Quercetin induces cell death in cervical cancer by reducing O-GlcNAcylation of adenosine monophosphate-activated protein kinase / 대한해부학회지

Hyper-O-GlcNAcylation is a general feature of cancer which contributes to various cancer phenotypes, including cell proliferation and cell growth. Quercetin, a naturally occurring dietary flavonoid, has been reported to reduce the proliferation and growth of cancer. Several reports of the anticancer effect of quercetin have been published, but there is no study regarding its effect on O-GlcNAcylation. The aim of this study was to investigate the anticancer effect of quercetin on HeLa cells and compare this with its effect on HaCaT cells. Cell viability and cell death were determined by MTT and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling assays. O-GlcNAcylation of AMP-activated protein kinase (AMPK) was examined by succinylated wheat germ agglutinin pulldown and immunoprecipitation. Immunofluorescence staining was used to detect the immunoreactivitiy of O-linked N-acetylglucosamine transferase (OGT) and sterol regulatory element binding protein 1 (SREBP-1). Quercetin decreased cell proliferation and induced cell death, but its effect on HaCaT cells was lower than that on HeLa cells. O-GlcNAcylation level was higher in HeLa cells than in HaCaT cells. Quercetin decreased the expression of global O-GlcNAcylation and increased AMPK activation by reducing the O-GlcNAcylation of AMPK. AMPK activation due to reduced O-GlcNAcylation of AMPK was confirmed by treatment with 6-diazo-5-oxo-L-norleucine. Our results also demonstrated that quercetin regulated SREBP-1 and its transcriptional targets. Furthermore, immunofluorescence staining showed that quercetin treatment decreased the immunoreactivities of OGT and SREBP-1 in HeLa cells. Our findings demonstrate that quercetin exhibited its anticancer effect by decreasing the O-GlcNAcylation of AMPK. Further studies are needed to explore how quercetin regulates O-GlcNAcylation in cancer.

Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939-Mediated Transcriptional Repression of Bcl-xL / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Studies have found that long noncoding RNA HEIH (lncRNA-HEIH) is upregulated and facilitates hepatocellular carcinoma tumor growth. However, its clinical significances, roles, and action mechanism in colorectal cancer (CRC) remains unidentified. MATERIALS AND METHODS: lncRNA-HEIH expression in CRC tissues and cell lines was measured by quantitative real-time polymerase chain reaction. Cell CountingKit-8, ethynyl deoxyuridine incorporation assay, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and nude mice xenografts assays were performed to investigate the roles of lncRNA-HEIH. RNA pull-down, RNA immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter assays were performed to investigate the action mechanisms of lncRNA-HEIH. RESULTS: In this study, we found that lncRNA-HEIH is significantly increased in CRC tissues and cell lines. lncRNA-HEIH expression is positively associated with tumor size, invasion depth, and poor prognosis of CRC patients. Enhanced expression of lncRNA-HEIH promotes CRC cell proliferation and decreases apoptosis in vitro, and promotes CRC tumor growth in vivo. Whereas knockdown of lncRNA-HEIH inhibits CRC cell proliferation and induces apoptosis in vitro, and suppresses CRC tumor growth in vivo. Mechanistically, lncRNA-HEIH physically binds to miR-939. The interaction between lncRNA-HEIH and miR-939 damages the binding between miR-939 and nuclear factor κB (NF-κB), increases the binding of NF-κB to Bcl-xL promoter, and promotes the transcription and expression of Bcl-xL. Moreover, Bcl-xL expression is positively associatedwith lncRNA-HEIH in CRC tissues. Blocking the interaction between lncRNA-HEIH and miR-939 abolishes the effects of lncRNA-HEIH on CRC tumorigenesis. CONCLUSION: This study demonstrated that lncRNA-HEIH promotes CRC tumorigenesis through counteracting miR-939-mediated transcriptional repression of Bcl-xL, and suggested that lncRNA-HEIH may serve as a prognostic biomarker and therapeutic target for CRC.

The Cytotoxic Effect of Oral Wet Wipes on Gingival Cells / 치위생과학회지

Wet wipes are being increasingly used because of their convenience. Particularly, oral wet wipes are useful for regular cleaning of a baby's mouth after birth. Therefore, the consumption of oral wet wipes has increased over the past few years and a variety of products are commercially available. However, product information on safety is not sufficiently provided and still raises doubts regarding adverse effects. To confirm the safety of wet wipes as an oral hygiene item and provide information for their use, we investigated the cytotoxicity of oral wet wipes and verified the underlying mechanism. The anti-bacterial effect of oral wet wipes was analyzed using the disk diffusion method. The cytotoxic effects of oral wet wipes were observed based on morphological changes using microscopy and determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in gingival epithelial cells and gingival fibroblasts. Evaluation of apoptosis by oral wet wipes was explored using propidium iodide flow cytometric analysis and a terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay. Apoptosis-related molecules were also analyzed using western blotting. Five types of oral wet wipes were tested, and two products from Fisher-Price and Dr. Kennedy revealed strong cytotoxic effects on gingiva epithelial cells and gingiva fibroblasts, although they also showed intense anti-bacterial effects on oral bacteria. Cell cycle arrest in the G2/M phase and apoptosis were observed based on treatment of extracts from Fisher-Price and Dr. KENNEDY. Relatively high TUNEL levels, reduction of proliferating cell nuclear antigen and cyclin-dependent kinase 4 expression, and fragmentation of poly (ADP-ribose) polymerase were also elucidated. These results suggest that commercial oral wet wipes could exert cytotoxic influences on oral tissue, although there are anti-bacterial effects, and careful attention is required, especially for infants and toddlers.

What should be done for men with sperm DNA fragmentation? / 대한생식의학회지

In an age when a small quantity of sperm can lead to pregnancy through in vitro fertilization or intracytoplasmic sperm injection, selecting healthy sperm is important. Sperm DNA fragmentation (SDF) is known to be higher in infertile men. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and the alkaline comet test are SDF tests that directly measure DNA damage and have shown closer correlations with assisted reproduction results than indirect tools such as the sperm chromatin structure assay or the sperm chromatic dispersion test. It is difficult; however, to endorse a single test as the best test overall; instead, it is best to select a testing method based on each patient's clinical condition and goals. In a couple struggling with infertility, if the male partner has a high level of SDF, he should aim to decrease SDF through lifestyle modifications, antioxidant treatment, and ensuring an appropriate duration of abstinence, and physicians need to treat the underlying diseases of such patients. If sperm DNA damage continues despite the patient's and physician's efforts, other methods, such as micromanipulation-based sperm selection or testicular sperm extraction, should be used to select healthy sperm with nuclear DNA integrity.

Effects of supplementation with antifreeze proteins on the follicular integrity of vitrified-warmed mouse ovaries: Comparison of two types of antifreeze proteins alone and in combination / 대한생식의학회지

OBJECTIVE: The aim of this study was to analyze the effect of supplementing vitrification and warming solutions with two types of antifreeze proteins (AFPs) and the combination thereof on the follicular integrity of vitrified-warmed mouse ovaries. METHODS: Ovaries (n=154) were obtained from 5-week-old BDF1 female mice (n=77) and vitrified using ethylene glycol and dimethyl sulfoxide with the supplementation of 10 mg/mL of Flavobacterium frigoris ice-binding protein (FfIBP), 10 mg/mL of type III AFP, or the combination thereof. Ovarian sections were examined by light microscopy after hematoxylin and eosin staining, and follicular intactness was assessed as a whole and according to the type of follicle. Apoptosis within the follicles as a whole was detected by a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay. RESULTS: The proportion of overall intact follicles was significantly higher in the type III AFP-supplemented group (60.5%) and the combination group (62.9%) than in the non-supplemented controls (43.8%, p<0.05 for each). The proportion of intact primordial follicles was significantly higher in the FfIBP-supplemented (90.0%), type III AFP-supplemented (92.3%), and combination (89.7%) groups than in the non-supplemented control group (46.2%, p<0.05 for each). The proportions of non-apoptotic follicles were similar across the four groups. CONCLUSION: Supplementation of the vitrification and warming solutions with FfIBP, type III AFP, or the combination thereof was equally beneficial for the preservation of primordial follicles in vitrified mouse ovaries.

Clinicopathologic Significance of Survivin Expression in Relation to CD133 Expression in Surgically Resected Stage II or III Colorectal Cancer

BACKGROUND: Cancer stem cells have been investigated as new targets for colorectal cancer (CRC) treatment. We recently reported that CD133+ colon cancer cells showed chemoresistance to 5-fluorouracil through increased survivin expression and proposed the survivin inhibitor YM155 as an effective therapy for colon cancer in an in vitro study. Here, we investigate the relationship between survivin and CD133 expression in surgically resected CRC to identify whether the results obtained in our in vitro study are applicable to clinical samples. METHODS: We performed immunohistochemical staining for survivin and CD133 in surgically resected tissue from 187 stage II or III CRC patients. We also comparatively analyzed apoptosis according to survivin and CD133 expression using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. RESULTS: The results of the Mantel-Haenszel test established a linear association between nuclear survivin and CD133 expression (p = .018), although neither had prognostic significance, according to immunohistochemical expression level. No correlation was found between survivin expression and the following pathological parameters: invasion depth, lymph node metastasis, or histologic differentiation (p > .05). The mean apoptotic index in survivin+ and CD133+ tumors was higher than that in negative tumors: 5.116 ± 4.894 in survivin+ versus 4.103 ± 3.691 in survivin– (p = .044); 5.165 ± 4.961 in CD133+ versus 4.231 ± 3.812 in CD133– (p = .034). CONCLUSIONS: As observed in our in vitro study, survivin expression is significantly related to CD133 expression. Survivin may be considered as a new therapeutic target for chemoresistant CRC.

Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination

Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples.

Therapeutic Effects of Umbilical Cord Blood Derived Mesenchymal Stem Cell-Conditioned Medium on Pulmonary Arterial Hypertension in Rats

BACKGROUND: Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) may have multiple therapeutic applications for cell based therapy including the treatment of pulmonary artery hypertension (PAH). As low survival rates and potential tumorigenicity of implanted cells could undermine the mesenchymal stem cell (MSC) cell-based therapy, we chose to investigate the use of conditioned medium (CM) from a culture of MSC cells as a feasible alternative. METHODS: CM was prepared by culturing hUCB-MSCs in three-dimensional spheroids. In a rat model of PAH induced by monocrotaline, we infused CM or the control unconditioned culture media via the tail-vein of 6-week-old Sprague-Dawley rats. RESULTS: Compared with the control unconditioned media, CM infusion reduced the ventricular pressure, the right ventricle/(left ventricle+interventricular septum) ratio, and maintained respiratory function in the treated animals. Also, the number of interleukin 1alpha (IL-1alpha), chemokine (C-C motif) ligand 5 (CCL5), and tissue inhibitor of metalloproteinase 1 (TIMP-1)-positive cells increased in lung samples and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique (TUNEL)-positive cells decreased significantly in the CM treated animals. CONCLUSIONS: From our in vivo data in the rat model, the observed decreases in the TUNEL staining suggest a potential therapeutic benefit of the CM in ameliorating PAH-mediated lung tissue damage. Increased IL-1alpha, CCL5, and TIMP-1 levels may play important roles in this regard.

Icariin promotes self-renewal of neural stem cells: an involvement of extracellular regulated kinase signaling pathway / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effects and underlying molecular mechanisms of icariin (ICA) on self-renewal and differentiation of neural stem cells (NSCs).</p><p><b>METHODS</b>NSCs were derived from forebrains of mice embryos by mechanical dissociation into single cell suspension. The self-renewal of NSCs was measured by neurosphere formation assay. The proliferation of NSCs was detected by water-soluble tetrazolium (WST) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Protein expression of neuron-specific marker tubulin-βIII(TuJ1) and astrocyte-specific marker glial fibrillary acidic protein (GFAP) were measured by immunofluorescence and Western blotting. Using microarray, the differentially expressed genes (DEGs) were screened between NSCs with or without ICA treatment. The signaling pathways enriched by these DEGs and their role in mediating effects of ICA were analyzed.</p><p><b>RESULTS</b>ICA significantly promoted neurosphere formation of NSCs cultured in growth protocol in a dose-dependent manner and achieved the maximum effects at 100 nmol/L. ICA also increased optical absorbance value and EdU incorporation into nuclei of NSCs. ICA had no significant effects on the percentage of TuJ1 or GFAP-positive cells, and TuJ1 or GFAP protein expression in NSCs cultured in differentiation protocol. A total of 478 genes were found to be differentially regulated. Among signaling pathways significantly enriched by DEGs, mitogen activated protein kinase (MAPK) pathway was of interest. Blockade of extracellular signal-regulated kinase (ERK)/MAPK, other than p38/MAPK subfamily pathway partially abolished effects of ICA on neurosphere formation and EdU incorporation of NSCs.</p><p><b>CONCLUSION</b>ICA can promote the selfrenewal of NSCs at least partially through ERK/MAPK signaling pathway.</p>

Dense distribution of macrophages in flexor aspects of the hand and foot of mid-term human fetuses / 대한해부학회지

In the developing human musculoskeletal system, cell death with macrophage accumulation occurs in the thigh muscle and interdigital area. To comprehensively clarify the distribution of macrophages, we immunohistochemically examined 16 pairs of upper and lower extremities without the hip joint (left and right sides) obtained from 8 human fetuses at approximately 10-15 weeks of gestation. Rather than in muscles, CD68-positive macrophages were densely distributed in loose connective tissues of the flexor aspects of the extremities, especially in the wrist, hand and foot. In contrast, no or fewer macrophages were evident in the shoulder and the extensor aspects of the extremities. The macrophages were not concentrated at the enthesis of the tendon and ligament, but tended to be arranged along other connective tissue fibers. Deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling revealed apoptosis in the hand lumbricalis muscles, but not in the area of macrophage accumulation. Likewise, podoplanin-positive lymphatic vessels were not localized to areas of macrophage accumulation. Re-organization of the connective tissue along and around the flexor tendons of the hand and foot, such as development of the bursa or tendon sheath at 10-15 weeks, might require the phagocytotic function of macrophages, although details of the mechanism remain unknown.

Neuroprotective effects of L-carnitine against oxygen-glucose deprivation in rat primary cortical neurons / 소아과

PURPOSE: Hypoxic-ischemic encephalopathy is an important cause of neonatal mortality, as this brain injury disrupts normal mitochondrial respiratory activity. Carnitine plays an essential role in mitochondrial fatty acid transport and modulates excess acyl coenzyme A levels. In this study, we investigated whether treatment of primary cultures of rat cortical neurons with L-carnitine was able to prevent neurotoxicity resulting from oxygen-glucose deprivation (OGD). METHODS: Cortical neurons were prepared from Sprague-Dawley rat embryos. L-Carnitine was applied to cultures just prior to OGD and subsequent reoxygenation. The numbers of cells that stained with acridine orange (AO) and propidium iodide (PI) were counted, and lactate dehydrogenase (LDH) activity and reactive oxygen species (ROS) levels were measured. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were performed to evaluate the effect of L-carnitine (1 microM, 10 microM, and 100 microM) on OGD-induced neurotoxicity. RESULTS: Treatment of primary cultures of rat cortical neurons with L-carnitine significantly reduced cell necrosis and prevented apoptosis after OGD. L-Carnitine application significantly reduced the number of cells that died, as assessed by the PI/AO ratio, and also reduced ROS release in the OGD groups treated with 10 microM and 100 microM of L-carnitine compared with the untreated OGD group (P<0.05). The application of L-carnitine at 100 microM significantly decreased cytotoxicity, LDH release, and inhibited apoptosis compared to the untreated OGD group (P<0.05). CONCLUSION: L-Carnitine has neuroprotective benefits against OGD in rat primary cortical neurons in vitro.

Effects of Heat Shock and Antioxidant on the Growth Plate of Unloaded Rats / 대한정형외과연구학회지

PURPOSE: This study was performed to examine any histopathological changes occurring in the growth plate when the rats were subjected to be deprived of normal weight bearing using the hindlimb suspension model, and to search for any countermeasures for improving and/or recovering the chondrocyte activities within the growth plate. MATERIALS AND METHODS: Sixty male Sprague-Dawley rats, aged 6 weeks, were divided into 10 groups each: Group I-control to unloading; Group II-unloading 3 weeks only; Group III-unloading+application of heat shock; Group IV-unloading+application of antioxidant; Group V-unloading+application of heat shock and antioxidant; Group VI-control to reloading; Group VII-reloading 1 week only; Group VIII-reloading+application of heat shock; Group IX-reloading+application of antioxidant; Group X-reloading+application of heat shock and antioxidant. The animals were double labeled with 5-Bromo-2'-deoxydiuridin (BrdU) and BrdU immunohistochemistry was performed for the cellular kinetic analysis. Transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay was done for the investigation of apoptotic changes in the growth plate, and the positive cells were counted in each zones of the growth plate in both TUNEL and BrdU immunohistochemistry. Heat shock protein (HSP), indian hedgehog (Ihh), and vascular endothelial growth factor (VEGF) were immunolocalized to assess the chondrocytic activities in terms of production of extracellular matrix protein. RESULTS: Non-weight bearing induced a reduction of height of the growth plate, reduced cellular proliferation of chondrocytes, reduced expression of Ihh and VEGF, and altered expression of heat shock protein. When heat shock and/or antioxidant were applied to the unloaded and reloaded rats, only rats in the group of application of both heat shock and antioxidant showed normal cellular activities in terms of cellular proliferation and the production of extracellular matrix protein. CONCLUSION: The present results suggest that application of heat shock and antioxidant would be a countermeasure for the restoration of chondrocytic activities when the normal weight-bearing is deprived of.

The Clinical Features and Pathophysiology of Acute Radiation Dermatitis in Patients Receiving Tomotherapy

BACKGROUND: Radiation therapy (RT) including tomotherapy has been widely used to treat primary tumors, as well as to alleviate the symptoms of metastatic cancers. OBJECTIVE: The primary purpose of this study was to examine the characteristics of the clinical features and pathophysiological mechanisms associated with acute radiation dermatitis in cancer patients that received tomotherapy, and compare the results to patients treated by conventional radiation therapy. METHODS: The study population consisted of 11 patients that were referred to the dermatology department because of radiation dermatitis after receiving tomotherapy; all patients were evaluated for clinical severity. The patients were assessed and identified using the National Cancer Institute Common Toxicity Criteria version (CTC) 3.0. We performed biopsies of the skin lesions that were examined for apoptosis using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL) assay and stained immunohistochemically with monoclonal antibodies to CD8, CD4 and TGF-beta. As a positive control, patients with radiation dermatitis treated with conventional radiation therapy were also studied. RESULTS: The results of the clinical features of the skin of tomotherapy patients were the following: grade 1 (36%), grade 2 (55%) and other changes (9%). Among the population that had skin lesions due to acute radiation dermatitis, the mean number of positive cells per high power field (HPF) was the following: there were 30.50+/-.50 TUNEL- positive cells, 34.60+/-12.50 CD8+ T cells, 5.19+/-3.17 CD4+T cells and 9.95+/-1.33 TGF-beta positive cells measured per HPF. The mean number of positive cells per HPF for the patients that received conventional radiation therapy was: TUNLEL-positive cells in 7.5+/-1.64, CD8-, CD4- and TGF-beta-positive cells in 12.50+/-3.73, 3.16+/- 1.47, 6.50+/-1.97. CONCLUSION: We found that the number of TUNEL-positive cells and CD8+ T cells were higher in the lesions of patients receiving tomotherapy compared to the lesions of the patients receiving conventional radiation therapy. These findings suggest that tomotherapy without dose modification may cause significantly more severe forms of radiation dermatitis by apoptosis and cytotoxic immune responses than conventional radiation therapy.

The Different Effects of Testicular Torsion on the Contralateral Testis between Pubertal and Adult Rats

PURPOSE: Our study aimed to determine whether the severity of damage to the contralateral testis by ipsilateral testicular torsion/detorsion in pubertal rats, which have an incomplete blood-testis barrier, is different from that in adult rats. MATERIALS AND METHODS: We divided pubertal (6 weeks, n=17) and adult (10 weeks, n=17) Sprague-Dawley (SD) rats into group S (sham; n=5), group O (orchiectomy; n=6), and group D (detorsion; n=6). After 4 hours' torsion of the ipsilateral testis, we applied orchiectomy (group O) and detorsion (group D) depending on the group and compared the histopathologic changes and germ cell apoptosis of the contralateral testis at the age of 13 weeks. RESULTS: In each age group, increased interstitial area, edema, and germ cell sloughing were observed in group D. The mean seminiferous tubule diameter decreased more in group D than in group S or O in each age group (p<0.05). The mean germ cell layer thickness and number of spermatids per tubule decreased more in group D than in group S or O in each age group; additionally, in group D, values decreased more in pubertal rats than in adult ones (p<0.05, respectively). The mean numbers of terminal deoxyuridine nick-end labeling (TUNEL)-positive cells were less than 1.0 in groups S and O, which was smaller than in group D (p<0.05); additionally, in group D, this value tended to be higher in pubertal rats than in adult ones (p=0.057). CONCLUSIONS: SD rats with a detorsioned testis had more severe damage to the contralateral testis than did those undergoing orchiectomy of the torsioned testis. Also, when comparing the severity of damage to the contralateral testis after ipsilateral torsion/detorsion between pubertal and adult rats, rats at a pubertal age, when most testicular torsions occur in clinical situations, had more severe damage than did those at an adult age.

Expression of Survivin in Gastric Carcinoma and its Relation to Tumor Cell Proliferation and Apoptosis

BACKGROUND: Survivin, a novel antiapoptotic gene has been linked with tumor development and progression in various human carcinomas including gastric carcinomas. The aim of this study was to evaluate the expression of survivin in gastric carcinoma and its correlation with tumor cell proliferation and apoptosis. METHODS: Expression of survivin was evaluated immunohistochemically in 84 surgically resected gastric carcinomas. Tumor cell apoptosis was evaluated with terminal deoxynucleotidyl transferase (TdT) mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL), and Ki-67 immunostaining was used for evaluation of tumor cell proliferation. RESULTS: Expression of survivin was noted in 53.6% of the gastric carcinomas, and was significantly associated with depth of invasion, status of lymph node metastasis or tumor stage (p=0.022, 0.034, 0.040, respectively). There was an inverse correlation between survivin expression and apoptotic index (p=0.015). But there was no significant correlation between survivin expression and Ki-67 labeling index (p=0.430). CONCLUSIONS: These results suggest that survivin expression may contribute to tumor development and progression by inhibiting apoptosis in human gastric carcinoma.

Trophoblast Apoptosis and BRCA1 Expression in Fetal Growth Restriction Pregnancy / 대한산부인과학회지

OBJECTIVE: To know the role of trophoblast apoptosis and BRCA1 on pregnancy of intra-uterine fetal growth restriction (FGR). BRCA1 is regarded as not only a tumor suppressor gene but regulator of cell growth, proliferation and differentiation. We tested the hypothesis that enhanced trophoblast apoptosis and expression of BRCA1 reduced placental mass, which caused placental dysfunction and FGR. METHODS: Placentas were obtained from women with pregnancies complicated by FGR (n=10) and from women with controlled normal pregnancies at different gestational ages (n=15). After placental sections were isolated, In Situ DNA 3'-end labelling analysis as well as detection of cytokeratin 18 cleavage products for apoptosis were measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and counter staining with hematoxylin and eosin. The same sections were examined by means of immunohistochemistry and Western immunoblotting for identifying the expression of BRCA1 protein. RESULTS: TUNEL staining exhibited consistently higher levels of nuclear staining and more cytokeratin 18 cleavage products in trophoblasts from FGR than normal growth pregnancy. In the trophoblasts of FGR, the BRCA1 expression and the staining intensity were more augmented on Western blotting and immunohistochemistry than normal pregnancies. There was no remarkable difference of BRCA1 expression according to gestational ages. CONCLUSION: The expression of BRCA1 and apoptosis is up-regulated in human placental villi from pregnancies complicated by FGR. It suggests that BRCA1 may play a role in trophoblast proliferation and differentiation and be related to the pathophysiologic mechanism of FGR. We speculated that the clinical conditions associated with enhanced BRCA1-mediated apoptosis in trophoblasts and thereby contribute to placental dysfunction and FGR.

Disparity between MR Imaging and Histochemical Grading in Human Intervertebral Disc Degeneration

OBJECTIVE: In order to establish the index of degeneration, the authors performed a histochemical study with Safranin-O staining and investigated the occurrence of apoptosis in the human intervertebral disc. METHODS: Eighteen intervertebral disc specimens surgically extracted from the patients and two additional specimens from the autopsied cases were stained with Safranin-O for proteoglycan according to a standard protocol. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling(TUNEL) was used to detect the fragmented DNA known to be associated with apoptotic cell death and classification scheme was formulated for categorization of the degree of Safranin-O staining (normal, moderate reduction, faint) by modification of Makin's histological-histochemical grading. The Kruskal-Wallis H test and Chi-square test were used for statistical analysis. RESULTS: The statistical results showed a significant difference in the mean age between "normal" Safranin-O staining group and the others (19.3 versus 55, 43.4, p=0.021). However, there was no statistically significant correlation between Safranin-O staining and MR grading of disc degeneration. Only six of eighteen surgical specimens and none in autopsies showed positive apoptotic cells in TUNEL staining. CONCLUSION: The determination of the degree of degeneration in surgically obtained disc tissue per se by histochemical staining or by the degree of apoptosis that corresponds to its morphologic change was not feasible.

The Time Evolution of Cerebral Apoptosis in the Permanent Middle Cerebral Artery Occlusion Model in Rats

OBJECTIVE: The purpose of this study is to determine the time evolution and distribution of cerebral apoptosis using the middle cerebral artery occlusion model in rats. METHODS: A total of twenty four male rats - with 2, 3, 4, 6, 8, 12, 24 and 48 hours of middle cerebral artery occlusion respectively - were studied. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling(TUNEL) method was used for the observation of the apoptotic cells. The apoptotic ratio was calculated and the distribution of apoptosis was inspected in the pyriform cortex, basal ganglia and middle cerebral artery territory cortex. The rats were divided into three groups(Group I: 2~4 hours of occlusion, Group II: 6~12 hours of occlusion, Group III: 24~48 hours of occlusion). RESULTS: In this study, the proportion of apoptosis increased with the duration of middle cerebral artery occlusion and reached a maximum after about 12 hours of middle cerebral artery occlusion. The mean values of the apoptotic ratio were 30.7+/-11.3% in group I, 60.8+/-2.6% in group II and 48.7+/-0.7% in group III. The distribution of apoptosis differed in the pyriform cortex, basal ganglia and middle cerebral artery territory cortex according to the duration of time of the middle cerebral artery occlusion. CONCLUSION: In the middle cerebral artery occlusion model of the rats, apoptosis is found to increase according to the occlusion time, reaching a peak after 6 hours, and the distribution of apoptosis changed from the pyriform cortex to the basal ganglia and middle cerebral artery territory cortex.

Placental Apoptosis from Pregnancies Complicated by Fetal Growth Restriction or Preeclampsia: Expression of Bcl-2, Bax and p53 / 대한산부인과학회지

OBJECTIVE: The purpose of this study was to investigate the degree of apoptosis in placentas with pregnancy complicated by fetal growth restriction or preeclampsia as compared with normal term pregnancy placentas and to evaluate the expression of proapoptotic proteins Bax, p53 and antiapoptotic protein Bcl-2 in their placentas. METHODS: Placentas were obtained from 40 normal term pregnancies and from 30 pregnancies complicated by fetal growth restriction or preeclampsia admitted for delivery to the department of Obstetrics and Gynecology of Inha university hospital from January 1 to November 30, 2003. Placental sections were examined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining, indicative of apoptosis. The expressions of Bcl-2, Bax, p53 in their placentas were analyzed by immunohistochemical staining. RESULTS: Increased apoptosis was found in the trophoblast layer of villi from pregnancies complicated by fetal growth restriction or preeclampsia (n=30) compared to normal pregnancies (n=40) (0.93 +/- 0.54: 1.5 +/- 1.2) (p=0.014, t-test). There was no statistical significance between the two groups in expressions of Bcl-2, Bax, p53 (p=0.073, p=0.424, p=0.208, chi-square test). CONCLUSION: These results suggest that placental apoptosis may play a role in the pathogenesis of fetal growth restriction or preeclampsia. But placental apoptosis does not seem to be mediated by Bcl-2, Bax, p53 in trophoblasts.

The Serial Microscopic Changes of Cell Proliferative and Apoptotic Phenomenon in Obstructed Ureters in the Rat / 대한비뇨기과학회지

Purpose: Obstructive uropathy due to a ureteral obstruction is one of the most common diseases of the urinary tract, and can lead to severe renal injury and ureteral damage. This study performed to elucidate the histological findings and serial changes in the apoptotic and proliferative phenomena in the pathogenesis of ureteral damage during the course of obstructive uropathy in ligated rat ureters. Materials and Methods: After unilateral ligation of the ureter, each group of five Sprague-Dawley rats was sacrificed, and examined 1, 5, 10, 15, 20, 25, 30 and 35 days after ligation: five rats with normal ureters were also examined as controls. The cell proliferation and apoptosis were detected with proliferating cell nuclear antigen (PCNA) immunohistochemistry and a terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) in situ nick-end labeling (TUNEL) study, respectively, in 45 Sprague-Dawley rats. Results: The epithelial layer was thickened in the 5 day-obstructed ureters (DOUs). The severity of thickening of the fibrous and smooth muscle layers progressed consistently to the 15 DOUs, which was maintained until day 35. The expression of PCNA in the epithelial layer was present in every ureter, with a significant increase of labeled cells in the 1 and 5 DOUs. The expressions of PCNA in the fibrous and smooth muscle layers were present from day 10 after ligation and maintained until day 20, but then significantly declined at 25 DOUs. TUNEL-positive cells were shown in the epithelial layer in the 10, 15, 20, 25, 30 and 35 DOUs. The mean numbers of TUNEL-positive cells in the 20, 25 and 30 DOUs were significantly higher than those in the 10 DOUs, and reached their peak in the 25 DOUs. Positive cells were shown in the fibrous and smooth muscle layers in the 25, 30 and 35 DOUs. Conclusions: Apoptosis and cell proliferation may play an important role in the pathogenesis of ureteral damage in obstructed ureters. The peak of apoptosis was shown in the 25 DOUs.