• Applied Microscopy
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• v.28 no.1
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• pp.107-119
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• 1998

This study was designed to investigate the appearence and the characteristics of the apoptotic cells and the process of the joint cavity formation in mouse knee joint. Fetal mouse knee joints from 15 to 19 days of gestation were used. Paraffin-embedded serial sections, stained with H & E for light microscopic observation, Epon 812 embedded thin sections for electron microscopic observation and Lowicryl HM 20 embedded thin sections for immune-electron microscopic observation were prepared. Monoclonal antibodies to $\beta-tubulin$ and polyclonal antibodies to tissue transglutaminase were used for immune-electron microscopic study. The results obtained were as follows. 1. At 15 days of gestation, blood vessels, which have invaded in the mesenchymal cells, were present in the synovium, to form the joint cavity in the future. 2. At 16 days of gestation, the joint cleft was first appeared and several RBCs were present in the joint cleft. The invasion of blood vessels into the joint cleft was continuing, and apoptotic cells were present in the inner cell layer, adjacent to the joint cleft. Necrotic cells were also present in the outer cell layer; they were present 18 days of gestation, but apoptotic cells did not appear after 17 days of gestation. 3. In the apoptotic cells, transglutaminase were localized around vacuoles and the marginal site of the cytoplasm. 4. In the apoptotic cells, tubulin was around the endoplasmic reticulum and the marginal site of the cytoplasm. In the late stage of apoptotic cells, tubulin was localized diffusely in the cytoplasm. Tubulin was also strongly labeled around in the cytoplasm of the neighboring cell at which the apoptotic body was phagocytosed. Tubulin labeled particles were apparently increased in the seperated apoptotic bodies. On the basis of the above findings, it is proposed that during the development of the mouse knee joint, blood vessel invasion first occurs and then apoptosis and cell necrosis follow it. In the apoptotic cell, present in the synovium of the developing knee joint of the mouse. it is suggested that the redistribution of tubulin is associated with apoptotic process. And transglutaminase overexpressed in the apoptotic cell.

• The Journal of Korean Medicine
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• v.26 no.4
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• pp.12-21
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• 2005

Objectives: In the present study, we investigated whether an aqueous extract of Armeniacae semen induces apoptotic neuronal cell death upon mouse N2a neuroblastoma cells. Methods: 1. Cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTI) assay. 2. For in situ detection of apoptotic cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, 4,6-diamidino-2-phenylindole (DAPI) staining. 3. The fraction of cells was revealed by flow cytometric analysis used that. 4. For detection of apoptotic DNA cleavage, DNA fragmentation assay was performed. 5. For detection of bax and bcl-2, Western blot analysis was performed. 6. Caspase enzyme activity was measured using caspase-3 assay. Results: From the present results, N2a neuroblastoma cells treated with Armeniacae semen extract exhibited several characteristics of apoptosis. A treatment of Armeniacae semen extract was shown to increase the expression of Bax, a proapoptotic protein, and the treatment decreased the expression of Blc2, an anti-apoptotic protein. In addition, Armeniacae semen extract increased the caspase-3 enzyme activity. Conclusions: The present results show that Armeniacae semen extract induces apoptotic cell death in mouse N2a neuroblastoma cells.

• Journal of fish pathology
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• v.16 no.1
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• pp.39-49
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• 2003

Cytosolic oxidation by 4-hydroxy-2-nonenal (4HNE) and tert-butyl hydroperoxide (t-BHP) results in cell death of bovine aortic endothelial cells (BAEC). In this study, we have investigated the roles of antioxidants such as 2,3,6-tribromo-4,5-dihydroxy benzyl methyl ether (TDB) and phloroglucinol in preventing cell death. After treatment with oxidants for 6h, cells became compact and showed nuclear condensation, which were characteristics of early apoptosis. After l2h treatment, morphologic features including severe cytoplasm condensation, membrane blebbing, and apoptotic bodies were prominent and these findings were interpreted as characteristics of late-apoptosis. When the apoptotic cells were treated with antioxidants for 12h, both early and late apoptotic cells did show no significant change. After oxidant treated cells were incubated with antioxidant for 24h, the characteristics of early-apoptosis were eliminated but cells in lateapoptosis could not return to normal cells. These results suggest that TDB and phloroglucinol prevent the cells from dying through apoptosis induced by 4HNE and t-BHP in early stage.

• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.1
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• pp.187-192
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• 2006

Herbal medicines have been utilized to treat a variety of diseases, including cancer. Several species of the family rubiaceae have been reported to have antitumor activity. In this study, we report the cytotoxicity and antitumor activity exhibited dy the methanol extracts prepared from Rubia radix (RRME), Uncaria gambir (UGME) and Oldenlandia diffusa (ODME) (family: Rubiaceae) against human promyleloid leukemia cell line, HL-60. The cytotoxicity of RRME (2~20 ${\mu}g/ml$), UGME (20~200 ${\mu}g/ml$) and ODME (20~200 ${\mu}g/ml$) were assessed dy the MTT reduction assay. IC50 values for RRME, UGME and ODME were 11.0, 99.5 and 106.1 ${\mu}g/ml$, respectively. When the HL-60 cells were treated with RRME (10 ${\mu}g/ml$), UGME (120 ${\mu}g/ml$) and ODME (140 ${\mu}g/ml$) for 24 h, several apoptotic characteristics such as DNA fragmentation and morphologic changes were observed. Furthermore, flow cytometric analysis was peformed to determine the percent of apoptotic cells. The poupulation of sub-G1 hypodiploid cells was increased 37.49% in RRME treatment, 12.49% in UGME treatment and 7.21% in ODME treatment compared with untreated control cells (2.64%). To further confirm apoptotic cell death, we assayed caspase-3, -8 and -9 activities in RRME, UGME and ODME-treated cells. After treatment of RRME, UGME and ODME for 12 h, caspase-3, -8 and -9 activities significantly increased.compared to untreated control cells. These results show that RRME, UGME and ODME induced apoptotic cell death in HL-60 cells and may have a possibility of potential antitumor activities.

• Applied Microscopy
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• v.40 no.2
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• pp.81-88
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• 2010

The fine structural characteristics of the apoptotic cells in the cutaneous epithelium of the anuran tadpole of the black-spotted frog, Rana nigromaculata was examined using the TUNEL (Terminal deoxynucleotidyl transferase-mediated biotinylated d-Uridine triphosphate Nick End Labeling) staining technique and TEM (transmission electron microscopy) observations. The cutaneous epithelium of the tadpole was composed of stratified cuboidal cells and the apoptotic cell death was observed continuously during the tail degeneration stages from the Shumway stage number 31 to 33. The early apoptotic cells shown in the epithelium demonstrated condensation and margination of the chromatin material at the nuclear periphery, and nuclear breakdown and cytoplasmic condensation were followed. Subsequent cytoplasmic degeneration of the apoptotic cell were produced by membrane-bounded cell fragments with relatively well preserved organelles. Following the processes of autophagic degradation, the late apoptotic cells being phagocytosed by other surrounding cells. These nearby cells, presumptive intraepithelial macrophages, contain a variety of lysosomal residual bodies which fuses with other cell organelles or other cytoplasmatic material to form secondary lysosomes. They are soon transformed into lamellar shaped vesicles and finally disappeared during the process of degradation.

• Molecules and Cells
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• v.21 no.1
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• pp.1-6
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• 2006

Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is a mitochondrial pro-apoptotic protein that has a single Bcl-2 homology 3 (BH3) domain and a COOH-terminal transmembrane (TM) domain. Although it belongs to the Bcl-2 family and can heterodimerize with Bcl-2, its pro-apoptotic activity is distinct from those of other members of the Bcl-2 family. For example, cell death mediated by BNIP3 is independent of caspases and shows several characteristics of necrosis. Furthermore, the TM domain, but not the BH3 domain, is required for dimerization, mitochondrial targeting and pro-apoptotic activity. BNIP3 plays an important role in hypoxia-induced death of normal and malignant cells. Its expression is markedly increased in the hypoxic regions of some solid tumors and appears to be regulated by hypoxia-inducible factor (HIF), which binds to a site on the BNIP3 promoter. Silencing, followed by methylation, of the BNIP3 gene occurs in a significant proportion of cancer cases, especially in pancreatic cancers. BNIP3 also has a role in the death of cardiac myocytes in ischemia. Further studies of BNIP3 should provide insight into hypoxic cell death and may contribute to improved treatment of cancers and cardiovascular diseases.

• Parasites, Hosts and Diseases
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• v.42 no.4
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• pp.205-208
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• 2004

Neutrophils are important effector cells against protozoan extracellular parasite Entamoeba histolytica, which causes amoebic colitis and liver abscess in human beings. Apoptotic cell death of neutrophils is an important event in the resolution of inflammation and parasite's survival in vivo. This study was undertaken to investigate the ultrastructural aspects of apoptotic cells during neutrophil death triggered by Entamoeba histolytica. Isolated human neutrophils from the peripheral blood were incubated with or without live trophozoites of E. histolytica and examined by transmission electron microscopy (TEM). Neutrophils incubated with E. histolytica were observed to show apoptotic characteristics, such as compaction of the nuclear chromatin and swelling of the nuclear envelop. In contrast, neutrophils incubated in the absence of the amoeba had many protrusions of irregular cell surfaces and heterogenous nuclear chromatin. Therefore, it is suggested that Entamoeba-induced neutrophil apoptosis contribute to prevent unwanted tissue inflammation and damage in the amoeba-invaded lesions in vivo.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.183-187
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• 2009

In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro fertilization (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22h post insemination were treated at different concentration of actinomycin D (0, 5, 50 and 500 ng/ml in NCSU medium). Treated embryos were incubated at $39^{\circ}C$ in 5% $CO_2$, 5% $O_2$ for 8h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2days and BL development rate at 7days after in vitro culture. A significantly lower rate of cleavage was found in the 500 ng/ml group compared to others (500 ng/ml vs. 0, 5, 50 ng/ml; 27.8 % vs. 50.0%, 41.2%, 35.9%), and BL formation rate in 500 ng/ml was lower than that of others (500 ng/ml vs. 0, 5, 50 ng/ml; 8.0% vs. 12.6%, 11.2%, 12.6%). In experiment 2, to evaluate apoptotic cells, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng/ml actinomycin D). A high number of BL derived control group contained at least one apoptotic cell. Actinomycin D treated BLs responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and increase in the incidence of apoptotic cell death. In 500 ng/ml group, the incidence of apoptosis increased at 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is useful tool for the apoptosis study of porcine preimplantation embryos.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.4
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• pp.265-271
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• 2012

HoxB4, a homeodomain-containing transcription factor, is involved in the expansion of hematopoietic stem cells and progenitor cells in vivo and in vitro, and plays a key role in regulating the balance between hematopoietic stem cell renewal and cell differentiation. However, the biological activity of HoxB4 in other cells has not been reported. In this study, we investigated the effect of overexpressed HoxB4 on cell survival under various conditions that induce death, using the Ba/F3 cell line. Analysis of phenotypical characteristics showed that HoxB4 overexpression in Ba/F3 cells reduced cell size, death, and proliferation rate. Moreover, the progression from early to late apoptotic stages was inhibited in Ba/F3 cells subjected to HoxB4 overexpression under removal of interleukin-3-mediated signal, leading to the induction of cell cycle arrest at the G2/M phase and attenuated cell death by Fas protein stimulation in vitro. Furthermore, apoptotic cell death induced by doxorubicin-treated G2/M phase cell-cycle arrest also decreased with HoxB4 overexpression in Ba/F3 cells. From these data, we suggest that HoxB4 may play an important role in the regulation of pro-B cell survival under various apoptotic death environments.

• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.6
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• pp.1459-1466
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• 2006

The water extract of Hwansodan has been traditionally used for treatment of ischemic brain damage in oriental medicine. However, little is known about the mechanism by which the water extract of Hwansodan rescues cells from neurodegenerative disease. PC12 pheochromocytoma cells have been used extensively as a model for studying the cellular and molecular mechanisms of neuronal cell damages. Under deprivation of growth factor and ischemic injury, PC12 cells spontaneously undergoes apoptotic cell death. Serum and glucose deprivation markedly decreased the viability of PC12 cells, which was characterized with apparent apoptotic features such as membrane blebbing as well as fragmentation of genomic DNA and nuclei. However, the aqueous extract of Hwansodan significantly reduced serum and glucose deprivation-induced cell death and apoptotic characteristics through reduction of intracellular peroxide generation. Pretreatment of Hwansodan also ingibited the activation of caspase-3, in turn, degradation of ICAD/DFF45 was completely abolished in serum and glucose deprivated cells. Furthermore, pretreatment of Hwansodan obviously increased heme oxygenase 1 (HO-1) expression in PC12 cells. Taken together, the data suggest that the protective effects of Hwansodan against serum and glucose deprivation induced oxidative injuries may be achieved through the scavenging of reactive oxygene species accompanying with HO-1 induction.