Screening and Detection of Apoptosis
ABSTRACT Since programmed cell death was first described by the electron microscopic cellular changes demonstrating an organized form of cell death over 30 years ago, it has undergone a great deal of scrutiny as a potential target for several diseases including cancer. The techniques for the study of apoptosis have evolved accordingly. Methodologies for the study of apoptosis were examined by a MEDLINE search of the English-language literature and are summarized in this review. This review discusses the various ways to study apoptosis with specific assays, reagents, and molecules. The particular advantages and disadvantages of each method are reviewed.
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- "Monoclonal antibodies are available against all the caspases for immunohistochemistry , ELISA, and even some for flow cytometry (i.e., active caspase 3) . Each method that is able to detect and verify apoptosis has advantages and disadvantages, and it is best to confirm apoptosis with multiple complementary techniques . Inhibition of apoptosis seems to improve hepatocyte survival and prevent reperfusion injury. "
ABSTRACT: Background. The aim of this experimental study was to investigate the role of apigenin in liver apoptosis, in an experimental model of hepatic ischemia-reperfusion in rats. Materials and Methods. Forty-eight Wistar rats (apigenin and control groups), 14 to 16 weeks old and weighing 220 to 350 g, were used. They were all subjected to hepatic ischemia by occlusion of the hepatic artery and portal vein for 45 minutes and reperfusion was followed for 60, 120, and 240 minutes. Apigenin was administrated intraperitoneally. Liver tissues were used for the detection of apoptosis by TUNEL assay and caspase 3 antibodies. Expression analysis of Fas/FasL genes was evaluated by real time PCR. Results. The expression analysis of Fas and FasL genes was increasing during reperfusion (significantly in the group of 240 minutes of reperfusion). It was in the same group that apigenin decreased Fas receptor levels and inhibited apoptosis as confirmed by TUNEL assay and caspase 3 antibodies. Conclusions. The effects of apigenin in the Fas/FasL mediated pathway of apoptosis, in the hepatic ischemia-reperfusion, seem to have a protective result on the hepatic cell.BioMed Research International 07/2014; 2014:157216. DOI:10.1155/2014/157216 · 2.71 Impact Factor
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- "Pancreatic beta cell apoptosis plays a crucial role in the pathogenesis of Type 1 Diabetes mellitus (T1DM) (Atkinson, 2005; Eizirik and Mandrup-Poulsen, 2001; Kim and Lee, 2009). This process is initiated by two main pathways: the " extrinsic " or death receptor and the " intrinsic " or mitochondrial apoptosis pathway (Huerta et al., 2007; Millan and Huerta, 2009). The intrinsic death pathway involves loss of mitochondrial homeostasis, particularly of the outer mitochondrial membrane integrity, and subsequently the release of mitochondrial pro-apoptotic factors including cytochrome c. "
ABSTRACT: Pro-inflammatory cytokines are key mediators in the selective and progressive destruction of insulin-producing beta cells during type 1 diabetes development. However, the mechanisms of cytokine-induced beta cell apoptosis are not fully understood. This study demonstrates that pro-inflammatory cytokines strongly modified the expression of the anti-apoptotic protein Bcl-2 and the pro-apoptotic BH3-only proteins Bad, Bim, and Bid in primary rat islets and insulin-producing RINm5F cells. Overexpression of mitochondrially located catalase (MitoCatalase) specifically increased basal Bcl-2 and decreased basal Bax expression, suppressed cytokine-mediated reduction of Bcl-2, and thereby prevented the release of cytochrome c, Smac/DIABLO and the activation of caspase-9 and -3. Thus, cytokine-mediated decrease of Bcl-2 expression and the sequentially changed Bax/Bcl-2 ratio are responsible for the release of pro-apoptotic mitochondrial factors, activation of caspase-9, and ultimately caspase-3. These results indicate that activation of the intrinsic/mitochondrial apoptosis pathway is essential for cytokine-induced beta cell death and the mitochondrial generation of reactive oxygen species, in particular mitochondrial hydrogen peroxide, differentially regulates the Bax/Bcl-2 ratio.Molecular and Cellular Endocrinology 10/2010; 332(1-2):88-96. DOI:10.1016/j.mce.2010.09.017 · 4.24 Impact Factor
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- "This may depend on cleavage-directed activation of one or more signaling pathways , a feature observed during caspase-mediated differentiation of skeletal muscle myoblasts and epithelial cell types (Fernando et al., 2005). Another important aspect of the present results is that higher mRNA levels of Caspase 3 do not indicate higher Caspase 3 activity within the cell, because Caspases are secreted as inactive procaspases (Huerta et al., 2007). In this regard, measuring protein levels seems to be needed to confirm the real impact within a testis exposed to xenoestrogens. "
ABSTRACT: Apoptosis inhibition has been reported in the male reproductive tract of teleost fish exposed to 17beta-estrogen or estrogen-like compounds. In order to understand the molecular mechanisms of cell death inhibition, this study examined 2 genes involved in the apoptotic pathway, Bcl-2 and Caspase 3, an anti-apoptotic and a pro-apoptotic genes, respectively. Partial cDNA sequences of Bcl-2 and Caspase 3 were cloned from gudgeon (Gobio gobio), a common European cyprinid fish. To follow mRNA levels of Bcl-2 and Caspase 3 under xenoestrogen exposure, we first performed an in vitro experiment on fish testis exposed to the most potent xenoestrogen found in the environment, ethinylestradiol (EE2). We further studied mRNA expression of both genes in the testis of fish exposed to xenoestrogens in situ. In the in vitro experiment, fragments of gudgeon testis were exposed for 21 days to 10(-3), 10(-2), 10(-1), 1 and 10 microg/L of EE2, as well as to positive (10(-1) microg/L of E2) and ethanol control medium. Results showed a significant induction of Bcl-2 mRNA at 10(-1) microg/L (p<0.05). Surprisingly, Caspase 3, a cell death effector, displayed the same profile as observed for the anti-apoptotic gene Bcl-2. In the experiment on wild gudgeon exposed from birth to an estrogenic sewage treatment plant effluent, the mRNA expression of Bcl-2 and Caspase 3 in feminized fish (ovotestis) was not significantly different due to high variability of expression between individuals. At the current state of knowledge on spermatogenesis disruption in teleost fish, in vitro studies seem better adapted than in situ investigations to enlighten the molecular pathway of apoptosis inhibition in testis exposed to xenoestrogens.Aquatic toxicology (Amsterdam, Netherlands) 03/2010; 98(3):304-10. DOI:10.1016/j.aquatox.2010.02.016 · 3.51 Impact Factor