Transferase-mediated dUTP-digoxigenin Nick End Labeling Research Articles

Polydatin improves the developmental competence of bovine embryos in vitro via induction of sirtuin 1 (Sirt1).

The aim of the present study was to investigate the beneficial effect of polydatin (PD), the glycoside form of resveratrol, on embryo development in vitro. Oocytes were aspirated from ovaries of Korean Hanwoo cows and cultured until Day 8 in a humidified atmosphere of 5% CO2 in air at 38.5°C. Protein and gene expression levels were determined through confocal microscopy and reverse transcription-polymerase chain reaction respectively, whereas the number of total and apoptotic cells in Day 8 blastocysts was determined using Hoechst 33342 staining and terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling. Of the different concentrations of PD (0.5, 1.0 and 2.0µM) added to the IVM medium, only 1.0µM PD significantly improved blastocyst development. Immunofluorescence analysis confirmed that protein levels of sirtuin 1 (Sirt1) increased significantly (P<0.05) after PD treatment, whereas levels of reactive oxygen species (ROS) were significantly (P<0.05) decreased, as evidenced by reductions in 8-oxoguanine immunoreactivity. Similarly, protein levels of nuclear factor (NF)-κB and cyclo-oxygenase (COX)-2 were significantly (P<0.05) lower in the PD-treated group than in the control group. Treatment with 1.0µM PD reduced gene expression of BCL2-associated X protein, inducible nitric oxide synthase, COX2 and Nfkb, but increased the expression of Sirt1, supporting the immunofluorescence data. PD possesses antioxidant activity and is useful for embryo development in vitro. We conclude that supplementation of IVM medium with PD improves embryo developmental competence via Sirt1.

Sclerotic Effect of Oxytetracycline on the Submandibular Gland: An Experimental Model

Oxytetracycline has been suggested as an alternate therapy for chronic recurrent sialadenitis and sialorrhea. We conducted an experimental study to investigate the sclerotic effect of this drug on the submandibular gland by histopathologic methods. Our subjects were 20 New Zealand white rabbits, which were divided into two groups of 10. The right submandibular gland of the rabbits in the active-treatment group was injected with 0.3 ml of oxytetracycline (100 mg/ml), and that of the controls was injected with saline. Four weeks after the injections, all the glands were removed. Histopathologic studies, including hematoxylin and eosin and Masson trichrome staining, were carried out. The glands were evaluated for tissue inflammation, congestion, fibrosis, edema, lipomatosis, and atrophy. To investigate apoptosis, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) immunohistochemical staining was used. In the study group, inflammation (n = 9), congestion (n = 9), fibrosis (n = 6), edema (n = 6), and lipomatosis (n = 4) were observed; in the sham group, only lipomatosis was seen (n = 5). The TUNEL assay results for acinar cells were 4.51 ± 1.41% in the oxytetracycline group and 2.08 ± 1.76% in the control group (p = 0.006); the corresponding figures for the duct cells were 7.05 ± 0.87% and 3.10 ± 2.26% (p = 0.001). Based on our findings, we conclude that oxytetracycline might be a viable alternative for the treatment of chronic recurrent sialadenitis and sialorrhea. However, more research in this area is needed.

Is β-cell aging involved in the pathogenesis of diabetes?

β-Cells at different stages have different functions and capacity for proliferation, regenerative and apoptosis. The aim of the present study was to investigate whether there are changes in β-cell phonotype in the development of diabetes to identify potential β-cell targets to prevent the progression of diabetes. A cross-sectional study was performed on pancreatic tissues obtained from 80 patients classified into three groups: 25 with type 2 diabetes (T2D), 25 with impaired fasting glucose (IFG), and 30 non-diabetics (ND). The ratio of the insulin-positive area to pancreatic area was used as an indirect marker of β-cell mass. Insulin-positive duct cells and scattered β-cells were defined as newly generated β-cells, whereas insulin/neurogenin 3 (Ngn3), insulin/v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MafA) and insulin/P16 double-positive cells were defined as immature, mature, and senescent β-cells, respectively; Ki67 was used as a marker of cell proliferation, and terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) was used as a marker of cell apoptosis. Data were analyzed using the Kruskal-Wallis test. There were no significant differences in β-cell mass, the prevalence of insulin-positive duct cells, scattered β-cells, or insulin/Ngn3, insulin/MafA, and Insulin/Ki67 double-positive cells among groups. The incidence of insulin/P16 double-positive cells was significantly higher in T2D than ND. β-Cell apoptosis was significantly higher in T2D and IFG than ND. The senescence and apoptosis of β-cells may be involved in the course of diabetes.

Thrombospondin-1 expression may be implicated in liver atrophic mechanism due to obstructed portal venous flow.

Liver is an amazing organ that can undergo regenerative and atrophic changes inversely, depending on blood flow conditions. Although the regenerative mechanism has been extensively studied, the atrophic mechanism remains to be elucidated. To assess the molecular mechanism of liver atrophy due to reduced portal blood flow, we analyzed the gene expressions between atrophic and hypertrophic livers induced by portal vein embolization in three human liver tissues using microarray analyses. Thrombospondin (TSP)-1 is an extracellular protein and a negative regulator of liver regeneration through its activation of the transforming growth factor-β/Smad signaling pathway. TSP-1 was extracted as the most upregulated gene in atrophic liver compared to hypertrophic liver due to portal flow obstruction in human. Liver atrophic and hypertrophic changes were confirmed by HE and proliferating cell nuclear antigen staining and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling. In an in vivo model with portal ligation, TSP-1 and phosphorylated Smad2 expression were continuously induced at 6h and thereafter in the portal ligated liver, whereas the induction was transient at 6h in the portal non-ligated liver. Indeed, while cell proliferation represented by proliferating cell nuclear antigen expression at 48h was induced in the portal ligated liver, the sinusoidal dilatation and hepatocyte cell death with terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling was detectable at 48h in the portal ligated liver. Obstructed portal flow induces persistent TSP-1 expression and transforming growth factor-β/Smad signal activation in atrophic liver. Thrombospondin-1 may be implicated in the liver atrophic change due to obstructed portal flow as a pro-atrophic factor.

Hypoxia-inducible factor-2α promotes hepatocyte apoptosis during cholestasis.

Hypoxia-inducible factor-2α (HIF-2α) has been reported to play an important role in a host of pathophysiological processes, including cellular survival. This study explores the role of HIF-2α in cholestasis-mediated hepatocyte apoptosis. Hypoxia-inducible factor-2α expression was measured by immunohistochemistry and confocal microscopy. Hepatic apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling. The cholestatic mouse model was treated with bile duct ligation. The c-myc, p53, and Bax protein levels were measured with Western blot analysis. In pediatric and murine cholestatic liver tissues, HIF-2α protein was widely expressed in the nucleus of parenchymal cells as well as in stromal cells. Hepatocyte HIF-2α expression was significantly elevated at the early stage of pediatric cholestasis and decreased at the late stage. In both in vivo and in vitro murine studies, HIF-2α deletion could alleviate cholestasis-mediated hepatocyte apoptosis and regulate the expression of c-myc, p53, and Bax proteins. These findings implied the contribution of HIF-2α to cholestasis-mediated hepatocyte apoptosis.

The effect of 7 days of letrozole pretreatment combined with misoprostol on the expression of progesterone receptor and apoptotic factors of placental and decidual tissues from first-trimester abortion: a randomized controlled trial

ObjectiveTo evaluate if letrozole-induced suppression of estradiol reduces progesterone receptor expression and apoptosis in the first-trimester placenta. Study DesignWe performed a double-blinded, randomized, placebo-controlled trial. We randomized 20 women requesting first-trimester abortion with gestation up to 63 days to receive either letrozole 10 mg daily or placebo pretreatment for 7 days before administrating 400 mcg of vaginal misoprostol followed by suction abortion. We collected the placental and decidual tissues on which we performed immunohistochemical staining for progesterone receptor and apoptotic markers (active caspase 3, caspase 3, Bcl2, CD95, fas ligand) and determined H-scores of each based on the intensities of staining. We performed terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay for apoptosis in the samples of four women to confirm the findings from apoptotic markers. ResultsWe excluded one woman in the letrozole group from the analysis because she had passage of abortus after taking letrozole, leaving 19 women (9 in the letrozole group, 10 in the placebo group) for analysis. There was no significant difference in the H-scorings of progesterone receptor and apoptotic markers, as well as proportion of apoptotic cells on TUNEL assay between the two groups. The H-scores for the progesterone receptor were 8.17±2.67 (mean±SD) in the letrozole group and 9.01±2.82 in the placebo group (p=0.36). ConclusionWe did not detect a difference in the expression of progesterone receptor and apoptotic markers in placental and decidual tissues after letrozole pretreatment for 7 days in first-trimester abortion. ImplicationsWe did not confirm the hypothesis that letrozole reduces progesterone receptor expression and induces apoptosis in the first-trimester placenta. Further studies are required to allow better understanding of the mechanism by which estrogen suppression following the use of letrozole can lead to improved abortion rate in the first trimester.

Sperm DNA fragmentation in cryopreserved samples from subjects with different cancers.

Sperm cryopreservation is widely used by cancer patients undergoing chemo- or radiotherapy. Evidence suggests that IVF outcome with cryopreserved spermatozoa from cancer patients is less successful. To determine whether sperm DNA fragmentation (SDF) is involved in the lower fertilising ability of cryopreserved spermatozoa of cancer patients, SDF was evaluated in thawed spermatozoa from 78 men affected by different cancers and 53 men with non-cancer pathologies. SDF was assessed by the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL), propidium iodide (PI), flow cytometry procedure, which allows determination of two different cell populations (PIbrighter and PIdimmer) and thus to determine the percentage of DNA fragmented sperm in both. PIdimmer spermatozoa are totally unviable, whereas PIbrighter spermatozoa with SDF may be motile and morphologically normal, having higher biological relevance in the reproductive process. We found that the proportion of DNA fragmented PIbrighter cells was significantly higher in thawed spermatozoa from cancer than non-cancer patients. Moreover, a positive correlation was found between the degree of DNA fragmentation and sperm motility in the PIbrighter population of spermatozoa from cancer patients that wasn't seen in non-cancer patients. The results of the present study suggest that higher SDF levels may contribute to the lower IVF success of cryopreserved spermatozoa from cancer patients and that evaluation of SDF could complement genetic counselling as part of the routine management of cancer patients who seek fertility preservation.

TallyHO obese female mice experience poor reproductive outcomes and abnormal blastocyst metabolism that is reversed by metformin.

Obese women experience worse reproductive outcomes than normal weight women, specifically infertility, pregnancy loss, fetal malformations and developmental delay of offspring. The aim of the present study was to use a genetic mouse model of obesity to recapitulate the human reproductive phenotype and further examine potential mechanisms and therapies. New inbred, polygenic Type 2 diabetic TallyHO mice and age-matched control C57BL/6 mice were superovulated to obtain morula or blastocyst stage embryos that were cultured in human tubal fluid (HTF) medium. Deoxyglucose uptake was determined for individual insulin-stimulated blastocysts. Apoptosis was detected by confocal microscopy using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay and Topro-3 nuclear dye. Embryos were scored for TUNEL-positive as a percentage of total nuclei. AMP-activated protein kinase (AMPK) activation, tumour necrosis factor (TNF)-α expression and adiponectin expression were analysed by western immunoblot and confocal immunofluorescent microscopy. Lipid accumulation was assayed by BODIPY. Comparisons were made between TallyHO morulae cultured to blastocyst embryos in either HTF medium or HTF medium with 25 μg mL(-1) metformin. TallyHO mice developed whole body abnormal insulin tolerance, had decreased litter sizes and increased non-esterified fatty acid levels. Blastocysts from TallyHO mice exhibited increased apoptosis, decreased insulin sensitivity and decreased AMPK. A possible cause for the insulin resistance and abnormal AMPK phosphorylation was the increased TNF-α expression and lipid accumulation, as detected by BODIPY, in TallyHO blastocysts and decreased adiponectin. Culturing TallyHO morulae with the AMPK activator metformin led to a reversal of all the abnormal findings, including increased AMPK phosphorylation, improved insulin-stimulated glucose uptake and normalisation of lipid accumulation. Women with obesity and insulin resistance experience poor pregnancy outcomes. Previously we have shown in mouse models of insulin resistance that AMPK activity is decreased and that activators of AMPK reverse poor embryo outcomes. Here, we show for the first time using a genetically altered obese model, not a diet-induced model, that metformin reverses many of the adverse effects of obesity at the level of the blastocyst. Expanding on this we determine that activation of AMPK via metformin reduces lipid droplet accumulation, presumably by eliminating the inhibitory effects of TNF-α, resulting in normalisation of fatty acid oxidation and HADH2 (hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit) activity. Metformin exposure in vitro was able to partially reverse these effects, at the level of the blastocyst, and may thus be effective in preventing the adverse effects of obesity on pregnancy and reproductive outcomes.

Multisite phosphorylation of Bcl-2 via protein kinase Cδ facilitates apoptosis of hypertrophic cardiomyocytes.

Activated protein kinase Cδ (PKCδ) associated with cardiac hypertrophy moves from the cytoplasm to the mitochondria and subsequently triggers the apoptotic signalling pathway. The underlying mechanisms remain unknown. The aim of the present study was to investigate whether mitochondrial translocation of PKCδ phosphorylates multiple sites of Bcl-2, resulting in an imbalance between Bcl-2 and Bak or Bax, thus enhancing the susceptibility of hypertrophic cardiomyocytes to angiotensin II (AngII)-induced apoptosis. Chronic pressure overload was induced by transverse aortic constriction (TAC) in rats. The apoptotic rate increased in hypertrophied cardiomyocytes. In AngII-treated hearts (10 nmol/L, 60 min), there was an increase in the number of TERMINAL deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL)-positive cells; PKCδ inhibition with 500 nmol/L δV1-1 for 60 min prevented the AngII-induced increase in apoptosis. In the hypertrophied myocardium, PKCδ expression increased, whereas that of Bcl-2 decreased compared with the synchronous control. Treatment of hearts with 10 nmol/L AngII for 60 min activated PKCδ and induced translocation of PKCδ to the mitochondria, where activated PKCδ facilitated the phosphorylation of Bcl-2 at serine-87 and serine-70 sites. The multisite phosphorylated Bcl-2 was released from the mitochondria, and exhibited reduced affinity for Bak and Bax. The imbalance between Bcl-2 and Bak/Bax induced the release of mitochondrial cytochrome c and then activated the caspase 3 apoptotic pathway during AngII stimulation (10 nmol/L, 60 min) of hypertrophied cardiomyocytes. Inhibition of PKCδ reduced these effects of AngII. The results suggest that PKCδ can counteract the anti-apoptotic effect of Bcl-2 and may promote cardiomyocyte apoptosis through multisite phosphorylation of Bcl-2 in hypertrophied cardiomyocytes.

Cisplatin-induced alterations in the functional spermatogonial stem cell pool and niche in C57/BL/6J mice following a clinically relevant multi-cycle exposure

A typical clinical cis-diamminedichloroplatinum(II) (cisplatin) dosing regimen consists of repeated treatment cycles followed by a recovery period. While effective, this dosing structure results in a prolonged, often permanent, infertility in men. Spermatogonial stem cells (SSCs) are theoretically capable of repopulating the seminiferous tubules after exposure has ceased. We propose that an altered spermatogonial environment during recovery from the initial treatment cycle drives an increase in SSC mitotic cell activity, rendering the SSC pool increasingly susceptible to cisplatin-induced injury from subsequent cycles. To test this hypothesis, the undifferentiated spermatogonia population and niche of the adult mouse (C57/BL/6J) were examined during the recovery periods of a clinically-relevant cisplatin exposure paradigm. Histological examination revealed a disorganization of spermatogenesis correlating with the number of exposure cycles. Quantification of terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end labeling (TUNEL) staining indicated an increase in apoptotic frequency following exposure. Immunohistochemical examination of Foxo1 and incorporated BrdU showed an increase in the undifferentiated spermatogonial population and mitotic activity in the recovery period in mice exposed to one cycle, but not two cycles of cisplatin. Immunohistochemical investigation of glial cell line-derived neurotrophic factor (GDNF) revealed an increase in production along the basal Sertoli cell membrane throughout the recovery period in all treatment groups. Taken together, these data establish that the impact of cisplatin exposure on the functional stem cell pool and niche correlates with: (1) the number of dosing cycles; (2) mitotic activity of early germ cells; and (3) alterations in the basal Sertoli cell GDNF expression levels after cisplatin-induced testicular injury.

Assessment of DNA damage in goat preantral follicles after vitrification of the ovarian cortex.

Effective methods for gamete preservation should have low impact on DNA integrity. The present study investigated the effects of vitrification of goat ovarian tissues on the occurrence of DNA fragmentation and DNA double-stand breaks using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay and detection of phosphorylated histone H2AX (γH2AX), respectively. Goat ovaries were collected at a local abattoir and 12 tissue fragments were prepared from each ovarian pair. Tissue fragments were used as fresh control samples or were cultured in vitro, vitrified or vitrified and cultured. Vitrification was performed using the Ovarian Tissue Cryosystem. Fragments from all groups (control and treatments) were processed for histology, transmission electron microscopy, TUNEL assay and immunofluorescence. Compared with fresh control samples, a lower percentage of morphologically normal follicles was detected in the vitrification followed by culture treatment group (P<0.05). Normal follicular ultrastructure was observed in all groups. Immunofluorescence revealed the presence of γH2AX foci in few oocytes and ovarian stromal cells. TUNEL-positive follicles were found in samples without significant differences among groups (P>0.05). In conclusion, the vitrification protocol used in the present study did not increase DNA damage in preantral follicles enclosed in goat ovarian tissues.

Small incision lenticule extraction (SMILE) and femtosecond laser LASIK: comparison of corneal wound healing and inflammation

AimTo evaluate and compare early corneal wound healing and inflammatory responses after small incision lenticule extraction (SMILE) versus femtosecond laser laser in situ keratomileusis (LASIK).MethodsThirty-six eyes of 36 rabbits underwent...

Anti-oxidative stress effect of red ginseng in the brain is mediated by peptidyl arginine deiminase type IV (PADI4) repression via estrogen receptor (ER) β up-regulation

Aim of the studyGinseng has been used as an anti-stress agent, and its active ingredient, ginsenoside, is similar in structure to estrogen. However, the effect of ginseng on the stressed brain is not completely understood. The aim of this study is to understand systematically how red ginseng (RG) affects gene expressions in the brain of immobilization (IMO) stressed mice to elucidate its underlying mechanism. Materials and methodsFor in vivo experiments, mice were stressed by immobilization for 30, 45, or 60min, and gene expression in the mice brain was analyzed by microarray and system biology. Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) staining, and gene expression by Western blot or qPCR. For in vitro study, the SK-N-SH neuroblastoma cells were stressed by H2O2 exposure. The resultant cytotoxicity was measured by MTT assay, and gene expression by Western blot, ELISA, or qPCR. ResultsMicroarray analysis of genes in IMO stressed mice brains showed that RG administration prior to IMO stress downregulated >40 genes including peptidyl arginine deiminase type 4 (PADI4). Interestingly, PADI4 was up-regulated by various stresses such as H2O2, acrylamide, and tunicamycin in neuroblastoma SK-N-SH cells but inhibited by RG. IMO stress and in vitro H2O2 stress depressed the estrogen receptor (ER)-β expression but not ERα. However, RG treatment increased ERβ expression both in vivo and in vitro. Comparative analysis regarding the networks by systems biology revealed that TNF-α plays a critical role in IMO stress, and the cell death associated network was much higher than other categories. Consistently, the IMO stress induced TNF-α and Cox-2 expressions, malondialdehyde (MDA), and cell death in the brain, whereas RG administration inhibited these inductions in vivo. siRNA and transient expression studies revealed that ERβ inhibited the PADI4 expression. ConclusionPADI4 could be used as an oxidative stress marker. RG seems to inhibit oxidative stress-inducible PADI4 by up-regulating ERβ expression in the brain thus protecting brain cells from apoptosis.

Aldosterone induces p21-regulated apoptosis via increased synthesis and secretion of tumour necrosis factor-α in human proximal tubular cells

1. Aldosterone has been shown to mediate p21-dependent cellular senescence in rat kidney proximal tubules in vivo and in cultured human proximal tubular cells. The p21-induced senescent cells express higher levels of apoptotic cytokines, such as tumour necrosis factor (TNF)-α compared with non-senescent cells. The aim of the present study was to investigate the hypothesis that aldosterone increases proximal tubular apoptosis by increasing the secretion of apoptosis-inducing factors through a p21-dependent mechanism. 2. Human proximal tubular cells were incubated with aldosterone (10 nmol/L) and cell senescence was detected by senescence-associated β-galactosidase staining and expression of p21. Apoptosis was analysed by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling and annexin/propidium iodide staining, whereas p21 localization was determined by immunofluorescence. 3. Exposure of cells to aldosterone for 3 or 5 days increased senescence-associated β-galactosidase staining, p21 and TNF-α mRNA expression and secretion of TNF-α into the culture medium. These changes were abolished by gene silencing of p21. Aldosterone failed to increase the number of apoptotic cells on day 3, but did increase them on day 5. A neutralizing antibody against TNF-α prevented the aldosterone-induced apoptotic changes. Aldosterone did not affect localization of p21. 4. These findings indicate that aldosterone increases TNF-α synthesis and secretion in proximal tubular cells via p21/senescence-dependent cell phenotypic changes and that the TNF-α secreted plays an important role as a paracrine factor in mediating cell apoptosis, indicating a possible involvement in aldosterone-induced renal damage.

Arachidonic acid supplementation during gestational, lactational and post-weaning periods prevents retinal degeneration induced in a rodent model

Fatty acids and their derivatives play a role in the response to retinal injury. The effects of dietary arachidonic acid (AA) supplementation on N-methyl-N-nitrosourea (MNU)-induced retinal degeneration was investigated in young Lewis rats during the gestational, lactational and post-weaning periods. Dams were fed 0·1, 0·5 or 2·0% AA diets or a basal (< 0·01% AA) diet. On postnatal day 21 (at weaning), male pups received a single intraperitoneal injection of 50 mg MNU/kg or vehicle, and were fed the same diet as their mother for 7 d. Retinal apoptosis was analysed by the terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick-end labelling (TUNEL) assay 24 h after the MNU treatment, and retinal morphology was examined 7 d post-MNU. Histologically, all rats that received MNU and were fed the basal and 0·1% AA diets developed retinal degeneration characterised by the loss of photoreceptor cells (disappearance of the outer nuclear layer and the photoreceptor layer) in the central retina. The 0·5 and 2·0% AA diets rescued rats from retinal damage. Morphometrically, in parallel with the AA dose (0·5 and 2·0% AA), the photoreceptor ratio significantly increased and the retinal damage ratio decreased in the central retina, compared with the corresponding ratios in basal diet-fed rats. In parallel with the increase in serum and retinal AA levels and the AA:DHA ratio, the apoptotic index in the central retina was dose-dependently decreased in rats fed the 0·5 and 2·0% AA diets. In conclusion, an AA-rich diet during the gestation, lactation and post-weaning periods rescued young Lewis rats from MNU-induced retinal degeneration via the inhibition of photoreceptor apoptosis. Therefore, an AA-enriched diet in the prenatal and postnatal periods may be an important strategy to suppress the degree of photoreceptor injury in humans.

Mechanisms mediating propofol protection of pulmonary epithelial cells against lipopolysaccharide‐induced cell death

Propofol (2,6-diisopropylphenol) is an anaesthetic agent with anti-oxidant properties. The aim of the present study was to determine whether propofol can protect pulmonary epithelial (A549) cells against lipopolysaccharide (LPS)-induced cell death and, if so, the mechanisms involved. The effects of LPS alone and in combination with propofol on A549 cell death were investigated. Cell viability was determined using the colourimetric 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apoptotic A549 cells were detected by flow cytometry, as propidium iodide-negative and annexin-V-positive cells, and terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL). Mitochondrial membrane potential (MMP), caspase 9 activity, Ca(2+) concentrations and reactive oxygen species (ROS) were analysed by immunofluorescent methods. Aconitase 2 (ACO2), microtubule-associated light chain 3 (LC3) and beclin-1 levels were evaluated using reverse transcription-polymerase chain reaction and/or western blot analysis. Exposure of A549 cells to 1-50μg/mL LPS for 3-24h resulted in the concentration- and time-dependent induction of cell death. Cell apoptosis accounted for approximately 77% of cell death induced by LPS. Propofol (5-150μmol/L) concentration-dependently inhibited LPS-induced A549 cell death. This protective effect of propofol was accompanied by prevention of LPS-induced mitochondrial dysfunction (reductions in MMP, ACO2 expression and ATP) and was associated with the inhibition of LPS-induced activation of apoptotic signals (caspase 9 activity, ROS overproduction and Ca(2+) accumulation). In addition, propofol blocked LPS-induced overexpression of the autophagy-associated proteins LC3 and beclin-1. The data indicate that propofol protects A549 cells against LPS-induced apoptosis, and probably autophagy, by blocking LPS-induced activation of ROS/caspase 9 pathways and upregulation of LC3 and beclin-1, respectively.

Markers of potential malignancy in chronic hyperplastic candidiasis

To examine the presence of markers associated with malignancy, including p53, p21 cyclin-dependent kinase inhibitor 1A, murine double minutes-2, and others, in chronic hyperplastic candidiasis. Immunohistochemical methods were used to examine the expression of p53, murine double minutes-2, p21 cyclin-dependent kinase inhibitor 1A, metallothionein, and proliferating cell nuclear antigen in 42 chronic hyperplastic candidiasis lesions and 11 non-infected control tissues. Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling was used to examine apoptosis, which was correlated with p53 expression. These markers were measured in lesions of chronic hyperplastic candidiasis that did not show any epithelial dysplasia or histological signs of malignancy. p53 scores were higher in chronic hyperplastic candidiasis than in controls (P = 0.0046). Murine double-minutes 2 levels were not elevated. p21 cyclin-dependent kinase inhibitor 1A was increased in parabasal (P < 0.0001) and basal epithelial cells. Chronic hyperplastic candidiasis lesions showed a similar basal/parabasal metallothionein staining pattern to that seen in normal squamous epithelium. Proliferating cell nuclear antigen was increased (P = 0.0007), as was apoptosis (P = 0.0033). Increased p53 in oral chronic hyperplastic candidiasis suggests an increased potential for malignant change in the epithelium, above that of normal tissues. Further functional investigation is required, as well as clinical follow-up studies.

RETRACTED: Dynamic changes of mitochondrial fission proteins after transient cerebral ischemia in mice

With fusion or fission, mitochondria alter their morphology in response to various physiological and pathological stimuli resulting in either elongated, tubular, interconnected or fragmented form. Immunohistochemistry and Western blot analyses were performed at 2, 7, 14 and 28 d after 90 min of transient middle cerebral artery occlusion (tMCAO) in mice. The present study showed that mitochondrial fission protein fission 1 (Fis1) and phosphorylated dynamin-related protein 1 (P-Drp1) both progressively increased with the peak at 14 d after tMCAO. Double immunofluorescent analysis showed the number of double positive cells with Fis1/Drp1 reduced between 2 and 28 d after 90 min of tMCAO, and also showed some double positive cells with Fis1/terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) in the peri-infract regions at 2d after the reperfusion. The present study suggests a progressive activation of mitochondrial fission proteins Fis1 and P-Drp1 in relation to apoptotic process in neural cells of the peri-infract regions after tMCAO.

Dynamic changes of mitochondrial fusion and fission proteins after transient cerebral ischemia in mice

With fusion or fission, mitochondria alter their morphology in response to various physiological and pathological stimuli, resulting in elongated, tubular, interconnected, or fragmented forms. Immunohistochemistry and Western blot analysis were performed at 2 days, 7 days, 14 days, and 28 days after 90 min of transient middle cerebral artery occlusion (tMCAO) in mice. This study showed that mitochondrial fission protein dynamin-related protein 1 (Drp1) and fusion protein optic atrophy 1 (Opa1) were both upregulated in the ischemic penumbra, with the peak at 2 days after tMCAO, whereas phosphorylated-Drp1 (P-Drp1) progressively increased with a peak at 14 days after tMCAO. Double-immunofluorescence analysis showed many Drp1/cytochrome c oxidase subunit l (COX1) double-positive cells and Opa1/COX1 double-positive cells in the ischemic penumbra and also showed some double-positive cells with Drp1/terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and Opa1/TUNEL in the ischemic penumbra. In contrast, both Drp1 and Opa1 showed progressive decreases until 2 days after tMCAO in the ischemic core because of necrotic brain damage. The present study suggests that there was a continuous mitochondrial fission and fusion during these periods in the ischemic penumbra after tMCAO, probably in an effort toward mitophagy and cellular survival.

Effect of taurine on acinar cell apoptosis and pancreatic fibrosis in dibutyltin dichloride-induced chronic pancreatitis.

The relationship between pancreatic fibrosis and apoptosis of pancreatic acinar cells has not been fully elucidated. We reported that taurine had an anti-fibrotic effect in a dibutyltin dichloride (DBTC)-chronic pancreatitis model. However, the effect of taurine on apoptosis of pancreatic acinar cells is still unclear. Therefore, we examined apoptosis in DBTC-chronic pancreatitis and in the AR42J pancreatic acinar cell line with/without taurine. Pancreatic fibrosis was induced by a single administration of DBTC. Rats were fed a taurine-containing diet or a normal diet and were sacrificed at day 5. The AR42J pancreatic acinar cell line was incubated with/without DBTC with taurine chloramines. Apoptosis was determined by using terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. The expression of Bad and Bcl-2 proteins in the AR42J cells lysates was detected by Western blot analysis. The apoptotic index of pancreatic acinar cells in DBTC-administered rats was significantly increased. Taurine treatment inhibited pancreatic fibrosis and apoptosis of acinar cells induced by DBTC. The number of TUNEL-positive cells in the AR42J pancreatic acinar cell lines was significantly increased by the addition of DBTC. Incubation with taurine chloramines ameliorated these changes. In conclusion, taurine inhibits apoptosis of pancreatic acinar cells and pancreatitis in experimental chronic pancreatitis.