Quantifying the ultrastructure changes of air-dried and irradiated human amniotic membrane using atomic force microscopy: a preliminary study

Air-dried and sterilized amnion has been widely used as a dressing to treat burn and partial thickness wounds. Sterilisation at the standard dose of 25 kGy was reported to cause changes in the morphological structure as observed under the scanning electron microscope. This study aimed to quantify the changes in the ultrastructure of the air-dried amnion after gamma-irradiated at several doses by using atomic force microscope. Human placentae were retrieved from mothers who had undergone cesarean elective surgery. Amnion separated from chorion was processed and air-dried for 16 h. It was cut into 10?×?10 mm, individually packed and exposed to gamma irradiation at 5, 15, 25 and 35 kGy. Changes in the ultrastructural images of the amnion were quantified in term of diameter of the epithelial cells, size of the intercellular gap and membrane surface roughness. The longest diameter of the amnion cells reduced significantly after radiation (p?<?0.01) however the effect was not dose dependent. No significant changes in the shortest diameter after radiation, except at 35 kGy which decreased significantly when compared to 5 kGy (p?<?0.01). The size of the irradiated air-dried amnion cells reduced in the same direction without affecting the gross ultrastructure. At 15 kGy the intercellular gap decreased significantly (p?<?0.01) with Ra and Rq, values reflecting surface roughness, were significantly the highest (p?<?0.01). Changes in the ultrastructure quantified by using atomic force microscope could complement results from other microscopic techniques.     

In vitro effect of FGIN-1-27, a ligand to 18 kDa mitochondrial translocator protein,in human osteoblast-like cells

Ligands of 18 kDa mitochondrial translocator protein (TSPO) differ in their cellular effects. We hypothesize that different TSPO ligands might exert different cellular responses. Therefore, following previous studies that showed different cellular responses to two specific TSPO ligands, PK 11195 and protoporphyrin IX, in human osteoblast-like cells in vitro, we now report the cellular response to another specific TSPO ligand, FGIN-1-27 (10^?5 M) (MW 436 kDa), in order to characterize the effects of each TSPO ligand. We found in primary culture of the human osteoblast-like cells that cell numbers were decreased by an average of 30 % (p?<?0.001) following exposure to 10^?5 M of FGIN-1-27 in comparison to vehicle controls. Cellular [¹⁸F]-FDG incorporation and ATP content were suppressed, by an average of 43 % (p?<?0.001) and 83 % (p?<?0.001), respectively. Mitochondrial mass and ΔΨm increased by an average of 26 % (p?<?0.01) and 425 % (p?<?0.0001) respectively. Lactate dehydrogenase activity was enhanced in culture media by 60 % (p?<?0.05), indicating overall cell death, while no increase in apoptotic levels was observed. Cellular proliferation, as determined by BrdU assay, was not affected. Synthesis of mRNA of TSPO, VDAC 1, and hexokinase 2 decreased in 0.3, 0.3 and 0.5 fold respectively, with accompanying decreases in protein expression of TSPO and Voltage Dependent Anion Channel 1 by 23 % (p?<?0.001) and 98 % (p?<?0.001), respectively, but without changes in hexokinase 2 protein expression. Thus it appears that 10^?5 M FGIN-1-27 reduces cell viability, cell metabolism, and mitochondrial function. Previously we found similar effects of PK 11195 on mitochondrial function and cell metabolism and of protoporphyrin IX on cell death in primary osteoblast-like cells.     

Expression of tumor necrosis factor-alpha and vascular endothelial growth factor in different zones of fetal membranes: a possible relation to onset of labor

This study aimed to explore whether the altered expression of tumor necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF) and apoptotic changes in mid zone (MZ) and rupture zone (RZ) of fetal membranes (FM) are regulatory mechanisms associated with labor at term. Fifteen FM specimens were collected after vaginal deliveries and 13 specimens after elective caesarian section. Histological and immunohistochemical analysis were employed. Area percent of TNF-α and VEGF immunostaining and apoptotic index (AI) were evaluated using image analysis. The statistical data revealed significantly higher area % for TNF-α, VEGF immunoexpression and AI in labor compared to non-labor specimens (p < 0.0001). There was a significantly higher percentage of TNF-α immunoexpressed area in MZ compared with RZ in both groups (p < 0.0001). VEGF expression in RZ of both groups proved nearly double or triple the area % of expression relative to MZ with highly significant difference (p < 0.0001). quantitative analysis revealed near two fold increase in the AI in RZ (13.42 % ± 1.2 in labor; 11.20 % ± 0.96 in non-labor groups) when compared to MZ (7.20 % ± 0.6 in labor; 5.08 % ± 0.76 in non-labor groups) with highly significant zonal difference (p < 0.0001). Correlation analysis revealed significant correlation between apoptotic indices and area % of TNF-α (r = 0.575, p = 0.002 in non-labor; r = 0.652, p < 0.0001 in labor) and VEGF (r = 0.795, p < 0.0001 in non-labor; r = 0.668, p < 0.0001 in labor). In conclusion, Apoptosis may be regulated by TNF-α and VEGF expression in FM at labor. MZ is a step back from RZ and could participate actively in rupture of the FM during labor. TNF-α and VEGF increase with onset of labor and differentially expressed in the RZ and the MZ. These findings call for further study with tissue cultures or animal models.     

Lactobacillus plantarum 299v Prevents Caspase-Dependent Apoptosis In Vitro

Selective microbes used as probiotics can enhance epithelial cell protection. We have previously shown that a Lactobacillus plantarum strain 299v (Lp299v) has the ability to induce mucin genes. In the current study, we utilized a cytokine model of inflammation in cell culture to study the modulation of apoptosis by this probiotic. HT-29 cells were pre-incubated with the Lp299v or L. plantarum strain adh- (Lpadh-), a non-adherent derivative of Lp299v. Cells were challenged with a mixture of cytokines (TNF-α, IFN-γ, and IL-1a) to imitate conditions of inflammation. To assess for cell death, we evaluated TUNEL, multi-caspase, and caspase-3 and caspase-7 activity assays. There was a marked decrease in apoptosis as measured by TUNEL⁺ cells in samples pre-treated with Lp299v (18.7 ± 4.1%, p < 0.01) and Lpadh- (16.6 ± 3.2%, p < 0.05) prior to cytokine exposure when compared to cells (43.6 ± 6.2%) exposed to the cytokine mixture. Lp299v pre-incubation with HT-29 cells reduced caspase⁺ cells in the multi-caspase activity assay (3.6 ± 0.6%, p < 0.05) compared to cells exposed to cytokines (68.9 ± 5.1%) whereas Lpadh- did not (46.8 ± 17.5%, p > 0.05). Similarly, caspase-3, caspase-7 activity was also reduced by Lp299v. Selected probiotics may confer an exogenous protective effect at the mucosal–luminal interface for intestinal epithelial cells via alteration of caspase-dependent apoptotic pathways.     

Exosomes derived from RPE cells under oxidative stress mediate inflammation and apoptosis of normal RPE cells through Apaf1/caspase-9 axis

This study aims to explore the effects of exosomes, secreted by retinal pigment epithelial (RPE) cells under oxidative stress (OS), on apoptosis and inflammation of normal RPE cells. Exosomes secreted by normal RPE cells (named as exo) and rotenone (2.5 µmol/L) stimulated RPE cells (named as rot-exo) were isolated and extracted by multi-step differential centrifugation for morphology observation under a transmission electron microscopy. pcDNA3.1a, pcDNA3.1a-Apaf1, and p3xFlag-CMV-caspase-9 plasmids were constructed and transfected into ARPE-19 cells. Exosomes secreted by ARPE-19 cells were injected into the vitreous body of rats to verify the effect of Apaf1 and caspase-9 on cell apoptosis and inflammation. Co-immunoprecipitation was applied to clarify the interaction of Apaf1 with caspase-9. Exosomes secreted by rotenone stimulated ARPE-19 cells could induce cell apoptosis, oxidative injury, and inflammation in ARPE-19 cells. Exosomes secreted under OS can damage retinal functions of rats and have upregulated expression of Apaf1. Overexpression of Apaf1 in exosomes secreted under OS can cause the inhibition of cell proliferation, the increase of cell apoptosis and elicitation of inflammatory response in ARPE-19 cells. Exosomes derived from ARPE-19 cells under OS regulate Apaf1 expression to increase cell apoptosis and to induce oxidative injury and inflammatory response through a caspase-9 apoptotic pathway.     

The Effects and Mechanism of miR-92a and miR-126 on Myocardial Apoptosis in Mouse Ischemia-Reperfusion Model

Our objective was to explore the effects of miR-92a and miR-126 on myocardial apoptosis in mouse ischemia-reperfusion model and further investigate the underlying mechanisms. Eighteen Kunming mice were selected and randomly divided into sham operation group and ischemia-reperfusion group with nine mice in each group. Cardiac muscle tissue was stained with Evans blue to confirm myocardial infarction and ischemia. Annexin V/PI double staining was used to detect the apoptotic rate of myocardial cells, and terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) was used to detect the number of apoptotic cells; Western blot was used to detect expression of Caspase 3 to evaluate the apoptosis of mouse myocardial cells; qRT-PCR was used to detect expression of miR-92a and miR-126 in mouse myocardium, and Western blot was used to detect expression of HSP70 in two groups. Evans blue staining results showed that there was a large area of ischemia in myocardium of ischemia-reperfusion mice with marked infarction, suggesting successful establishment of the model. In sham operation group, myocardial cells were mostly normal cells. Annexin V/PI double staining of flow cytometry result showed that the apoptotic rate was 5.9 % in sham operation group and 37.0 % in ischemia-reperfusion group, respectively. Apoptosis detection results showed that apoptotic index (AI) of myocardial cells in ischemia-reperfusion mice was significantly higher than in sham operation group. In addition, qRT-PCR results showed that miR-92a expression in ischemia-reperfusion group was significantly higher than in sham operation group (F = 32.302, P = 0.000), and miR-126 expression in ischemia-reperfusion group was significantly lower than in sham operation group (F = 41.125, P = 0.000). Moreover, HSP70 detected by Western blot showed that HSP expression in ischemia-reperfusion group was significantly lower than in sham operation group. The change of miR-92a was in accordance with AI of myocardial cells. However, the change of miR-126 is in contrary with AI of myocardial cells, which may be related to the HSP70 expression in myocardial cells.     

The role of bronchial epithelial cell apoptosis in the pathogenesis of COPD

There is an increased airway inflammation in the pathogenesis of chronic obstructive pulmonary disease (COPD), and it has been suggested that there may also be problem in the apoptosis and renewal of cells. However, there are limited human airway cell studies, in particular those from larger airways such as bronchi. We cultured primary human bronchial epithelial cells (HBECs) from bronchial explants of smokers (n = 6) without COPD and smokers with COPD (n = 8). Apoptosis was studied by fluorescence activated cell sorting. qRT-PCR was used to assess mRNA expression for proteins involving apoptosis including p21^CIP1/WAF1, p53, caspase-8 and caspase-9. Although there was no difference in the rate of viable cells between cells from smokers and COPDs, the level of early apoptotic cells was significantly increased in COPD cells [mean ± standard error of mean (SEM) = 4.86 ± 3.2 %, p = 0.015] as compared to smokers (mean ± SEM = 2.71 ± 1.62 %). In contrast, the rate of late apoptotic cells was significantly decreased in COPD cells (mean ± SEM = 9.82 ± 5.71 %) comparing to smokers (mean ± SEM = 15.21 ± 5.08 %, p = 0.003). Although expression of mRNA for p21^CIP1/WAF1 and caspase-9 was similar in both groups, p53 and caspase-8 mRNA expression was significantly greater in COPD cells. These findings suggest that HBEC apoptosis is increased in COPD, and that this involves p53 and caspase-8 pathways.     

SASH1 regulates proliferation, apoptosis, and invasion of osteosarcoma cell

SASH1, a member of the SLY-family of signal adapter proteins, is a candidate tumor suppressor in breast and colon cancer. The SASH1 protein possesses both the SH3 and SAM domains, indicating that it may play an important role in intracellular signal transduction. Reduced expression of SASH1 is closely related to tumor growth, invasion, metastasis, and poor prognosis. However, the biological role of SASH1 remains unknown in osteosarcoma. To unravel the function of SASH1, we explored the expression of SASH1 in osteosarcoma tissues and its correlation to the clinical pathology of osteosarcoma and analyzed the relationship between SASH1 expression and cell cycle, apoptosis and invasion of osteosarcoma MG-63 cells, using the flow cytometry analysis and transwell invasion chamber experiments. Furthermore, the effect of SASH1 on the expression of cyclin D1, caspase-3, matrix metalloproteinase (MMP)-9 were observed by western blot. Our results showed that the expression rate of SASH1 mRNA in osteosarcoma tissues was significantly lower than that in normal bone tissue (p = 0.000), that the expression rate of SASH1 mRNA in the carcinoma tissues from patients with lung metastasis was significantly lower than that from patients without lung metastasis (p = 0.041), and that the expression rate of SASH1 mRNA also decreased with increasing Enneking stage (p = 0.032). However, the mRNA expression of SASH1 in osteosarcoma was independent of the patient’s gender, age, and tumor size (p = 0.983, 0.343, 0.517, respectively). The SASH1 protein displayed a down-regulation in osteosarcoma tissues compared to normal bone tissue (p = 0.000), displayed a down-regulation in osteosarcoma tissues from patients with lung metastasis compared to from patients without lung metastasis (p = 0.000), and displayed a gradual decrease with increasing Enneking stage (p = 0.000). In addition, the MG-63 cells from pcDNA3.1-SASH1 group exhibited significantly reduced cell viability, proliferation, and invasive ability compared to the empty vector group and blank control group (p = 0.023, 0.001, respectively), and there was no difference between the empty vector group and blank control group. The pcDNA3.1-SASH1 group displayed significantly more apoptotic cells than the empty vector group and blank control group (p = 0.004). The expression of cyclin D1, MMP-9 displayed a down-regulation in MG-63 cells from pcDNA3.1-SASH1 group compared to the empty vector group and blank control group (p = 0.000, 0.001, respectively) and the expression levels of caspase-3 displayed an up-regulation in MG-63 cells from pcDNA3.1-SASH1 group compared to the empty vector group and blank control group (p = 0.000). Taken together, these data indicated that the overexpression of SASH1 might be associated with the inhibition of growth, proliferation, and invasion of MG-63 cells and the promotion of apoptosis of MG-63 cells.     

Antioxidant and Neuroprotective Effects of Scrophularia striata Extract Against Oxidative Stress-Induced Neurotoxicity

In this study, the neuroprotective effect of Scrophularia striata Boiss (Scrophulariaceae) extract, a plant growing in northeastern of Iran, against oxidative stress-induced neurocytotoxicity in PC12 was evaluated. The PC12 cell line pretreated with different concentrations (10, 50, 100, and 200 μg/ml) of the extract and then treated with H₂O₂ to induce oxidative stress and neurotoxicity. Survival of the cells, reactive oxygen species (ROS) generation, and apoptosis were measured using MTT assay, fluorescent probe 2′,7′-dichlorofluorescein diacetate, and annexin V/propidium iodide, respectively. Moreover, the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) was used to evaluate the antioxidant capacity of the plant extract. Phytochemical assay by thin layer chromatography showed that the main components, including phenolic compounds, phenyl propanoids and flavonoids, were presented in the S. striata extract. The extract in concentrations of 50–200 μg/ml protected PC12 cells from H₂O₂-induced toxicity. The survival of the cells at concentration of 200 μg/ml was 64 % compared to that of H₂O₂ alone-treated cells (48 %) (p < 0.001). The extract also dose-dependently reduced intracellular ROS production (p < 0.001). Moreover, the extract showed antioxidative effects and decreased apoptotic cells. Collectively, these findings indicated the ability of S. striata to decrease ROS generation and cell apoptosis and also suggest the presence of the neuroprotective agents in this plant.     

DNA methylation of apoptosis genes in rectal cancer predicts patient survival and tumor recurrence

Deregulation of the apoptotic pathway, one of the hallmarks of tumor growth and -progression, has been shown to have prognostic value for tumor recurrence in rectal cancer. In order to develop clinically relevant biomarkers, we studied the methylation status of promoter regions of key apoptosis genes in rectal cancer patients, using methylation-sensitive restriction enzymes. DNA was extracted from fresh-frozen tumor tissues of 49 stage I-III rectal cancer patients and 10 normal rectal tissues. The results of this pilot study were validated in 88 stage III tumor tissues and 18 normal rectal tissues. We found that methylation of the intrinsic apoptotic pathway genes Apaf1, Bcl2 and p53 correlated with the apoptotic status (M30) of the tumor. Combined survival analyses of these three genes, based on the number of genes showing high methylation (all low, 1 high, 2 high or all high), showed shorter patient survival and recurrence-free periods with an increasing number of methylated markers. Multivariate analyses showed significant differences for overall survival (p = 0.01; HR = 0.28 (0.09–0.83)), cancer-specific survival (p = 0.004; HR = 0.13 (0.03–0.67)) and distant recurrence-free survival (p = 0.001; HR = 0.22(0.05–0.94)). The shortest survival was observed for patients showing low methylation of all markers, which—as was expected—correlated with high apoptosis (M30), but also with high proliferation (Ki-67). The study of epigenetic regulation of apoptosis genes provides more insight in the tumorigenic process in rectal cancer and might be helpful in further refining treatment regimens for individual patients.     

A novel inhibitor of proteasome deubiquitinating activity renders tumor cells sensitive to TRAIL-mediated apoptosis by natural killer cells and T cells

The proteasome inhibitor bortezomib simultaneously renders tumor cells sensitive to killing by natural killer (NK) cells and resistant to killing by tumor-specific T cells. Here, we show that b-AP15, a novel inhibitor of proteasome deubiquitinating activity, sensitizes tumors to both NK and T cell-mediated killing. Exposure to b-AP15 significantly increased the susceptibility of tumor cell lines of various origins to NK (p < 0.0002) and T cell (p = 0.02)-mediated cytotoxicity. Treatment with b-AP15 resulted in increased tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-2 expression (p = 0.03) and decreased cFLIP expression in tumor cells in vitro. In tumor-bearing SCID/Beige mice, treatment with b-AP15 followed by infusion of either human NK cells or tumor-specific T cells resulted in a significantly delayed tumor progression compared with mice treated with NK cells (p = 0.006), T cells (p < 0.0001) or b-AP15 alone (p = 0.003). Combined infusion of NK and T cells in tumor-bearing BALB/c mice following treatment with b-AP15 resulted in a significantly prolonged long-term survival compared with mice treated with b-AP15 and NK or T cells (p ≤ 0.01). Our findings show that b-AP15-induced sensitization to TRAIL-mediated apoptosis could be used as a novel strategy to augment the anticancer effects of adoptively infused NK and T cells in patients with cancer.     

Investigation of the Effects of 2.1 GHz Microwave Radiation on Mitochondrial Membrane Potential (ΔΨ m), Apoptotic Activity and Cell Viability in Human Breast Fibroblast Cells

In the present study we aimed to investigate the effects of 2.1 GHz Wideband Code Division Multiple Access (W-CDMA) modulated Microwave (MW) Radiation on cell survival and apoptotic activity of human breast fibroblast cells. The cell cultures were exposed to W-CDMA modulated MW at 2.1 GHz at a SAR level of 0.607 W/kg for 4 and 24 h. The cell viability was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. The percentage of apoptotic cells was analyzed by Annexin V-FITC and PI staining. 5,5′,6,6′-Tetrachloro-1,1′,3,3′- tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to measure Mitochondrial Membrane Potential (ΔΨ _m). sFasL and Fas/APO-1 protein levels were determined by ELISA method. 2.1 GHz MW radiation was shown to be able to inhibit cell proliferation and induce apoptosis in human breast fibroblast cells. The cell viability of MW-exposed cells was decreased significantly. The percentages of Annexin V-FITC positive cells were higher in MW groups. ΔΨ _m was decreased significantly due to MW radiation exposure. However, neither sFas nor FasL level was significantly changed in MW-exposed fibroblast cells. The results of this study showed that 2.1 GHz W-CDMA modulated MW radiation-induced apoptotic cell death via the mitochondrial pathway.     

Altered Mitochondria Morphology and Cell Metabolism in Apaf1-Deficient Cells

Background

Apaf1 (apoptotic protease activating factor 1) is the central component of the apoptosome, a multiprotein complex that activates procaspase-9 after cytochrome c release from the mitochondria in the intrinsic pathway of apoptosis. Other cellular roles, including a pro-survival role, have also been described for Apaf1, while the relative contribution of each function to cell death, but also to cell homeostatic conditions, remain to be clarified.

Methodology and Principal Findings

Here we examined the response to apoptosis induction of available embryonic fibroblasts from Apaf1 knockout mice (MEFS KO Apaf1). In the absence of Apaf1, cells showed mitochondria with an altered morphology that affects cytochrome c release and basal metabolic status.

Conclusions

We analysed mitochondrial features and cell death response to etoposide and ABT-737 in two different Apaf1-deficient MEFS, which differ in the immortalisation protocol. Unexpectedly, MEFS KO Apaf1 immortalised with the SV40 antigen (SV40IM-MEFS Apaf1) and those which spontaneously immortalised (SIM-MEFS Apaf1) respond differently to apoptotic stimuli, but both presented relevant differences at the mitochondria when compared to MEFS WT, indicating a role for Apaf1 at the mitochondria.     

The histone methyltransferase ESET is required for the survival of spermatogonial stem/progenitor cells in mice

Self-renewal and differentiation of spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout a male''s life. SSC transplantation will be a valuable solution for young male patients to preserve their fertility. As SSCs in the collected testis tissue from the patients are very limited, it is necessary to expansion the SSCs in vitro. Previous studies suggested that histone methyltransferase ERG-associated protein with SET domain (ESET) represses gene expression and is essential for the maintenance of the pool of embryonic stem cells and neurons. The objective of this study was to determine the role of ESET in SSCs using in vitrocell culture and germ cell transplantation. Cell transplantation assay showed that knockdown of ESET reduced the number of seminiferous tubules with spermatogenesis when compared with that of the control. Knockdown of ESET also upregulated the expression of apoptosis-associated genes (such as P53, Caspase9, Apaf1), whereas inhibited the expression of apoptosis-suppressing genes (such as Bcl2l1, X-linked inhibitor of apoptosis protein). In addition, suppression of ESET led to increase in expression of Caspase9 and activation of Caspase3 (P17) as well as cleavage of poly (ADP-ribose) polymerase. Among the five ESET-targeting genes (Cox4i2, spermatogenesis and oogenesis Specific Basic Helix-Loop-Helix 2, Nobox, Foxn1 and Dazl) examined by ChIP assay, Cox4i2 was found to regulate SSC apoptosis by the rescue experiment. BSP analyses further showed that DNA methylation in the promoter loci of Cox4i2was influenced by ESET, indicating that ESET also regulated gene expression through DNA methylation in addition to histone methylation. In conclusion, we found that ESET regulated SSC apoptosis by suppressing of Cox4i2 expression through histone H3 lysine 9 tri-methylation and DNA methylation. The results obtained will provide unique insights that would broaden the research on SSC biology and contribute to the treatment of male infertility.     

Induction of apoptosis in human glioma cell lines of various grades through the ROS-mediated mitochondrial pathway and caspase activation by <Emphasis Type="Italic">Rhaponticum carthamoides</Emphasis> transformed root extract

The placenta plays a major role in embryo-fetal defects and intrauterine growth retardation after maternal alcohol consumption. Our aims were to determine the oxidative status and cellular and molecular oxidative stress effects on uterine myometrium and trophoblast-decidual tissue following perigestational alcohol intake at early organogenesis. CF-1 female mice were administered with 10% alcohol in drinking water for 17 days prior to and up to day 10 of gestation. Control females received ethanol-free water. Treated mice had smaller implantation sites compared to controls (p < 0.05), diminished maternal vascular lumen, and irregular/discontinuous endothelium of decidual vessels. The trophoblast giant cell layer was disorganized and presented increased abnormal nuclear frequency. The myometrium of treated females had reduced nitrite content, increased superoxide dismutase activity, and reduced glutathione (GSH) content (p < 0.05). However, the trophoblast-decidual tissue of treated females had increased nitrite content (p < 0.05), increased GSH level (p < 0.001), increased thiobarbituric acid-reactive substance concentration (p < 0.001), higher 3-nitrotyrosine immunoreaction, and increased apoptotic index (p < 0.05) compared to controls. In summary, perigestational alcohol ingestion at organogenesis induced oxidative stress in the myometrium and trophoblast-decidual tissue, mainly affecting cells and macromolecules of trophoblast and decidual tissues around early organogenesis, in CF-1 mouse, and suggests that oxidative-induced abnormal early placental formation probably leads to risk of prematurity and fetal growth impairment at term.     

The apoptotic protease-activating factor 1-mediated pathway of apoptosis is dispensable for negative selection of thymocytes

Negative selection is a process to delete potentially autoreactive clones in developing thymocytes. Programmed cell death or apoptosis is thought to play an important role in this selection process. In this study, we investigated the role of apoptotic protease-activating factor 1 (Apaf1), a mammalian homologue of CED-4, in programmed cell death during the negative selection in thymus. There was no developmental abnormality in thymocytes from newborn Apaf1(-/-) mice in terms of CD4 and CD8 expression pattern and thymocyte number. Clonal deletion by endogenous male H-Y Ag of Apaf1-deficient thymocytes with transgenic expression of H-Y Ag-specific TCRs (H-Y Tg/Apaf1(-/-) thymocytes) was normally observed in lethally irradiated wild-type mice reconstituted with fetal liver-derived hemopoietic stem cells. Clonal deletion induced in vitro by a bacterial superantigen was also normal in fetal thymic organ culture. Thus, Apaf1-mediated pathway of apoptosis is dispensable for the negative selection of thymocytes. However, H-Y Tg/Apaf1(-/-) thymocytes showed partial resistance to H-Y peptide-induced deletion in vitro as compared with H-Y Tg/Apaf1(+/-) thymocytes, implicating the Apaf1-mediated apoptotic pathway in the negative selection in a certain situation. In addition, the peptide-induced deletion was still observed in H-Y Tg/Apaf1(-/-) thymocytes in the presence of a broad spectrum caspase inhibitor, z-VAD-fmk, suggesting the presence of caspase-independent cell death pathway playing roles during the negative selection. We assume that mechanisms for the negative selection are composed of several cell death pathways to avoid failure of elimination of autoreactive clones.     

Ghrelin and NUCB2/nesfatin-1 are expressed in the same gastric cell and differentially correlated with body mass index in obese subjects

The orexigenic peptide ghrelin and the anorexigenic peptide nesfatin-1 are expressed by the same endocrine cell of the rat stomach, the X/A-like cell. However, data in humans are lacking, especially under conditions of obesity. We collected gastric tissue of obese patients undergoing sleeve gastrectomy and investigated the expression of nesfatin-1 and ghrelin in the gastric oxyntic mucosa by immunofluorescence. Nesfatin-1 immunoreactivity was detected in the human oxyntic mucosa in cells with an endocrine phenotype. A major portion of nesfatin-1 immunoreactive cells (78 %) co-localized with ghrelin indicating the occurrence in human X/A-like cells. In patients with very high body mass index (BMI 55–65 kg/m²), the number of nesfatin-1 immunoreactive cells/low-power field was significantly higher than in obese patients with lower BMI (40–50 kg/m², 118 ± 10 vs. 82 ± 11, p < 0.05). On the other hand, the number of ghrelin immunoreactive cells was significantly reduced in obese patients with higher compared to lower BMI (96 ± 12 vs. 204 ± 21, p < 0.01). Also the ghrelin-acylating enzyme ghrelin-O-acyltransferase decreased with increasing BMI. In conclusion, nesfatin-1 immunoreactivity is also co-localized with ghrelin in human gastric X/A-like cells giving rise to a dual role of this cell type with differential effects on stimulation and inhibition of appetite dependent on the peptide released. The expression of these two peptides is differentially regulated under obese conditions with an increase of nesfatin-1 and a decrease of ghrelin immunoreactivity with rising BMI pointing towards an adaptive change of expression that may counteract further body weight increase.     

X/XO or H2O2 induced IPEC-J2 cell as a new in vitro model for studying apoptosis in post-weaning piglets

We previously demonstrated that intestinal epithelial cell apoptosis in weaned piglets is much more serious than that observed in sucking piglets and is related to oxidative stress during weaning. It is difficult to study the apoptosis mechanisms only using in vivo methods because of the limit of existing research technology. An in vitro cellular system is required for piglet intestinal epithelial cell apoptosis research. In this study, a non-tumorigenic epithelial cell line, IPEC-J2 cells, was employed as a cell model. Hydrogen peroxide and xanthine/xanthine oxidase (X/XO) were both used and compared for apoptosis modeling. The concentrations of hydrogen peroxide and XO were selected and verified using cell viability analysis, the comet assay and flow cytometry. Intracellular ROS were measured using fluorescent probes. Additionally, the expression levels of the apoptosis-related genes Fas, Bcl-2, P53, Caspase 3, Caspase 8, and Caspase 9 were analyzed using quantitative RT-PCR. The results indicated the optimal modeling method is a final concentration of 0.5 mM H₂O₂ incubated with IPEC-J2 cells for 1 h at 37 °C in 5 % CO₂ for hydrogen peroxide-induced apoptosis modeling, and a final concentration of 250 μM X/50 U/L XO incubated with IPEC-J2 cells for 6 h at 37 °C in 5 % CO₂ for X/XO-induced apoptosis modeling. For the apoptotic pathway, the X/XO modeling method is more similar to 21 days weaning piglets. Therefore, we suggest that X/XO modeling with IPEC-J2 cells be used as an in vitro cell culture model for weaning piglet intestinal epithelial cell apoptosis.     

Mentha piperita as a pivotal neuro-protective agent against gamma irradiation induced DNA fragmentation and apoptosis

Ionizing radiation is classified as a potent carcinogen, and its injury to living cells, in particular to DNA, is due to oxidative stress enhancing apoptotic cell death. Our present study aimed to characterize and semi-quantify the radiation-induced apoptosis in CNS and the activity of Mentha extracts as neuron-protective agent. Our results through flow cytometry exhibited the significant disturbance and arrest in cell cycle in % of M1: SubG1 phase, M2: G0/1 phase of diploid cycle, M3: S phase and M4: G2/M phase of cell cycle in brain tissue (p < 0.05). Significant increase in % of apoptosis and P53 protein expression as apoptotic biomarkers were coincided with significant decrease in Bcl₂ as an anti-apoptotic marker. The biochemical analysis recorded a significant decrease in the levels of reduced glutathione, superoxide dismutase, deoxyribonucleic acid (DNA) and ribonucleic acid contents. Moreover, numerous histopathological alterations were detected in brain tissues of gamma irradiated mice such as signs of chromatolysis in pyramidal cells of cortex, nuclear vacuolation, numerous apoptotic cell, and neural degeneration. On the other hand, gamma irradiated mice pretreated with Mentha extract showed largely an improvement in all the above tested parameters through a homeostatic state for the content of brain apoptosis and stabilization of DNA cycle with a distinct improvement in cell cycle analysis and antioxidant defense system. Furthermore, the aforementioned effects of Mentha extracts through down-regulation of P53 expression and up-regulation of Bcl₂ domain protected brain structure from extensive damage. Therefore, Mentha extract seems to have a significant role to ameliorate the neuronal injury induced by gamma irradiation.