerebellar neurons in normal conditions, and that some are localized in the synapses.

• Korean Journal of Veterinary Research
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• 제36권4호
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• pp.845-858
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• 1996

The purpose of this study was to establish early diagnostic methods for the detection of Aujeszky's disease viral antigens and nucleic acid in nasal cells, and buffy coats from experimentally infected living pigs by a combination of immunocytochemistry, in situ hybridization with digoxigenin(DIG)-labled probe and electron microscopy. Forty days old piglets were inoculated intranasally with $10^{7.0}TCID_{50}$ of Aujeszky's disease virus (ADV, NYJ-1-87 strain). The viral antigens and nucleic acid of ADV were detected in nasal cells, and buffy coat for 20 days after inoculation by immunocytochemistry, in situ hybridization with DIG-labeled probe and electron microscopical method. The results were compared with conventional methods such as a porcine Aujeszky's disease serodiagnostic(PAD) kit, neutralization test(NT) and virus isolation. 1. The viral antigens, nucleic acids and capsids of ADV were detected in nasal cells, buffy coats from 3 days to 20 days after inoculation by immunocytochemistry, in situ hybridization with DIG-labeled probe and electron microscopy, respectively. 2. When viral antigens were detected by the immunocytochemical technique, a diffuse brown deposit was observed in the nucleus and cytoplasm of nasal cells, buffy coats and PK-15 cells under a microscope. 3. DIG-labeled DNA probe was prepared by amplification of conserved sequence of recombinant ADV-gp50 clone with polymerase chain reacction. When ADV-DNA was detected by ISH with DIG-labeled probe, purplish blue pigmentation were observed in the nuclei and cytoplasms of ADV-infected cells under a microscope. Positive signals were observed in nasal cells and in the buffy coat and PK-15 cells at the first day after inoculation. 4. Where ADV-capsids were detected by transmission electron microscopical method, aggregation of capsids was observed in the nuclei and cytoplasms of nasal cells, buffy coats and PK-15 cells. The results suggested that these methods were considered as the highly sensitive and reliable tools for rapid and confirmative diagnosis of Aujeszky's disease in living pigs.

• Clinical and Experimental Pediatrics
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• 제45권2호
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• pp.247-255
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• 2002

Purpose : This study was undertaken to obtain basic data about the megakaryocyte colony formation of fetal liver cells by using immunocytochemical staining and ex vivo culture with growth factors. Methods : The mononuclear cells were isolated from fetal liver and bone marrow with idiopathic thrombocytopenic purpura(ITP) and pancytopenia. These mononuclear cells were cultured in $MegaCult^{TM}-C$(Stem Cell Tech, Canada) media in the presence of growth factors and CFU-Megakaryocyte( CFU-Mk) colonies were counted on day 12. The expansion of CD34+ and CD41+ cell was analyzed by flow cytometry after 5 days incubation using flask culture. Results : The numbers of CFU-Mk colonies of mononuclear cells obtained from fetal liver in the 11th week gestational age were more than those in the 19th week specimens; growth factors could not enhance the colony expansion in all cases. Total numbers of CFU-Mk colony of fetal liver cells were higher than bone marrow from ITP or pancytopenia groups. The numbers of pure or large CFU-Mk colonies of fetal liver cells were also higher than bone marrow specimens. The rate of CD34+ cell expression of fetal liver was increased after flask culture and the enhancement effect of epression was seen only in cases which added thrombopoietin. The rate of CD41+ cell expression of fetal liver was increased after incubation, but the enhancement effect of growth factors was unclear. Conclusion : This study revealed good results about the megakaryocyte colony assay of fetal liver mononuclear cells using $MegaCult^{TM}-C$ media. This study suggests that the fetal liver could be a good source of megakaryocytic progenitor cells for clinical application in hematopoietic stem cell transplantation.

• Journal of the korean academy of Pediatric Dentistry
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• 제29권2호
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• pp.243-254
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• 2002

For the purpose of evaluating the biocompatability of 3 kinds of metallic materials frequently used in pediatric dentistry (stainless steel crown, orthodontic band, orthodontic wire), cellular and molecular studies, including cell growth and proliferation, screening of cell death with determination of types whether necrosis or apoptosis and changes in expressions of related signaling molecules were examined, using cultured human gingival fibroblasts (HGF-1), HGF-1 was cultured in Dulbecco's modified Eagle's medium. among which the 3rd to 6th generations of HGF-1 were used. The specimen were divided into stainless steel crown (R), band (B) and wire (W). The immunocytochemical study was done for the detection of anti-PCNA (proliferating cell nuclear antigen) labeling. With extracted protein, western blot was done for the detection of ERK1/2, JNK, and p38, using individual antibodies. Cultured cells proliferated, remarkably till 7 day and slightly at 11 day. There was no statistical significance in the counts of proliferating HGF-1 between control and experimental groups (p>0.05). Relative growth rates were no statistically significant difference between control and experimental groups (p>0.05). PCNA labeling indexes showing similar patterns in control and experimental groups. The expressions of ERK1 and ERK2, p38 were similar in control and experimental groups. The expression of JNK increased at 1st day, slightly decreased at 4th day and markedly increased at 7th and 11 day. Although the patterns of control and experimental groups were similar, the increased expressions of JNK at late period suggest a possible stress due to inhibited cell growth and proliferation, and worse culture condition. Conclusively, the 3 kinds of metal specimens used in this study did not induce cellular and molecular hazards during short term culture of HGF-1. But, for the better clinical stability, the establishment of long period culture and animal experiment was thought necessary.

• Applied Microscopy
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• 제30권4호
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• pp.377-387
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• 2000

There are a few tanycytes between the general ependymal cells lining the ependymal layer of the brain ventricle. These cells are considered as modified ependymal cells which possess a long basal process. Tanycytes are known to have an ability to communicate by absorbing substances from cerebrospinal fluid and transporting them to the blood vessels and/or to the neurons in the CNS. The third and fourth ventricular tanycytes were mainly studied as subjects but it's rare to find reports about the tanycytes of the area postrema. Glial fibrillary acidic protein is an intermediate filament protein that is expressed especially in astrocytes of the CNS. But GFAP is also found in filament of the tanycytes and its process. Therefore this study was carried out for the examination of the GFAP immunoreactive tanycytes lining the area postrema of the bat, and we also examined the ultrastructure of tanycytes using electron microscope. GFAP immunoreactive tanycytes were located in the caudal portion of the fourth ventricle, and especially mainly in the transitional zone between the floor of the caudal fourth ventricle and ependymal layer lining the area postrema. A few GFAP immunoreactive tanycytes were also found in the ependymal layer lining the area postrema, and some groups of tanycytes were found in the ependymal layer of the area postrema near the floor of the caudal fourth ventricle , The processes of tanycytes were stained deeply with anti-GFAP antibody. Especially the GFAP immunoreactive tanycytes lining the area postrema had very long processes that cross the whole width of the area postrema. In the electron microscope, the cell body of ependymal tanycyte was located on the ependymal layer and had a long basal process. Intermediate filaments were observed around the nucleus and well developed in the process of tanycrte. Longitudinal oriented long mitochondria and a few lipid droplets were also found in this process. After immunocytocheical staining, the gold particles were found only in the intermediate filaments. We could not determine the function of the tanycytes in the area postrema. Thus, further investigation is required to determine the functional relationship between the tanycytes and the area postrema in hibernating animal, the bat.