No abstract available.
Animals* ; Brain* ; Hypothermia* ; In Situ Nick-End Labeling* ; Models, Animal*

PURPOSE: The purposes of this study were to examine whether meniscal degeneration in human osteoarthritis(OA) was related with the occurrence of apoptosis, the expression of nitrotyrosine and Fas. MATERIALS AND METHODS: Menisci were obtained from OA patients undergoing total knee replacement arthroplasty and from normal subjects who were operated an above knee amputaton. According to histologic degeneration, menisci were graded to normal, grade 1(mild), grade 2(moderate), and grade 3(severe). Apoptotic cells were identified by TUNEL method and electron microscopy. Meniscal sections were analyzed by immunohistochemistry for the presence of nitrotyrosine and Fas expression. RESULTS: The number of apoptotic cells were significantly increased in OA meniscus compared with normal meniscus(p < 0.05). The number of apoptotic cells were increased with tissue degeneration. On electron microscopy, the typical chromatin condensation in the OA meniscus was shown in apoptotic cell. The number of Fas-expressing cells was significantly higher in the OA meniscus(p < 0.05). Nitrotyrosine immuno reactivity was prominent in the degenerative menisci(p < 0.05). Fas and nitrotyrosine expression were increased with degree of tissue degeneration. An increase in number of apoptotic cells was correlated with tissue degeneration but not with age . CONCLUSION: Apoptosis was suggested as one of the causes in the tissue degeneration of the human OA meniscus. The development of apoptosis in the meniscus may be related with Fas and nitrotyrosine expression but not with age.
Apoptosis* ; Arthroplasty ; Arthroplasty, Replacement, Knee ; Chromatin ; Humans* ; Immunohistochemistry ; In Situ Nick-End Labeling ; Knee* ; Microscopy, Electron

We studied the mechanism and inhibition of cell death by exposure to UV alone or combination of UV exposure and intracellular zinc depletion with TPEN in cultured human retinal pigment epithelial cells (RPE). Cell death was quantified by measuring LDH release from injured cells. RPE were exposed to UV at 253.7 nm for 1~20 minutes.The 2~5 minutes UV exposure was duration-dependently cytotoxic, whereas 1minute exposure was minimally so.And exposure to TPEN induced concentration-dependent cell death at 1~4 micrometer range ;0.5 micrometer TPEN was minimally toxic.Then, cultures were exposed to varying exposure durations of UV in the absence or presence of 0.5 micrometer TPEN.At any point, the presence of TPEN markedly increased UV toxicity.In contrast, cell membrane-impermeable zinc chelator showed no toxicity.On the other hand, addition of a protein synthesis inhibitor or caspase inhibitor was markedly protective.In addition, RPE injury with exposure to combination of UV 1 min and TPEN 0.25 micrometer was accompanied by TUNEL and Hoechst staining positivity indicating that the toxicity is mainly apoptosis.Electron microscopic examinations revealed that nuclear fragmentation occurred even in sublethal UV or TPEN exposure, suggesting that the injury process already began at these condition consistently with the death being apoptosis. The present study has shown that combination of known risk factors may act synergistically to induce ARMD etc.The RPE injury induced by low dose UV and minimal intracellular zinc depletion was inhibited with protein synthesis inhibitor or caspase inhibitor, so these results suggested the possibility of prevention or treatment of RPE dysfunction.
Apoptosis ; Cell Death ; Epithelial Cells* ; Hand ; Humans* ; In Situ Nick-End Labeling ; Retinaldehyde* ; Risk Factors ; Zinc*

PURPOSE: Replication-competent adenoviruses (Ads) are promising new modalities for the treatment of cancer. Selective replication of a viral agent in tumor may lead to improved efficacy over non-replicating Ads due to viral multiplication, lysis of the infected cancer cell and spread to surrounding cells. In our previous studies it was shown that the E1B 55 kD-deleted Ad (YKL-1) exhibits tumor specific replication and cell lysis, but with reduced cytolytic effects compared to the wild type adenovirus (Int J Cancer 2000;88: 454-463). Thus, improving the potency of oncolytic Ads remains an important goal for cancer gene therapy. To increase the oncolytic ability of YKL-1, an adenovirus death protein (ADP) gene was reintroduced under the control of a CMV or MLP promoter at the E3 region of the YKL-1, generating an YKL-cADP and YKL-mADP, respectively. MATERIALS AND METHODS: The in vitro cytolytic effect of ADP expressing Ads was evaluated by MTT assay, and the induction of apoptosis by ADP expressing Ads was examined by TUNEL analysis. Finally, the antitumor effect of ADP expressing Ads was demonstrated in C33A xenograft tumor model. RESULTS: The YKL-cADP exerted a markedly enhanced cytolytic effect against H460 and SK-Hep1 cancer cell lines. The TUNEL assay indicated that the ADP-mediated cytotoxicity was largely driven by apoptosis. Finally, the YKL-cADP showed a superior antitumor effect than the YKL-1 or YKL-mADP in C33A xenografts. CONCLUSION: These lines of evidence demonstrate that the YKL-cADP induces efficient cell lysis, which is critical for the addition of therapeutic value to replicating Ads in cancer gene therapy.
Adenosine Diphosphate ; Adenoviridae* ; Apoptosis ; Cell Line ; Genes, Neoplasm ; Heterografts ; In Situ Nick-End Labeling

PURPOSE: To investigate the efficacy of an amniotic membrane contact lens on corneal epithelial wound healing. METHODS: We made a model with a corneal epithelial wound by applying 6 mm round filter paper soaked with 1 N NaOHonto the central cornea in 24 eyes of 12 rabbits. The rabbits were divided into three groups: AMCL (amniotic membrane contact lens), T-AMT (temporary amniotic membrane transplantation) and the control group. We evaluated corneal wound healing every postoperative day using a digital photo slitlamp and fluorescein dye. The corneas were harvested for histopathologic studies after seven days and analyzed with hematoxylin-eosin (H & E) stain and TUNEL staining. RESULTS: The average wound healing time was similar between the amniotic membrane contact lens and the temporary amniotic membrane transplantation group. The number of the infiltrated PMNs (polymorphonuclear cells) was 8.8+/-2.58, 8.6+/-2.19 and 48.6+/-7.12 in the AMCL, T-AMT and control groups, respectively. Apoptotic keratocytes were 3.8+/-1.1, 3.6+/-1.09 and 23.2+/-5.06 in the AMCL, T-AMT and control groups, respectively. In the AMCL and T-AMT groups, the number of infiltrated PMNs and apoptotic keratocytes were significantly less than those the control group (p<0.05). There were not significant differences in the number of PMNs and apoptotic cells in the AMCL and the T-AMT groups. CONCLUSIONS: Amniotic membrane contact lenses have the benefits of being an easily applied method and having a wound healing ability comparable to that possible with conventional suture methods.
Amnion ; Contact Lenses ; Cornea ; Eye ; Fluorescein ; In Situ Nick-End Labeling ; Membranes ; Rabbits ; Sutures ; Transplants ; Wound Healing

PURPOSE: This study was performed to investigate apoptosis by radiation in the developing fetal rat brain. MATERIALS AND METHODS: Fetal brains were irradiated in utero between the 17th and 19th days of fetal life (E17-19) by linear accelerator. A dose of irradiation ranging from 1 Gy to 4 Gy was used to evaluate dose dependency. To test time dependency the rats were irradiated with 2 Gy and then the fetal brain specimens were removed at variable time course; 1, 3, 6, 12 and 24 hours after the onset of irradiation. Immunohistochemical staining using in situ TdT-mediated dUTP nick end labelling (TUNEL) technique was used for apoptotic cells. The cerebral cortex, including three zones of cortical zone (CZ), intermediate zone (IZ), and ventricular zone (VZ), was examined. RESULTS: TUNEL positive cells revealed typical features of apoptotic cells under light microscope in the fetal rat cerebral cortex. Apoptotic cells were not found in the cerebral cortex of non-irradiated fetal rats, but did appear in the entire cerebral cortex after 1 Gy irradiation, and were more extensive at the ventricular and intermediate zones than at the cortical zone. The extent of apoptosis was increased with increasing doses of radiation. Apoptosis reached the peak at 6 hours after the onset of 2 Gy irradiation and persisted until 24 hours. CONCLUSION: Typical morphologic features of apoptosis by irradiation were observed in the developing fetal rat cerebral cortex. It was more extensive at the ventricular and intermediate zones than at the cortical zone, which suggested that stem cells or early differentiating cells are more radiosensitive than differentiated cells of the cortical zone.
Animals ; Apoptosis* ; Brain ; Cerebral Cortex* ; In Situ Nick-End Labeling ; Particle Accelerators ; Rats* ; Stem Cells

PURPOSE: Gene-attenuated replication-competent adenoviruses are emerging as a promising new modality for the treatment of cancer. In an effort to continually improve upon cancer gene therapy, we have modified gene- attenuated replication-competent adenoviruses so as to cause them to replicate efficiently and lyse the infected cancer cells more effectively. MATERIALS AND METHODS: We modified the E1 region of the adenovirus (Ad) systematically, generating Ad-deltaE1B19, Ad-deltaE1B55, Ad-deltaE1B19/55, and Ad-WT. The cytopathic effects (CPE) and viral replication of these four gene modified adenoviruses were compared, and the morphology and DNA fragmentation of the infected cells was evaluated. RESULTS: Among the constructed adenoviruses, E1B 19kD-inactivated adenovirus (Ad-deltaE1B19) was the most potent, inducing the largest-sized plaques and markedCPE. Moreover, cells infected with Ad-deltaE1B19 showed complete cell lysis with disintegrated cellular structure whereas cells infected with Ad-WT maintained intact cellular and nuclear membrane with properly structured organelles. TUNEL assay was also used to monitor DNA integrity, and a more profound induction of apoptosis was observed in the Ad-deltaE1B19 infected cells in comparison to wild type adenovirus infected cells. CONCLUSION: We demonstrate that the inactivation of the E1B19kD gene in a replicating adenovirus leads to increased CPE, rapid viral release, improved cell-to-cell viral spread and increased induction of apoptosis.
Adenoviridae* ; Apoptosis ; Cellular Structures ; DNA ; DNA Fragmentation ; Genes, Neoplasm* ; In Situ Nick-End Labeling ; Nuclear Envelope ; Organelles

OBJECTIVE: This study is designed to investigate how apoptosis is presented and how the genes of p53 and bcl-2 are expressed depending on graded injury in experimental spinal cord injury. METHODS: Experimental spinal cord injury was made on rats with weight drop method. Two different amounts of impact were applied on rat spinal cord. Rats were categorized into three groups (control; five rats, mild injury; five rats, severe injury; five rats). Fourty eight hours following cord injury, cord specimen was harvested from injury epicenter. TUNEL staining was done for apoptotic detection and immunohistochemical staining for p53 and bcl-2 expression. Positively stained cells were counted and mean values were compared among three groups. RESULTS: TUNEL positive cells increased depending on injury severity(p=0.027). The p53 positive cells increased in both injury groups compared to control group(p=0.001). Bcl-2 positive cells decreased as injury amount increased(p=0.002). The p53 expression increased in proportion to TUNEL staining in correlation curve in white matter(correlation coefficient, 0.387). The bcl-2 expression was inversely proportional to TUNEL staining and steeper decrease was found in gray matter than in white matter (correlation coefficient, -0.875). CONCLUSION: Apoptosis increases as the injury grading elevated within 20gm-cm of impact. The p53 seems to promote apoptosis in white matter, but do not show proportional relationship with injury amount. Bcl-2 appeared to be protective to cell death due to apoptosis.
Animals ; Apoptosis* ; Cell Death ; Contusions* ; In Situ Nick-End Labeling ; Rats* ; Spinal Cord Injuries ; Spinal Cord*

The apoptotic effect of bacteria-derived beta-glucan was investigated in human colon cancer cells SNU-C4 using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, reverse transcription-polymerase chain reaction (RT-PCR) expressions of Bcl-2, Bax, and Caspase-3 genes, and assay of caspase-3 enzyme activity. beta-Glucan of 10, 50, and 100 microg/mL decreased cell viability in a dose-dependent manner with typical apoptotic characteristics, such as morphological changes of chromatin condensation and apoptotic body formation from TUNEL assay. In addition, beta-glucan (100 microgram/mL) decreased the expression of Bcl-2 by 0.6 times, whereas the expression of Bax and Caspase-3 were increased by 3.1 and 2.3 times, respectively, compared to untreated control group. Furthermore, the caspase-3 activity in the beta-glucan-treated group was significantly increased compared to those in control group (P < 0.05). Bacterial derived beta-glucan could be used as an effective compound inducing apoptosis in human colon cancer.
Apoptosis ; Caspase 3 ; Cell Survival ; Chromatin ; Colon ; Colonic Neoplasms ; DNA Nucleotidylexotransferase ; Humans ; In Situ Nick-End Labeling

OBJECTIVE: To investigate the change of placental apoptosis and the expression of their mediator in preeclampsia women. METHODS: Placental tissues from 10 cases of preeclampsia and 15 cases of normal pregnancy were analyzed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling(TUNEL) staining. Expressions of bcl-2, bax, caspase-3 was also assessed using immunohistochemistry. RESULTS: In TUNEL staining, the number of apoptotic nuclei were significantly increased in the trophoblast of preeclampsia than normal pregnancy. Bcl-2 was mainly expressed in syncytiotrophoblast and bax was mainly expressed in cytotrophoblast. Bcl-2 expression was decreased and bax expression was increased in the preeclampsia than normal, but the difference was not significant. Caspase-3 was mainly expressed in the cytotrophoblast and expression was significantly increased in the preeclampsia than normal pregnancy(p<0.05). CONCLUSION: Placental apoptosis, especially accompanied with increased expression of caspase-3 in cytotrophoblast, might be related with in the pathogenesis of preeclampsia.
Apoptosis* ; Caspase 3 ; Deoxyuridine ; Female ; Humans ; Immunohistochemistry ; In Situ Nick-End Labeling ; Placenta ; Pre-Eclampsia* ; Pregnancy ; Trophoblasts