scholarly journals Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

10.3791/57345 ◽  
2018 ◽  
Author(s):  
Laura A. Brannelly ◽  
Alexandra A. Roberts ◽  
Lee F. Skerratt ◽  
Lee Berger
Keyword(s):  
Cell Death ◽  
Epidermal Cell ◽  
Caspase 3 ◽  
Terminal Transferase

The P-MAPA immunomodulator partially prevents apoptosis induced by Zika virus infection in THP-1 cells

2020 ◽  
Vol 21 ◽  
Author(s):  
Morganna C. Lima ◽  
Elisa A. N. Azevedo ◽  
Clarice N. L. de Morais ◽  
Larissa I. O. de Sousa ◽  
Bruno M. Carvalho ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Virus Infection ◽  
Virus Replication ◽  
Zika Virus ◽  
Mammalian Cells ◽  
Caspase 3 ◽  
Neurological Complications ◽  
Zika Virus Infection

Background: Zika virus is an emerging arbovirus of global importance. ZIKV infection is associated with a range of neurological complications such as the Congenital Zika Syndrome and Guillain Barré Syndrome. Despite the magnitude of recent outbreaks, there is no specific therapy to prevent or to alleviate disease pathology. Objective: To investigate the role of P-MAPA immunomodulator in Zika-infected THP-1 cells. Methods: THP-1 cells were subjected at Zika virus infection (Multiplicity of Infection = 0.5) followed by treatment with P-MAPA for until 96 hours post-infection. After that, the cell death was analyzed by annexin+/ PI+ and caspase 3/ 7+ staining by flow cytometry. In addition, the virus replication and cell proliferation were accessed by RT-qPCR and Ki67 staining, respectively. Results: We demonstrate that P-MAPA in vitro treatment significantly reduces Zika virus-induced cell death and caspase-3/7 activation on THP-1 infected cells, albeit it has no role in virus replication and cell proliferation. Conclusions: Our study reveals that P-MAPA seems to be a satisfactory alternative to inhibits the effects of Zika virus infection in mammalian cells.

scholarly journals Transcriptome Analysis Reveals HgCl2 Induces Apoptotic Cell Death in Human Lung Carcinoma H1299 Cells through Caspase-3-Independent Pathway

2021 ◽  
Vol 22 (4) ◽  
pp. 2006
Author(s):  
Mi Jin Kim ◽  
Jinhong Park ◽  
Jinho Kim ◽  
Ji-Young Kim ◽  
Mi-Jin An ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Transcriptome Analysis ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Expression Patterns ◽  
Nitrogen Compound ◽  
Apoptotic Cell Death ◽  
Mercury Chloride ◽  
Rna Seq

Mercury is one of the detrimental toxicants that can be found in the environment and exists naturally in different forms; inorganic and organic. Human exposure to inorganic mercury, such as mercury chloride, occurs through air pollution, absorption of food or water, and personal care products. This study aimed to investigate the effect of HgCl2 on cell viability, cell cycle, apoptotic pathway, and alters of the transcriptome profiles in human non-small cell lung cancer cells, H1299. Our data show that HgCl2 treatment causes inhibition of cell growth via cell cycle arrest at G0/G1- and S-phase. In addition, HgCl2 induces apoptotic cell death through the caspase-3-independent pathway. Comprehensive transcriptome analysis using RNA-seq indicated that cellular nitrogen compound metabolic process, cellular metabolism, and translation for biological processes-related gene sets were significantly up- and downregulated by HgCl2 treatment. Interestingly, comparative gene expression patterns by RNA-seq indicated that mitochondrial ribosomal proteins were markedly altered by low-dose of HgCl2 treatment. Altogether, these data show that HgCl2 induces apoptotic cell death through the dysfunction of mitochondria.

scholarly journals Cell Death and Metabolic Stress in Gymnodinium catenatum Induced by Allelopathy

Toxins ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 506
Author(s):  
Leyberth José Fernández-Herrera ◽  
Christine Johanna Band-Schmidt ◽  
Tania Zenteno-Savín ◽  
Ignacio Leyva-Valencia ◽  
Claudia Judith Hernández-Guerrero ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Protein Content ◽  
Caspase 3 ◽  
Cellular Stress ◽  
Allelopathic Effect ◽  
Sod Activity ◽  
Free Media ◽  
O2 Production ◽  
Gymnodinium Catenatum

Allelopathy between phytoplankton species can promote cellular stress and programmed cell death (PCD). The raphidophyte Chattonella marina var. marina, and the dinoflagellates Margalefidinium polykrikoides and Gymnodinium impudicum have allelopathic effects on Gymnodinium catenatum; however, the physiological mechanisms are unknown. We evaluated whether the allelopathic effect promotes cellular stress and activates PCD in G. catenatum. Cultures of G. catenatum were exposed to cell-free media of C. marina var. marina, M. polykrikoides and G. impudicum. The mortality, superoxide radical (O2●−) production, thiobarbituric acid reactive substances (TBARS) levels, superoxide dismutase (SOD) activity, protein content, and caspase-3 activity were quantified. Mortality (between 57 and 79%) was registered in G. catenatum after exposure to cell-free media of the three species. The maximal O2●− production occurred with C. marina var. marina cell-free media. The highest TBARS levels and SOD activity in G. catenatum were recorded with cell-free media from G. impudicum. The highest protein content was recorded with cell-free media from M. polykrikoides. All cell-free media caused an increase in the activity of caspase-3. These results indicate that the allelopathic effect in G. catenatum promotes cell stress and caspase-3 activation, as a signal for the induction of programmed cell death.

Two steroidal saponins from Camassia cusickii induce L1210 cell death through the apoptotic mechanism

2001 ◽  
Vol 79 (11) ◽  
pp. 953-958 ◽  
Author(s):  
Ellyawati Candra ◽  
Kimihiro Matsunaga ◽  
Hironori Fujiwara ◽  
Yoshihiro Mimaki ◽  
Yutaka Sashida ◽  
...  
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Chromatin Condensation ◽  
Flow Cytometric Analysis ◽  
Apoptotic Cell Death ◽  
Morphological Observation ◽  
Steroidal Saponin ◽  
Steroidal Saponins ◽  
L1210 Cells

Two steroidal saponins, tigogenin hexasaccharide-1 (TGHS-1, (25R)-5α-spirostan-3β-yl 4-O-[2-O-[3-O- (α-L-rhamnopyranosyl)-β-D-glucopyranosyl]-3-O-[4-O-(α-L-rhamnopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranosyl]- β-D-galactopyranoside) and tigogenin hexasaccharide-2 (TGHS-2, (25R)-5α-spirostan-3β-yl 4-O-[2-O-[3-O- (β-D-glucopyranosyl)-β-D-glucopyranosyl]-3-O-[4-O-(α-L-rhamnopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranosyl]- β-D-galactopyranoside), were isolated from the fresh bulbs of Camassia cusickii. In murine leukemic L1210 cells, both compounds showed cytotoxicity with an EC50 value of 0.06 µM. The morphological observation revealed that TGHS-1 and TGHS-2 induced shrinkage in cell soma and chromatin condensation, suggesting apoptotic cell death. The cell death was confirmed to be apoptosis by Annexin V binding to phosphatidylserine in the cell membrane and excluding propidium iodide. A typical apoptotic DNA ladder and the cleavage of caspase-3 were observed after treatment with TGHS-1 and TGHS-2. In the presence of both the compounds, cells with sub-G1 DNA content were detected by flow cytometric analysis, indicating that TGHS-1 and TGHS-2 (each EC50 value of 0.1 µM) are the most powerful apoptotic saponins known. These results suggest that TGHS-1 and TGHS-2 induce apoptotic cell death through caspase-3 activation.Key words: steroidal saponin, tigogenin hexasaccharide, apoptosis, DNA fragmentation, murine leukemic L1210 cells.

Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway

2004 ◽  
Vol 287 (4) ◽  
pp. H1730-H1739 ◽  
Author(s):  
Ron Zohar ◽  
Baoqian Zhu ◽  
Peter Liu ◽  
Jaro Sodek ◽  
C. A. McCulloch
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Cardiac Fibroblasts ◽  
Chromatin Condensation ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tunel Staining ◽  
Wild Type ◽  
Nuclear Fragmentation ◽  
A Cell

Reperfusion-induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN−/−) mice were treated in vitro with H2O2to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide (PI) was increased from 5 to 60% in OPN−/−cells but was increased to only 20% in WT cells. In contrast, after 1–8 h of treatment with H2O2, the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained cells was more than twofold higher in WT than OPN−/−cells. Electron microscopy of WT cells treated with H2O2showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H2O2-treated OPN−/−cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN−/−and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN−/−cells by ∼30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PI-permeable OPN−/−cells was not altered. Restoration of OPN expression in OPN−/−fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H2O2treatment. Thus H2O2-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death.

scholarly journals Neuronal caspase-3 signaling: not only cell death

2009 ◽  
Vol 17 (7) ◽  
pp. 1104-1114 ◽  
Author(s):  
M D'Amelio ◽  
V Cavallucci ◽  
F Cecconi
Keyword(s):  
Cell Death ◽  
Caspase 3

scholarly journals AnIn VivoZebrafish Model for Interrogating ROS-Mediated Pancreaticβ-Cell Injury, Response, and Prevention

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Abhishek A. Kulkarni ◽  
Abass M. Conteh ◽  
Cody A. Sorrell ◽  
Anjali Mirmira ◽  
Sarah A. Tersey ◽  
...  
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Cell Injury ◽  
Zebrafish Model ◽  
Chemical Genetic ◽  
Regulated Cell Death ◽  
Specific Manner ◽  
High Doses

It is well known that a chronic state of elevated reactive oxygen species (ROS) in pancreaticβ-cells impairs their ability to release insulin in response to elevated plasma glucose. Moreover, at its extreme, unmitigated ROS drives regulated cell death. This dysfunctional state of ROS buildup can result both from genetic predisposition and environmental factors such as obesity and overnutrition. Importantly, excessive ROS buildup may underlie metabolic pathologies such as type 2 diabetes mellitus. The ability to monitor ROS dynamics inβ-cells in situ and to manipulate it via genetic, pharmacological, and environmental means would accelerate the development of novel therapeutics that could abate this pathology. Currently, there is a lack of models with these attributes that are available to the field. In this study, we use a zebrafish model to demonstrate that ROS can be generated in aβ-cell-specific manner using a hybrid chemical genetic approach. Using a transgenic nitroreductase-expressing zebrafish line,Tg(ins:Flag-NTR)s950, treated with the prodrug metronidazole (MTZ), we found that ROS is rapidly and explicitly generated inβ-cells. Furthermore, the level of ROS generated was proportional to the dosage of prodrug added to the system. At high doses of MTZ, caspase 3 was rapidly cleaved,β-cells underwent regulated cell death, and macrophages were recruited to the islet to phagocytose the debris. Based on our findings, we propose a model for the mechanism of NTR/MTZ action in transgenic eukaryotic cells and demonstrate the robust utility of this system to model ROS-related disease pathology.

Abstract WP62: Enhanced Neuroprotection of Normobaric Oxygenation with Ethanol Conferred by Apoptosis Reduction in Acute Ischemic Stroke

Stroke ◽  
2013 ◽  
Vol 44 (suppl_1) ◽  
Author(s):  
Sweena Parmar ◽  
Xiaokun Geng ◽  
Changya Peng ◽  
Murali Guthikonda ◽  
Yuchuan Ding
Keyword(s):  
Cell Death ◽  
Ischemic Stroke ◽  
Acute Ischemic Stroke ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Neuroprotective Effect ◽  
Apoptotic Cell Death ◽  
Ethanol Administration ◽  
Sprague Dawley ◽  
Apoptotic Proteins

Objectives: Normobaric oxygenation (NBO) has been shown to provide neuroprotection in vivo and in vitro . Yet, a recent Phase 2 clinical trial investigating NBO therapy in acute ischemic stroke was terminated due to questionable therapeutic benefit. NBO therapy alone may be insufficient to produce improved outcomes. In our recent study, we demonstrated a strong neuroprotective effect of ethanol at a dose of 1.5 g/kg (equivalent to the human legal driving limit). In this study, we sought to identify whether low-dose ethanol administration enhances the neuroprotection offered by NBO and whether combined administration of NBO with ethanol is associated with reduced apoptosis. Methods: Sprague-Dawley rats were subjected to right middle cerebral artery occlusion (MCAO) for 2 h, followed by reperfusion. Ischemic animals received either an intraperitoneal injection of 1.0 g/kg ethanol, 2 h of 100% NBO, or both ethanol and NBO. The Cell Death Detection ELISA Assay (Roche) was performed to determine apoptotic cell death at 24 h after reperfusion. Levels of pro-apoptotic (Caspase-3, Bcl-2-associated X-BAX, and Apoptosis-Inducing Factor-AIF) and anti-apoptotic proteins (Bcl-2 and Bcl-xL) were determined by Western blot analysis at 3 and 24 h after reperfusion. Results: As expected, untreated ischemic rats had the highest apoptotic cell death. Combined NBO/ethanol therapy decreased cell death by 48%, as compared to 29% with ethanol and 22% with NBO. Similarly, combined NBO/ethanol therapy promoted the greatest expression of anti-apoptotic factors and the lowest expression of pro-apoptotic proteins at 3 h after reperfusion. This effect was maintained at 24 h and even more pronounced for AIF and Caspase-3. Conclusions: Given singularly, NBO and ethanol improved the degree of cell death, decreased the expression of pro-apoptotic proteins, and increased the expression of anti-apoptotic proteins. Yet, when administered together, their effects largely compounded. These results suggest a synergistic neuroprotection offered by NBO with ethanol, which may be attributed at least in part to their shared role in modulating neuronal apoptosis.

scholarly journals Interplay between Endoplasmic Reticular Stress and Survivin in Colonic Epithelial Cells

Cells ◽  
2018 ◽  
Vol 7 (10) ◽  
pp. 171 ◽  
Author(s):  
Rohit Gundamaraju ◽  
Ravichandra Vemuri ◽  
Wai Chin Chong ◽  
Stephen Myers ◽  
Shaghayegh Norouzi ◽  
...  
Keyword(s):  
Cell Death ◽  
Cancer Cells ◽  
Inflammatory Markers ◽  
Caspase 3 ◽  
Annexin V ◽  
Parp Cleavage ◽  
Cellular Survival ◽  
Unfolded Protein ◽  
Proliferative Assay ◽  
The Unfolded Protein Response

Sustained endoplasmic reticular stress (ERS) is implicated in aggressive metastasis of cancer cells and increased tumor cell proliferation. Cancer cells activate the unfolded protein response (UPR), which aids in cellular survival and adaptation to harsh conditions. Inhibition of apoptosis, in contrast, is a mechanism adopted by cancer cells with the help of the inhibitor of an apoptosis (IAP) class of proteins such as Survivin to evade cell death and gain a proliferative advantage. In this study, we aimed to reveal the interrelation between ERS and Survivin. We initially verified the expression of Survivin in Winnie (a mouse model of chronic ERS) colon tissues by using immunohistochemistry (IHC) and immunofluorescence (IF) in comparison with wild type Blk6 mice. Additionally, we isolated the goblet cells and determined the expression of Survivin by IF and protein validation. Tunicamycin was utilized at a concentration of 10 µg/mL to induce ERS in the LS174T cell line and the gene expression of the ERS markers was measured. This was followed by determination of inflammatory cytokines. Inhibition of ERS was carried out by 4Phenyl Butyric acid (4PBA) at a concentration of 10 mM to assess whether there was a reciprocation effect. The downstream cell death assays including caspase 3/7, Annexin V, and poly(ADP-ribose) polymerase (PARP) cleavage were evaluated in the presence of ERS and absence of ERS, which was followed by a proliferative assay (EdU click) with and without ERS. Correspondingly, we inhibited Survivin by YM155 at a concentration of 100 nM and observed the succeeding ERS markers and inflammatory markers. We also verified the caspase 3/7 assay. Our results demonstrate that ERS inhibition not only significantly reduced the UPR genes (Grp78, ATF6, PERKandXBP1) along with Survivin but also downregulated the inflammatory markers such as IL8, IL4, and IL6, which suggests a positive correlation between ERS and the inhibition of apoptosis. Furthermore, we provided evidence that ERS inhibition promoted apoptosis in LS174T cells and shortened the proliferation rate. Moreover, Survivin inhibition by YM155 led to a comparable effect as that of ERS inhibition, which includes attenuation of ERS genes and inflammatory markers as well as the promotion of programmed cell death via the caspase 3/7 pathway. Together, our results propose the interrelation between ERS and inhibition of apoptosis assigning a molecular and therapeutic target for cancer treatment.

Reactive oxygen species and caspase activation mediate silica-induced apoptosis in alveolar macrophages

2001 ◽  
Vol 280 (1) ◽  
pp. L10-L17 ◽  
Author(s):  
Han-Ming Shen ◽  
Zhuo Zhang ◽  
Qi-Feng Zhang ◽  
Choon-Nam Ong
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Death ◽  
Alveolar Macrophages ◽  
Caspase 3 ◽  
Reactive Oxygen ◽  
Parp Cleavage ◽  
Oxygen Species ◽  
Caspase Activation ◽  
Caspase 9 ◽  
Induced Apoptosis

Alveolar macrophages (AMs) are the principal target cells of silica and occupy a key position in the pathogenesis of silica-related diseases. Silica has been found to induce apoptosis in AMs, whereas its underlying mechanisms involving the initiation and execution of apoptosis are largely unknown. The main objective of the present study was to examine the form of cell death caused by silica and the mechanisms involved. Silica-induced apoptosis in AMs was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay and cell cycle/DNA content analysis. The elevated level of reactive oxygen species (ROS), caspase-9 and caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage in silica-treated AMs were also determined. The results showed that there was a temporal pattern of apoptotic events in silica-treated AMs, starting with ROS formation and followed by caspase-9 and caspase-3 activation, PARP cleavage, and DNA fragmentation. Silica-induced apoptosis was significantly attenuated by a caspase-3 inhibitor, N-acetyl-Asp-Glu-Val-Asp aldehyde, and ebselen, a potent antioxidant. These findings suggest that apoptosis is an important form of cell death caused by silica exposure in which the elevated ROS level that results from silica exposure may act as an initiator, leading to caspase activation and PARP cleavage to execute the apoptotic process.

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