The role of apoptotic cell death in Drosophila melanogaster radioinduced aging

International Nuclear Information System (INIS)

Moskalev, A.A.; Zajnullin, V.G.

2001-01-01

The attempt is made to estimate a role of programmed cell death (apoptosis) in radioinduced life span alteration and aging. It was shown with the use of mutant Drosophila melanogaster laboratory strains that the dysfunction of a reaper-dependent apoptosis pathway together with the action of ionizing radiation and/or apoptosis inductor etoposide could to lead to change of life span and a pace of aging. In Drosophila strain with defect of proapoptosis gene reaper the increase of life span after irradiation and etoposide treatment was observed. At the same time the strain with overexpression of a protease dcp-1 gene and the strain with the defect of antiapoptosis diap-1/th gene decreased the life span after irradiation and etoposide treatment. The obtained facts are discussed from a position of participation of apoptosis deregulation in radioinduced and natural aging of whole organisms [ru

Drosophila spaghetti and doubletime link the circadian clock and light to caspases, apoptosis and tauopathy.

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John C Means

2015-05-01

Full Text Available While circadian dysfunction and neurodegeneration are correlated, the mechanism for this is not understood. It is not known if age-dependent circadian dysfunction leads to neurodegeneration or vice-versa, and the proteins that mediate the effect remain unidentified. Here, we show that the knock-down of a regulator (spag of the circadian kinase Dbt in circadian cells lowers Dbt levels abnormally, lengthens circadian rhythms and causes expression of activated initiator caspase (Dronc in the optic lobes during the middle of the day or after light pulses at night. Likewise, reduced Dbt activity lengthens circadian period and causes expression of activated Dronc, and a loss-of-function mutation in Clk also leads to expression of activated Dronc in a light-dependent manner. Genetic epistasis experiments place Dbt downstream of Spag in the pathway, and Spag-dependent reductions of Dbt are shown to require the proteasome. Importantly, activated Dronc expression due to reduced Spag or Dbt activity occurs in cells that do not express the spag RNAi or dominant negative Dbt and requires PDF neuropeptide signaling from the same neurons that support behavioral rhythms. Furthermore, reduction of Dbt or Spag activity leads to Dronc-dependent Drosophila Tau cleavage and enhanced neurodegeneration produced by human Tau in a fly eye model for tauopathy. Aging flies with lowered Dbt or Spag function show markers of cell death as well as behavioral deficits and shortened lifespans, and even old wild type flies exhibit Dbt modification and activated caspase at particular times of day. These results suggest that Dbt suppresses expression of activated Dronc to prevent Tau cleavage, and that the circadian clock defects confer sensitivity to expression of activated Dronc in response to prolonged light. They establish a link between the circadian clock factors, light, cell death pathways and Tau toxicity, potentially via dysregulation of circadian neuronal remodeling in

Effects of 2,4-D and DCP on the DHT-induced androgenic action in human prostate cancer cells.

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Kim, Hyun-Jung; Park, Young In; Dong, Mi-Sook

2005-11-01

2,4-Dichlorophenoxyacetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (DCP) are used extensively in agriculture as herbicides, and are suspected of potential endocrine disruptor activity. In a previous study, we showed that these compounds exhibited synergistic androgenic effects by co-treatment with testosterone in the Hershberger assay. To elucidate the mechanisms of the synergistic effects of these compounds on the androgenicity of testosterone, the androgenic action of 2,4-D and DCP was characterized using a mammalian detection system in prostate cancer cell lines. In in vitro assay systems, while 2,4-D or DCP alone did not show androgenic activity, 2,4-D or DCP with 5alpha-dihydroxytestosterone (DHT) exhibited synergistic androgenic activities. Co-treatment of 10 nM 2,4-D or DCP with 10 nM DHT was shown to stimulate the cell proliferation by 1.6-fold, compared to 10 nM DHT alone. In addition, in transient transfection assays, androgen-induced transactivation was also increased to a maximum of 32-fold or 1.28-fold by co-treatment of 2,4-D or DCP with DHT, respectively. However, 2,4-D and DCP exerted no effects on either mRNA or protein levels of AR. In a competitive AR binding assay, 2,4-D and DCP inhibited androgen binding to AR, up to 50% at concentrations of approximately 0.5 microM for both compounds. The nuclear translocation of green fluorescent protein-AR fusion protein in the presence of DHT was promoted as the result of the addition of 2,4-D and DCP. Collectively, these results that 2,4-D and DCP enhanced DHT-induced AR transcriptional activity might be attributable, at least in part, to the promotion of AR nuclear translocation.

NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus.

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Ando, Kiyohiro; Parsons, Melissa J; Shah, Richa B; Charendoff, Chloé I; Paris, Sheré L; Liu, Peter H; Fassio, Sara R; Rohrman, Brittany A; Thompson, Ruth; Oberst, Andrew; Sidi, Samuel; Bouchier-Hayes, Lisa

2017-06-05

The PIDDosome (PIDD-RAIDD-caspase-2 complex) is considered to be the primary signaling platform for caspase-2 activation in response to genotoxic stress. Yet studies of PIDD-deficient mice show that caspase-2 activation can proceed in the absence of PIDD. Here we show that DNA damage induces the assembly of at least two distinct activation platforms for caspase-2: a cytoplasmic platform that is RAIDD dependent but PIDD independent, and a nucleolar platform that requires both PIDD and RAIDD. Furthermore, the nucleolar phosphoprotein nucleophosmin (NPM1) acts as a scaffold for PIDD and is essential for PIDDosome assembly in the nucleolus after DNA damage. Inhibition of NPM1 impairs caspase-2 processing, apoptosis, and caspase-2-dependent inhibition of cell growth, demonstrating that the NPM1-dependent nucleolar PIDDosome is a key initiator of the caspase-2 activation cascade. Thus we have identified the nucleolus as a novel site for caspase-2 activation and function. © 2017 Ando et al.

Caspase-1 from the silkworm, Bombyx mori, is involved in Bombyx mori nucleopolyhedrovirus infection.

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Wang, Qiang; Ju, Xiaoli; Chen, Liang; Chen, Keping

2017-03-01

Caspase-1 is one of the effector caspases in mammals that plays a central role in apoptosis. However, the lepidopteran caspase-1, especially the Bombyx mori caspase-1 (Bm-caspase-1), has not been investigated in detail. In this study, Bm-caspase-1 was identified from an expressed sequence tag database in B. mori by BLAST search. The open reading frame of Bm-caspase-1 contained 879 nucleotides and encoded 293 amino acids with a predicted molecular mass of 33 kDa. Bm-caspase-1 contained two consensus amino acid motifs of caspase cleavage sites, DEGDA and TETDG. Caspase activity assays revealed significant proteolytic activity of the Ac-DEVD-pNA substrate. Bm-caspase-1 can be detected in all tissues and developmental stages by a semi quantitative polymerase chain reaction assay. More importantly, the expression level of Bm-caspase-1 is increased upon baculovirus infection and up-regulated in BmNPV-resistant silkworms. Taken together, these results indicate that Bm-caspase-1 plays an important role during baculovirus infection.

Triglyceride-induced macrophage cell death is triggered by caspase-1.

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Son, Sin Jee; Rhee, Ki-Jong; Lim, Jaewon; Kim, Tae Ue; Kim, Tack-Joong; Kim, Yoon Suk

2013-01-01

Triglyceride (TG) induces macrophage cell death which contributes to the development of atherosclerosis. We confirmed that exogenous TG accumulates in human THP-1 macrophages and causes cell death. TG treated THP-1 macrophages exhibited no change in tumor necrosis factor (TNF)-α, interleukin (IL)-18, macrophage inflammatory protein (MIP)-1α, and IL-1R1 receptor mRNA expression. However, there was a marked decrease in IL-1β mRNA expression but an increase in IL-1β protein secretion. Decreased expression of IL-1β mRNA and increased secretion of IL-1β protein was not the direct cause of cell death. Until now, TG was assumed to induce necrotic cell death in macrophages. Since caspase-1 is known to be involved in activation and secretion of IL-1β protein and pyroptotic cell death, next we determined whether caspase-1 is associated with TG-induced macrophage cell death. We found an increase in caspase-1 activity in TG-treated THP-1 macrophages and inhibition of caspase-1 activity using a specific inhibitor partially rescued cell death. These results suggest activation of the pyroptotic pathway by TG. This is the first report implicating the activation of caspase-1 and the triggering of the pyroptosis pathway in TG-induced macrophage cell death.

75 FR 38802 - DCP Raptor Pipeline, LLC; Notice of Baseline Filing

Science.gov (United States)

2010-07-06

... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission [Docket No. PR10-42-000] DCP Raptor Pipeline, LLC; Notice of Baseline Filing June 28, 2010. Take notice that on June 22, 2010, DCP Raptor Pipeline, LLC submitted a baseline filing of its Statement of Operating Conditions for services provided...

echinus, required for interommatidial cell sorting and cell death in the Drosophila pupal retina, encodes a protein with homology to ubiquitin-specific proteases

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Gorski Sharon M

2007-07-01

Full Text Available Abstract Background Programmed cell death is used to remove excess cells between ommatidia in the Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death. Results We generated a number of new echinus alleles, some likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases. These proteins cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss. Conclusion The echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions

76 FR 76711 - DCP Raptor Pipeline, LLC; Notice of Motion for Extension of Rate Case Filing Deadline

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2011-12-08

... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission [Docket No. PR09-32-003] DCP Raptor..., 2011, DCP Raptor Pipeline, LLC (Raptor) filed a request for an extension consistent with the Commission... pipelines to extend the cycle for such reviews from three to five years.\\1\\ Therefore, Raptor requests that...

Mononuclear Phagocyte-Derived Microparticulate Caspase-1 Induces Pulmonary Vascular Endothelial Cell Injury.

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Srabani Mitra

Full Text Available Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS and impacts disease progression. Lung endothelial injury has traditionally been focused on the role of neutrophil trafficking to lung vascular integrin receptors induced by proinflammatory cytokine expression. Although much is known about the pathogenesis of cell injury and death in ALI/ARDS, gaps remain in our knowledge; as a result of which there is currently no effective pharmacologic therapy. Enzymes known as caspases are essential for completion of the apoptotic program and secretion of pro-inflammatory cytokines. We hypothesized that caspase-1 may serve as a key regulator of human pulmonary microvascular endothelial cell (HPMVEC apoptosis in ALI/ARDS. Our recent experiments confirm that microparticles released from stimulated monocytic cells (THP1 induce lung endothelial cell apoptosis. Microparticles pretreated with the caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, were unable to induce cell death of HPMVEC, suggesting the role of caspase-1 or its substrate in the induction of HPMVEC cell death. Neither un-induced microparticles (control nor direct treatment with LPS induced apoptosis of HPMVEC. Further experiments showed that caspase-1 uptake into HPMVEC and the induction of HPMVEC apoptosis was facilitated by caspase-1 interactions with microparticulate vesicles. Altering vesicle integrity completely abrogated apoptosis of HPMVEC suggesting an encapsulation requirement for target cell uptake of active caspase-1. Taken together, we confirm that microparticle centered caspase-1 can play a regulator role in endothelial cell injury.

Simultaneous Analysis of 3-MCPD and 1,3-DCP in Asian Style Sauces Using QuEChERS Extraction and Gas Chromatography���Triple Quadrupole Mass Spectrometry

OpenAIRE

Genualdi, Susan; Nyman, Patricia; DeJager, Lowri

2017-01-01

Acid hydrolyzed vegetable protein (aHVP) is used for flavoring a wide variety of foods and also in the production of nonfermented soy sauce. During the production of aHVP, chloropropanols including 3-monochloropropane-1,2-diol (3-MCPD) and 1,3 dichloropropane-2-ol (1,3-DCP) can be formed through the reaction of the hydrochloric acid catalyst and residual fat and the reaction of 3-MCPD with acetic acid, respectively. 3-MCPD is a carcinogen, and 1,3-DCP has been classified as a genotoxic carcin...

Caspase-3 controls AML1-ETO-driven leukemogenesis via autophagy modulation in a ULK1-dependent manner.

Science.gov (United States)

Man, Na; Tan, Yurong; Sun, Xiao-Jian; Liu, Fan; Cheng, Guoyan; Greenblatt, Sarah M; Martinez, Camilo; Karl, Daniel L; Ando, Koji; Sun, Ming; Hou, Dan; Chen, Bingyi; Xu, Mingjiang; Yang, Feng-Chun; Chen, Zhu; Chen, Saijuan; Nimer, Stephen D; Wang, Lan

2017-05-18

AML1-ETO (AE), a fusion oncoprotein generated by t(8;21), can trigger acute myeloid leukemia (AML) in collaboration with mutations including c-Kit, ASXL1/2, FLT3, N-RAS, and K-RAS. Caspase-3, a key executor among its family, plays multiple roles in cellular processes, including hematopoietic development and leukemia progression. Caspase-3 was revealed to directly cleave AE in vitro, suggesting that AE may accumulate in a Caspase-3-compromised background and thereby accelerate leukemogenesis. Therefore, we developed a Caspase-3 knockout genetic mouse model of AML and found that loss of Caspase-3 actually delayed AML1-ETO9a (AE9a)-driven leukemogenesis, indicating that Caspase-3 may play distinct roles in the initiation and/or progression of AML. We report here that loss of Caspase-3 triggers a conserved, adaptive mechanism, namely autophagy (or macroautophagy), which acts to limit AE9a-driven leukemia. Furthermore, we identify ULK1 as a novel substrate of Caspase-3 and show that upregulation of ULK1 drives autophagy initiation in leukemia cells and that inhibition of ULK1 can rescue the phenotype induced by Caspase-3 deletion in vitro and in vivo. Collectively, these data highlight Caspase-3 as an important regulator of autophagy in AML and demonstrate that the balance and selectivity between its substrates can dictate the pace of disease. © 2017 by The American Society of Hematology.

Caspase-1 deficiency reduces intestinal and hepatic triglyceride-rich lipoprotein secretion

NARCIS (Netherlands)

Diepen, van Janna A.; Stienstra, Rinke; Hooiveld, Guido; Willems van Dijk, Ko; Rensen, Patrick C.

2013-01-01

Background and Aims: Inflammasome-mediated caspase-1 activity regulates the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18. Recently, we showed that caspase-1 deficiency strongly reduces high fat diet-induced adiposity although the mechanism is still unclear.

Evaluation of automated serum des-gamma-carboxyprothrombin (DCP) assays for detecting hepatocellular carcinoma.

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Choi, Jonghyeon; Park, Yongjung; Kim, Jeong-Ho; Kim, Hyon-Suk

2011-12-01

We evaluated two new autoanalyzers, μTAS and Lumipulse for des-γ-carboxyprothrombin (DCP) assay. Analytical performance was evaluated, and the upper reference limit of the 97.5th percentile for DCP was re-established using sera from 140 healthy individuals. DCP levels were determined by the two autoanalyzers and EIA in a total of 239 sera from HCC patients (n=120) and those without HCC (n=119). Total imprecision of the two automated assays was Lumipulse. There were proportional and constant biases between the results from the autoanalyzers and those from EIA. The two newly developed DCP assays showed high analytical performance, but re-establishment of reference limits would be necessary. The new analyzers could be useful for clinical laboratories because of convenience of operation and wide AMRs. Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Engineering a light-activated caspase-3 for precise ablation of neurons in vivo.

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Smart, Ashley D; Pache, Roland A; Thomsen, Nathan D; Kortemme, Tanja; Davis, Graeme W; Wells, James A

2017-09-26

The circuitry of the brain is characterized by cell heterogeneity, sprawling cellular anatomy, and astonishingly complex patterns of connectivity. Determining how complex neural circuits control behavior is a major challenge that is often approached using surgical, chemical, or transgenic approaches to ablate neurons. However, all these approaches suffer from a lack of precise spatial and temporal control. This drawback would be overcome if cellular ablation could be controlled with light. Cells are naturally and cleanly ablated through apoptosis due to the terminal activation of caspases. Here, we describe the engineering of a light-activated human caspase-3 (Caspase-LOV) by exploiting its natural spring-loaded activation mechanism through rational insertion of the light-sensitive LOV2 domain that expands upon illumination. We apply the light-activated caspase (Caspase-LOV) to study neurodegeneration in larval and adult Drosophila Using the tissue-specific expression system (UAS)-GAL4, we express Caspase-LOV specifically in three neuronal cell types: retinal, sensory, and motor neurons. Illumination of whole flies or specific tissues containing Caspase-LOV-induced cell death and allowed us to follow the time course and sequence of neurodegenerative events. For example, we find that global synchronous activation of caspase-3 drives degeneration with a different time-course and extent in sensory versus motor neurons. We believe the Caspase-LOV tool we engineered will have many other uses for neurobiologists and others for specific temporal and spatial ablation of cells in complex organisms.

Simultaneous Analysis of 3-MCPD and 1,3-DCP in Asian Style Sauces Using QuEChERS Extraction and Gas Chromatography–Triple Quadrupole Mass Spectrometry

Science.gov (United States)

Genualdi, Susan; Nyman, Patricia; DeJager, Lowri

2017-01-01

Acid hydrolyzed vegetable protein (aHVP) is used for flavoring a wide variety of foods and also in the production of nonfermented soy sauce. During the production of aHVP, chloropropanols including 3-monochloropropane-1,2-diol (3-MCPD) and 1,3 dichloropropane-2-ol (1,3-DCP) can be formed through the reaction of the hydrochloric acid catalyst and residual fat and the reaction of 3-MCPD with acetic acid, respectively. 3-MCPD is a carcinogen, and 1,3-DCP has been classified as a genotoxic carcinogen. The European Union (EU) has set a maximum concentration of 0.02 mg/kg of 3-MCPD in aHVP, and the Food and Drug Administration (FDA) set a guidance limit of 1 mg/kg of 3-MCPD in aHVP. 1,3-DCP is not an approved food additive, and the Joint FAO/WHO Expert Committee on Food Additives (JEFCA) has set a limit at 0.005 mg/kg, which is close to the estimated method detection limit. Currently there are few analytical methods for the simultaneous determination of 3-MCPD and 1,3-DCP without derivatization due to differences in their physical chemical properties and reactivity. A new method was developed using QuEChERS (quick, easy, cheap, effective, rugged, and safe) with direct analysis of the extract without derivatization using gas chromatography–triple quadrupole mass spectrometry (GC-QQQ). Additionally, a market sampling of 60 soy sauce samples was performed in 2015 to determine if concentrations have changed since the FDA limit was set in 2008. The sampling results were compared between the new QuEChERS method and a method using phenylboronic acid (PBA) as a derivatizing agent for 3-MCPD analysis. The concentrations of 3-MCPD detected in soy sauce samples collected in 2015 (3-MCPD below current regulatory limits PMID:28064506

Simultaneous Analysis of 3-MCPD and 1,3-DCP in Asian Style Sauces Using QuEChERS Extraction and Gas Chromatography-Triple Quadrupole Mass Spectrometry.

Science.gov (United States)

Genualdi, Susan; Nyman, Patricia; DeJager, Lowri

2017-02-01

Acid hydrolyzed vegetable protein (aHVP) is used for flavoring a wide variety of foods and also in the production of nonfermented soy sauce. During the production of aHVP, chloropropanols including 3-monochloropropane-1,2-diol (3-MCPD) and 1,3 dichloropropane-2-ol (1,3-DCP) can be formed through the reaction of the hydrochloric acid catalyst and residual fat and the reaction of 3-MCPD with acetic acid, respectively. 3-MCPD is a carcinogen, and 1,3-DCP has been classified as a genotoxic carcinogen. The European Union (EU) has set a maximum concentration of 0.02 mg/kg of 3-MCPD in aHVP, and the Food and Drug Administration (FDA) set a guidance limit of 1 mg/kg of 3-MCPD in aHVP. 1,3-DCP is not an approved food additive, and the Joint FAO/WHO Expert Committee on Food Additives (JEFCA) has set a limit at 0.005 mg/kg, which is close to the estimated method detection limit. Currently there are few analytical methods for the simultaneous determination of 3-MCPD and 1,3-DCP without derivatization due to differences in their physical chemical properties and reactivity. A new method was developed using QuEChERS (quick, easy, cheap, effective, rugged, and safe) with direct analysis of the extract without derivatization using gas chromatography-triple quadrupole mass spectrometry (GC-QQQ). Additionally, a market sampling of 60 soy sauce samples was performed in 2015 to determine if concentrations have changed since the FDA limit was set in 2008. The sampling results were compared between the new QuEChERS method and a method using phenylboronic acid (PBA) as a derivatizing agent for 3-MCPD analysis. The concentrations of 3-MCPD detected in soy sauce samples collected in 2015 (3-MCPD below current regulatory limits.

7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity.

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Wypijewska, Anna; Bojarska, Elzbieta; Lukaszewicz, Maciej; Stepinski, Janusz; Jemielity, Jacek; Davis, Richard E; Darzynkiewicz, Edward

2012-10-09

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' → 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' → 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Both the caspase CSP-1 and a caspase-independent pathway promote programmed cell death in parallel to the canonical pathway for apoptosis in Caenorhabditis elegans.

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Daniel P Denning

Full Text Available Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3, of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell

SfDredd, a Novel Initiator Caspase Possessing Activity on Effector Caspase Substrates in Spodoptera frugiperda.

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Yang, Zhouning; Zhou, Ke; Liu, Hao; Wu, Andong; Mei, Long; Liu, Qingzhen

2016-01-01

Sf9, a cell line derived from Spodoptera frugiperda, is an ideal model organism for studying insect apoptosis. The first notable study that attempted to identify the apoptotic pathway in Sf9 was performed in 1997 and included the discovery of Sf-caspase-1, an effector caspase of Sf9. However, it was not until 2013 that the first initiator caspase in Sf9, SfDronc, was discovered, and the apoptotic pathway in Sf9 became clearer. In this study, we report another caspase of Sf9, SfDredd. SfDredd is highly similar to insect initiator caspase Dredd homologs. Experimentally, recombinant SfDredd underwent autocleavage and exhibited different efficiencies in cleavage of synthetic caspase substrates. This was attributed to its caspase activity for the predicted active site mutation blocked the above autocleavage and synthetic caspase substrates cleavage activity. SfDredd was capable of not only cleaving Sf-caspase-1 in vitro but also cleaving Sf-caspase-1 and inducing apoptosis when it was co-expressed with Sf-caspase-1 in Sf9 cells. The protein level of SfDredd was increased when Sf9 cells were treated by Actinomycin D, whereas silencing of SfDredd reduced apoptosis and Sf-caspase-1 cleavage induced by Actinomycin D treatment. These results clearly indicate that SfDredd functioned as an apoptotic initiator caspase. Apoptosis induced in Sf9 cells by overexpression of SfDredd alone was not as obvious as that induced by SfDronc alone, and the cleavage sites of Sf-caspase-1 for SfDredd and SfDronc are different. In addition, despite sharing a sequence homology with initiator caspases and possessing weak activity on initiator caspase substrates, SfDredd showed strong activity on effector caspase substrates, making it the only insect caspase reported so far functioning similar to human caspase-2 in this aspect. We believe that the discovery of SfDredd, and its different properties from SfDronc, will improve the understanding of apoptosis pathway in Sf9 cells.

Roles of inflammatory caspases during processing of zebrafish interleukin-1β in Francisella noatunensis infection

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Vojtech, Lucia N.; Scharping, Nichole; Woodson, James C.; Hansen, John D.

2012-01-01

The interleukin-1 family of cytokines are essential for the control of pathogenic microbes but are also responsible for devastating autoimmune pathologies. Consequently, tight regulation of inflammatory processes is essential for maintaining homeostasis. In mammals, interleukin-1 beta (IL-1β) is primarily regulated at two levels, transcription and processing. The main pathway for processing IL-1β is the inflammasome, a multiprotein complex that forms in the cytosol and which results in the activation of inflammatory caspase (caspase 1) and the subsequent cleavage and secretion of active IL-1β. Although zebrafish encode orthologs of IL-1β and inflammatory caspases, the processing of IL-1β by activated caspase(s) has never been examined. Here, we demonstrate that in response to infection with the fish-specific bacterial pathogen Francisella noatunensis, primary leukocytes from adult zebrafish display caspase-1-like activity that results in IL-1β processing. Addition of caspase 1 or pancaspase inhibitors considerably abrogates IL-1β processing. As in mammals, this processing event is concurrent with the secretion of cleaved IL-1β into the culture medium. Furthermore, two putative zebrafish inflammatory caspase orthologs, caspase A and caspase B, are both able to cleave IL-1β, but with different specificities. These results represent the first demonstration of processing and secretion of zebrafish IL-1β in response to a pathogen, contributing to our understanding of the evolutionary processes governing the regulation of inflammation.                   

Caspase-1 but Not Caspase-11 Is Required for NLRC4-Mediated Pyroptosis and Restriction of Infection by Flagellated Legionella Species in Mouse Macrophages and In Vivo.

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Cerqueira, Daiane M; Pereira, Marcelo S F; Silva, Alexandre L N; Cunha, Larissa D; Zamboni, Dario S

2015-09-01

Gram-negative bacteria from the Legionella genus are intracellular pathogens that cause a severe form of pneumonia called Legionnaires' disease. The bacteria replicate intracellularly in macrophages, and the restriction of bacterial replication by these cells is critical for host resistance. The activation of the NAIP5/NLRC4 inflammasome, which is readily triggered in response to bacterial flagellin, is essential for the restriction of bacterial replication in murine macrophages. Once activated, this inflammasome induces pore formation and pyroptosis and facilitates the restriction of bacterial replication in macrophages. Because investigations related to the NLRC4-mediated restriction of Legionella replication were performed using mice double deficient for caspase-1 and caspase-11, we assessed the participation of caspase-1 and caspase-11 in the functions of the NLRC4 inflammasome and the restriction of Legionella replication in macrophages and in vivo. By using several species of Legionella and mice singly deficient for caspase-1 or caspase-11, we demonstrated that caspase-1 but not caspase-11 was required for pore formation, pyroptosis, and restriction of Legionella replication in macrophages and in vivo. By generating F1 mice in a mixed 129 × C57BL/6 background deficient (129 × Casp-11(-/-) ) or sufficient (129 × C57BL/6) for caspase-11 expression, we found that caspase-11 was dispensable for the restriction of Legionella pneumophila replication in macrophages and in vivo. Thus, although caspase-11 participates in flagellin-independent noncanonical activation of the NLRP3 inflammasome, it is dispensable for the activities of the NLRC4 inflammasome. In contrast, functional caspase-1 is necessary and sufficient to trigger flagellin/NLRC4-mediated restriction of Legionella spp. infection in macrophages and in vivo. Copyright © 2015 by The American Association of Immunologists, Inc.

Incorporation of 210Pb and 210Po to Poultry through the Addition of Dicalcium Phosphate (DCP) to the Diet

International Nuclear Information System (INIS)

Casacuberta, N.; Masque, P.; Garcia-Orellana, J.; Gasa, J.; Anguita, M.

2008-01-01

Due to the replacement of calcium by uranium in the phosphorite, sedimentary phosphate rock contains high concentrations of 238 U (i.e. from 1500 Bq·kg -1 in Morocco to 4000 Bq·kg -1 in Tanzania ores). Dicalcium Phosphate (DCP) is produced by the wet acid digestion of the phosphorite, and is used as a source of calcium and phosphorus for livestock feed supplement. If the phosphorite acid digestion is made with hydrochloric acid, DCP may present specific activities of about 10 3 Bq·kg -1 of 238 U and some of its decay chain daughters. In particular, due to its radiological implications, the presence of 210 Pb and 210 Po in DCP is of special relevance. The aim of this work was to investigate the potential incorporation of these radionuclides to poultry through its diet. Three different diets were therefore prepared with different contents of both DCP and 210 Pb and 210 Po. Diet A was used as a blank, and had a 2.5% in weight of monocalcium phosphate (MCP); diet B, with a 5% in weight of DCP; and diet C, with a 2.5% of DCP. Concentrations of 210 Pb were 0.93, 101.4 and 51.2 Bq·kg -1 ; whereas concentrations of 210 Po were 0.92, 74 and 36 Bq·kg -1 of food for diets A, B and C, respectively. Accumulation of 210 Pb and 210 Po was analysed at several times during poultry growth in samples of bone, liver, kidney, muscle, excrements as well as entire animals, with a total of 30 broilers fed with the 3 different diets. Results showed clear enhancements in the accumulation of both 210 Pb and 210 Po in chicken for diets B and C, and in particular in liver and bone. However, total accumulation of radionuclides in chicken, and especially in edible parts, is low compared to its expulsion through excrements. These results are interpreted in terms of the potential dose through consumption of chicken

The Inflammasome Drives GSDMD-Independent Secondary Pyroptosis and IL-1 Release in the Absence of Caspase-1 Protease Activity.

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Schneider, Katharina S; Groß, Christina J; Dreier, Roland F; Saller, Benedikt S; Mishra, Ritu; Gorka, Oliver; Heilig, Rosalie; Meunier, Etienne; Dick, Mathias S; Ćiković, Tamara; Sodenkamp, Jan; Médard, Guillaume; Naumann, Ronald; Ruland, Jürgen; Kuster, Bernhard; Broz, Petr; Groß, Olaf

2017-12-26

Inflammasomes activate the protease caspase-1, which cleaves interleukin-1β and interleukin-18 to generate the mature cytokines and controls their secretion and a form of inflammatory cell death called pyroptosis. By generating mice expressing enzymatically inactive caspase-1 C284A , we provide genetic evidence that caspase-1 protease activity is required for canonical IL-1 secretion, pyroptosis, and inflammasome-mediated immunity. In caspase-1-deficient cells, caspase-8 can be activated at the inflammasome. Using mice either lacking the pyroptosis effector gasdermin D (GSDMD) or expressing caspase-1 C284A , we found that GSDMD-dependent pyroptosis prevented caspase-8 activation at the inflammasome. In the absence of GSDMD-dependent pyroptosis, the inflammasome engaged a delayed, alternative form of lytic cell death that was accompanied by the release of large amounts of mature IL-1 and contributed to host protection. Features of this cell death modality distinguished it from apoptosis, suggesting it may represent a distinct form of pro-inflammatory regulated necrosis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Caspase-1-dependent and -independent cell death pathways in Burkholderia pseudomallei infection of macrophages.

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Antje Bast

2014-03-01

Full Text Available The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1β and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1β secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1β, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1

Fenton-like oxidation of 2,4-DCP in aqueous solution using iron-based nanoparticles as the heterogeneous catalyst.

Science.gov (United States)

Li, Renchao; Gao, Ying; Jin, Xiaoying; Chen, Zuliang; Megharaj, Mallavarapu; Naidu, Ravendra

2015-01-15

In this report, various iron-based nanoparticles (nZVI, n-Ni/Fe, n-Pd/Fe) were used for both heterogeneous Fenton oxidation of 2,4-dichlorophenol (2,4-DCP) and reductive dechlorination of 2,4-DCP in order to understand their roles in the Fenton oxidation and the reductive degradation of 2,4-DCP. The dechlorination efficiency of 2,4-DCP using nZVI, n-Ni/Fe, n-Fe/Pd and Fe(2)(+) was 6.48%, 6.80%, 15.95%, 5.02%, while Fenton oxidation efficiency of 2,4-DCP was 57.87%, 34.23%, 27.94%, 19.61% after 180 min, respectively. The new findings included a higher dechlorination using n-Fe/Pd due to Pd effective catalysis and the effective heterogeneous Fenton oxidation using nZVI depending on reductive dechlorination and heterogeneous Fenton oxidation occurs simultaneously. However, nZVI as the potential catalyst for heterogeneous Fenton was observed, and SEM, EDS and XRD demonstrate that change on the nZVI surface occurred due to the Fe(2+) leaching, and Total Organic Carbon (TOC) (30.71%) shows that 2,4-DCP was degraded. Furthermore, the experiment indicates that the pH values and concentration of 2,4-DCP significantly impacted on the heterogeneous Fenton oxidation of 2,4-DCP and the data fits well with the pseudo first-order kinetic model, which was a diffusion-controlled reaction. Finally, a possible mechanism for degradation of 2,4-DCP was proposed. Copyright © 2014 Elsevier Inc. All rights reserved.

Caspase-1 from Human Myeloid-Derived Suppressor Cells Can Promote T Cell-Independent Tumor Proliferation.

Science.gov (United States)

Zeng, Qi; Fu, Juan; Korrer, Michael; Gorbounov, Mikhail; Murray, Peter J; Pardoll, Drew; Masica, David L; Kim, Young J

2018-05-01

Immunosuppressive myeloid-derived suppressive cells (MDSCs) are characterized by their phenotypic and functional heterogeneity. To better define their T cell-independent functions within the tumor, sorted monocytic CD14 + CD11b + HLA-DR low/- MDSCs (mMDSC) from squamous cell carcinoma patients showed upregulated caspase-1 activity, which was associated with increased IL1β and IL18 expression. In vitro studies demonstrated that mMDSCs promoted caspase-1-dependent proliferation of multiple squamous carcinoma cell lines in both human and murine systems. In vivo , growth rates of B16, MOC1, and Panc02 were significantly blunted in chimeric mice adoptively transferred with caspase-1 null bone marrow cells under T cell-depleted conditions. Adoptive transfer of wild-type Gr-1 + CD11b + MDSCs from tumor-bearing mice reversed this antitumor response, whereas caspase-1 inhibiting thalidomide-treated MDSCs phenocopied the antitumor response found in caspase-1 null mice. We further hypothesized that MDSC caspase-1 activity could promote tumor-intrinsic MyD88-dependent carcinogenesis. In mice with wild-type caspase-1, MyD88-silenced tumors displayed reduced growth rate, but in chimeric mice with caspase-1 null bone marrow cells, MyD88-silenced tumors did not display differential tumor growth rate. When we queried the TCGA database, we found that caspase-1 expression is correlated with overall survival in squamous cell carcinoma patients. Taken together, our findings demonstrated that caspase-1 in MDSCs is a direct T cell-independent mediator of tumor proliferation. Cancer Immunol Res; 6(5); 566-77. ©2018 AACR . ©2018 American Association for Cancer Research.

Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1β maturation

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Zamboni Dario S

2010-10-01

Full Text Available Abstract Background Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1β and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold using caspase-1 deficient mice (casp1-/-. Results Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE2 and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL-1β and cyclooxygenase (COX-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNFα and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1β and PGE2 did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-α and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1β was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE2 production in the paw of WT mice, but was reduced in casp1-/- mice. Conclusion These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1β maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE2 production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

Abrogation of the presenilin 1/beta-catenin interaction and preservation of the heterodimeric presenilin 1 complex following caspase activation.

Science.gov (United States)

Tesco, G; Kim, T W; Diehlmann, A; Beyreuther, K; Tanzi, R E

1998-12-18

beta-Catenin has previously been shown to interact with presenilin 1 (PS1) in transfected cells. Here we report that beta-catenin co-immunoprecipitates with the endogenous C-terminal fragment of presenilin 1 (PS1-CTF) but not with the endogenous CTF of presenilin 2 (PS2-CTF) in H4 human neuroglioma cells. During staurosporine (STS)-induced cell death, beta-catenin and PS1-CTF undergo a caspase-mediated cleavage. After 12 h of STS treatment, the beta-catenin.PS1-CTF interaction is abrogated. While PS1-CTF immunoprecipitated with all caspase-cleaved species of beta-catenin, beta-catenin holoprotein did not co-immunoprecipitate with the "alternative" caspase-derived PS1-CTF (PS1-aCTF). Thus, the abrogation of the beta-catenin.PS1-CTF complex was due to caspase cleavage of PS1-CTF. beta-Catenin co-immunoprecipitated with PS1-NTF, but only when PS1-NTF was associated with PS1-CTF. Even though PS1-NTF.CTF complex stability was not altered by caspase cleavage, its ability to bind beta-catenin was abolished. Thus, while the PS1-NTF.CTF complex is preserved after caspase cleavage, it may no longer be fully functional.

DCP Series

Directory of Open Access Journals (Sweden)

Philip Stearns

2011-06-01

Full Text Available Photo essay. A collection of Images produced by intentionally corrupting the circuitry of a Kodak DC280 2 MP digitalcamera. By rewiring the electronics of a digital camera, glitched images are produced in a manner that parallels chemically processing unexposed film or photographic paper to produce photographic images without exposure to light. The DCP Series of Digital Images are direct visualizations of data generated by a digital camera as it takes a picture. Electronic processes associated with the normal operations of the camera, which are usually taken for granted, are revealed through an act of intervention. The camera is turned inside­out through complexes of short­circuits, selected by the artist, transforming the camera from a picture taking device to a data capturing device that renders raw data (electronic signals as images. In essence, these images are snap­shots of electronic signals dancing through the camera's circuits, manually rerouted, written directly to the on­board memory device. Rather than seeing images of the world through a lens, we catch a glimpse of what the camera sees when it is forced to peer inside its own mind.

Caspase-1 deficiency in mice reduces intestinal triglyceride absorption and hepatic triglyceride secretion

NARCIS (Netherlands)

Diepen, J.A. van; Stienstra, R.; Vroegrijk, I.O.C.M.; Berg, S.A.A. van den; Salvatori, D.; Hooiveld, G.J.; Kersten, S.; Tack, C.J.; Netea, M.G.; Smit, J.W.A.; Joosten, L.A.B.; Havekes, L.M.; Dijk, K.W. van; Rensen, P.C.N.

2013-01-01

Caspase-1 is known to activate the proinflammatory cytokines IL-1β and IL-18. Additionally, it can cleave other substrates, including proteins involved in metabolism. Recently, we showed that caspase-1 deficiency in mice strongly reduces high-fat diet-induced weight gain, at least partly caused by

Carbon ion beam triggers both caspase-dependent and caspase-independent pathway of apoptosis in HeLa and status of PARP-1 controls intensity of apoptosis.

Science.gov (United States)

Ghorai, Atanu; Sarma, Asitikantha; Bhattacharyya, Nitai P; Ghosh, Utpal

2015-04-01

High linear energy transfer (LET) carbon ion beam (CIB) is becoming very promising tool for various cancer treatments and is more efficient than conventional low LET gamma or X-rays to kill malignant or radio-resistant cells, although detailed mechanism of cell death is still unknown. Poly (ADP-ribose) polymerase-1 (PARP-1) is a key player in DNA repair and its inhibitors are well-known as radio-sensitizer for low LET radiation. The objective of our study was to find mechanism(s) of induction of apoptosis by CIB and role of PARP-1 in CIB-induced apoptosis. We observed overall higher apoptosis in PARP-1 knocked down HeLa cells (HsiI) compared with negative control H-vector cells after irradiation with CIB (0-4 Gy). CIB activated both intrinsic and extrinsic pathways of apoptosis via caspase-9 and caspase-8 activation respectively, followed by caspase-3 activation, apoptotic body, nucleosomal ladder formation and sub-G1 accumulation. Apoptosis inducing factor translocation into nucleus in H-vector but not in HsiI cells after CIB irradiation contributed caspase-independent apoptosis. Higher p53 expression was observed in HsiI cells compared with H-vector after exposure with CIB. Notably, we observed about 37 % fall of mitochondrial membrane potential, activation of caspase-9 and caspase-3 and mild activation of caspase-8 without any detectable apoptotic body formation in un-irradiated HsiI cells. We conclude that reduction of PARP-1 expression activates apoptotic signals via intrinsic and extrinsic pathways in un-irradiated cells. CIB irradiation further intensified both intrinsic and extrinsic pathways of apoptosis synergistically along with up-regulation of p53 in HsiI cells resulting overall higher apoptosis in HsiI than H-vector.

Diosgenin induces apoptosis in IGF-1-stimulated human thyrocytes through two caspase-dependent pathways

International Nuclear Information System (INIS)

Mu, Shumin; Tian, Xingsong; Ruan, Yongwei; Liu, Yuantao; Bian, Dezhi; Ma, Chunyan; Yu, Chunxiao; Feng, Mei; Wang, Furong; Gao, Ling; Zhao, Jia-jun

2012-01-01

Highlights: ► Diosgenin induces apoptosis in IGF-1-treated thyrocytes through two caspase pathways. ► Diosgenin inhibits FLIP and activates caspase-8 in FAS related-pathway. ► Diosgenin increases ROS, regulates the ratio of Bax/Bcl-2 in mitochondrial pathway. -- Abstract: Insulin-like growth factor-1 (IGF-1) is a growth factor of the thyroid that has been shown in our previous study to possess proliferative and antiapoptotic effects in FRTL-5 cell lines through the upregulation of cyclin D and Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-inhibitory protein (FLIP). Diosgenin, a natural steroid sapogenin from plants, has been shown to induce apoptosis in many cell lines, with the exception of thyroid cells. In this report, we investigated the apoptotic effect and mechanism of diosgenin in IGF-1-stimulated primary human thyrocytes. Primary human thyrocytes were preincubated with or without IGF-1 for 24 h and subsequently exposed to varying concentrations of diosgenin for different times. We found that diosgenin induced apoptosis in human thyrocytes pretreated with IGF-1 in a dose-dependent manner through the activation of caspase cascades. Moreover, diosgenin inhibited FLIP and activated caspase-8 in the FAS-related apoptotic pathway. Diosgenin increased the production of ROS, regulated the balance of Bax and Bcl-2 and cleaved caspase-9 in the mitochondrial apoptotic pathway. These results indicate that diosgenin induces apoptosis in IGF-1-stimulated primary human thyrocytes through two caspase-dependent pathways.

IQGAP1 is important for activation of caspase-1 in macrophages and is targeted by Yersinia pestis type III effector YopM.

Science.gov (United States)

Chung, Lawton K; Philip, Naomi H; Schmidt, Valentina A; Koller, Antonius; Strowig, Till; Flavell, Richard A; Brodsky, Igor E; Bliska, James B

2014-07-01

YopM is a leucine-rich repeat (LRR)-containing effector in several Yersinia species, including Yersinia pestis and Y. pseudotuberculosis. Different Yersinia strains encode distinct YopM isoforms with variable numbers of LRRs but conserved C-terminal tails. A 15-LRR isoform in Y. pseudotuberculosis YPIII was recently shown to bind and inhibit caspase-1 via a YLTD motif in LRR 10, and attenuation of YopM(-) YPIII was reversed in mice lacking caspase-1, indicating that caspase-1 inhibition is a major virulence function of YopM(YPIII). To determine if other YopM proteins inhibit caspase-1, we utilized Y. pseudotuberculosis strains natively expressing a 21-LRR isoform lacking the YLTD motif (YopM(32777)) or ectopically expressing a Y. pestis 15-LRR version with a functional (YopM(KIM)) or inactivated (YopM(KIM) D271A) YLTD motif. Results of mouse and macrophage infections with these strains showed that YopM(32777), YopM(KIM), and YopM(KIM) D271A inhibit caspase-1 activation, indicating that the YLTD motif is dispensable for this activity. Analysis of YopM(KIM) deletion variants revealed that LRRs 6 to 15 and the C-terminal tail are required to inhibit caspase-1 activation. YopM(32777), YopM(KIM), and YopM(KIM) deletion variants were purified, and binding partners in macrophage lysates were identified. Caspase-1 bound to YopM(KIM) but not YopM(32777). Additionally, YopM(KIM) bound IQGAP1 and the use of Iqgap1(-/-) macrophages revealed that this scaffolding protein is important for caspase-1 activation upon infection with YopM(-) Y. pseudotuberculosis. Thus, while multiple YopM isoforms inhibit caspase-1 activation, their variable LRR domains bind different host proteins to perform this function and the LRRs of YopM(KIM) target IQGAP1, a novel regulator of caspase-1, in macrophages. Importance: Activation of caspase-1, mediated by macromolecular complexes termed inflammasomes, is important for innate immune defense against pathogens. Pathogens can, in turn, subvert

Radiation levels in samples of dicalcium phosphate (DCP) and bovine and poultry rations by gamma-ray spectrometry

International Nuclear Information System (INIS)

Luz Filho, Isaias Venancio da; Scheibel, Viviane; Appoloni, Carlos Roberto

2009-01-01

The objective of the present work is to determine the radiation levels found in bovine rations and in the dicalcium phosphate (DCP) feed supplement. Knowledge of the radiation levels in samples of rations and DCP for cattle is important, because they are directly and indirectly-part of the human diet. In order to obtain this data, gamma-ray spectrometry technique was used, employing a HPGe detector of 66% of relative efficiency, with an energy resolution of 2.03 KeV for the 60 Co 1332.46 KeV line. The radioactive activity of the radionuclides 40 K, 137 Cs, 238 U, 232 Th and its respective decay series was measured. The accommodation recipient of the samples was a Marinelli beaker of 1 L. The 238 U series activities were calculated through 214 Pb and 214 Bi activities, and the 232 Th series' activity was calculated through the 228 Ac, 212 Pb, 212 Bi and 208 Tl values. The DCP samples and the rations measured in this work were produced in Londrina city, Brazil, in the second semester of 2007. Among the rations, the largest 40 K and 228 Ra activities were found in the ration sample for milk cattle, 402 ± 14 and 1.71 ± 0.10 Bq/kg, respectively. The ration for beef cattle yielded the largest activity for 226 Ra, 1.51 + 0.93 Bq/kg. In the DCP sample, the activities for the 238 U, 40 K and 232 Th were respectively 83 ± 26; 46.6 ± 2.8 and 7.79 ± 0.70 Bq/kg. The MDA values for 137 Cs varied from 0.037 to 0.29 Bq/kg. (author)

Membrane receptor-mediated apoptosis and caspase activation in the differentiated EoL-1 eosinophilic cell line.

Science.gov (United States)

Al-Rabia, Mohammed W; Blaylock, Morgan G; Sexton, Darren W; Walsh, Garry M

2004-06-01

Caspases are key molecules in the control of apoptosis, but relatively little is known about their contribution to eosinophil apoptosis. We examined caspase-3, -8, and -9 activities in receptor ligation-dependent apoptosis induction in the differentiated human eosinophilic cell line EoL-1. Differentiated EoL-1 exhibited bi-lobed nuclei, eosinophil-associated membrane receptors, and basic granule proteins. Annexin-V fluorescein isothiocyanate binding to EoL-1 revealed significant (PEoL-1 but had no effect on constitutive (baseline) apoptosis at 16 and 20 h. Caspase activity was analyzed using the novel CaspaTag trade mark technique and flow cytometry. EoL-1 treated with pan-CD45, CD45RA, CD45RB, and CD95 mAb exhibited caspase-3 and -9 activation at 12 h post-treatment, which increased at 16 and 20 h. Activated caspase-8 was detected 12 and 16 h after ligation with CD45, CD45RA, CD45RB, and CD95 mAb followed by a trend toward basal levels at 20 h. CD69 ligation resulted in caspase-3 activation, a modest but significant activation of caspase-8, and a loss in mitochondrial transmembrane potential but had no significant effect on activation of caspase-9. Thus, the intrinsic and extrinsic caspase pathways are involved in controlling receptor ligation-mediated apoptosis induction in human eosinophils, findings that may aid the development of a more targeted, anti-inflammatory therapy for asthma.

Diosgenin induces apoptosis in IGF-1-stimulated human thyrocytes through two caspase-dependent pathways

Energy Technology Data Exchange (ETDEWEB)

Mu, Shumin [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Hospital Affiliated to Shandong Traditional Chinese Medicine University, Jinan 250011 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China); Tian, Xingsong; Ruan, Yongwei [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Liu, Yuantao [The Second Hospital of Shandong University, Jinan 250033 (China); Bian, Dezhi [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Jining Medical College, Jining 272013 (China); Ma, Chunyan [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Yu, Chunxiao [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China); Feng, Mei [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Wang, Furong [Shandong University of Traditional Chinese Medicine, Jinan 250011 (China); Gao, Ling [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China); Zhao, Jia-jun, E-mail: jjzhao@medmail.com.cn [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China)

2012-02-10

Highlights: Black-Right-Pointing-Pointer Diosgenin induces apoptosis in IGF-1-treated thyrocytes through two caspase pathways. Black-Right-Pointing-Pointer Diosgenin inhibits FLIP and activates caspase-8 in FAS related-pathway. Black-Right-Pointing-Pointer Diosgenin increases ROS, regulates the ratio of Bax/Bcl-2 in mitochondrial pathway. -- Abstract: Insulin-like growth factor-1 (IGF-1) is a growth factor of the thyroid that has been shown in our previous study to possess proliferative and antiapoptotic effects in FRTL-5 cell lines through the upregulation of cyclin D and Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-inhibitory protein (FLIP). Diosgenin, a natural steroid sapogenin from plants, has been shown to induce apoptosis in many cell lines, with the exception of thyroid cells. In this report, we investigated the apoptotic effect and mechanism of diosgenin in IGF-1-stimulated primary human thyrocytes. Primary human thyrocytes were preincubated with or without IGF-1 for 24 h and subsequently exposed to varying concentrations of diosgenin for different times. We found that diosgenin induced apoptosis in human thyrocytes pretreated with IGF-1 in a dose-dependent manner through the activation of caspase cascades. Moreover, diosgenin inhibited FLIP and activated caspase-8 in the FAS-related apoptotic pathway. Diosgenin increased the production of ROS, regulated the balance of Bax and Bcl-2 and cleaved caspase-9 in the mitochondrial apoptotic pathway. These results indicate that diosgenin induces apoptosis in IGF-1-stimulated primary human thyrocytes through two caspase-dependent pathways.

DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1.

Science.gov (United States)

Wong, Jing Ting; Akhbar, Farzanah; Ng, Amanda Yunn Ee; Tay, Mandy Li-Ian; Loi, Gladys Jing En; Pek, Jun Wei

2017-10-02

Stable intronic sequence RNAs (sisRNAs) are by-products of splicing and regulate gene expression. How sisRNAs are regulated is unclear. Here we report that a double-stranded RNA binding protein, Disco-interacting protein 1 (DIP1) regulates sisRNAs in Drosophila. DIP1 negatively regulates the abundance of sisR-1 and INE-1 sisRNAs. Fine-tuning of sisR-1 by DIP1 is important to maintain female germline stem cell homeostasis by modulating germline stem cell differentiation and niche adhesion. Drosophila DIP1 localizes to a nuclear body (satellite body) and associates with the fourth chromosome, which contains a very high density of INE-1 transposable element sequences that are processed into sisRNAs. DIP1 presumably acts outside the satellite bodies to regulate sisR-1, which is not on the fourth chromosome. Thus, our study identifies DIP1 as a sisRNA regulatory protein that controls germline stem cell self-renewal in Drosophila.Stable intronic sequence RNAs (sisRNAs) are by-products of splicing from introns with roles in embryonic development in Drosophila. Here, the authors show that the RNA binding protein DIP1 regulates sisRNAs in Drosophila, which is necessary for germline stem cell homeostasis.

Involvement of caspase-2 and caspase-9 in endoplasmic reticulum stress-induced apoptosis: A role for the IAPs

International Nuclear Information System (INIS)

Cheung, Herman H.; Lynn Kelly, N.; Liston, Peter; Korneluk, Robert G.

2006-01-01

Dysregulation of apoptosis is involved in a wide spectrum of disease ranging from proliferative to degenerative disorders. An emerging area of study in apoptosis is the critical contribution of the endoplasmic reticulum (ER) in both mitochondrial and ER specific apoptosis pathways. Here we show that brefeldin A and tunicamycin-mediated ER stress lead to caspase-dependent apoptosis involving caspase-2. Confocal microscopy and subcellular fractionation indicate that caspase-2 is localized to the ER, and following ER stress, the processing of caspase-2 and -9 is an early event preceding the activation of caspase-3 and -7 and the cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP). Inhibition and silencing of either caspase-2 or caspase-9 suppress ER stress-induced apoptosis, as demonstrated by annexin V binding. Similarly, transduction with an adenovirus encoding either Inhibitors of Apoptosis (IAP) protein HIAP1/c-IAP2 or HIAP2/c-IAP1 also suppresses ER stress-induced apoptosis. However, among HIAP1, HIAP2 and XIAP, only HIAP2 binds and inhibits caspase-2. Our results thus indicate a novel mechanism by which HIAP2 can regulate ER-initiated apoptosis by modulating the activity of caspase-2

Radiation levels in samples of dicalcium phosphate (DCP) and bovine and poultry rations by gamma-ray spectrometry

Energy Technology Data Exchange (ETDEWEB)

Luz Filho, Isaias Venancio da; Scheibel, Viviane; Appoloni, Carlos Roberto, E-mail: isaiasfilhojr@yahoo.com.b [Universidade Estadual de Londrina (UEL), PR (Brazil). Dept. de Fisica

2009-07-01

The objective of the present work is to determine the radiation levels found in bovine rations and in the dicalcium phosphate (DCP) feed supplement. Knowledge of the radiation levels in samples of rations and DCP for cattle is important, because they are directly and indirectly-part of the human diet. In order to obtain this data, gamma-ray spectrometry technique was used, employing a HPGe detector of 66% of relative efficiency, with an energy resolution of 2.03 KeV for the {sup 60}Co 1332.46 KeV line. The radioactive activity of the radionuclides {sup 40}K, {sup 137}Cs, {sup 238}U, {sup 232}Th and its respective decay series was measured. The accommodation recipient of the samples was a Marinelli beaker of 1 L. The {sup 238}U series activities were calculated through {sup 214}Pb and {sup 214}Bi activities, and the {sup 232}Th series' activity was calculated through the {sup 228}Ac, {sup 212}Pb, {sup 212}Bi and {sup 208}Tl values. The DCP samples and the rations measured in this work were produced in Londrina city, Brazil, in the second semester of 2007. Among the rations, the largest {sup 40}K and {sup 228}Ra activities were found in the ration sample for milk cattle, 402 +- 14 and 1.71 +- 0.10 Bq/kg, respectively. The ration for beef cattle yielded the largest activity for {sup 226}Ra, 1.51 + 0.93 Bq/kg. In the DCP sample, the activities for the {sup 238}U, {sup 40}K and {sup 232}Th were respectively 83 +- 26; 46.6 +- 2.8 and 7.79 +- 0.70 Bq/kg. The MDA values for {sup 137}Cs varied from 0.037 to 0.29 Bq/kg. (author)

Identification of Serratia marcescens SE1 and determination of its Herbicide 2,2-dichloropropionate (2,2-DCP Degradation Potential

Directory of Open Access Journals (Sweden)

Abel, E.

2012-01-01

Full Text Available Aims: The goal of the study is to isolate species of bacteria that capable of utilizing 2,2-dichloropropionic acid (2,2-DCP as sole carbon source from soil sample collected from surrounding lake water located in Universiti Teknologi Malaysia, Skudai, Johor. Methodology and Results: Genomic DNA from bacterium SE1 was extracted and PCR amplification was carried out using universal primers, Fd1 (5’ - AGA GTT TGA TCC TGGCTC AG - 3’ and rP1 (5’- ACG GTC ATA CCT TGT TAC GAC TT - 3’ before sending for sequencing. The 16S rDNA nucleotide sequences were compared with Basic Local Alignment Search Tool nucleotide (BLASTn and further analyzed using phylogenetic tree of Neighbour-Joining method (MEGA 5. Phylogenetic analysis indicated that SE1 strain clearly shared 97% homology to the genus of Serratia marcescens and therefore designated as Serratia marcescens sp. SE1. SE1 exhibited the ability to utilize 2,2-DCP as sole carbon source at 20 mM concentration with cell doubling time of 5 h and maximum chloride ion release of 38 μmolCl-/mL. This result suggests that the dehalogenase enzyme present in the bacteria has high affinity towards the substrate. Based on morphological and partial biochemical characteristics, strain SE1 was a non-motile Gram negative bacterium with red colonies, that gave a catalase positive reaction. Conclusion, significance and impact of study: A better understanding of dehalogenases enzyme produce by this S. marcescens sp. SE1 in general will be useful to be used as bioremediation tools for environmental management. This is the first reported case that Serratia sp. has the ability to degrade halogenated compound.

Functional Analysis of Drosophila NF1

National Research Council Canada - National Science Library

Bernards, Andre

2005-01-01

...) for Ras, yet homozygous loss of a highly conserved Drosophila NF1 ortholog results in several phenotypes that are insensitive to manipulating Ras signal transduction, but rescued by increasing...

Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death

Energy Technology Data Exchange (ETDEWEB)

Woo, Sang Hyeok; Seo, Sung-Keum [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); An, Sungkwan; Choe, Tae-Boo [Department of Microbiological Engineering, Kon-Kuk University, Gwangjin-gu, Seoul (Korea, Republic of); Hong, Seok-Il [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Lee, Yun-Han, E-mail: yhlee87@yuhs.ac [Department of Radiation Oncology, College of Medicine, Yonsei University, 250 Seongsan-no, Seodaemun-gu, Seoul (Korea, Republic of); Park, In-Chul, E-mail: parkic@kcch.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of)

2014-10-24

Highlights: • Caspase-1 mediates doxorubicin-induced downregulation of cyclin A1. • Active caspase-1 effectively cleaved cyclin A1 at D165. • Cyclin A1 expression is involved in DNA damage-induced cell death. - Abstract: Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lung cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.

High Ca2+ Influx During Traumatic Brain Injury Leads to Caspase-1-Dependent Neuroinflammation and Cell Death.

Science.gov (United States)

Abdul-Muneer, P M; Long, Mathew; Conte, Adriano Andrea; Santhakumar, Vijayalakshmi; Pfister, Bryan J

2017-08-01

We investigated the hypothesis that high Ca 2+ influx during traumatic brain injury induces the activation of the caspase-1 enzyme, which triggers neuroinflammation and cell apoptosis in a cell culture model of neuronal stretch injury and an in vivo model of fluid percussion injury (FPI). We first established that stretch injury causes a rapid increase in the intracellular Ca 2+ level, which activates interleukin-converting enzyme caspase-1. The increase in the intracellular Ca 2+ level and subsequent caspase-1 activation culminates into neuroinflammation via the maturation of IL-1β. Further, we analyzed caspase-1-mediated apoptosis by TUNEL staining and PARP western blotting. The voltage-gated sodium channel blocker, tetrodotoxin, mitigated the stretch injury-induced neuroinflammation and subsequent apoptosis by blocking Ca 2+ influx during the injury. The effect of tetrodotoxin was similar to the caspase-1 inhibitor, zYVAD-fmk, in neuronal culture. To validate the in vitro results, we demonstrated an increase in caspase-1 activity, neuroinflammation and neurodegeneration in fluid percussion-injured animals. Our data suggest that neuronal injury/traumatic brain injury (TBI) can induce a high influx of Ca 2+ to the cells that cause neuroinflammation and cell death by activating caspase-1, IL-1β, and intrinsic apoptotic pathways. We conclude that excess IL-1β production and cell death may contribute to neuronal dysfunction and cognitive impairment associated with TBI.

The Drosophila gene CG9918 codes for a pyrokinin-1 receptor

DEFF Research Database (Denmark)

Cazzamali, Giuseppe; Torp, Malene; Hauser, Frank

2005-01-01

The database from the Drosophila Genome Project contains a gene, CG9918, annotated to code for a G protein-coupled receptor. We cloned the cDNA of this gene and functionally expressed it in Chinese hamster ovary cells. We tested a library of about 25 Drosophila and other insect neuropeptides......, and seven insect biogenic amines on the expressed receptor and found that it was activated by low concentrations of the Drosophila neuropeptide, pyrokinin-1 (TGPSASSGLWFGPRLamide; EC50, 5 x 10(-8) M). The receptor was also activated by other Drosophila neuropeptides, terminating with the sequence PRLamide...... (Hug-gamma, ecdysis-triggering-hormone-1, pyrokinin-2), but in these cases about six to eight times higher concentrations were needed. The receptor was not activated by Drosophila neuropeptides, containing a C-terminal PRIamide sequence (such as ecdysis-triggering-hormone-2), or PRVamide (such as capa...

Expression of Caspase-1 in breast cancer tissues and its effects on cell proliferation, apoptosis and invasion.

Science.gov (United States)

Sun, Yanxia; Guo, Yingzhen

2018-05-01

The present study aimed to detect the expression of Caspase-1 in the tumor tissues and tumor-adjacent tissues of patients with breast cancer, and to investigate the effects of Caspase-1 on the proliferation, apoptosis and invasion of breast cancer cells. Reverse transcription-quantitative polymerase chain reaction was used to detect Caspase-1 mRNA expression in breast cancer tissues and tumor-adjacent tissues from patients. Additionally, the human breast cancer MDA-MB-231 cell line was treated with the Caspase-1 small molecule inhibitor Ac-YVAD-CMK, following which the changes to Caspase-1 protein expression were detected via western blotting. The MTT method detected the changes to cell proliferation, flow cytometry detected the rate of apoptosis, and a Transwell assay was employed to assess invasion. Caspase-1 mRNA expression was significantly decreased in the breast cancer tissues of patients, compared with in the tumor-adjacent tissues, a difference that was statistically significant (P<0.05). Treatment with the Ac-YVAD-CMK markedly decreased the protein expression of Caspase-1 in MDA-MB-231 cells, and the difference was statistically significant (P<0.05). Following this treatment of Ac-YVAD-CMK cells, the proliferation and invasion abilities markedly increased, while the apoptotic levels significantly decreased (P<0.05). In conclusion, the expression of Caspase-1 is low in breast cancer tissues, which may promote the proliferation and invasion of breast cancer cells and could be closely associated with the occurrence and development of breast cancer.

Differential regulation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages.

Directory of Open Access Journals (Sweden)

Toshihiko Suzuki

2007-08-01

Full Text Available Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms of this activation remain poorly understood. We demonstrate here that caspase-1 activation and IL-1beta processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD-like receptor (NLR family, and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC. We also show that Ipaf was critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC was dispensable. Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells. Treatment of macrophages with 3-methyladenine, an inhibitor of autophagy, enhanced pyroptosis induced by Shigella infection, suggesting that autophagy protects infected macrophages from pyroptosis. Thus, Ipaf plays a critical role in caspase-1 activation induced by Shigella independently of flagellin. Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial-host interactions.

Evolution of mitochondrial cell death pathway: Proapoptotic role of HtrA2/Omi in Drosophila

International Nuclear Information System (INIS)

Igaki, Tatsushi; Suzuki, Yasuyuki; Tokushige, Naoko; Aonuma, Hiroka; Takahashi, Ryosuke; Miura, Masayuki

2007-01-01

Despite the essential role of mitochondria in a variety of mammalian cell death processes, the involvement of mitochondrial pathway in Drosophila cell death has remained unclear. To address this, we cloned and characterized DmHtrA2, a Drosophila homolog of a mitochondrial serine protease HtrA2/Omi. We show that DmHtrA2 normally resides in mitochondria and is up-regulated by UV-irradiation. Upon receipt of apoptotic stimuli, DmHtrA2 is translocated to extramitochondrial compartment; however, unlike its mammalian counterpart, the extramitochondrial DmHtrA2 does not diffuse throughout the cytosol but stays near the mitochondria. RNAi-mediated knock-down of DmHtrA2 in larvae or adult flies results in a resistance to stress stimuli. DmHtrA2 specifically cleaves Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), a cellular caspase inhibitor, and induces cell death both in vitro and in vivo as potent as other fly cell death proteins. Our observations suggest that DmHtrA2 promotes cell death through a cleavage of DIAP1 in the vicinity of mitochondria, which may represent a prototype of mitochondrial cell death pathway in evolution

Artemisinin induces ROS-mediated caspase3 activation in ASTC-a-1 cells

Science.gov (United States)

Xiao, Feng-Lian; Chen, Tong-Sheng; Qu, Jun-Le; Liu, Cheng-Yi

2010-02-01

Artemisinin (ART), an antimalarial phytochemical from the sweet wormwood plant or a naturally occurring component of Artemisia annua, has been shown a potential anticancer activity by apoptotic pathways. In our report, cell counting kit (CCK-8) assay showed that treatment of human lung adenocarcinoma (ASTC-a-1) cells with ART effectively increase cell death by inducing apoptosis in a time- and dose-dependent fashion. Hoechst 33258 staining was used to detect apoptosis as well. Reactive oxygen species (ROS) generation was observed in cells exposed to ART at concentrations of 400 μM for 48 h. N-acetyl-L-cysteine (NAC), an oxygen radical scavenger, suppressed the rate of ROS generation and inhibited the ART-induced apoptosis. Moreover, AFC assay (Fluorometric assay for Caspase3 activity) showed that ROS was involved in ART-induced caspase3 acitvation. Taken together, our data indicate that ART induces ROS-mediated caspase3 activation in a time-and dose-dependent way in ASCT-a-1 cells.

Stalagmite carbon isotopes and dead carbon proportion (DCP) in a near-closed-system situation: An interplay between sulphuric and carbonic acid dissolution

Science.gov (United States)

Bajo, Petra; Borsato, Andrea; Drysdale, Russell; Hua, Quan; Frisia, Silvia; Zanchetta, Giovanni; Hellstrom, John; Woodhead, Jon

2017-08-01

In this study, the 'dead carbon proportion' (DCP) calculated from combined U-Th and radiocarbon analyses was used to explore the carbon isotope systematics in Corchia Cave (Italy) speleothems, using the example of stalagmite CC26 which grew during the last ∼12 ka. The DCP values in CC26 are among the highest ever recorded in a stalagmite, spanning the range 44.8-68.8%. A combination of almost closed-system conditions and sulphuric acid dissolution (SAD) are proposed as major drivers in producing such a high DCP with minor contribution from old organic matter from the deep vadose zone. The long-term decrease in both DCP and δ13C most likely reflects post-glacial soil recovery above the cave, with a progressive increase of soil CO2 contribution to the total dissolved inorganic carbon (DIC). Pronounced millennial-scale shifts in DCP and relatively small coeval but antipathetic changes in δ13C are modulated by the effects of hydrological variability on open and closed-system dissolution, SAD and prior calcite precipitation. Hence, the DCP in Corchia Cave speleothems represents an additional proxy for rainfall amount.

Acute fasting inhibits central caspase-1 activity reducing anxiety-like behavior and increasing novel object and object location recognition.

Science.gov (United States)

Towers, Albert E; Oelschlager, Maci L; Patel, Jay; Gainey, Stephen J; McCusker, Robert H; Freund, Gregory G

2017-06-01

Inflammation within the central nervous system (CNS) is frequently comorbid with anxiety. Importantly, the pro-inflammatory cytokine most commonly associated with anxiety is IL-1β. The bioavailability and activity of IL-1β are regulated by caspase-1-dependent proteolysis vis-a-vis the inflammasome. Thus, interventions regulating the activation or activity of caspase-1 should reduce anxiety especially in states that foster IL-1β maturation. Male C57BL/6j, C57BL/6j mice treated with the capase-1 inhibitor biotin-YVAD-cmk, caspase-1 knockout (KO) mice and IL-1R1 KO mice were fasted for 24h or allowed ad libitum access to food. Immediately after fasting, caspase-1 activity was measured in brain region homogenates while activated caspase-1 was localized in the brain by immunohistochemistry. Mouse anxiety-like behavior and cognition were tested using the elevated zero maze and novel object/object location tasks, respectively. A 24h fast in mice reduced the activity of caspase-1 in whole brain and in the prefrontal cortex, amygdala, hippocampus, and hypothalamus by 35%, 25%, 40%, 40%, and 40% respectively. A 24h fast also reduced anxiety-like behavior by 40% and increased novel object and object location recognition by 21% and 31%, respectively. IL-1β protein, however, was not reduced in the brain by fasting. ICV administration of YVAD decreased caspase-1 activity in the prefrontal cortex and amygdala by 55%, respectively leading to a 64% reduction in anxiety like behavior. Importantly, when caspase-1 KO or IL1-R1 KO mice are fasted, no fasting-dependent reduction in anxiety-like behavior was observed. Results indicate that fasting decrease anxiety-like behavior and improves memory by a mechanism tied to reducing caspase-1 activity throughout the brain. Copyright © 2017 Elsevier Inc. All rights reserved.

Caspase 1 activation is protective against hepatocyte cell death by up-regulating beclin 1 protein and mitochondrial autophagy in the setting of redox stress.

Science.gov (United States)

Sun, Qian; Gao, Wentao; Loughran, Patricia; Shapiro, Rick; Fan, Jie; Billiar, Timothy R; Scott, Melanie J

2013-05-31

Caspase 1 activation can be induced by oxidative stress, which leads to the release of the proinflammatory cytokines IL1β and IL18 in myeloid cells and a potentially damaging inflammatory response. However, little is known about the role of caspase 1 in non-immune cells, such as hepatocytes, that express and activate the inflammasome but do not produce a significant amount of IL1β/IL18. Here we demonstrate that caspase 1 activation protects against cell death after redox stress induced by hypoxia/reoxygenation in hepatocytes. Mechanistically, we show that caspase 1 reduces mitochondrial respiration and reactive oxygen species by increasing mitochondrial autophagy and subsequent clearance of mitochondria in hepatocytes after hypoxia/reoxygenation. Caspase 1 increases autophagic flux through up-regulating autophagy initiator beclin 1 during redox stress and is an important cell survival factor in hepatocytes. We find that during hemorrhagic shock with resuscitation, an in vivo mouse model associated with severe hepatic redox stress, caspase 1 activation is also protective against liver injury and excessive oxidative stress through the up-regulation of beclin 1. Our findings suggest an alternative role for caspase 1 activation in promoting adaptive responses to oxidative stress and, more specifically, in limiting reactive oxygen species production and damage in cells and tissues where IL1β/IL18 are not highly expressed.

Caspase 1 Activation Is Protective against Hepatocyte Cell Death by Up-regulating Beclin 1 Protein and Mitochondrial Autophagy in the Setting of Redox Stress*

Science.gov (United States)

Sun, Qian; Gao, Wentao; Loughran, Patricia; Shapiro, Rick; Fan, Jie; Billiar, Timothy R.; Scott, Melanie J.

2013-01-01

Caspase 1 activation can be induced by oxidative stress, which leads to the release of the proinflammatory cytokines IL1β and IL18 in myeloid cells and a potentially damaging inflammatory response. However, little is known about the role of caspase 1 in non-immune cells, such as hepatocytes, that express and activate the inflammasome but do not produce a significant amount of IL1β/IL18. Here we demonstrate that caspase 1 activation protects against cell death after redox stress induced by hypoxia/reoxygenation in hepatocytes. Mechanistically, we show that caspase 1 reduces mitochondrial respiration and reactive oxygen species by increasing mitochondrial autophagy and subsequent clearance of mitochondria in hepatocytes after hypoxia/reoxygenation. Caspase 1 increases autophagic flux through up-regulating autophagy initiator beclin 1 during redox stress and is an important cell survival factor in hepatocytes. We find that during hemorrhagic shock with resuscitation, an in vivo mouse model associated with severe hepatic redox stress, caspase 1 activation is also protective against liver injury and excessive oxidative stress through the up-regulation of beclin 1. Our findings suggest an alternative role for caspase 1 activation in promoting adaptive responses to oxidative stress and, more specifically, in limiting reactive oxygen species production and damage in cells and tissues where IL1β/IL18 are not highly expressed. PMID:23589298

Natural Radioactivity In Poultry Rations And DCP For Bovine Nutrition

International Nuclear Information System (INIS)

Luz-Filho, Isaias V.; Scheibel, Viviane; Appoloni, Carlos R.

2011-01-01

The aim of this study is to determine the level of radioactivity present in samples of poultry rations and dicalcium phosphate (DCP) used for cattle feed. Knowledge of these levels is of fundamental importance, because part of this radioactivity will possibly be transferred to humans. The radiation found in such samples is due to the presence of radioactive series of 238 U and 232 Th and 40 K. Measurements were performed with a 66% HPGe detector at the Laboratory of Applied Nuclear Physics, State University of Londrina. The measured samples were commercialized in Londrina, Brazil, in the second half of 2007. The accommodation recipient of the samples was a 1 L Marinelli beaker. Poultry rations were divided into two types: for young chickens and adult chickens. Among these, the ration for adult chickens showed the highest values for the activities of 226 Ra and 228 Ra, 0.23±0.17 and 0.493±0.091 Bq/kg respectively. But the ration for young chickens showed the highest activity for the 40 K, 304±15 Bq/kg. The DCP sample showed a much higher value for the series of 238 U and 232 Th, 83±26 and 7.79±0.70 Bq/kg, respectively. However, the 40 K activity in this sample was about 5 or 6 times lower than samples for poultry feed, reaching 46.6±2.8 Bq/kg.

Malaria-induced NLRP12/NLRP3-dependent caspase-1 activation mediates inflammation and hypersensitivity to bacterial superinfection.

Directory of Open Access Journals (Sweden)

Marco A Ataide

2014-01-01

Full Text Available Cyclic paroxysm and high fever are hallmarks of malaria and are associated with high levels of pyrogenic cytokines, including IL-1β. In this report, we describe a signature for the expression of inflammasome-related genes and caspase-1 activation in malaria. Indeed, when we infected mice, Plasmodium infection was sufficient to promote MyD88-mediated caspase-1 activation, dependent on IFN-γ-priming and the expression of inflammasome components ASC, P2X7R, NLRP3 and/or NLRP12. Pro-IL-1β expression required a second stimulation with LPS and was also dependent on IFN-γ-priming and functional TNFR1. As a consequence of Plasmodium-induced caspase-1 activation, mice produced extremely high levels of IL-1β upon a second microbial stimulus, and became hypersensitive to septic shock. Therapeutic intervention with IL-1 receptor antagonist prevented bacterial-induced lethality in rodents. Similar to mice, we observed a significantly increased frequency of circulating CD14(+CD16(-Caspase-1(+ and CD14(dimCD16(+Caspase-1(+ monocytes in peripheral blood mononuclear cells from febrile malaria patients. These cells readily produced large amounts of IL-1β after stimulation with LPS. Furthermore, we observed the presence of inflammasome complexes in monocytes from malaria patients containing either NLRP3 or NLRP12 pyroptosomes. We conclude that NLRP12/NLRP3-dependent activation of caspase-1 is likely to be a key event in mediating systemic production of IL-1β and hypersensitivity to secondary bacterial infection during malaria.

A Crohn's disease variant in Atg16l1 enhances its degradation by caspase 3

Science.gov (United States)

Murthy, Aditya; Li, Yun; Peng, Ivan; Reichelt, Mike; Katakam, Anand Kumar; Noubade, Rajkumar; Roose-Girma, Merone; Devoss, Jason; Diehl, Lauri; Graham, Robert R.; van Lookeren Campagne, Menno

2014-02-01

Crohn's disease is a debilitating inflammatory bowel disease (IBD) that can involve the entire digestive tract. A single-nucleotide polymorphism (SNP) encoding a missense variant in the autophagy gene ATG16L1 (rs2241880, Thr300Ala) is strongly associated with the incidence of Crohn's disease. Numerous studies have demonstrated the effect of ATG16L1 deletion or deficiency; however, the molecular consequences of the Thr300Ala (T300A) variant remains unknown. Here we show that amino acids 296-299 constitute a caspase cleavage motif in ATG16L1 and that the T300A variant (T316A in mice) significantly increases ATG16L1 sensitization to caspase-3-mediated processing. We observed that death-receptor activation or starvation-induced metabolic stress in human and murine macrophages increased degradation of the T300A or T316A variants of ATG16L1, respectively, resulting in diminished autophagy. Knock-in mice harbouring the T316A variant showed defective clearance of the ileal pathogen Yersinia enterocolitica and an elevated inflammatory cytokine response. In turn, deletion of the caspase-3-encoding gene, Casp3, or elimination of the caspase cleavage site by site-directed mutagenesis rescued starvation-induced autophagy and pathogen clearance, respectively. These findings demonstrate that caspase 3 activation in the presence of a common risk allele leads to accelerated degradation of ATG16L1, placing cellular stress, apoptotic stimuli and impaired autophagy in a unified pathway that predisposes to Crohn's disease.

TLR3 mediates release of IL-1β and cell death in keratinocytes in a caspase-4 dependent manner.

Science.gov (United States)

Grimstad, Øystein; Husebye, Harald; Espevik, Terje

2013-10-01

Inflammation and timely cell death are important elements in host defence and healing processes. Keratinocytes express high levels of Toll-like receptor 3 (TLR3), and stimulation of the receptor with its ligand polyinosinic-polycytidylic acid (polyI:C) is a powerful signal for release of a variety of proinflammatory cytokines. Caspase-4 is required for maturation of pro-IL-1β through activation of caspase-1 in keratinocytes. TLR3 in keratinocytes was stimulated with polyI:C. Induction of messenger RNA of pro-IL-1β and inflammasomal components was measured using quantitative polymerase chain reaction methodology. Protein expression of IL-1β was analysed with ELISA and Western blot techniques. Activation of apoptotic caspases was measured with flow cytometry, and cytotoxicity was determined. TLR3 induced release of substantial amounts of pro-IL-1β in keratinocytes. NLRP3 or ASC dependent processing of IL-1β into its cleaved bioactive form was found to be minimal. The release of IL-1β was due to polyI:C induced cell death that occurred through a caspase-4 dependent manner. Caspase-1 did not seem to be involved in the polyI:C induced cytotoxicity despite that TLR3 stimulation induced activation of caspase-1. In addition, the apoptotic caspases -8, -9 and -3/7 were activated by polyI:C. TLR3 stimulation in keratinocytes induces a caspase-4 dependent release of pro-IL-1β, but further processing to active IL-1β is limited. Furthermore, TLR3 stimulation results in pyroptotic- and apoptotic cell death. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

TUG1 promotes lens epithelial cell apoptosis by regulating miR-421/caspase-3 axis in age-related cataract.

Science.gov (United States)

Li, Guoxing; Song, Huiyang; Chen, Lei; Yang, Weihua; Nan, Kaihui; Lu, Peirong

2017-07-01

Age-related cataract is among the most common chronic disorders of ageing and the apoptosis of lens epithelial cells contributes to non-congenital cataract development. We amid to explore the role of TUG1 and miR-421 in the age-related cataract. The expression level of TUG1, miR-421 and caspase-3 were detected by RT-qPCR. The apoptotic-related protein, caspase-3, Bax and blc-2 were analyzed by western blot. We performed ultraviolet (UV) irradiation to induce SAR01/04 cell apoptosis which was analyzed by flow cytometry. RIP pull-down and luciferase reporter assay were used to verified the combination and regulating among TUG1, miR-421 and caspase-3. Here, we observed that the expression level of TUG1 and caspase-3 in the anterior lens capsules of age-related cataract were significantly higher and miR-421 was significantly lower than that in the normal anterior lens capsules. The apoptosis-related protein, caspase-3, Bax and blc-2 were abnormal expression in the anterior lens capsules of age-related cataract tissue. Our data showed that the expression level of TUG1 and caspase-3 and cell apoptosis rate in SAR01/04 cells treated with UV irradiation was remarkably higher than that in the control. TUG1 negatively regulated miR-421 expression and promoted UV irradiation-induced SAR01/04 cell apoptosis. However, miR-421 inhibitor and pcDNA-caspase-3 could reverse the action of the SRA01/04 cell apoptosis by si-TUG1, which suggested TUG1 promoted UV irradiation-induced apoptosis through downregulating miR-421 expression. Furthermore, this study confirmed TUG1 could been in combination with miR-421, and TUG1 and caspase-3 were both a directly target of miR-421. TUG1 modulated lens epithelial cell apoptosis through miR-421/caspase-3 axis. These findings will offer a novel insight into the pathogenesis of cataract. Copyright © 2017 Elsevier Inc. All rights reserved.

Applying the dynamic cone penetrometer (DCP) design method to low volume roads

CSIR Research Space (South Africa)

Paige-Green, P

2011-07-01

Full Text Available The Dynamic Cone Penetrometer (DCP) has been in use since the 1950s for various applications in pavement investigation. During the 1980s, Kleyn and Van Zyl described a method for upgrading unsealed roads to light sealed road standard based...

Caspase-1 inhibitor regulates humoral responses in experimental autoimmune myasthenia gravis via IL-6- dependent inhibiton of STAT3.

Science.gov (United States)

Wang, Cong-Cong; Zhang, Min; Li, Heng; Li, Xiao-Li; Yue, Long-Tao; Zhang, Peng; Liu, Ru-Tao; Chen, Hui; Li, Yan-Bin; Duan, Rui-Sheng

2017-08-24

We have previously demonstrated that Cysteinyl aspartate-specific proteinase-1 (caspase-1) inhibitor ameliorates experimental autoimmune myasthenia gravis (EAMG) by inhibited cellular immune response, via suppressing DC IL-1 β, CD4 + T and γdT cells IL-17 pathways. In this study, we investigated the effect of caspase-1 inhibitor on humoral immune response of EAMG and further explore the underlying mechanisms. An animal model of MG was induced by region 97-116 of the rat AChR α subunit (R97-116 peptide) in Lewis rats. Rats were treated with caspase-1 inhibitor Ac-YVAD-cmk intraperitoneally (i.p.) every second day from day 13 after the first immunization. Flow cytometry, western blot, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were performed to evaluate the neuroprotective effect of caspase-1 inhibitor on humoral immune response of EAMG. The results showed that caspase-1 inhibitor reduced the relative affinity of anti-R97-116 IgG, suppressed germinal center response, decreased follicular helper T cells, and increased follicular regulatory T cells and regulatory B cells. In addition, we found that caspase-1 inhibitor inhibited humoral immunity response in EAMG rats via suppressing IL-6-STAT3-Bcl-6 pathways. These results suggest that caspase-1 inhibitor ameliorates EAMG by regulating humoral immune response, thus providing new insights into the development of myasthenia gravis and other autoimmune diseases therapies. Copyright © 2017 Elsevier B.V. All rights reserved.

75 FR 9201 - DCP Raptor Pipeline, LLC; Notice of Compliance Filing

Science.gov (United States)

2010-03-01

... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission [Docket No. PR09-32-002] DCP Raptor... Raptor Pipeline, LLC (Raptor) filed its Statement of Operating Conditions in compliance with the January 27, 2010 Letter Order (January 27th Letter Order) in Docket Nos. PR09-32-000 and PR09-32-001. Raptor...

Caspase activation increases beta-amyloid generation independently of caspase cleavage of the beta-amyloid precursor protein (APP).

Science.gov (United States)

Tesco, Giuseppina; Koh, Young Ho; Tanzi, Rudolph E

2003-11-14

The amyloid precursor protein (APP) undergoes "alternative" proteolysis mediated by caspases. Three major caspase recognition sites have been identified in the APP, i.e. one at the C terminus (Asp720) and two at the N terminus (Asp197 and Asp219). Caspase cleavage at Asp720 has been suggested as leading to increased production of Abeta. Thus, we set out to determine which putative caspase sites in APP, if any, are cleaved in Chinese hamster ovary cell lines concurrently with the increased Abeta production that occurs during apoptosis. We found that cleavage at Asp720 occurred concurrently with caspase 3 activation and the increased production of total secreted Abeta and Abeta1-42 in association with staurosporine- and etoposide-induced apoptosis. To investigate the contribution of caspase cleavage of APP to Abeta generation, we expressed an APP mutant truncated at Asp720 that mimics APP caspase cleavage at the C-terminal site. This did not increase Abeta generation but, in contrast, dramatically decreased Abeta production in Chinese hamster ovary cells. Furthermore, the ablation of caspase-dependent cleavage at Asp720, Asp197, and Asp219 (by site-directed mutagenesis) did not prevent enhanced Abeta production following etoposide-induced apoptosis. These findings indicate that the enhanced Abeta generation associated with apoptosis does not require cleavage of APP at its C-terminal (Asp720) and/or N-terminal caspase sites.

Role of HIF-1α and CASPASE-3 in cystogenesis of odontogenic cysts and tumors.

Science.gov (United States)

da Costa, Natacha M M; de Siqueira, Adriane S; Ribeiro, André L R; da Silva Kataoka, Maria S; Jaeger, Ruy G; de Alves-Júnior, Sérgio M; Smith, Andrew M; de Jesus Viana Pinheiro, João

2018-01-01

Odontogenic cysts and tumors are the most relevant lesions that affect the gnathic bones. These lesions have in common the formation of cystic areas and this common feature may suggest involvement of similar mechanisms. The hypoxia inducible factor 1 alpha (HIF-1α), a responsive protein to hypoxia and caspase-3, an irreversible apoptosis marker, may contribute to cyst formation. Thus, this study aimed to investigate the immunoexpression of these proteins in odontogenic cysts and tumors. Twenty cases of ameloblastoma, keratocystic odontogenic tumor (KOT) (n = 20), radicular cyst (RC) (n = 18), dentigerous cyst (DC) (n = 11), calcifying cystic odontogenic tumor (n = 8), and dental follicle (DF) (n = 10) were used to investigate HIF-1α and caspase-3 expression in sequential serial cuts by immunohistochemistry. HIF-1α was overexpressed in RC, DC, and ameloblastoma when compared with DF. The basal and sometimes the lower suprabasal layer showed no or very low expression in DC, KOT, and ameloblastoma, the last also showing strong expression in solid epithelial areas and initial cystic formation regions. Caspase-3 was found to be overexpressed in all lesions, with the highest expression in odontogenic cysts compared to tumors. HIF-1α and caspase-3 were localized in similar areas of the same lesions, especially in the epithelium surrounding cystic formations. This study showed distinct immunoexpression of HIF-1α and caspase-3 in odontogenic cyst and tumors, with higher expression observed in odontogenic cysts. These findings suggest a possible correlation between hypoxia, apoptosis, and cystogenesis, leading to understand the mechanisms responsible to cystic formation in odontogenic lesions.

Alendronate augments interleukin-1{beta} release from macrophages infected with periodontal pathogenic bacteria through activation of caspase-1

Energy Technology Data Exchange (ETDEWEB)

Xue, Deng; Tamai, Riyoko [Division of Oral Bacteriology, Department of Oral Medical Science, Ohu University School of Dentistry, 31-1 Misumido, Tomitamachi, Koriyama, Fukushima 963-8611 (Japan); Endo, Yasuo [Department of Molecular Regulation, Graduate School of Dentistry, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Kiyoura, Yusuke [Division of Oral Bacteriology, Department of Oral Medical Science, Ohu University School of Dentistry, 31-1 Misumido, Tomitamachi, Koriyama, Fukushima 963-8611 (Japan)

2009-02-15

Nitrogen-containing bisphosphonates (NBPs) are anti-bone-resorptive drugs with inflammatory side effects that include osteomyelitis and osteonecrosis of the jaw. Oral bacteria have been considered to be a trigger for these NBP-associated jaw bone diseases. The present study examined the effects of alendronate (a typical NBP) and clodronate (a non-NBP) on the production of proinflammatory cytokines by macrophages infected with Porphyromonas gingivalis and Tannerella forsythia, which are important pathogens of periodontal diseases. Pretreatment with alendronate augmented IL-1{beta}, but not TNF{alpha}, production by macrophages infected with P. gingivalis or T. forsythia. This augmentation of IL-1{beta} production was inhibited by clodronate. Furthermore, caspase-1, a promoter of IL-1{beta} production, was activated by treatment with alendronate, and caspase-1 inhibitor reduced the production of IL-1{beta} induced by alendronate and P. gingivalis. These results suggest that NBPs augment periodontal pathogenic bacteria-induced IL-1{beta} release via caspase-1 activation, and this phenomenon may contribute to the development of NBP-associated inflammatory side effects including jaw osteomyelitis. Co-treatment with clodronate may prevent and/or reduce these inflammatory effects induced by NBPs.

Alendronate augments interleukin-1β release from macrophages infected with periodontal pathogenic bacteria through activation of caspase-1

International Nuclear Information System (INIS)

Deng Xue; Tamai, Riyoko; Endo, Yasuo; Kiyoura, Yusuke

2009-01-01

Nitrogen-containing bisphosphonates (NBPs) are anti-bone-resorptive drugs with inflammatory side effects that include osteomyelitis and osteonecrosis of the jaw. Oral bacteria have been considered to be a trigger for these NBP-associated jaw bone diseases. The present study examined the effects of alendronate (a typical NBP) and clodronate (a non-NBP) on the production of proinflammatory cytokines by macrophages infected with Porphyromonas gingivalis and Tannerella forsythia, which are important pathogens of periodontal diseases. Pretreatment with alendronate augmented IL-1β, but not TNFα, production by macrophages infected with P. gingivalis or T. forsythia. This augmentation of IL-1β production was inhibited by clodronate. Furthermore, caspase-1, a promoter of IL-1β production, was activated by treatment with alendronate, and caspase-1 inhibitor reduced the production of IL-1β induced by alendronate and P. gingivalis. These results suggest that NBPs augment periodontal pathogenic bacteria-induced IL-1β release via caspase-1 activation, and this phenomenon may contribute to the development of NBP-associated inflammatory side effects including jaw osteomyelitis. Co-treatment with clodronate may prevent and/or reduce these inflammatory effects induced by NBPs

Inhibition of Histone Deacetylases Permits Lipopolysaccharide-Mediated Secretion of Bioactive IL-1β via a Caspase-1-Independent Mechanism.

Science.gov (United States)

Stammler, Dominik; Eigenbrod, Tatjana; Menz, Sarah; Frick, Julia S; Sweet, Matthew J; Shakespear, Melanie R; Jantsch, Jonathan; Siegert, Isabel; Wölfle, Sabine; Langer, Julian D; Oehme, Ina; Schaefer, Liliana; Fischer, Andre; Knievel, Judith; Heeg, Klaus; Dalpke, Alexander H; Bode, Konrad A

2015-12-01

Histone deacetylase (HDAC) inhibitors (HDACi) are clinically approved anticancer drugs that have important immune-modulatory properties. We report the surprising finding that HDACi promote LPS-induced IL-1β processing and secretion in human and murine dendritic cells and murine macrophages. HDACi/LPS-induced IL-1β maturation and secretion kinetics differed completely from those observed upon inflammasome activation. Moreover, this pathway of IL-1β secretion was dependent on caspase-8 but was independent of the inflammasome components NACHT, LRR, and PYD domains-containing protein 3, apoptosis-associated speck-like protein containing a carboxyl-terminal caspase-recruitment domain, and caspase-1. Genetic studies excluded HDAC6 and HDAC10 as relevant HDAC targets in this pathway, whereas pharmacological inhibitor studies implicated the involvement of HDAC11. Treatment of mice with HDACi in a dextran sodium sulfate-induced colitis model resulted in a strong increase in intestinal IL-1β, confirming that this pathway is also operative in vivo. Thus, in addition to the conventional inflammasome-dependent IL-1β cleavage pathway, dendritic cells and macrophages are capable of generating, secreting, and processing bioactive IL-1β by a novel, caspase-8-dependent mechanism. Given the widespread interest in the therapeutic targeting of IL-1β, as well as the use of HDACi for anti-inflammatory applications, these findings have substantial clinical implications. Copyright © 2015 by The American Association of Immunologists, Inc.

Association of caspase-1 polymorphisms with Chagas cardiomyopathy among individuals in Santa Cruz, Bolivia.

Science.gov (United States)

Fu, Katherine Yih-Jia; Zamudio, Roxana; Henderson-Frost, Jo; Almuedo, Alex; Steinberg, Hannah; Clipman, Steven Joseph; Duran, Gustavo; Marcus, Rachel; Crawford, Thomas; Alyesh, Daniel; Colanzi, Rony; Flores, Jorge; Gilman, Robert Hugh; Bern, Caryn

2017-01-01

Trypanosoma cruzi (Tc) infection is usually acquired in childhood in endemic areas, leading to Chagas disease, which progresses to Chagas cardiomyopathy in 20-30% of infected individuals over decades. The pathogenesis of Chagas cardiomyopathy involves the host inflammatory response to T. cruzi, in which upstream caspase-1 activation prompts the cascade of inflammatory chemokines/cytokines, cardiac remodeling, and myocardial dysfunction. The aim of the present study was to examine the association of two caspase-1 single nucleotide polymorphisms (SNPs) with cardiomyopathy. We recruited infected (Tc+, n = 149) and uninfected (Tc-, n = 87) participants in a hospital in Santa Cruz, Bolivia. Cardiac status was classified (I, II, III, IV) based on Chagas cardiomyopathy-associated electrocardiogram findings and ejection fractions on echocardiogram. Genotypes were determined using Taqman probes via reverse transcription-polymerase chain reaction of peripheral blood DNA. Genotype frequencies were analyzed according to three inheritance patterns (dominant, recessive, additive) using logistic regression adjusted for age and sex. The AA allele for the caspase-1 SNP rs501192 was more frequent in Tc+ cardiomyopathy (classes II, III, IV) patients compared to those with a normal cardiac status (class I) [odds ratio (OR) = -2.18, p = 0.117]. This trend approached statistical significant considering only Tc+ patients in class I and II (OR = -2.64, p = 0.064). Caspase-1 polymorphisms may play a role in Chagas cardiomyopathy development and could serve as markers to identify individuals at higher risk for priority treatment.

Rational approach to identify newer caspase-1 inhibitors using pharmacophore based virtual screening, docking and molecular dynamic simulation studies.

Science.gov (United States)

Patel, Shivani; Modi, Palmi; Chhabria, Mahesh

2018-05-01

Caspase-1 is a key endoprotease responsible for the post-translational processing of pro-inflammatory cytokines IL-1β, 18 & 33. Excessive secretion of IL-1β leads to numerous inflammatory and autoimmune diseases. Thus caspase-1 inhibition would be considered as an important therapeutic strategy for development of newer anti-inflammatory agents. Here we have employed an integrated virtual screening by combining pharmacophore mapping and docking to identify small molecules as caspase-1 inhibitors. The ligand based 3D pharmacophore model was generated having the essential structural features of (HBA, HY & RA) using a data set of 27 compounds. A validated pharmacophore hypothesis (Hypo 1) was used to screen ZINC and Minimaybridge chemical databases. The retrieved virtual hits were filtered by ADMET properties and molecular docking analysis. Subsequently, the cross-docking study was also carried out using crystal structure of caspase-1, 3, 7 and 8 to identify the key residual interaction for specific caspase-1 inhibition. Finally, the best mapped and top scored (ZINC00885612, ZINC72003647, BTB04175 and BTB04410) molecules were subjected to molecular dynamics simulation for accessing the dynamic structure of protein after ligand binding. This study identifies the most promising hits, which can be leads for the development of novel caspase-1 inhibitors as anti-inflammatory agents. Copyright © 2018 Elsevier Inc. All rights reserved.

RNA silencing of Mcl-1 enhances ABT-737-mediated apoptosis in melanoma: role for a caspase-8-dependent pathway.

Science.gov (United States)

Keuling, Angela M; Felton, Kathleen E A; Parker, Arabesque A M; Akbari, Majid; Andrew, Susan E; Tron, Victor A

2009-08-17

Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma's striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10) activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity. Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.

Caspase-12 and the inflammatory response to Yersinia pestis.

Science.gov (United States)

Ferwerda, Bart; McCall, Matthew B B; de Vries, Maaike C; Hopman, Joost; Maiga, Boubacar; Dolo, Amagana; Doumbo, Ogobara; Daou, Modibo; de Jong, Dirk; Joosten, Leo A B; Tissingh, Rudi A; Reubsaet, Frans A G; Sauerwein, Robert; van der Meer, Jos W M; van der Ven, André J A M; Netea, Mihai G

2009-09-01

Caspase-12 functions as an antiinflammatory enzyme inhibiting caspase-1 and the NOD2/RIP2 pathways. Due to increased susceptibility to sepsis in individuals with functional caspase-12, an early-stop mutation leading to the loss of caspase-12 has replaced the ancient genotype in Eurasia and a significant proportion of individuals from African populations. In African-Americans, it has been shown that caspase-12 inhibits the pro-inflammatory cytokine production. We assessed whether similar mechanisms are present in African individuals, and whether evolutionary pressures due to plague may have led to the present caspase-12 genotype population frequencies. No difference in cytokine induction through the caspase-1 and/or NOD2/RIP2 pathways was observed in two independent African populations, among individuals with either an intact or absent caspase-12. In addition, stimulations with Yersinia pestis and two other species of Yersinia were preformed to investigate whether caspase-12 modulates the inflammatory reaction induced by Yersinia. We found that caspase-12 did not modulate cytokine production induced by Yersinia spp. Our experiments demonstrate for the first time the involvement of the NOD2/RIP2 pathway for recognition of Yersinia. However, caspase-12 does not modulate innate host defense against Y. pestis and alternative explanations for the geographical distribution of caspase-12 should be sought.

Prediction of Liquefaction Potential of Dredge Fill Sand by DCP and Dynamic Probing

International Nuclear Information System (INIS)

Alam, Md. Jahangir; Azad, Abul Kalam; Rahman, Ziaur

2008-01-01

From many research it is proved that liquefaction potential of sand is function of mainly relative density and confining pressure. During routine site investigations, high-quality sampling and laboratory testing of sands are not feasible because of inevitable sample disturbance effects and budgetary constraints. On the other hand quality control of sand fill can be done by determining in situ density of sand in layer by layer which is expensive and time consuming. In this paper TRL DCP (Transportation Research Laboratory Dynamic Cone Penetration) and DPL (Dynamic Probing Light) are calibrated to predict the relative density of sand deposit. For this purpose sand of known relative density is prepared in a calibration chamber which is a mild steel cylinder with diameter 0.5 m and height 1.0 m. Relative density of sand is varied by controlling height of fall and diameter of hole of sand discharge bowl. After filling, every time DPL and DCP tests are performed and for every blow the penetration of cone is recorded. N10 is then calculated from penetration records. Thus a database is compiled where N10 and relative densities are known. A correlation is made between N 10 and relative density for two types of sand. A good correlation of N 10 and relative density is found

Caspase-12 and the inflammatory response to Yersinia pestis.

NARCIS (Netherlands)

Ferwerda, B.; McCall, M.B.B.; Vries, M.C. de; Hopman, J.C.W.; Maiga, B.; Dolo, A.; Doumbo, O.; Daou, M.; Jong, D.J. de; Joosten, L.A.B.; Tissingh, R.A.; Reubsaet, F.A.; Sauerwein, R.W.; Meer, J.W.M. van der; Ven, A.J.A.M. van der; Netea, M.G.

2009-01-01

BACKGROUND: Caspase-12 functions as an antiinflammatory enzyme inhibiting caspase-1 and the NOD2/RIP2 pathways. Due to increased susceptibility to sepsis in individuals with functional caspase-12, an early-stop mutation leading to the loss of caspase-12 has replaced the ancient genotype in Eurasia

RNA silencing of Mcl-1 enhances ABT-737-mediated apoptosis in melanoma: role for a caspase-8-dependent pathway.

Directory of Open Access Journals (Sweden)

Angela M Keuling

Full Text Available BACKGROUND: Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma's striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10 activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.

Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2

Science.gov (United States)

Andersen, Joshua L; Johnson, Carrie E; Freel, Christopher D; Parrish, Amanda B; Day, Jennifer L; Buchakjian, Marisa R; Nutt, Leta K; Thompson, J Will; Moseley, M Arthur; Kornbluth, Sally

2009-01-01

The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage- and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1–cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1–cyclin B1 activity must be overcome for apoptosis to occur. PMID:19730412

NF-1 Dependent Gene Regulation in Drosophila Melanogaster

National Research Council Canada - National Science Library

Zhong, Yi

2004-01-01

.... We have used an Affymetrix whole genome chip, containing all 13,500 genes of the fruit fly Drosophila, to identify 93 genes with altered expression patterns in flies that have no NF1 protein compared...

O-GlcNAc regulates NEDD4-1 stability via caspase-mediated pathway

International Nuclear Information System (INIS)

Jiang, Kuan; Bai, Bingyang; Ta, Yajie; Zhang, Tingling; Xiao, Zikang; Wang, Peng George; Zhang, Lianwen

2016-01-01

O-GlcNAc modification of cytosolic and nuclear proteins regulates essential cellular processes such as stress responses, transcription, translation, and protein degradation. Emerging evidence indicates O-GlcNAcylation has a dynamic interplay with ubiquitination in cellular regulation. Here, we report that O-GlcNAc indirectly targets a vital E3 ubiquitin ligase enzyme of NEDD4-1. The protein level of NEDD4-1 is accordingly decreased following an increase of overall O-GlcNAc level upon PUGNAc or glucosamine stimulation. O-GlcNAc transferase (OGT) knockdown, overexpression and mutation results confirm that the stability of NEDD4-1 is negatively regulated by cellular O-GlcNAc. Moreover, the NEDD4-1 degradation induced by PUGNAc or GlcN is significantly inhibited by the caspase inhibitor. Our study reveals a regulation mechanism of NEDD4-1 stability by O-GlcNAcylation. - Highlights: • Reduced NEDD4-1 correlates with increased overall O-GlcNAc level. • OGT negatively regulates NEDD4-1 stability. • O-GlcNAc regulates NEDD4-1 through caspase-mediated pathway.

Drosophila arginine methyltransferase 1 (DART1) is an ecdysone receptor co-repressor

International Nuclear Information System (INIS)

Kimura, Shuhei; Sawatsubashi, Shun; Ito, Saya; Kouzmenko, Alexander; Suzuki, Eriko; Zhao, Yue; Yamagata, Kaoru; Tanabe, Masahiko; Ueda, Takashi; Fujiyama, Sari; Murata, Takuya; Matsukawa, Hiroyuki; Takeyama, Ken-ichi; Yaegashi, Nobuo

2008-01-01

Histone arginine methylation is an epigenetic marker that regulates gene expression by defining the chromatin state. Arginine methyltransferases, therefore, serve as transcriptional co-regulators. However, unlike other transcriptional co-regulators, the physiological roles of arginine methyltransferases are poorly understood. Drosophila arginine methyltransferase 1 (DART1), the mammalian PRMT1 homologue, methylates the arginine residue of histone H4 (H4R3me2). Disruption of DART1 in Drosophila by imprecise P-element excision resulted in low viability during metamorphosis in the pupal stages. In the pupal stage, an ecdysone hormone signal is critical for developmental progression. DART1 interacted with the nuclear ecdysone receptor (EcR) in a ligand-dependent manner, and co-repressed EcR in intact flies. These findings suggest that DART1, a histone arginine methyltransferase, is a co-repressor of EcR that is indispensable for normal pupal development in the intact fly

Divergent modulation of neuronal differentiation by caspase-2 and -9.

Directory of Open Access Journals (Sweden)

Giuseppa Pistritto

Full Text Available Human Ntera2/cl.D1 (NT2 cells treated with retinoic acid (RA differentiate towards a well characterized neuronal phenotype sharing many features with human fetal neurons. In view of the emerging role of caspases in murine stem cell/neural precursor differentiation, caspases activity was evaluated during RA differentiation. Caspase-2, -3 and -9 activity was transiently and selectively increased in differentiating and non-apoptotic NT2-cells. SiRNA-mediated selective silencing of either caspase-2 (si-Casp2 or -9 (si-Casp9 was implemented in order to dissect the role of distinct caspases. The RA-induced expression of neuronal markers, i.e. neural cell adhesion molecule (NCAM, microtubule associated protein-2 (MAP2 and tyrosine hydroxylase (TH mRNAs and proteins, was decreased in si-Casp9, but markedly increased in si-Casp2 cells. During RA-induced NT2 differentiation, the class III histone deacetylase Sirt1, a putative caspase substrate implicated in the regulation of the proneural bHLH MASH1 gene expression, was cleaved to a ∼100 kDa fragment. Sirt1 cleavage was markedly reduced in si-Casp9 cells, even though caspase-3 was normally activated, but was not affected (still cleaved in si-Casp2 cells, despite a marked reduction of caspase-3 activity. The expression of MASH1 mRNA was higher and occurred earlier in si-Casp2 cells, while was reduced at early time points during differentiation in si-Casp9 cells. Thus, caspase-2 and -9 may perform opposite functions during RA-induced NT2 neuronal differentiation. While caspase-9 activation is relevant for proper neuronal differentiation, likely through the fine tuning of Sirt1 function, caspase-2 activation appears to hinder the RA-induced neuronal differentiation of NT2 cells.

Oxidized low density lipoprotein induced caspase-1 mediated pyroptotic cell death in macrophages: implication in lesion instability?

Directory of Open Access Journals (Sweden)

Jing Lin

Full Text Available BACKGROUND: Macrophage death in advanced lesion has been confirmed to play an important role in plaque instability. However, the mechanism underlying lesion macrophage death still remains largely unknown. METHODS AND RESULTS: Immunohistochemistry showed that caspase-1 activated in advanced lesion and co-located with macrophages and TUNEL positive reaction. In in-vitro experiments showed that ox-LDL induced caspase-1 activation and this activation was required for ox-LDL induced macrophages lysis, IL-1β and IL-18 production as well as DNA fragmentation. Mechanism experiments showed that CD36 and NLRP3/caspase-1/pathway involved in ox-LDL induced macrophage pyroptosis. CONCLUSION: Our study here identified a novel cell death, pyroptosis in ox-LDL induced human macrophage, which may be implicated in lesion macrophages death and play an important role in lesion instability.

MicroRNAs Targeting Caspase-3 and -7 in PANC-1 Cells

Directory of Open Access Journals (Sweden)

Jong Kook Park

2018-04-01

Full Text Available MicroRNAs (miRNAs, a critical part of the RNA silencing machinery, are known to play important regulatory roles in cancer. However, the consequence of miRNA deregulation in cancer is unknown for many miRNAs. Here, we define that miRNAs, miR-17-5p, miR-132-3p/-212-3p, and miR-337-3p are significantly up-regulated in the pancreatic ductal adenocarcinomas (PDAC compared to the normal and benign tissues. Furthermore, by using PANC-1 cells, we demonstrate that overexpressed miR-337-3p and miR-17-5p/miR-132-3p/-212-3p can regulate executioner caspases-3 and -7, respectively. In addition, over-expression of miRNAs, especially miR-337-3p, attenuates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL cytotoxicity in PANC-1 cells. Our findings unveil an important biological function for miRNAs up-regulated in PDAC in coordinately regulating caspases, potentially contributing to the malignant progression of PDAC.

MicroRNAs Targeting Caspase-3 and -7 in PANC-1 Cells.

Science.gov (United States)

Park, Jong Kook; Doseff, Andrea I; Schmittgen, Thomas D

2018-04-16

MicroRNAs (miRNAs), a critical part of the RNA silencing machinery, are known to play important regulatory roles in cancer. However, the consequence of miRNA deregulation in cancer is unknown for many miRNAs. Here, we define that miRNAs, miR-17-5p, miR-132-3p/-212-3p, and miR-337-3p are significantly up-regulated in the pancreatic ductal adenocarcinomas (PDAC) compared to the normal and benign tissues. Furthermore, by using PANC-1 cells, we demonstrate that overexpressed miR-337-3p and miR-17-5p/miR-132-3p/-212-3p can regulate executioner caspases-3 and -7, respectively. In addition, over-expression of miRNAs, especially miR-337-3p, attenuates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity in PANC-1 cells. Our findings unveil an important biological function for miRNAs up-regulated in PDAC in coordinately regulating caspases, potentially contributing to the malignant progression of PDAC.

Caspase-1 Specific Light-Up Probe with Aggregation-Induced Emission Characteristics for Inhibitor Screening of Coumarin-Originated Natural Products.

Science.gov (United States)

Lin, Hao; Yang, Haitao; Huang, Shuai; Wang, Fujia; Wang, Dong-Mei; Liu, Bin; Tang, Yi-Da; Zhang, Chong-Jing

2018-04-18

Caspase-1 is a key player in pyroptosis and inflammation. Caspase-1 inhibition is found to be beneficial to various diseases. Coumarin-originated natural products have an anti-inflammation function, but their direct inhibition effect to caspase-1 remains unexplored. To evaluate their interactions, the widely used commercial coumarin-based probe (Ac-YVAD-AMC) is not suitable, as the background signal from coumarin-originated natural products could interfere with the screening results. Therefore, fluorescent probes using a large Stokes shift could help solve this problem. In this work, we chose the fluorophore of tetraphenylethylene-thiophene (TPETH) with aggregation-induced emission characteristics and a large Stokes shift of about 200 nm to develop a molecular probe. Bioconjugation between TPETH and hydrophilic peptides (DDYVADC) through a thiol-ene reaction generated a light-up probe, C1-P3. The probe has little background signal in aqueous media and exerts a fluorescent turn-on effect in the presence of caspase-1. Moreover, when evaluating the inhibition potency of coumarin-originated natural products, the new probe could generate a true and objective result but not for the commercial probe (Ac-YVAD-AMC), which is evidenced by HPLC analysis. The quick light-up response and accurate screening results make C1-P3 very useful in fundamental study and inhibitior screening toward caspase-1.

Atrial natriuretic peptide down-regulates LPS/ATP-mediated IL-1β release by inhibiting NF-kB, NLRP3 inflammasome and caspase-1 activation in THP-1 cells.

Science.gov (United States)

Mezzasoma, Letizia; Antognelli, Cinzia; Talesa, Vincenzo Nicola

2016-02-01

Atrial natriuretic peptide (ANP) is an hormone/paracrine/autocrine factor regulating cardiovascular homeostasis by guanylyl cyclase natriuretic peptide receptor (NPR-1). ANP plays an important role also in regulating inflammatory and immune systems by altering macrophages functions and cytokines secretion. Interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine involved in a wide range of biological responses, including the immunological one. Unlike other cytokines, IL-1β production is rigorously controlled. Primarily, NF-kB activation is required to produce pro-IL-1β; subsequently, NALP3 inflammasome/caspase-1 activation is required to cleave pro-IL-1β into the active secreted protein. NALP3 is a molecular platform capable of sensing a large variety of signals and a major player in innate immune defense. Due to their pleiotropism, IL-1β and NALP3 dysregulation is a common feature of a wide range of diseases. Therefore, identifying molecules regulating IL-1β/NALP3/caspase-1 expression is an important step in the development of new potential therapeutic agents. The aim of our study was to evaluate the effect of ANP on IL-1β/NALP3/caspase-1 expression in LPS/ATP-stimulated human THP1 monocytes. We provided new evidence of the direct involvement of ANP/NPR-1/cGMP axis on NF-kB/NALP3/caspase-1-mediated IL-1β release and NF-kB-mediated pro-IL-1β production. In particular, ANP inhibited both NF-kB and NALP3/caspase-1 activation leading to pro- and mature IL-1β down-regulation. Our data, pointing out a modulatory role of this endogenous peptide on IL-1β release and on NF-kB/NALP3/caspase-1 activation, indicate an important anti-inflammatory and immunomodulatory effect of ANP via these mechanisms. We suggest a possible employment of ANP for the treatment of inflammatory/immune-related diseases and IL-1β/NALP3-associated disorders, affecting millions of people worldwide.

The SCF ubiquitin ligase Slimb controls Nerfin-1 turnover in Drosophila.

Science.gov (United States)

Lin, Xiaohui; Wang, Feng; Li, Yuanpei; Zhai, Chaojun; Wang, Guiping; Zhang, Xiaoting; Gao, Yang; Yi, Tao; Sun, Dan; Wu, Shian

2018-01-01

The C2H2 type zinc-finger transcription factor Nerfin-1 expresses dominantly in Drosophila nervous system and plays an important role in early axon guidance decisions and preventing neurons dedifferentiation. Recently, increasing reports indicated that INSM1 (homologue to nerfin-1 in mammals) is a useful marker for prognosis of neuroendocrine tumors. The dynamic expression of Nerfin-1 is regulated post-transcriptionally by multiple microRNAs; however, its post-translational regulation is still unclear. Here we showed that the protein turnover of Nerfin-1 is regulated by Slimb, the substrate adaptor of SCF Slimb ubiquitin ligase complex. Mechanistically, Slimb associates with Nerfin-1 and promotes it ubiquitination and degradation in Drosophila S2R + cells. Furthermore, we determined that the C-terminal half of Nerfin-1 (Nerfin-1 CT ) is required for its binding to Slimb. Genetic epistasis assays showed that Slimb misexpression antagonizes, while knock-down enhances the activity of Nerfin-1 CT in Drosophila eyes. Our data revealed a new link to understand the underlying mechanism for Nerfin-1 turnover in post-translational level, and provided useful insights in animal development and disease treatment by manipulating the activity of Slimb and Nerfin-1. Copyright © 2017 Elsevier Inc. All rights reserved.

Optimum design of sustainable sealed low volume roads using the Dynamic Cone Penetrometer (DCP)

CSIR Research Space (South Africa)

Paige-Green, P

2012-09-01

Full Text Available in a reduction in the need to import large quantities of virgin material. Appropriate testing with the simple DCP test device can be used to assess the in situ conditions including material quality and moisture regimes along the road alignment...

Cadmium induces Ca2+ mediated, calpain-1/caspase-3-dependent apoptosis in primary cultured rat proximal tubular cells.

Science.gov (United States)

Wang, Hong; Zhai, Nianhui; Chen, Ying; Xu, Haibin; Huang, Kehe

2017-07-01

Calcium, as a ubiquitous second messenger, governs a large array of cellular processes and is necessary for cell survival. More recently, it was observed that the cytosolic Ca 2+ concentration ([Ca 2+ ] c ) elevation could induce apoptosis in primary cultured rat proximal tubular (rPT) cells exposed to cadmium (Cd), but the concrete mechanism is still unclear. This study was designed to investigate the signal pathway involved in [Ca 2+ ] c elevation-mediated apoptosis. The results confirmed the elevation of [Ca 2+ ] c by confocal microscopy and enhancement of the apoptosis by Hoechst 33258 staining and flow cytometer when rPT cells were exposed to Cd for 12h. Then we demonstrated that Cd enhanced the protein levels of active calpain-1 and caspase-3 in rPT cells. Pretreatment with a cytosolic Ca 2+ chelator, 1,2-Bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), markedly blocked the up-regulation of active calpain-1 and caspase-3 and inhibited the apoptosis induced by Cd. Further, rPT cells were pretreated with a cell-permeable selective calpain-1 inhibitor, 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) and caspase-3 inhibitor, N-Acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO), respectively. PD150606 significantly attenuated the up-regulation of active caspase-3 and the apoptosis induced by Cd. As expected, inhibition of active caspase-3 by Ac-DEVD-CHO decreased the apoptosis induced by Cd. Taken together, it could be concluded that [Ca 2+ ] c elevation did act as a pro-apoptotic signal in Cd-induced cytotoxicity of rPT cells, triggered calpain-1 and caspase-3 activation in turn, and induced apoptosis of rPT cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Caspase-9 mediates synaptic plasticity and memory deficits of Danish dementia knock-in mice: caspase-9 inhibition provides therapeutic protection

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Tamayev Robert

2012-12-01

Full Text Available Abstract Background Mutations in either Aβ Precursor protein (APP or genes that regulate APP processing, such as BRI2/ITM2B and PSEN1/PSEN2, cause familial dementias. Although dementias due to APP/PSEN1/PSEN2 mutations are classified as familial Alzheimer disease (FAD and those due to mutations in BRI2/ITM2B as British and Danish dementias (FBD, FDD, data suggest that these diseases have a common pathogenesis involving toxic APP metabolites. It was previously shown that FAD mutations in APP and PSENs promote activation of caspases leading to the hypothesis that aberrant caspase activation could participate in AD pathogenesis. Results Here, we tested whether a similar mechanism applies to the Danish BRI2/ITM2B mutation. We have generated a genetically congruous mouse model of FDD, called FDDKI, which presents memory and synaptic plasticity deficits. We found that caspase-9 is activated in hippocampal synaptic fractions of FDDKI mice and inhibition of caspase-9 activity rescues both synaptic plasticity and memory deficits. Conclusion These data directly implicate caspase-9 in the pathogenesis of Danish dementia and suggest that reducing caspase-9 activity is a valid therapeutic approach to treating human dementias.

Wavelength Discrimination in Drosophila Suggests a Role of Rhodopsin 1 in Color Vision

OpenAIRE

Garbers, Christian; Wachtler, Thomas

2016-01-01

Among the five photoreceptor opsins in the eye of Drosophila, Rhodopsin 1 (Rh1) is expressed in the six outer photoreceptors. In a previous study that combined behavioral genetics with computational modeling, we demonstrated that flies can use the signals from Rh1 for color vision. Here, we provide an in-depth computational analysis of wildtype Drosophila wavelength discrimination specifically considering the consequences of different choices of computations in the preprocessing of the behavi...

Expression levels of cleaved caspase-3 and caspase-3 in tumorigenesis and prognosis of oral tongue squamous cell carcinoma.

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Pei-Feng Liu

Full Text Available Apoptosis plays a dual role in cancer development and malignancy. The role of apoptosis-related caspases in cancer remains controversial, particularly in oral tongue squamous cell carcinoma (OTSCC. In this study, we examined the protein levels of cleaved caspase-3, caspase-3, caspase-8, and caspase-9 on tissue microarrays consisting of samples from 246 OTSCC patients by immunohistochemistry. Wilcoxon signed-rank test indicated that the protein levels of cleaved caspase-3, caspase-3, caspase-8, and caspase-9 in tumor tissues were significantly higher compared to those in adjacent normal tissues (all p<0.001. The expression level of caspase-8 in tumors was elevated in patients with lymph node invasion. Moreover, positive expression of cleaved caspase-3 was associated with shorter disease-free survival (DFS in OTSCC patients with moderate differentiation and lymph node invasion. Combination of either positive cleaved caspase-3 or higher caspase-3 expression or both was associated with poor DFS. Interestingly, stratification analysis showed that co-expression levels of positive cleaved caspase-3 or/and higher caspase-3 were associated with better disease-specific survival in patients with advanced stages of the disease, such as large tumor size and lymph node invasion, whereas it was associated with poor DFS in OTSCC patients with moderate cell differentiation and small tumor size. Taken together, cleaved caspase-3 and caspase-3/8/9 could be biomarkers for tumorigenesis in OTSCC patients. The co-expression level of cleaved caspase-3 and caspase-3 might be a prognostic biomarker for OTSCC patients, particular in those patients with certain tumor stages and cell differentiation status.

The Rho-family GTPase Rac1 regulates integrin localization in Drosophila immunosurveillance cells.

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Miguel J Xavier

Full Text Available BACKGROUND: When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila β-integrin Myospheroid (Mys is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of α and β subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and we have previously shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. We wanted to test the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response. RESULTS: In the current study we demonstrate that Rac1 is required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results we conclude that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization. SIGNIFICANCE: We show for the first time that the small GTPase Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila we quite often raise larvae at 29°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes.

Intervention with a caspase-1 inhibitor reduces obesity-associated hyperinsulinemia, non-alcoholic steatohepatitis and hepatic fibrosis in LDLR-/-.Leiden mice

NARCIS (Netherlands)

Morrison, M. C.; Mulder, P.; Salic, K.; Verheij, J.; Liang, W.; van Duyvenvoorde, W.; Menke, A.; Kooistra, T.; Kleemann, R.; Wielinga, P. Y.

BACKGROUND/OBJECTIVES: Non-alcoholic steatohepatitis (NASH) is a serious liver condition, closely associated with obesity and insulin resistance. Recent studies have suggested an important role for inflammasome/caspase-1 in the development of NASH, but the potential therapeutic value of caspase-1

Purification, crystallization and preliminary crystallographic characterization of the caspase-recruitment domain of human Nod1

International Nuclear Information System (INIS)

Srimathi, Thiagarajan; Robbins, Sheila L.; Dubas, Rachel L.; Seo, Jang-Hoon; Park, Young Chul

2006-01-01

The caspase-recruitment domain of the cytosolic pathogen receptor Nod1 was crystallized. X-ray diffraction data were collected to 1.9 Å resolution. The caspase-recruitment domain (CARD) is known to play an important role in apoptosis and inflammation as an essential protein–protein interaction domain. The CARD of the cytosolic pathogen receptor Nod1 was overexpressed in Escherichia coli and purified by affinity chromatography and gel filtration. The purified CARD was crystallized at 277 K using the microseeding method. X-ray diffraction data were collected to 1.9 Å resolution. The crystals belong to space group P3 1 or P3 2 , with unit-cell parameters a = b = 79.1, c = 80.9 Å. Preliminary analysis indicates that there is one dimeric CARD molecule in the asymmetric unit

Caspase-11 Plays a Protective Role in Pulmonary Acinetobacter baumannii Infection.

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Wang, Wei; Shao, Yue; Li, Shengjun; Xin, Na; Ma, Tingxian; Zhao, Chenghai; Song, Min

2017-10-01

Activation of caspase-11 by some Gram-negative bacteria triggers the caspase-1/interleukin 1β (IL-1β) pathway, independent of canonical inflammasomes. Acinetobacter baumannii is a Gram-negative, conditionally pathogenic bacterium that can cause severe pulmonary infection in hospitalized patients. A. baumannii was revealed to activate canonical and noncanonical inflammasome pathways in bone marrow-derived macrophages (BMDMs). Pulmonary infection of caspase-11 -/- mice with A. baumannii showed that caspase-11 deficiency impaired A. baumannii clearance, exacerbated pulmonary pathological changes, and enhanced susceptibility to A. baumannii These data indicate that the caspase-11-mediated innate immune response plays a crucial role in defending against A. baumannii . Copyright © 2017 American Society for Microbiology.

Primary enamel knot cell death in Apaf-1 and caspase-9 deficient mice

Czech Academy of Sciences Publication Activity Database

Šetková, Jana; Matalová, Eva; Sharpe, P. T.; Míšek, Ivan; Tucker, A. S.

2007-01-01

Roč. 52, 1 (2007), s. 15-19 ISSN 0003-9969 R&D Projects: GA AV ČR KJB500450503; GA ČR GA304/04/0101; GA MŠk OC B23.001 Institutional research plan: CEZ:AV0Z50450515 Keywords : dental apoptosis * apoptosome * apaf-1 knockout * caspase-9 knockout Subject RIV: FF - HEENT, Dentistry Impact factor: 1.554, year: 2007

A Biotin Switch-Based Proteomics Approach Identifies 14-3-3ζ as a Target of Sirt1 in the Metabolic Regulation of Caspase-2

Science.gov (United States)

Andersen, Joshua L.; Thompson, J. Will; Lindblom, Kelly R.; Johnson, Erika S.; Yang, Chih-Sheng; Lilley, Lauren R.; Freel, Christopher D.; Moseley, M. Arthur; Kornbluth, Sally

2011-01-01

While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a novel proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes 14-3-3ζ dissociation from caspase-2 in both egg extract and human cell lines. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation. PMID:21884983

Ethanol Extract of Evodia rutaecarpa Attenuates Cell Growth through Caspase-Dependent Apoptosis in Benign Prostatic Hyperplasia-1 Cells

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Eunsook Park

2018-04-01

Full Text Available The dried fruits of Evodia rutaecarpa Bentham have been used widely as a herbal medicine for the treatment of inflammatory disorders and abdominal pain. Benign prostatic hyperplasia (BPH is a nonmalignant disease characterized by overgrowth of prostates. Despite the pharmacological efficacy of the fruits of E. rutaecarpa against various diseases, their effects against BPH have not been reported. Here, we investigated the inhibitory activity of a 70% ethanol extract of E. rutaecarpa (EEER against BPH, and its underlying mechanisms regarding cell growth of BPH using BPH-1 cells. An in vitro 5α-reductase activity assay showed that EEER exhibited inhibitory activity against 5α-reductase. In BPH-1 cells, EEER treatment inhibited cell viability and reduced the expression of the proliferating cell nuclear antigen proliferating cell nuclear antigen (PCNA, cyclin D1, and phosphor-ERK1/2 proteins. Moreover, EEER also induced apoptosis, with chromatin condensation, apoptotic bodies, and internucleosomal DNA fragmentation. Regarding its underlying mechanisms, EEER exacerbated the activation of caspase-8 and caspase-3 in a concentration-dependent manner and eventually caused the cleavage of PARP. Taken together, these data demonstrated that EEER had a potent 5α-reductase inhibitory activity and that EEER treatment in BPH-1 cells inhibited cell viability via caspase-8- and caspase-3-dependent apoptosis. Therefore, EEER may be a potential phytotherapeutic agent for the treatment of BPH.

Sustained high serum caspase-3 concentrations and mortality in septic patients.

Science.gov (United States)

Lorente, L; Martín, M M; Pérez-Cejas, A; González-Rivero, A F; López, R O; Ferreres, J; Solé-Violán, J; Labarta, L; Díaz, C; Palmero, S; Jiménez, A

2018-02-01

Caspase-3 is the main executor of the apoptotic process. Higher serum caspase-3 concentrations in non-survivor compared to survivor septic patients have been found. The objectives of this work (with the increase of sample size to 308 patients, and the determination of serum caspase-3 concentrations also on days 4 and 8 of diagnosis of severe sepsis) were to know whether an association between serum caspase-3 concentrationss during the first week, degree of apoptosis, sepsis severity, and sepsis mortality exists. We collected serum samples of 308 patients with severe sepsis from eight intensive care units on days 1, 4 and 8 to measure concentrations of caspase-3 and caspase-cleaved cytokeratin (CCCK)-18 (to assess degree of apoptosis). End point was 30-day mortality. We found higher serum concentrations of caspase-3 and CCCK-18 in non-survivors compared to survivors on days 1 (p < 0.001), 4 (p < 0.001), and 8 (p < 0.001). We found an association between serum caspase-3 concentrations on days 1, 4 and 8 of severe sepsis diagnosis and serum CCCK-18 concentrations (p < 0.001), SOFA (p < 0.001), serum acid lactic concentrations (p < 0.001), and 30-day sepsis mortality (p < 0.001). The new findings of this work were that an association between serum caspase-3 concentrations during the first week, apoptosis degree, sepsis severity, and sepsis mortality exists.

Transient protective effect of caspase inhibitors in RCS rat.

Science.gov (United States)

Perche, O; Doly, M; Ranchon-Cole, I

2008-03-01

In most retinal degenerations in humans and in animal models, photoreceptor cells die by apoptosis. Although the biochemical features are similar in all apoptotic cells, different molecular events lead the cell to death. In the present study we used a rat model of inherited retinal degeneration, the RCS rats, to investigate the involvement of the proteases, caspases and/or calpains, in photoreceptor apoptosis. In the first experiments, rats were untreated or injected intravitreally at post natal day 27 (P27) with the large broad spectrum caspase inhibitor, ZVAD, the calpain inhibitor, MuhPhe, or with the vehicle, DMSO. Retinal status was evaluated at P35 and P42 by electroretinography, morphometry and apoptotic nuclei detection. DMSO and MuhPhe had no effect on RCS retinas as evidenced by equivalent loss of function and equivalent number of apoptotic cells than in untreated group. ZVAD transiently reduced apoptotic cells and preserved photoreceptor function at P35 but not at P42. These results suggest that caspases but not calpains are involved in retinal degeneration in the RCS. In the second experiments, RCS rats were injected twice at P27 and P35 with ZVAD or DMSO. Although ZVAD-treated retinas were preserved at P35 compared to the DMSO controls, the second injection of ZVAD did not extend the preserving effect to P42. Moreover, a single injection of ZVAD at P35 had no preserving effect at P42. All these data taken together suggest that caspases do not play a pivotal role after P35. In a fourth set of experiments, we used specific caspase inhibitors to elucidate which caspase was activated. The caspase-1/4 inhibitor (YVAD) or the caspase-3/7 inhibitor (DEVD) were injected intravitreally at P27 and retinal status was evaluated at P35 and P42. Electroretinograms and apoptotic nuclei detection demonstrated that YVAD and DEVD preserved photoreceptors at P35 but not at P42. These results suggest that both caspase-1/4 and caspase-3/7 play a major role in the apoptotic

3-Monochloro-1,2-propanediol (3-MCPD) induces apoptosis via mitochondrial oxidative phosphorylation system impairment and the caspase cascade pathway

International Nuclear Information System (INIS)

Peng, Xiaoli; Gan, Jing; Wang, Qian; Shi, Zhenqiang; Xia, Xiaodong

2016-01-01

3-Monochloro-1,2-propanediol (3-MCPD) is the most toxic chloropropanols compounds in foodstuff which mainly generated during thermal processing. Kidney is one of the primary target organs for 3-MCPD. Using human embryonic kidney cell (HEK293FT) as an in vitro model, we found that 3-MCPD caused concentration-dependent increase in cytoxicity as assessed by dye uptake, lactatedehydrogenase (LDH) leakage and MTT assays. HEK293FT cell treated with 3-MCPD suffered the decrease of mitochondrial membrane potential and the impairment of mitochondrial oxidative phosphorylation system, especially the reduced amount of mRNA expression and protein synthesis of electron transport chain complex II, complex IV, and complex III. More importantly, energy release (ATP synthesis) was significantly inhibited by 3-MCPD resulting from the down regulation expressions of ATP synthase (ATP6 and ATP8), as well as the loss of transmembrane potential required for synthesis of ATP. The decreased ratio of mitochondrial apoptogenic factors Bax/Bcl-2 and the cytochrome-c release from mitochondria to cytosol followed by the activation of apoptotic initiators caspase 9 and apoptotic executioners (caspase 3, caspase 6 and caspase 7) leading to apoptosis. The activation of caspase 8 and caspase 2 implied that there were probably other factors to induce the caspase-dependent apoptosis.

Drosophila PAF1 Modulates PIWI/piRNA Silencing Capacity.

Science.gov (United States)

Clark, Josef P; Rahman, Reazur; Yang, Nachen; Yang, Linda H; Lau, Nelson C

2017-09-11

To test the directness of factors in initiating PIWI-directed gene silencing, we employed a Piwi-interacting RNA (piRNA)-targeted reporter assay in Drosophila ovary somatic sheet (OSS) cells [1]. This assay confirmed direct silencing roles for piRNA biogenesis factors and PIWI-associated factors [2-12] but suggested that chromatin-modifying proteins may act downstream of the initial silencing event. Our data also revealed that RNA-polymerase-II-associated proteins like PAF1 and RTF1 antagonize PIWI-directed silencing. PAF1 knockdown enhances PIWI silencing of reporters when piRNAs target the transcript region proximal to the promoter. Loss of PAF1 suppresses endogenous transposable element (TE) transcript maturation, whereas a subset of gene transcripts and long-non-coding RNAs adjacent to TE insertions are affected by PAF1 knockdown in a similar fashion to piRNA-targeted reporters. Additionally, transcription activation at specific TEs and TE-adjacent loci during PIWI knockdown is suppressed when PIWI and PAF1 levels are both reduced. Our study suggests a mechanistic conservation between fission yeast PAF1 repressing AGO1/small interfering RNA (siRNA)-directed silencing [13, 14] and Drosophila PAF1 opposing PIWI/piRNA-directed silencing. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sox11 Reduces Caspase-6 Cleavage and Activity.

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Elaine Waldron-Roby

Full Text Available The apoptotic cascade is an orchestrated event, whose final stages are mediated by effector caspases. Regulatory binding proteins have been identified for caspases such as caspase-3, -7, -8, and -9. Many of these proteins belong to the inhibitor of apoptosis (IAP family. By contrast, caspase-6 is not believed to be influenced by IAPs, and little is known about its regulation. We therefore performed a yeast-two-hybrid screen using a constitutively inactive form of caspase-6 for bait in order to identify novel regulators of caspase-6 activity. Sox11 was identified as a potential caspase-6 interacting protein. Sox11 was capable of dramatically reducing caspase-6 activity, as well as preventing caspase-6 self- cleavage. Several regions, including amino acids 117-214 and 362-395 within sox11 as well as a nuclear localization signal (NLS all contributed to the reduction in caspase-6 activity. Furthermore, sox11 was also capable of decreasing other effector caspase activity but not initiator caspases -8 and -9. The ability of sox11 to reduce effector caspase activity was also reflected in its capacity to reduce cell death following toxic insult. Interestingly, other sox proteins also had the ability to reduce caspase-6 activity but to a lesser extent than sox11.

Cutting Edge: Eosinophils Undergo Caspase-1-Mediated Pyroptosis in Response to Necrotic Liver Cells.

Science.gov (United States)

Palacios-Macapagal, Daphne; Connor, Jane; Mustelin, Tomas; Ramalingam, Thirumalai R; Wynn, Thomas A; Davidson, Todd S

2017-08-01

Many chronic liver disorders are characterized by dysregulated immune responses and hepatocyte death. We used an in vivo model to study the immune response to necrotic liver injury and found that necrotic liver cells induced eosinophil recruitment. Necrotic liver induced eosinophil IL-1β and IL-18 secretion, degranulation, and cell death. Caspase-1 inhibitors blocked all of these responses. Caspase-1-mediated cell death with accompanying cytokine release is the hallmark of a novel form of cell death termed pyroptosis. To confirm this response in a disease model, we isolated eosinophils from the livers of Schistosoma mansoni -infected mice. S. mansoni eggs lodge in the hepatic sinusoids of infected mice, resulting in hepatocyte death, inflammation, and progressive liver fibrosis. This response is typified by massive eosinophilia, and we were able to confirm pyroptosis in the infiltrating eosinophils. This demonstrated that pyroptosis is a cellular pathway used by eosinophils in response to large-scale hepatic cell death. Copyright © 2017 by The American Association of Immunologists, Inc.

Serial killers: ordering caspase activation events in apoptosis.

Science.gov (United States)

Slee, E A; Adrain, C; Martin, S J

1999-11-01

Caspases participate in the molecular control of apoptosis in several guises; as triggers of the death machinery, as regulatory elements within it, and ultimately as a subset of the effector elements of the machinery itself. The mammalian caspase family is steadily growing and currently contains 14 members. At present, it is unclear whether all of these proteases participate in apoptosis. Thus, current research in this area is focused upon establishing the repertoire and order of caspase activation events that occur during the signalling and demolition phases of cell death. Evidence is accumulating to suggest that proximal caspase activation events are typically initiated by molecules that promote caspase aggregation. As expected, distal caspase activation events are likely to be controlled by caspases activated earlier in the cascade. However, recent data has cast doubt upon the functional demarcation of caspases into signalling (upstream) and effector (downstream) roles based upon their prodomain lengths. In particular, caspase-3 may perform an important role in propagating the caspase cascade, in addition to its role as an effector caspase within the death programme. Here, we discuss the apoptosis-associated caspase cascade and the hierarchy of caspase activation events within it.

Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin.

Science.gov (United States)

Clark, Ira E; Dodson, Mark W; Jiang, Changan; Cao, Joseph H; Huh, Jun R; Seol, Jae Hong; Yoo, Soon Ji; Hay, Bruce A; Guo, Ming

2006-06-29

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction has been implicated as an important trigger for Parkinson's disease-like pathogenesis because exposure to environmental mitochondrial toxins leads to Parkinson's disease-like pathology. Recently, multiple genes mediating familial forms of Parkinson's disease have been identified, including PTEN-induced kinase 1 (PINK1; PARK6) and parkin (PARK2), which are also associated with sporadic forms of Parkinson's disease. PINK1 encodes a putative serine/threonine kinase with a mitochondrial targeting sequence. So far, no in vivo studies have been reported for pink1 in any model system. Here we show that removal of Drosophila PINK1 homologue (CG4523; hereafter called pink1) function results in male sterility, apoptotic muscle degeneration, defects in mitochondrial morphology and increased sensitivity to multiple stresses including oxidative stress. Pink1 localizes to mitochondria, and mitochondrial cristae are fragmented in pink1 mutants. Expression of human PINK1 in the Drosophila testes restores male fertility and normal mitochondrial morphology in a portion of pink1 mutants, demonstrating functional conservation between human and Drosophila Pink1. Loss of Drosophila parkin shows phenotypes similar to loss of pink1 function. Notably, overexpression of parkin rescues the male sterility and mitochondrial morphology defects of pink1 mutants, whereas double mutants removing both pink1 and parkin function show muscle phenotypes identical to those observed in either mutant alone. These observations suggest that pink1 and parkin function, at least in part, in the same pathway, with pink1 functioning upstream of parkin. The role of the pink1-parkin pathway in regulating mitochondrial function underscores the importance of mitochondrial dysfunction as a central mechanism of Parkinson's disease

Tumor necrosis factor-alpha potentiates the cytotoxicity of amiodarone in Hepa1c1c7 cells: roles of caspase activation and oxidative stress.

Science.gov (United States)

Lu, Jingtao; Miyakawa, Kazuhisa; Roth, Robert A; Ganey, Patricia E

2013-01-01

Amiodarone (AMD), a class III antiarrhythmic drug, causes idiosyncratic hepatotoxicity in human patients. We demonstrated previously that tumor necrosis factor-alpha (TNF-α) plays an important role in a rat model of AMD-induced hepatotoxicity under inflammatory stress. In this study, we developed a model in vitro to study the roles of caspase activation and oxidative stress in TNF potentiation of AMD cytotoxicity. AMD caused cell death in Hepa1c1c7 cells, and TNF cotreatment potentiated its toxicity. Activation of caspases 9 and 3/7 was observed in AMD/TNF-cotreated cells, and caspase inhibitors provided minor protection from cytotoxicity. Intracellular reactive oxygen species (ROS) generation and lipid peroxidation were observed after treatment with AMD and were further elevated by TNF cotreatment. Adding water-soluble antioxidants (trolox, N-acetylcysteine, glutathione, or ascorbate) produced only minor attenuation of AMD/TNF-induced cytotoxicity and did not influence the effect of AMD alone. On the other hand, α-tocopherol (TOCO), which reduced lipid peroxidation and ROS generation, prevented AMD toxicity and caused pronounced reduction in cytotoxicity from AMD/TNF cotreatment. α-TOCO plus a pancaspase inhibitor completely abolished AMD/TNF-induced cytotoxicity. In summary, activation of caspases and oxidative stress were observed after AMD/TNF cotreatment, and caspase inhibitors and a lipid-soluble free-radical scavenger attenuated AMD/TNF-induced cytotoxicity.

Relationship between triterpenoid anticancer drug resistance, autophagy, and caspase-1 in adult T-cell leukemia

Directory of Open Access Journals (Sweden)

Tsukasa Nakanishi

2016-05-01

Full Text Available We previously reported that the inflammasome inhibitor cucurbitacin D (CuD induces apoptosis in human leukemia cell lines. Here, we investigated the effects of CuD and a B-cell lymphoma extra-large (Bcl-xL inhibitor on autophagy in peripheral blood lymphocytes (PBL isolated from adult T-cell leukemia (ATL patients. CuD induced PBL cell death in patients but not in healthy donors. This effect was not significantly inhibited by treatment with rapamycin or 3-methyladenine (3-MA. The Bcl-xL inhibitor Z36 induced death in primary cells from ATL patients including that induced by CuD treatment, effects that were partly inhibited by 3-MA. Similarly, cell death induced by the steroid prednisolone was enhanced in the presence of Z36. A western blot analysis revealed that Z36 also promoted CuD-induced poly(ADP ribose polymerase cleavage. Interestingly, the effects of CuD and Z36 were attenuated in primary ATL patient cells obtained upon recurrence after umbilical cord blood transplantation, as compared to those obtained before chemotherapy. Furthermore, cells from this patient expressed a high level of caspase-1, and treatment with caspase-1 inhibitor-enhanced CuD-induced cell death. Taken together, these results suggest that rescue from resistance to steroid drugs can enhance chemotherapy, and that caspase-1 is a good marker for drug resistance in ATL patients.

The correlation between MIB-1, AgNOR, and caspase-3 apoptosis with chemoradiotherapy response in cervical cancer

International Nuclear Information System (INIS)

Iin Kurnia; Devita Tetriana; Budiningsih Siregar; Irwan Ramli; Andriono; Setiawan Soetopo; Tjahya Kurjana; Maringan DL Tobing; Bethy Suryawathi

2013-01-01

Chemoradiotherapy is one of treatments for the locally advanced cervical cancer given by concurrent radiotherapy combined with chemotherapy in the same time. Chemoradiotherapy response is influenced by biological factor i.e. cell kinetic that consists of cell proliferation and death. In this research, the correlation between AgNOR, MIB-1 cell proliferation biomarker and the expression of apoptotic caspase-3 with chemoradiotherapy response of cervical cancer has been studied. Twenty one microscopic tissue samples were taken from cervical cancer biopsies before radiotherapy. The tissue samples were stained with AgNOR, whereas MIB-1 and apoptosis caspase-3 in the tissue samples were detected by immunochemistry technique. After the completion of chemoradiotherapy treatment, the clinical response was observed by pelvic control method. The result of this research show that there is no correlation between AgNOR, MIB-1 value with apoptosis (p>0.05) before chemoradiotherapy. Cell proliferation observed by AgNOR and MIB-1 before chemoradiotherapy indicate no correlation with chemoradiotherapy response, however the apoptotic expression shows positive correlation with chemoradiotherapy response. The index of caspase-3 apoptosis obtained from this research can be used for considering the chemoradiotherapy schedule for the cervical cancer patient. (author)

SfDronc, an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda.

Science.gov (United States)

Huang, Ning; Civciristov, Srgjan; Hawkins, Christine J; Clem, Rollie J

2013-05-01

Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with a molecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Enigmatic Roles of Caspases in Tumor Development

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Jäger, Richard; Zwacka, Ralf M., E-mail: ralf.zwacka@nuigalway.ie [National University of Ireland, Galway, National Centre for Biomedical Engineering Science and Apoptosis Research Centre, Molecular Therapeutics Group, Galway (Ireland)

2010-11-24

One function ascribed to apoptosis is the suicidal destruction of potentially harmful cells, such as cancerous cells. Hence, their growth depends on evasion of apoptosis, which is considered as one of the hallmarks of cancer. Apoptosis is ultimately carried out by the sequential activation of initiator and executioner caspases, which constitute a family of intracellular proteases involved in dismantling the cell in an ordered fashion. In cancer, therefore, one would anticipate caspases to be frequently rendered inactive, either by gene silencing or by somatic mutations. From clinical data, however, there is little evidence that caspase genes are impaired in cancer. Executioner caspases have only rarely been found mutated or silenced, and also initiator caspases are only affected in particular types of cancer. There is experimental evidence from transgenic mice that certain initiator caspases, such as caspase-8 and -2, might act as tumor suppressors. Loss of the initiator caspase of the intrinsic apoptotic pathway, caspase-9, however, did not promote cellular transformation. These data seem to question a general tumor-suppressive role of caspases. We discuss several possible ways how tumor cells might evade the need for alterations of caspase genes. First, alternative splicing in tumor cells might generate caspase variants that counteract apoptosis. Second, in tumor cells caspases might be kept in check by cellular caspase inhibitors such as c-FLIP or XIAP. Third, pathways upstream of caspase activation might be disrupted in tumor cells. Finally, caspase-independent cell death mechanisms might abrogate the selection pressure for caspase inactivation during tumor development. These scenarios, however, are hardly compatible with the considerable frequency of spontaneous apoptosis occurring in several cancer types. Therefore, alternative concepts might come into play, such as compensatory proliferation. Herein, apoptosis and/or non-apoptotic functions of caspases may

The Enigmatic Roles of Caspases in Tumor Development

International Nuclear Information System (INIS)

Jäger, Richard; Zwacka, Ralf M.

2010-01-01

One function ascribed to apoptosis is the suicidal destruction of potentially harmful cells, such as cancerous cells. Hence, their growth depends on evasion of apoptosis, which is considered as one of the hallmarks of cancer. Apoptosis is ultimately carried out by the sequential activation of initiator and executioner caspases, which constitute a family of intracellular proteases involved in dismantling the cell in an ordered fashion. In cancer, therefore, one would anticipate caspases to be frequently rendered inactive, either by gene silencing or by somatic mutations. From clinical data, however, there is little evidence that caspase genes are impaired in cancer. Executioner caspases have only rarely been found mutated or silenced, and also initiator caspases are only affected in particular types of cancer. There is experimental evidence from transgenic mice that certain initiator caspases, such as caspase-8 and -2, might act as tumor suppressors. Loss of the initiator caspase of the intrinsic apoptotic pathway, caspase-9, however, did not promote cellular transformation. These data seem to question a general tumor-suppressive role of caspases. We discuss several possible ways how tumor cells might evade the need for alterations of caspase genes. First, alternative splicing in tumor cells might generate caspase variants that counteract apoptosis. Second, in tumor cells caspases might be kept in check by cellular caspase inhibitors such as c-FLIP or XIAP. Third, pathways upstream of caspase activation might be disrupted in tumor cells. Finally, caspase-independent cell death mechanisms might abrogate the selection pressure for caspase inactivation during tumor development. These scenarios, however, are hardly compatible with the considerable frequency of spontaneous apoptosis occurring in several cancer types. Therefore, alternative concepts might come into play, such as compensatory proliferation. Herein, apoptosis and/or non-apoptotic functions of caspases may

3-Monochloro-1,2-propanediol (3-MCPD) induces apoptosis via mitochondrial oxidative phosphorylation system impairment and the caspase cascade pathway.

Science.gov (United States)

Peng, Xiaoli; Gan, Jing; Wang, Qian; Shi, Zhenqiang; Xia, Xiaodong

2016-11-30

3-Monochloro-1,2-propanediol (3-MCPD) is the most toxic chloropropanols compounds in foodstuff which mainly generated during thermal processing. Kidney is one of the primary target organs for 3-MCPD. Using human embryonic kidney cell (HEK293FT) as an in vitro model, we found that 3-MCPD caused concentration-dependent increase in cytoxicity as assessed by dye uptake, lactatedehydrogenase (LDH) leakage and MTT assays. HEK293FT cell treated with 3-MCPD suffered the decrease of mitochondrial membrane potential and the impairment of mitochondrial oxidative phosphorylation system, especially the reduced amount of mRNA expression and protein synthesis of electron transport chain complex II, complex IV, and complex III. More importantly, energy release (ATP synthesis) was significantly inhibited by 3-MCPD resulting from the down regulation expressions of ATP synthase (ATP6 and ATP8), as well as the loss of transmembrane potential required for synthesis of ATP. The decreased ratio of mitochondrial apoptogenic factors Bax/Bcl-2 and the cytochrome-c release from mitochondria to cytosol followed by the activation of apoptotic initiators caspase 9 and apoptotic executioners (caspase 3, caspase 6 and caspase 7) leading to apoptosis. The activation of caspase 8 and caspase 2 implied that there were probably other factors to induce the caspase-dependent apoptosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Low doze γ-irradiation influence on drosophila life span in different genetics background

International Nuclear Information System (INIS)

Moskalev, A.

2007-01-01

Complete text of publication follows. The main goal of this work was to study in Drosophila melanogaster the contribution of DNA damage sensing and repair, apoptosis and heat shock defence into life span and physical activity alteration after gamma-irradiation at low doze rate. In our experiments, the strains were exposed to chronic gamma-irradiation from a 226Ra source (50 R/h) at doze rate 0.17 cGy/h at pre-imago development stages only. The absorbed radiation dose per generation (from embryo to imago, 12 days) was 60 cGy. Life span estimation was prepared in adult males and females separately. We compared the life span of apoptotic (p53, DIAP-1, dApaf-1, Dcp-1, reaper, grim and hid), heat shock defence (HSP70, HSP23, HSF), DNA damage sensing (ATR) and repair (XPF, XPC, PCNA, DSB repair helicase homologs) mutants after chronic irradiation with the control. On the basis of our investigation we have concluded: 1) Low doze irradiation alter the life span depending on genetic background (mutant alleles, heterozygosity level and sex); 2) Age dynamics of physical activity positively correlates with the life span; 3) Longevity potential forms at early development stages; 4) DNA damage sensing, DNA repair, heat shock defence and apoptosis as aging preventing mechanisms play crucial role in radiation-induced life span hormesis.

Identification of four Drosophila allatostatins as the cognate ligands for the Drosophila orphan receptor DAR-2

DEFF Research Database (Denmark)

Lenz, C; Williamson, M; Hansen, G N

2001-01-01

The allatostatins are generally inhibitory insect neuropeptides. The Drosophila orphan receptor DAR-2 is a G-protein-coupled receptor, having 47% amino acid residue identity with another Drosophila receptor, DAR-1 (which is also called dros. GPCR, or DGR) that was previously shown...... to be the receptor for an intrinsic Drosophila A-type (cockroach-type) allatostatin. Here, we have permanently expressed DAR-2 in CHO cells and found that it is the cognate receptor for four Drosophila A-type allatostatins, the drostatins-A1 to -A4. Of all the drostatins, drostatin-A4 (Thr...... weakly in the brain. The Drosophila larval gut also contains about 20-30 endocrine cells, expressing the gene for the drostatins-A1 to -A4. We suggest, therefore, that DAR-2 mediates an allatostatin (drostatin)-induced inhibition of gut motility. This is the first report on the permanent and functional...

An Allergic Lung Microenvironment Suppresses Carbon Nanotube-Induced Inflammasome Activation via STAT6-Dependent Inhibition of Caspase-1.

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Kelly A Shipkowski

Full Text Available Multi-walled carbon nanotubes (MWCNTs represent a human health risk as mice exposed by inhalation display pulmonary fibrosis. Production of IL-1β via inflammasome activation is a mechanism of MWCNT-induced acute inflammation and has been implicated in chronic fibrogenesis. Mice sensitized to allergens have elevated T-helper 2 (Th2 cytokines, IL-4 and IL-13, and are susceptible to MWCNT-induced airway fibrosis. We postulated that Th2 cytokines would modulate MWCNT-induced inflammasome activation and IL-1β release in vitro and in vivo during allergic inflammation.THP-1 macrophages were primed with LPS, exposed to MWCNTs and/or IL-4 or IL-13 for 24 hours, and analyzed for indicators of inflammasome activation. C57BL6 mice were sensitized to house dust mite (HDM allergen and MWCNTs were delivered to the lungs by oropharyngeal aspiration. Mice were euthanized 1 or 21 days post-MWCNT exposure and evaluated for lung inflammasome components and allergic inflammatory responses.Priming of THP-1 macrophages with LPS increased pro-IL-1β and subsequent exposure to MWCNTs induced IL-1β secretion. IL-4 or IL-13 decreased MWCNT-induced IL-1β secretion by THP-1 cells and reduced pro-caspase-1 but not pro-IL-1β. Treatment of THP-1 cells with STAT6 inhibitors, either Leflunomide or JAK I inhibitor, blocked suppression of caspase activity by IL-4 and IL-13. In vivo, MWCNTs alone caused neutrophilic infiltration into the lungs of mice 1 day post-exposure and increased IL-1β in bronchoalveolar lavage fluid (BALF and pro-caspase-1 immuno-staining in macrophages and airway epithelium. HDM sensitization alone caused eosinophilic inflammation with increased IL-13. MWCNT exposure after HDM sensitization increased total cell numbers in BALF, but decreased numbers of neutrophils and IL-1β in BALF as well as reduced pro-caspase-1 in lung tissue. Despite reduced IL-1β mice exposed to MWCNTs after HDM developed more severe airway fibrosis by 21 days and had increased

Caspase-10 Is the Key Initiator Caspase Involved in Tributyltin-Mediated Apoptosis in Human Immune Cells

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Harald F. Krug

2012-01-01

Full Text Available Tributyltin (TBT is one of the most toxic compounds produced by man and distributed in the environment. A multitude of toxic activities have been described, for example, immunotoxic, neurotoxic, and endocrine disruptive effects. Moreover, it has been shown for many cell types that they undergo apoptosis after treatment with TBT and the cell death of immune cells could be the molecular background of its immunotoxic effect. As low as 200 nM up to 1 μM of TBT induces all signs of apoptosis in Jurkat T cells within 1 to 24 hrs of treatment. When compared to Fas-ligand control stimulation, the same sequence of events occurs: membrane blebbing, phosphatidylserine externalisation, the activation of the “death-inducing signalling complex,” and the following sequence of cleavage processes. In genetically modified caspase-8-deficient Jurkat cells, the apoptotic effects are only slightly reduced, whereas, in FADD-negative Jurkat cells, the TBT effect is significantly diminished. We could show that caspase-10 is recruited by the TRAIL-R2 receptor and apoptosis is totally prevented when caspase-10 is specifically inhibited in all three cell lines.

ATP Induces IL-1β Secretion in Neisseria gonorrhoeae-Infected Human Macrophages by a Mechanism Not Related to the NLRP3/ASC/Caspase-1 Axis

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Killen García

2016-01-01

Full Text Available Neisseria gonorrhoeae (Ngo has developed multiple immune evasion mechanisms involving the innate and adaptive immune responses. Recent findings have reported that Ngo reduces the IL-1β secretion of infected human monocyte-derived macrophages (MDM. Here, we investigate the role of adenosine triphosphate (ATP in production and release of IL-1β in Ngo-infected MDM. We found that the exposure of Ngo-infected MDM to ATP increases IL-1β levels about ten times compared with unexposed Ngo-infected MDM (P0.05 and caspase-1 (CASP1, P>0.05. In addition, ATP was not able to modify caspase-1 activity in Ngo-infected MDM but was able to increase pyroptosis (P>0.01. Notably ATP treatment defined an increase of positive staining for IL-1β with a distinctive intracellular pattern of distribution. Collectively, these data demonstrate that ATP induces IL-1β secretion by a mechanism not related to the NLRP3/ASC/caspase-1 axis and likely is acting at the level of vesicle trafficking or pore formation.

Involvement of major components from Sporothrix schenckii cell wall in the caspase-1 activation, nitric oxide and cytokines production during experimental sporotrichosis.

Science.gov (United States)

Gonçalves, Amanda Costa; Maia, Danielle Cardoso Geraldo; Ferreira, Lucas Souza; Monnazzi, Luis Gustavo Silva; Alegranci, Pâmela; Placeres, Marisa Campos Polesi; Batista-Duharte, Alexander; Carlos, Iracilda Zeppone

2015-02-01

Sporotrichosis is a chronic infection caused by the dimorphic fungus Sporothrix schenckii, involving all layers of skin and the subcutaneous tissue. The role of innate immune toll-like receptors 2 and 4 in the defense against this fungus has been reported, but so far, there were no studies on the effect of cell wall major components over the cytosolic oligo-merization domain (NOD)-like receptors, important regulators of inflammation and responsible for the maturation of IL-1β and IL-18, whose functions are dependents of the caspase-1 activation, that can participate of inflammasome. It was evaluated the percentage of activation of caspase-1, the production of IL-1β, IL-18, IL-17, IFN-γ and nitric oxide in a Balb/c model of S. schenckii infection. It was observed a decreased activity of caspase-1 during the fourth and sixth weeks of infection accompanied by reduced secretion of the cytokines IL-1β, IL-18 and IL-17 and high production of nitric oxide. IFN-γ levels were elevated during the entire time course of infection. This temporal reduction in caspase-1 activity coincides exactly with the reported period of fungal burden associated with a transitory immunosuppression induced by this fungus and detected in similar infection models. These results indicate the importance of interaction between caspase-1, cytokines IL-1β and IL-18 in the host defense against S. schenckii infection, suggesting a participation the inflammasome in this response.

Induction of Manduca sexta Larvae Caspases Expression in Midgut Cells by Bacillus thuringiensis Cry1Ab Toxin

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Helena Porta

2011-01-01

Full Text Available Bacillus thuringiensis produces crystal toxins known as Cry that are highly selective against important agricultural and human health-related insect pests. Cry proteins are pore-forming toxins that interact with specific receptors in the midgut cell membrane of susceptible larvae making pores that cause osmotic shock, leading finally to insect death. In the case of pore-forming toxins that are specific to mammalian cells, death responses at low doses may induce apoptosis or pyroptosis, depending on the cell type. The death mechanism induced by Cry toxins in insect midgut cells is poorly understood. Here, we analyze the caspases expression by RT-PCR analysis, showing that the initial response of Manduca sexta midgut cells after low dose of Cry1Ab toxin administration involves a fast and transient accumulation of caspase-1 mRNA, suggesting that pyroptosis was activated by Cry1Ab toxin as an initial response but was repressed later. In contrast, caspase-3 mRNA requires a longer period of time of toxin exposure to be activated but presents a sustained activation, suggesting that apoptosis may be a cell death mechanism induced also at low dose of toxin.

Activation of caspase-3 in radioinduced apoptosis in developing brain

International Nuclear Information System (INIS)

Michelin, Severino C.; Perez, Maria del R.; Gisone, Pablo; Dubner, Diana

2001-01-01

ICE/ced-3 related proteases (caspases) have been implicated in programmed cell death in a wide variety of cell types. However, their roles in radiation-induced cell death in cultures of mixed, neuronal and glial precursors cells are poorly understood. In order to further elucidate the molecular mechanisms underlying radiation induced death in this system; we have examined the ability of ionizing radiation to induce cell death and the caspase-3 activity. Survival decreased in a dose-dependent manner 24 h after a single 0,1 to 4 Gy dose of ionizing radiation. Irradiation resulted in a significant induction in caspase-3 activity, as measured by increased cleavage of colorimetric caspase substrates. Specific inhibitor of caspase-3, zDEVD-fmk, protected only partially from radiation induced cell death. These results demonstrate that cell death occurred despite of caspase-3 inhibition, and suggest that radio-induced cell death may occur by other mechanisms. (author)

Synaptic and genomic responses to JNK and AP-1 signaling in Drosophila neurons

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Bohmann Dirk

2005-06-01

Full Text Available Abstract Background The transcription factor AP-1 positively controls synaptic plasticity at the Drosophila neuromuscular junction. Although in motor neurons, JNK has been shown to activate AP-1, a positive regulator of growth and strength at the larval NMJ, the consequences of JNK activation are poorly studied. In addition, the downstream transcriptional targets of JNK and AP-1 signaling in the Drosophila nervous system have yet to be identified. Here, we further investigated the role of JNK signaling at this model synapse employing an activated form of JNK-kinase; and using Serial Analysis of Gene Expression and oligonucleotide microarrays, searched for candidate early targets of JNK or AP-1 dependent transcription in neurons. Results Temporally-controlled JNK induction in postembryonic motor neurons triggers synaptic growth at the NMJ indicating a role in developmental plasticity rather than synaptogenesis. An unexpected observation that JNK activation also causes a reduction in transmitter release is inconsistent with JNK functioning solely through AP-1 and suggests an additional, yet-unidentified pathway for JNK signaling in motor neurons. SAGE profiling of mRNA expression helps define the neural transcriptome in Drosophila. Though many putative AP-1 and JNK target genes arose from the genomic screens, few were confirmed in subsequent validation experiments. One potentially important neuronal AP-1 target discovered, CG6044, was previously implicated in olfactory associative memory. In addition, 5 mRNAs regulated by RU486, a steroid used to trigger conditional gene expression were identified. Conclusion This study demonstrates a novel role for JNK signaling at the larval neuromuscular junction and provides a quantitative profile of gene transcription in Drosophila neurons. While identifying potential JNK/AP-1 targets it reveals the limitations of genome-wide analyses using complex tissues like the whole brain.

Saikosaponin d induces cell death through caspase-3-dependent, caspase-3-independent and mitochondrial pathways in mammalian hepatic stellate cells

International Nuclear Information System (INIS)

Chen, Ming-Feng; Huang, S. Joseph; Huang, Chao-Cheng; Liu, Pei-Shan; Lin, Kun-I; Liu, Ching-Wen; Hsieh, Wen-Chuan; Shiu, Li-Yen; Chen, Chang-Han

2016-01-01

Saikosaponin d (SSd) is one of the main active triterpene saponins in Bupleurum falcatum. It has a steroid-like structure, and is reported to have pharmacological activities, including liver protection in rat, cell cycle arrest and apoptosis induction in several cancer cell lines. However, the biological functions and molecular mechanisms of mammalian cells under SSd treatment are still unclear. The cytotoxicity and apoptosis of hepatic stellate cells (HSCs) upon SSd treatment were discovered by MTT assay, colony formation assay and flow cytometry. The collage I/III, caspase activity and apoptotic related genes were examined by quantitative PCR, Western blotting, immunofluorescence and ELISA. The mitochondrial functions were monitored by flow cytometry, MitoTracker staining, ATP production and XF24 bioenergetic assay. This study found that SSd triggers cell death via an apoptosis path. An example of this path might be typical apoptotic morphology, increased sub-G1 phase cell population, inhibition of cell proliferation and activation of caspase-3 and caspase-9. However, the apoptotic effects induced by SSd are partially blocked by the caspase-3 inhibitor, Z-DEVD-FMK, suggesting that SSd may trigger both HSC-T6 and LX-2 cell apoptosis through caspase-3-dependent and independent pathways. We also found that SSd can trigger BAX and BAK translocation from the cytosol to the mitochondria, resulting in mitochondrial function inhibition, membrane potential disruption. Finally, SSd also increases the release of apoptotic factors. The overall analytical data indicate that SSd-elicited cell death may occur through caspase-3-dependent, caspase-3-independent and mitochondrial pathways in mammalian HSCs, and thus can delay the formation of liver fibrosis by reducing the level of HSCs

Ginsenoside Rh2 Induces Human Hepatoma Cell Apoptosisvia Bax/Bak Triggered Cytochrome C Release and Caspase-9/Caspase-8 Activation

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Xiao-Xi Guo

2012-11-01

Full Text Available Ginsenoside Rh2 (G-Rh2 has been shown to induce apoptotic cell death in a variety of cancer cells. However, the details of the signal transduction cascade involved in G-Rh2-induced cell death is unclear. In this manuscript we elucidate the molecular mechanism of G-Rh2-induced apoptosis in human hepatoma SK-HEP-1 cells by demonstrating that G-Rh2 causes rapid and dramatic translocation of both Bak and Bax, which subsequently triggers mitochondrial cytochrome c release and consequent caspase activation. Interestingly, siRNA-based gene inactivation of caspase-8 effectively delays caspase-9 activation and apoptosis induced by G-Rh2, indicating that caspase-8 also plays an important role in the G-Rh2-induced apoptosis program. Taken together, our results indicate that G-Rh2 employs a multi pro-apoptotic pathway to execute cancer cell death, suggesting a potential role for G-Rh2 as a powerful chemotherapeutic agent.

Prostaglandin F2alpha- and FAS-activating antibody-induced regression of the corpus luteum involves caspase-8 and is defective in caspase-3 deficient mice

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Flavell Richard A

2003-02-01

Full Text Available Abstract We recently demonstrated that caspase-3 is important for apoptosis during spontaneous involution of the corpus luteum (CL. These studies tested if prostaglandin F2α (PGF2α or FAS regulated luteal regression, utilize a caspase-3 dependent pathway to execute luteal cell apoptosis, and if the two receptors work via independent or potentially shared intracellular signaling components/pathways to activate caspase-3. Wild-type (WT or caspase-3 deficient female mice, 25–26 days old, were given 10 IU equine chorionic gonadotropin (eCG intraperitoneally (IP followed by 10 IU human chorionic gonadotropin (hCG IP 46 h later to synchronize ovulation. The animals were then injected with IgG (2 micrograms, i.v., the FAS-activating antibody Jo2 (2 micrograms, i.v., or PGF2α (10 micrograms, i.p. at 24 or 48 h post-ovulation. Ovaries from each group were collected 8 h later for assessment of active caspase-3 enzyme and apoptosis (measured by the TUNEL assay in the CL. Regardless of genotype or treatment, CL in ovaries collected from mice injected 24 h after ovulation showed no evidence of active caspase-3 or apoptosis. However, PGF2α or Jo2 at 48 h post-ovulation and collected 8 h later induced caspase-3 activation in 13.2 ± 1.8% and 13.7 ± 2.2 % of the cells, respectively and resulted in 16.35 ± 0.7% (PGF2α and 14.3 ± 2.5% TUNEL-positive cells when compared to 1.48 ± 0.8% of cells CL in IgG treated controls. In contrast, CL in ovaries collected from caspase-3 deficient mice whether treated with PGF2α , Jo2, or control IgG at 48 h post-ovulation showed little evidence of active caspase-3 or apoptosis. CL of WT mice treated with Jo2 at 48 h post-ovulation had an 8-fold increase in the activity of caspase-8, an activator of caspase-3 that is coupled to the FAS death receptor. Somewhat unexpectedly, however, treatment of WT mice with PGF2α at 48 h post-ovulation resulted in a 22-fold increase in caspase-8 activity in the CL, despite the fact

Diatom-derived oxylipins induce cell death in sea urchin embryos activating caspase-8 and caspase 3/7.

Science.gov (United States)

Ruocco, Nadia; Varrella, Stefano; Romano, Giovanna; Ianora, Adrianna; Bentley, Matt G; Somma, Domenico; Leonardi, Antonio; Mellone, Stefano; Zuppa, Antonio; Costantini, Maria

2016-07-01

Diatoms are an important class of unicellular algae that produce bioactive secondary metabolites with cytotoxic activity collectively termed oxylipins, including polyunsaturated aldehydes (PUAs), hydroxyacids (HEPEs), oxo-acids and epoxyalcohols. Previous results showed that at higher concentrations, the PUA decadienal induced apoptosis on copepods and sea urchin embryos via caspase-3 activation; at lower concentrations decadienal affected the expression levels of the caspase-8 gene in embryos of the sea urchin Paracentrotus lividus. In the present work, we studied the effects of other common oxylipins produced by diatoms: two PUAs (heptadienal and octadienal) and four hydroxyacids (5-, 9- 11- and 15-HEPE) on P. lividus cell death and caspase activities. Our results showed that (i) at higher concentrations PUAs and HEPEs induced apoptosis in sea urchin embryos, detected by microscopic observation and through the activation of caspase-3/7 and caspase-8 measured by luminescent assays; (ii) at low concentrations, PUAs and HEPEs affected the expression levels of caspase-8 and caspase-3/7 (isolated for the first time here in P. lividus) genes, detected by Real Time qPCR. These findings have interesting implications from the ecological point of view, given the importance of diatom blooms in nutrient-rich aquatic environments. Copyright © 2016 Elsevier B.V. All rights reserved.

PTP1B Inhibition Causes Rac1 Activation by Enhancing Receptor Tyrosine Kinase Signaling

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Ayako Tsuchiya

2014-04-01

Full Text Available Background/Aims: The present study investigated the signaling pathway underlying Rac1 activation induced by the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl-cyclopropyl]-octanoic acid (DCP-LA. Methods: Activity of protein tyrosine phosphatase 1B (PTP1B was assayed under cell-free conditions. Western blot was carried out to quantify phosphorylation of insulin receptor substrate-1 (IRS-1 and Akt in PC-12 cells. Rac1 activity was monitored in the föerster resonance energy transfer (FRET analysis using living and fixed PC-12 cells. Results: DCP-LA markedly suppressed PTP1B activity in a concentration (100 pM-100 µM-dependent manner. In the DCP-LA binding assay, fluorescein-conjugated DCP-LA produced a single fluorescent signal band at 60 kDa, corresponding to the molecule of PTP1B, and the signal was attenuated or abolished by co-treatment or pretreatment with non-conjugated DCP-LA. DCP-LA significantly enhanced nerve growth factor (NGF-stimulated phosphorylation of IRS-1 at Tyr1222 and Akt1/2 at Thr308/309 and Ser473/474 in PC-12 cells. In the FRET analysis, DCP-LA significantly enhanced NGF-stimulated Rac1 activation, which is abrogated by the phosphatidylinositol 3 kinase (PI3K inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase-1 (PDK1 inhibitor BX912, or the Akt inhibitor MK2206. Conclusion: The results of the present study show that DCP-LA-induced PTP1B inhibition, possibly through its direct binding, causes Rac1 activation by enhancing a pathway along a receptor tyrosine kinase (RTK/IRS-1/PI3K/Akt/Rac1 axis.

Nicotinamide Inhibits Ethanol-Induced Caspase-3 and PARP-1 Over-activation and Subsequent Neurodegeneration in the Developing Mouse Cerebellum.

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Ieraci, Alessandro; Herrera, Daniel G

2018-06-01

Fetal alcohol spectrum disorder (FASD) is the principal preventable cause of mental retardation in the western countries resulting from alcohol exposure during pregnancy. Ethanol-induced massive neuronal cell death occurs mainly in immature neurons during the brain growth spurt period. The cerebellum is one of the brain areas that are most sensitive to ethanol neurotoxicity. Currently, there is no effective treatment that targets the causes of these disorders and efficient treatments to counteract or reverse FASD are desirable. In this study, we investigated the effects of nicotinamide on ethanol-induced neuronal cell death in the developing cerebellum. Subcutaneous administration of ethanol in postnatal 4-day-old mice induced an over-activation of caspase-3 and PARP-1 followed by a massive neurodegeneration in the developing cerebellum. Interestingly, treatment with nicotinamide, immediately or 2 h after ethanol exposure, diminished caspase-3 and PARP-1 over-activation and reduced ethanol-induced neurodegeneration. Conversely, treatment with 3-aminobenzadine, a specific PARP-1 inhibitor, was able to completely block PARP-1 activation, but not caspase-3 activation or ethanol-induced neurodegeneration in the developing cerebellum. Our results showed that nicotinamide reduces ethanol-induced neuronal cell death and inhibits both caspase-3 and PARP-1 alcohol-induced activation in the developing cerebellum, suggesting that nicotinamide might be a promising and safe neuroprotective agent for treating FASD and other neurodegenerative disorders in the developing brain that shares similar cell death pathways.

SOLO: a meiotic protein required for centromere cohesion, coorientation, and SMC1 localization in Drosophila melanogaster.

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Yan, Rihui; Thomas, Sharon E; Tsai, Jui-He; Yamada, Yukihiro; McKee, Bruce D

2010-02-08

Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila melanogaster remains largely uncharacterized. We describe a novel protein, sisters on the loose (SOLO), which is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis.

Tissue-specific regulation of BMP signaling by Drosophila N-glycanase 1.

Science.gov (United States)

Galeone, Antonio; Han, Seung Yeop; Huang, Chengcheng; Hosomi, Akira; Suzuki, Tadashi; Jafar-Nejad, Hamed

2017-08-04

Mutations in the human N- glycanase 1 ( NGLY1 ) cause a rare, multisystem congenital disorder with global developmental delay. However, the mechanisms by which NGLY1 and its homologs regulate embryonic development are not known. Here we show that Drosophila Pngl encodes an N -glycanase and exhibits a high degree of functional conservation with human NGLY1. Loss of Pngl results in developmental midgut defects reminiscent of midgut-specific loss of BMP signaling. Pngl mutant larvae also exhibit a severe midgut clearance defect, which cannot be fully explained by impaired BMP signaling. Genetic experiments indicate that Pngl is primarily required in the mesoderm during Drosophila development. Loss of Pngl results in a severe decrease in the level of Dpp homodimers and abolishes BMP autoregulation in the visceral mesoderm mediated by Dpp and Tkv homodimers. Thus, our studies uncover a novel mechanism for the tissue-specific regulation of an evolutionarily conserved signaling pathway by an N -glycanase enzyme.

Plasma emission spectroscopy (DCP) for rare earths determination in waters from Morro do Ferro (MG) Brazil, after chromatographic preconcentration

International Nuclear Information System (INIS)

Figueiredo, A.M.R. de.

1987-01-01

Rare earth determinations in surface and well waters from Morro do Ferro was studied using chemical preconcentration methods of the group and plasma emission spectroscopy excited by direct current are - DCP. A method that combines retention in alumina is HF medium with ion exchange for the preconcentration of the group was developed in semi micro scale. DCP determination, in the sequencial mode by using mixed standards containing, Y, La, Ce, Nd, Pr, Sm, Eu, Ga, Tb, Dy, Er, Yb and Al, principal concentrate componentes was studied by analysis of the profile of each spectral line. Principal cations, anions, ph and Eh were determined. Semi-micro techniques were developed for anions preconcentration and for determination of sulphate, phosphate and carbonate. (M.J.C.) [pt

Plasmonic Ag-pillared rectorite as catalyst for degradation of 2,4-DCP in the H{sub 2}O{sub 2}-containing system under visible light irradiation

Energy Technology Data Exchange (ETDEWEB)

Chen, Yunfang [School of Chemistry and Environment, South China Normal University, Guangzhou 510006, Guangdong (China); Fang, Jianzhang, E-mail: fangjzh@scnu.edu.cn [School of Chemistry and Environment, South China Normal University, Guangzhou 510006, Guangdong (China); Guangdong Technology Research Center for Ecological Management and Remediation of Urban Water System, Guangzhou 510006 (China); Lu, Shaoyou [Shenzhen Center for Disease Control and Prevention, Shenzhen 518055 (China); Wu, Yan; Chen, Dazhi; Huang, Liyan [Institute of Engineering Technology of Guangdong Province, Key Laboratory of Water Environmental Pollution Control of Guangdong Province, Guangzhou 510440 (China); Cheng, Cong; Ren, Lu; Zhu, Ximiao [School of Chemistry and Environment, South China Normal University, Guangzhou 510006, Guangdong (China); Fang, Zhanqiang [School of Chemistry and Environment, South China Normal University, Guangzhou 510006, Guangdong (China); Guangdong Technology Research Center for Ecological Management and Remediation of Urban Water System, Guangzhou 510006 (China)

2015-10-30

Highlights: • The Ag-R catalyst was synthesized via a novel thermal decomposition method. • Ag-R catalyst possessed the synergistic effects of SPR and adsorption capacity. • The degradation of 2,4-DCP was evaluated in Ag-R/H{sub 2}O{sub 2}/visible light system. - Abstract: This study aims at photocatalytic degradation of 2,4-DCP with the assistance of H{sub 2}O{sub 2} in aqueous solution by a composite catalyst of Ag-rectorite. The catalysts were prepared via a novel thermal decomposition method followed after the cation-exchange process. The synthesized nano-materials were characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Brunauer–Emmett–Teller (BET) surface analyzer, Ultraviolet–visible light (UV–vis) absorption spectra, field-emission scanning electron microscope (FE-SEM) and transmission electron microscope (TEM). The different mechanisms of degradation process with or without visible light irradiation were discussed, respectively. Moreover, the degradation efficiency of 2,4-DCP wastewater under Ag-rectorite/H{sub 2}O{sub 2}/visible light system was investigated by series of experiments, concerning on effects of major operation factors, such as H{sub 2}O{sub 2} dosage and the initial pH value. The highest degradation rate was observed when adding 0.18 mL H{sub 2}O{sub 2} into 50 mL 2,4-DCP solution, and the optimal pH value was 4 for the reaction. Afterwards, total organic carbon (TOC) experiments were carried out to evaluate the mineralization ratio of 2,4-DCP.

Dynamic compression plate (DCP) fixation of propagating medial condylar fractures of the third metacarpal/metatarsal bone in 30 racehorses: retrospective analysis (1990-2005).

Science.gov (United States)

Goodrich, L R; Nixon, A J; Conway, J D; Morley, P S; Bladon, B M; Hogan, P M

2014-11-01

An in-depth review of dynamic compression plate (DCP) fixation of propagating medial condyle fractures of the third metacarpus or metatarsus has not been previously reported. To describe the technique, evaluate short-term outcome and long-term race performance of racehorses that underwent DCP fixation for repair of propagating or spiralling medial condylar fractures of the third metacarpal (McIII) or metatarsal (MtIII) bone. Retrospective case series. The surgical case records of 30 horses with propagating fractures of the medial condyle of McIII or MtIII were reviewed. Medical information included: age, breed, sex, presentation, how injury occurred (racing or training), surgical treatment and post operative complications. Racing information included: starts, top 3 placing and career earnings. Long propagating fractures of the medial condyle of Mc/tIII were identified in 23 Thoroughbred (TB) and 7 Standardbred (STB) racehorses. The fracture spiralled proximally in 22 of 30 cases (73%). Standardbreds had a higher propensity for hindlimb involvement (71%), whereas TBs tended to have more front limb involvement (61%). Twelve of 30 (40%) horses raced post surgery. Career earnings were significantly lower for TB horses with medial condylar fractures; $34,916 when compared with the national average of $60,841 (P≤0.03). Overall, horses having DCP fixation for medial condylar fractures had less starts post surgery (3.1 TBs and 5.8 STBs) compared with the national average (7 TBs and 17.3 STBs) and decreased lifetime starts 13.4 (TBs) compared with 17.3 nationally. Propagating medial condyle fractures can be repaired with plate fixation to potentially lessen the risk of catastrophic fracture destabilisation and return to racing can be expected in 40% of horses. Further prospective studies are warranted comparing lag screw fixation with DCP fixation for repair of severe medial condylar fractures of the metacarpus/metatarsus. © 2013 The Authors. Equine Veterinary Journal

Caspase Activation in Fetal Rat Brain Following Experimental Intrauterine Inflammation

Science.gov (United States)

Sharangpani, Aditi; Takanohashi, Asako; Bell, Michael J.

2009-01-01

Intrauterine inflammation has been implicated in developmental brain injuries, including the development of periventricular leukomalacia (PVL) and cerebral palsy (CP). Previous studies in our rat model of intrauterine inflammation demonstrated apoptotic cell death in fetal brains within the first 5 days after lipopolysaccharide (LPS) administration to mothers and eventual dysmyelination. Cysteine-containing, aspartate-specific proteases, or caspases, are proteins involved with apoptosis through both intracellular (intrinsic pathway) and extracellular (extrinsic pathway) mechanisms. We hypothesized that cell death in our model would occur mainly via activation of the extrinsic pathway. We further hypothesized that Fas, a member of the tumor necrosis factor receptor (TNFR) superfamily, would be increased and the death inducing signaling complex (DISC) would be detectable. Pregnant rats were injected intracervically with LPS at E15 and immunoblotting, immunohistochemical and immunoprecipitation analyses were performed. The presence of the activated form of the effector caspase (caspase-3) was observed 24 h after LPS administration. Caspase activity assays demonstrated rapid increases in (i) caspases-9 and -10 within 1 h, (ii) caspase-8 at 2 h and (iii) caspase-3 at 4 h. At 24 h after LPS, activated caspase-3+/Fas+ cells were observed within the developing white matter. Lastly, the DISC complex (caspase-8, Fas and Fas-associated Death Domain (FADD)) was observed within 30 min by immunoprecipitation. Apoptosis in our model occurs via both extrinsic and intrinsic pathways, and activation of Fas may play a role. Understanding the mechanisms of cell death in models of intrauterine inflammation may affect development of future strategies to mitigate these injuries in children. PMID:18289516

CB1R-Mediated Activation of Caspase-3 Causes Epigenetic and Neurobehavioral Abnormalities in Postnatal Ethanol-Exposed Mice

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Shivakumar Subbanna

2018-02-01

Full Text Available Alcohol exposure can affect brain development, leading to long-lasting behavioral problems, including cognitive impairment, which together is defined as fetal alcohol spectrum disorder (FASD. However, the fundamental mechanisms through which this occurs are largely unknown. In this study, we report that the exposure of postnatal day 7 (P7 mice to ethanol activates caspase-3 via cannabinoid receptor type-1 (CB1R in neonatal mice and causes a reduction in methylated DNA binding protein (MeCP2 levels. The developmental expression of MeCP2 in mice is closely correlated with synaptogenesis and neuronal maturation. It was shown that ethanol treatment of P7 mice enhanced Mecp2 mRNA levels but reduced protein levels. The genetic deletion of CB1R prevented, and administration of a CB1R antagonist before ethanol treatment of P7 mice inhibited caspase-3 activation. Additionally, it reversed the loss of MeCP2 protein, cAMP response element binding protein (CREB activation, and activity-regulated cytoskeleton-associated protein (Arc expression. The inhibition of caspase-3 activity prior to ethanol administration prevented ethanol-induced loss of MeCP2, CREB activation, epigenetic regulation of Arc expression, long-term potentiation (LTP, spatial memory deficits and activity-dependent impairment of several signaling molecules, including MeCP2, in adult mice. Collectively, these results reveal that the ethanol-induced CB1R-mediated activation of caspase-3 degrades the MeCP2 protein in the P7 mouse brain and causes long-lasting neurobehavioral deficits in adult mice. This CB1R-mediated instability of MeCP2 during active synaptic maturation may disrupt synaptic circuit maturation and lead to neurobehavioral abnormalities, as observed in this animal model of FASD.

Voltage dependent anion channel-1 regulates death receptor mediated apoptosis by enabling cleavage of caspase-8

International Nuclear Information System (INIS)

Chacko, Alex D; Liberante, Fabio; Paul, Ian; Longley, Daniel B; Fennell, Dean A

2010-01-01

Activation of the extrinsic apoptosis pathway by tumour necrosis factor related apoptosis inducing ligand (TRAIL) is a novel therapeutic strategy for treating cancer that is currently under clinical evaluation. Identification of molecular biomarkers of resistance is likely to play an important role in predicting clinical anti tumour activity. The involvement of the mitochondrial type 1 voltage dependent anion channel (VDAC1) in regulating apoptosis has been highly debated. To date, a functional role in regulating the extrinsic apoptosis pathway has not been formally excluded. We carried out stable and transient RNAi knockdowns of VDAC1 in non-small cell lung cancer cells, and stimulated the extrinsic apoptotic pathway principally by incubating cells with the death ligand TRAIL. We used in-vitro apoptotic and cell viability assays, as well as western blot for markers of apoptosis, to demonstrate that TRAIL-induced toxicity is VDAC1 dependant. Confocal microscopy and mitochondrial fractionation were used to determine the importance of mitochondria for caspase-8 activation. Here we show that either stable or transient knockdown of VDAC1 is sufficient to antagonize TRAIL mediated apoptosis in non-small cell lung cancer (NSCLC) cells. Specifically, VDAC1 is required for processing of procaspase-8 to its fully active p18 form at the mitochondria. Loss of VDAC1 does not alter mitochondrial sensitivity to exogenous caspase-8-cleaved BID induced mitochondrial depolarization, even though VDAC1 expression is essential for TRAIL dependent activation of the intrinsic apoptosis pathway. Furthermore, expression of exogenous VDAC1 restores the apoptotic response to TRAIL in cells in which endogenous VDAC1 has been selectively silenced. Expression of VDAC1 is required for full processing and activation of caspase-8 and supports a role for mitochondria in regulating apoptosis signaling via the death receptor pathway

Caspase-responsive smart gadolinium-based contrast agent for magnetic resonance imaging of drug-induced apoptosis.

Science.gov (United States)

Ye, Deju; Shuhendler, Adam J; Pandit, Prachi; Brewer, Kimberly D; Tee, Sui Seng; Cui, Lina; Tikhomirov, Grigory; Rutt, Brian; Rao, Jianghong

2014-10-01

Non-invasive detection of caspase-3/7 activity in vivo has provided invaluable predictive information regarding tumor therapeutic efficacy and anti-tumor drug selection. Although a number of caspase-3/7 targeted fluorescence and positron emission tomography (PET) imaging probes have been developed, there is still a lack of gadolinium (Gd)-based magnetic resonance imaging (MRI) probes that enable high spatial resolution detection of caspase-3/7 activity in vivo . Here we employ a self-assembly approach and develop a caspase-3/7 activatable Gd-based MRI probe for monitoring tumor apoptosis in mice. Upon reduction and caspase-3/7 activation, the caspase-sensitive nano-aggregation MR probe (C-SNAM: 1 ) undergoes biocompatible intramolecular cyclization and subsequent self-assembly into Gd-nanoparticles (GdNPs). This results in enhanced r 1 relaxivity-19.0 (post-activation) vs. 10.2 mM -1 s -1 (pre-activation) at 1 T in solution-and prolonged accumulation in chemotherapy-induced apoptotic cells and tumors that express active caspase-3/7. We demonstrate that C-SNAM reports caspase-3/7 activity by generating a significantly brighter T 1 -weighted MR signal compared to non-treated tumors following intravenous administration of C-SNAM, providing great potential for high-resolution imaging of tumor apoptosis in vivo .

Caspase-10 Negatively Regulates Caspase-8-Mediated Cell Death, Switching the Response to CD95L in Favor of NF-κB Activation and Cell Survival

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Sebastian Horn

2017-04-01

Full Text Available Formation of the death-inducing signaling complex (DISC initiates extrinsic apoptosis. Caspase-8 and its regulator cFLIP control death signaling by binding to death-receptor-bound FADD. By elucidating the function of the caspase-8 homolog, caspase-10, we discover that caspase-10 negatively regulates caspase-8-mediated cell death. Significantly, we reveal that caspase-10 reduces DISC association and activation of caspase-8. Furthermore, we extend our co-operative/hierarchical binding model of caspase-8/cFLIP and show that caspase-10 does not compete with caspase-8 for binding to FADD. Utilizing caspase-8-knockout cells, we demonstrate that caspase-8 is required upstream of both cFLIP and caspase-10 and that DISC formation critically depends on the scaffold function of caspase-8. We establish that caspase-10 rewires DISC signaling to NF-κB activation/cell survival and demonstrate that the catalytic activity of caspase-10, and caspase-8, is redundant in gene induction. Thus, our data are consistent with a model in which both caspase-10 and cFLIP coordinately regulate CD95L-mediated signaling for death or survival.

Organization and evolution of Drosophila terminin: similarities and differences between Drosophila and human telomeres

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Grazia Daniela Raffa

2013-05-01

Full Text Available Drosophila lacks telomerase and fly telomeres are elongated by occasional transposition of three specialized retroelements. Drosophila telomeres do not terminate with GC-rich repeats and are assembled independently of the sequence of chromosome ends. Recent work has shown that Drosophila telomeres are capped by the terminin complex, which includes the fast-evolving proteins HOAP, HipHop, Moi and Ver. These proteins are not conserves outside Drosophilidae and localize and function exclusively at telomeres, protecting them from fusion events. Other proteins required to prevent end-to-end fusion in flies include HP1, Eff/UbcD1, ATM, the components of the Mre11-Rad50-Nbs (MRN complex, and the Woc transcription factor. These proteins do not share the terminin properties; they are evolutionarily conserved non-fast-evolving proteins that do not accumulate only telomeres and do not serve telomere-specific functions. We propose that following telomerase loss, Drosophila rapidly evolved terminin to bind chromosome ends in a sequence-independent manner. This hypothesis suggests that terminin is the functional analog of the shelterin complex that protects human telomeres. The non-terminin proteins are instead likely to correspond to ancestral telomere-associated proteins that did not evolve as rapidly as terminin because of the functional constraints imposed by their involvement in diverse cellular processes. Thus, it appears that the main difference between Drosophila and human telomeres is in the protective complexes that specifically associate with the DNA termini. We believe that Drosophila telomeres offer excellent opportunities for investigations on human telomere biology. The identification of additional Drosophila genes encoding non-terminin proteins involved in telomere protection might lead to the discovery of novel components of human telomeres.

Drosophila DJ-1 decreases neural sensitivity to stress by negatively regulating Daxx-like protein through dFOXO.

Directory of Open Access Journals (Sweden)

Soojin Hwang

2013-04-01

Full Text Available DJ-1, a Parkinson's disease (PD-associated gene, has been shown to protect against oxidative stress in Drosophila. However, the molecular mechanism underlying oxidative stress-induced phenotypes, including apoptosis, locomotive defects, and lethality, in DJ-1-deficient flies is not fully understood. Here we showed that Daxx-like protein (DLP, a Drosophila homologue of the mammalian Death domain-associated protein (Daxx, was upregulated under oxidative stress conditions in the loss-of-function mutants of Drosophila DJ-1β, a Drosophila homologue of DJ-1. DLP overexpression induced apoptosis via the c-Jun N-terminal kinase (JNK/Drosophila forkhead box subgroup O (dFOXO pathway, whereas loss of DLP increased resistance to oxidative stress and UV irradiation. Moreover, the oxidative stress-induced phenotypes of DJ-1β mutants were dramatically rescued by DLP deficiency, suggesting that enhanced expression of DLP contributes to the DJ-1β mutant phenotypes. Interestingly, we found that dFOXO was required for the increase in DLP expression in DJ-1β mutants and that dFOXO activity was increased in the heads of DJ-1β mutants. In addition, subcellular localization of DLP appeared to be influenced by DJ-1 expression so that cytosolic DLP was increased in DJ-1β mutants. Similarly, in mammalian cells, Daxx translocation from the nucleus to the cytosol was suppressed by overexpressed DJ-1β under oxidative stress conditions; and, furthermore, targeted expression of DJ-1β to mitochondria efficiently inhibited the Daxx translocation. Taken together, our findings demonstrate that DJ-1β protects flies against oxidative stress- and UV-induced apoptosis by regulating the subcellular localization and gene expression of DLP, thus implying that Daxx-induced apoptosis is involved in the pathogenesis of DJ-1-associated PD.

Inhibitor specificity of recombinant and endogenous caspase-9.

Science.gov (United States)

Ryan, Ciara A; Stennicke, Henning R; Nava, Victor E; Burch, Jennifer B; Hardwick, J Marie; Salvesen, Guy S

2002-01-01

Apoptosis triggered through the intrinsic pathway by radiation and anti-neoplastic drugs is initiated by the activation of caspase-9. To elucidate control mechanisms in this pathway we used a range of synthetic and natural reagents. The inhibitory potency of acetyl-Asp-Glu-Val-Asp-aldehyde ('Ac-DEVD-CHO'), benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone ('Z-VAD-FMK') and the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis protein ('XIAP') against recombinant caspase-9 were predictive of the efficacy of these compounds in a cell-free system. However, the viral proteins CrmA and p35, although potent inhibitors of recombinant caspase-9, had almost no ability to block caspase-9 in this system. These findings were also mirrored in cell expression studies. We hypothesize that the viral inhibitors CrmA and p35 are excluded from reacting productively with the natural form of active caspase-9 in vivo, making the potency of inhibitors highly context-dependent. This is supported by survival data from a mouse model of apoptosis driven by Sindbis virus expressing either p35 or a catalytic mutant of caspase-9. These results consolidate previous findings that CrmA is a potent inhibitor of caspase-9 in vitro, yet fails to block caspase-9-mediated cell death. PMID:12067274

Structural Insights into DD-Fold Assembly and Caspase-9 Activation by the Apaf-1 Apoptosome.

Science.gov (United States)

Su, Tsung-Wei; Yang, Chao-Yu; Kao, Wen-Pin; Kuo, Bai-Jiun; Lin, Shan-Meng; Lin, Jung-Yaw; Lo, Yu-Chih; Lin, Su-Chang

2017-03-07

Death domain (DD)-fold assemblies play a crucial role in regulating the signaling to cell survival or death. Here we report the crystal structure of the caspase recruitment domain (CARD)-CARD disk of the human apoptosome. The structure surprisingly reveals that three 1:1 Apaf-1:procaspase-9 CARD protomers form a novel helical DD-fold assembly on the heptameric wheel-like platform of the apoptosome. The small-angle X-ray scattering and multi-angle light scattering data also support that three protomers could form an oligomeric complex similar to the crystal structure. Interestingly, the quasi-equivalent environment of CARDs could generate different quaternary CARD assemblies. We also found that the type II interaction is conserved in all DD-fold complexes, whereas the type I interaction is found only in the helical DD-fold assemblies. This study provides crucial insights into the caspase activation mechanism, which is tightly controlled by a sophisticated and highly evolved CARD assembly on the apoptosome, and also enables better understanding of the intricate DD-fold assembly. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expression of caspase-3 predicts prognosis in advanced noncardia gastric cancer.

Science.gov (United States)

Amptoulach, Sousana; Lazaris, Andreas C; Giannopoulou, Ioanna; Kavantzas, Nikolaos; Patsouris, Efstratios; Tsavaris, Nikolaos

2015-01-01

There is strong evidence that tumor growth is not only a result of uncontrolled cell proliferation but also of decreased apoptosis. Caspase-3 is a member of interleukin-1 beta-converting enzyme which is involved in the induction of apoptosis. Data on the expression of caspase-3 in patients with gastric cancer and its association with patient outcome are somewhat contradictory. We aimed to investigate the potential relation of the expression of caspase-3 protein with response to therapy and overall survival in patients with advanced noncardia gastric cancer. Tumor tissue samples collected from 359 consecutive patients with gastric cancer stage IV were retrospectively analyzed for the expression of caspase-3 in the primary tumor. The DNA apoptotic index assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling method. All patients were followed up until death. Caspase-3 was expressed in 43.5 % of tumors. Caspase-3 expression compared to no expression was related with a higher DNA apoptotic index (p gastric cancer is a predictor of poor response to treatment and survival. Further studies are needed to fully elucidate the prognostic value of caspase-3 expression in these patients.

Biological effects of radon in Drosophila; Efectos biologicos del radon en Drosophila

Energy Technology Data Exchange (ETDEWEB)

Pimentel P, A E; Tavera D, L; Cruces M, M P; Arceo M, C; Rosa D, M.E. de la

1992-04-15

The main objective of this investigation, is to study the biological effects of the Radon-222 at low dose in 'Drosophila melanogaster'. It is necessary to mention that these effects will analyze from the genetic point of view for: 1) To evaluate in which form the Radon-222 to low dose it influences in some genetic components of the adaptation in Drosophila, such as: fecundity, viability egg-adult and sex proportion. 2) To evaluate which is the genetic effect that induces the Radon to low dose by means of the SMART technique in Drosophila melanogaster, and this way to try of to identify which is the possible mechanism that causes the genetic damage to somatic level. The carried out investigation was divided in three stages: 1. Tests to the vacuum resistance. 2. Test of somatic mutation, and 3. Determination of the presence of radon daughters on the adult of Drosophila. It is necessary to point out that all the experiments were made by triplicate and in each one of them was placed detectors in preset places. Those obtained results are presented inside the 4 charts included in the present work. (Author)

Biological effects of radon in Drosophila; Efectos biologicos del radon en Drosophila

Energy Technology Data Exchange (ETDEWEB)

Pimentel P, A.E.; Tavera D, L.; Cruces M, M.P.; Arceo M, C.; Rosa D, M.E. de la

1992-04-15

The main objective of this investigation, is to study the biological effects of the Radon-222 at low dose in 'Drosophila melanogaster'. It is necessary to mention that these effects will analyze from the genetic point of view for: 1) To evaluate in which form the Radon-222 to low dose it influences in some genetic components of the adaptation in Drosophila, such as: fecundity, viability egg-adult and sex proportion. 2) To evaluate which is the genetic effect that induces the Radon to low dose by means of the SMART technique in Drosophila melanogaster, and this way to try of to identify which is the possible mechanism that causes the genetic damage to somatic level. The carried out investigation was divided in three stages: 1. Tests to the vacuum resistance. 2. Test of somatic mutation, and 3. Determination of the presence of radon daughters on the adult of Drosophila. It is necessary to point out that all the experiments were made by triplicate and in each one of them was placed detectors in preset places. Those obtained results are presented inside the 4 charts included in the present work. (Author)

Molecular cloning, functional expression, and gene silencing of two Drosophila receptors for the Drosophila neuropeptide pyrokinin-2

DEFF Research Database (Denmark)

Rosenkilde, Carina; Cazzamali, Giuseppe; Williamson, Michael

2003-01-01

The database of the Drosophila Genome Project contains the sequences of two genes, CG8784 and CG8795, predicted to code for two structurally related G protein-coupled receptors. We have cloned these genes and expressed their coding parts in Chinese hamster ovary cells. We found that both receptors...... can be activated by low concentrations of the Drosophila neuropeptide pyrokinin-2 (CG8784, EC(50) for pyrokinin-2, 1x10(-9)M; CG8795, EC(50) for pyrokinin-2, 5 x 10(-10)M). The precise role of Drosophila pyrokinin-2 (SVPFKPRLamide) in Drosophila is unknown, but in other insects, pyrokinins have...... embryos and first instar larvae. In addition to the two Drosophila receptors, we also identified two probable pyrokinin receptors in the genomic database from the malaria mosquito Anopheles gambiae. The two Drosophila pyrokinin receptors are, to our knowledge, the first invertebrate pyrokinin receptors...

Suppression of human T cell proliferation by the caspase inhibitors, z-VAD-FMK and z-IETD-FMK is independent of their caspase inhibition properties

Energy Technology Data Exchange (ETDEWEB)

Lawrence, C.P. [Medical Research Council Toxicology Unit, Hodgkin Building, Lancaster Road, University of Leicester, Leicester LE1 9HN (United Kingdom); Chow, S.C., E-mail: chow.sek.chuen@monash.edu [School of Science, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, 46150 Selangor Darul Ehsan (Malaysia)

2012-11-15

The caspase inhibitors, benzyloxycarbony (Cbz)-l-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) and benzyloxycarbonyl (Cbz)-Ile-Glu (OMe)-Thr-Asp (OMe)-FMK (z-IETD-FMK) at non-toxic doses were found to be immunosuppressive and inhibit human T cell proliferation induced by mitogens and IL-2 in vitro. Both caspase inhibitors were shown to block NF-κB in activated primary T cells, but have little inhibitory effect on the secretion of IL-2 and IFN-γ during T cell activation. However, the expression of IL-2 receptor α-chain (CD25) in activated T cells was inhibited by both z-VAD-FMK and z-IETD-FMK, whereas the expression of the early activated T cell marker, CD69 was unaffected. During primary T cell activation via the antigen receptor, both caspase-8 and caspase-3 were activated and processed to their respective subunits, but neither caspase inhibitors had any effect on the processing of these two caspases. In sharp contrast both caspase inhibitors readily blocked apoptosis and the activation of caspases during FasL-induced apoptosis in activated primary T cells and Jurkat T cells. Collectively, the results demonstrate that both z-VAD-FMK and z-IETD-FMK are immunosuppressive in vitro and inhibit T cell proliferation without blocking the processing of caspase-8 and caspase-3. -- Highlights: ► Caspase-8 and caspase-3 were activated during T cell activation and proliferation. ► T cell proliferation was blocked by caspase inhibitors. ► Caspase activation during T cell proliferation was not block by caspase inhibitors.

Possible involvement of caspase-6 and -7 but not caspase-3 in the regulation of hypoxia-induced apoptosis in tube-forming endothelial cells

International Nuclear Information System (INIS)

Eguchi, Ryoji; Tone, Shigenobu; Suzuki, Akio; Fujimori, Yoshihiro; Nakano, Takashi; Kaji, Kazuhiko; Ohta, Toshiro

2009-01-01

We recently reported that a broad-spectrum caspase inhibitor zVAD-fmk failed, while p38 inhibitor SB203580 succeeded, to prevent chromatin condensation and nuclear fragmentation induced by hypoxia in tube-forming HUVECs. In this study, we investigated the reasons for zVAD-fmk's inability to inhibit these morphological changes at the molecular level. The inhibitor effectively inhibited DNA ladder formation and activation of caspase-3 and -6, but it surprisingly failed to inhibit caspase-7 activation. On the other hand, SB203580 successfully inhibited all of these molecular events. When zLEHD-fmk, which specifically inhibits initiator caspase-9 upstream of caspase-3, was used, it inhibited caspase-3 activation but failed to inhibit caspase-6 and -7 activation. It also failed to inhibit hypoxia-induced chromatin condensation, nuclear fragmentation and DNA ladder formation. Taken together, our results indicate that, during hypoxia, caspase-7 is responsible for chromatin condensation and nuclear fragmentation while caspase-6 is responsible for DNA ladder formation

Chlamydia abortus Pmp18.1 Induces IL-1β Secretion by TLR4 Activation through the MyD88, NF-κB, and Caspase-1 Signaling Pathways.

Science.gov (United States)

Pan, Qing; Zhang, Qiang; Chu, Jun; Pais, Roshan; Liu, Shanshan; He, Cheng; Eko, Francis O

2017-01-01

The polymorphic membrane protein D (Pmp18D) is a 160-kDa outer membrane protein that is conserved and plays an important role in Chlamydia abortus pathogenesis. We have identified an N-terminal fragment of Pmp18D (designated Pmp18.1) as a possible subunit vaccine antigen. In this study, we evaluated the vaccine potential of Pmp18.1 by investigating its ability to induce innate immune responses in dendritic cells and the signaling pathway(s) involved in rPmp18.1-induced IL-1β secretion. We next investigated the immunomodulatory impact of VCG, in comparison with the more established Th1-promoting adjuvants, CpG and FL, on rPmp18.1-mediated innate immune activation. Finally, the effect of siRNA targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in DCs on IL-1β cytokine secretion was also investigated. Bone marrow-derived dendritic cells (BMDCs) were stimulated with rPmp18.1 in the presence or absence of VCG or CpG or FL and the magnitude of cytokines produced was assessed using a multiplex cytokine ELISA assay. Expression of costimulatory molecules and Toll-like receptors (TLRs) was analyzed by flow cytometry. Quantitation of intracellular levels of myeloid differentiation factor 88 (MyD88), nuclear factor kappa beta (NF-κB p50/p65), and Caspase-1 was evaluated by Western immunoblotting analysis while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The results showed DC stimulation with rPmp18.1 provoked the secretion of proinflammatory cytokines and upregulated expression of TLRs and co-stimulatory molecules associated with DC maturation. These responses were significantly ( p ≤ 0.001) enhanced by VCG but not CpG or FL. In addition, rPmp18.1 activated the expression of MyD88, NF-κB p50, and Caspase-1 as well as the nuclear expression of NF-κB p65 in treated DCs. Furthermore, targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in BMDCs with siRNA significantly reduced their expression levels, resulting in decreased IL-1β cytokine

Chlamydia abortus Pmp18.1 Induces IL-1β Secretion by TLR4 Activation through the MyD88, NF-κB, and Caspase-1 Signaling Pathways

Directory of Open Access Journals (Sweden)

Qing Pan

2017-12-01

Full Text Available The polymorphic membrane protein D (Pmp18D is a 160-kDa outer membrane protein that is conserved and plays an important role in Chlamydia abortus pathogenesis. We have identified an N-terminal fragment of Pmp18D (designated Pmp18.1 as a possible subunit vaccine antigen. In this study, we evaluated the vaccine potential of Pmp18.1 by investigating its ability to induce innate immune responses in dendritic cells and the signaling pathway(s involved in rPmp18.1-induced IL-1β secretion. We next investigated the immunomodulatory impact of VCG, in comparison with the more established Th1-promoting adjuvants, CpG and FL, on rPmp18.1-mediated innate immune activation. Finally, the effect of siRNA targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in DCs on IL-1β cytokine secretion was also investigated. Bone marrow-derived dendritic cells (BMDCs were stimulated with rPmp18.1 in the presence or absence of VCG or CpG or FL and the magnitude of cytokines produced was assessed using a multiplex cytokine ELISA assay. Expression of costimulatory molecules and Toll-like receptors (TLRs was analyzed by flow cytometry. Quantitation of intracellular levels of myeloid differentiation factor 88 (MyD88, nuclear factor kappa beta (NF-κB p50/p65, and Caspase-1 was evaluated by Western immunoblotting analysis while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The results showed DC stimulation with rPmp18.1 provoked the secretion of proinflammatory cytokines and upregulated expression of TLRs and co-stimulatory molecules associated with DC maturation. These responses were significantly (p ≤ 0.001 enhanced by VCG but not CpG or FL. In addition, rPmp18.1 activated the expression of MyD88, NF-κB p50, and Caspase-1 as well as the nuclear expression of NF-κB p65 in treated DCs. Furthermore, targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in BMDCs with siRNA significantly reduced their expression levels, resulting in decreased IL-1�

A Yersinia effector with enhanced inhibitory activity on the NF-κB pathway activates the NLRP3/ASC/caspase-1 inflammasome in macrophages.

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Ying Zheng

2011-04-01

Full Text Available A type III secretion system (T3SS in pathogenic Yersinia species functions to translocate Yop effectors, which modulate cytokine production and regulate cell death in macrophages. Distinct pathways of T3SS-dependent cell death and caspase-1 activation occur in Yersinia-infected macrophages. One pathway of cell death and caspase-1 activation in macrophages requires the effector YopJ. YopJ is an acetyltransferase that inactivates MAPK kinases and IKKβ to cause TLR4-dependent apoptosis in naïve macrophages. A YopJ isoform in Y. pestis KIM (YopJ(KIM has two amino acid substitutions, F177L and K206E, not present in YopJ proteins of Y. pseudotuberculosis and Y. pestis CO92. As compared to other YopJ isoforms, YopJ(KIM causes increased apoptosis, caspase-1 activation, and secretion of IL-1β in Yersinia-infected macrophages. The molecular basis for increased apoptosis and activation of caspase-1 by YopJ(KIM in Yersinia-infected macrophages was studied. Site directed mutagenesis showed that the F177L and K206E substitutions in YopJ(KIM were important for enhanced apoptosis, caspase-1 activation, and IL-1β secretion. As compared to YopJ(CO92, YopJ(KIM displayed an enhanced capacity to inhibit phosphorylation of IκB-α in macrophages and to bind IKKβ in vitro. YopJ(KIM also showed a moderately increased ability to inhibit phosphorylation of MAPKs. Increased caspase-1 cleavage and IL-1β secretion occurred in IKKβ-deficient macrophages infected with Y. pestis expressing YopJ(CO92, confirming that the NF-κB pathway can negatively regulate inflammasome activation. K+ efflux, NLRP3 and ASC were important for secretion of IL-1β in response to Y. pestis KIM infection as shown using macrophages lacking inflammasome components or by the addition of exogenous KCl. These data show that caspase-1 is activated in naïve macrophages in response to infection with a pathogen that inhibits IKKβ and MAPK kinases and induces TLR4-dependent apoptosis. This pro

Pripper: prediction of caspase cleavage sites from whole proteomes

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Salmi Jussi

2010-06-01

Full Text Available Abstract Background Caspases are a family of proteases that have central functions in programmed cell death (apoptosis and inflammation. Caspases mediate their effects through aspartate-specific cleavage of their target proteins, and at present almost 400 caspase substrates are known. There are several methods developed to predict caspase cleavage sites from individual proteins, but currently none of them can be used to predict caspase cleavage sites from multiple proteins or entire proteomes, or to use several classifiers in combination. The possibility to create a database from predicted caspase cleavage products for the whole genome could significantly aid in identifying novel caspase targets from tandem mass spectrometry based proteomic experiments. Results Three different pattern recognition classifiers were developed for predicting caspase cleavage sites from protein sequences. Evaluation of the classifiers with quality measures indicated that all of the three classifiers performed well in predicting caspase cleavage sites, and when combining different classifiers the accuracy increased further. A new tool, Pripper, was developed to utilize the classifiers and predict the caspase cut sites from an arbitrary number of input sequences. A database was constructed with the developed tool, and it was used to identify caspase target proteins from tandem mass spectrometry data from two different proteomic experiments. Both known caspase cleavage products as well as novel cleavage products were identified using the database demonstrating the usefulness of the tool. Pripper is not restricted to predicting only caspase cut sites, but it gives the possibility to scan protein sequences for any given motif(s and predict cut sites once a suitable cut site prediction model for any other protease has been developed. Pripper is freely available and can be downloaded from http://users.utu.fi/mijopi/Pripper. Conclusions We have developed Pripper, a tool for

Zinc-mediated Allosteric Inhibition of Caspase-6*

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Velázquez-Delgado, Elih M.; Hardy, Jeanne A.

2012-01-01

Zinc and caspase-6 have independently been implicated in several neurodegenerative disorders. Depletion of zinc intracellularly leads to apoptosis by an unknown mechanism. Zinc inhibits cysteine proteases, including the apoptotic caspases, leading to the hypothesis that zinc-mediated inhibition of caspase-6 might contribute to its regulation in a neurodegenerative context. Using inductively coupled plasma optical emission spectroscopy, we observed that caspase-6 binds one zinc per monomer, under the same conditions where the zinc leads to complete loss of enzymatic activity. To understand the molecular details of zinc binding and inhibition, we performed an anomalous diffraction experiment above the zinc edge. The anomalous difference maps showed strong 5σ peaks, indicating the presence of one zinc/monomer bound at an exosite distal from the active site. Zinc was not observed bound to the active site. The zinc in the exosite was liganded by Lys-36, Glu-244, and His-287 with a water molecule serving as the fourth ligand, forming a distorted tetrahedral ligation sphere. This exosite appears to be unique to caspase-6, as the residues involved in zinc binding were not conserved across the caspase family. Our data suggest that binding of zinc at the exosite is the primary route of inhibition, potentially locking caspase-6 into the inactive helical conformation. PMID:22891250

Fas-Induced Apoptosis of Renal Cell Carcinoma is Mediated by Apoptosis Signal-Regulating Kinase 1 via Mitochondrial Damage-Dependent Caspase-8 Activation

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Mohamed Hassan

2009-01-01

Full Text Available Renal cell carcinoma (RCC is a prototype of a chemo refractory tumour. It remains the most lethal of the common urologic cancers and is highly resistant to conventional therapy. Here, we confirmed the efficiency of anti-Fas monoclonal antibody (CH11 as alternative therapeutic approach for the treatment of RCC and investigated the molecular mechanism(s, whereby CH11 induces apoptosis of RCC cells. The present study shows an essential role for apoptosis signal-regulating kinase 1 (ASK1, together with both c-jun-N-terminal kinase (JNK and p38 pathways, and caspase-8 in this process. Furthermore, CH11-dependent induction of the ASK1–JNK/p38 pathways was found to activate the transcription factors AP-1 and ATF-2, and FADD-caspase-8-Bid signalling, resulting in the translocation of both Bax and Bak proteins, and subsequently mitochondrial dysregulation that is characterized by the loss of mitochondrial membrane potential (ΔΨm, cytochrome c release and cleavage of caspase-9, caspase-3 and PARP. Thus, the described molecular mechanisms of CH11-induced apoptosis suggest the reliability of Fas activation as an alternative therapeutic approach for the treatment of patients with advanced renal cell carcinoma.

Heterologous expression of mammalian Plk1 in Drosophila reveals divergence from Polo during late mitosis

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Pearson, John; Godinho, Susana A.; Tavares, Alvaro; Glover, David M.

2006-01-01

Drosophila Polo kinase is the founder member of a conserved kinase family required for multiple stages of mitosis. We assessed the ability of mouse Polo-like kinase 1 (Plk1) to perform the multiple mitotic functions of Polo kinase, by expressing a Plk1-GFP fusion in Drosophila. Consistent with the previously reported localization of Polo kinase, Plk1-GFP was strongly localized to centrosomes and recruited to the centromeric regions of condensing chromosomes during early mitosis. However, in contrast to a functional Polo-GFP fusion, Plk1-GFP failed to localize to the central spindle midzone in both syncytial embryo mitosis and the conventional mitoses of cellularized embryos and S2 cells. Moreover, unlike endogenous Polo kinase and Polo-GFP, Plk1-GFP failed to associate with the contractile ring. Expression of Plk1-GFP enhanced the lethality of hypomorphic polo mutants and disrupted the organization of the actinomyosin cytoskeleton in a dominant-negative manner. Taken together, our results suggest that endogenous Polo kinase has specific roles in regulating actinomyosin rearrangements during Drosophila mitoses that its mammalian counterpart, Plk1, cannot fulfill. Consistent with this hypothesis, we observed defects in the cortical recruitment of myosin and myosin regulatory light chain in Polo deficient cells

Caspases : more than just killers?

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Los, Marek Jan; Stroh, C.; Janicke, R. U.; Engels, I. H.; Schulze-Osthoff, K.

2001-01-01

Proteases of the caspase family constitute the central executioners of apoptosis, Several recent observations suggest that caspases and apoptosis-regulatory molecules exert important functions beyond that of cell death, including the control of T-cell proliferation and cell-cycle progression. Here, Los and colleagues propose a model that directly connects cell suicide mechanisms to the regulation of cell-cycle progression.

Caspases: more than just killers?

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Los, M; Stroh, C; Jänicke, R U; Engels, I H; Schulze-Osthoff, K

2001-01-01

Proteases of the caspase family constitute the central executioners of apoptosis. Several recent observations suggest that caspases and apoptosis-regulatory molecules exert important functions beyond that of cell death, including the control of T-cell proliferation and cell-cycle progression. Here, Los and colleagues propose a model that directly connects cell suicide mechanisms to the regulation of cell-cycle progression.

Positive Selection Drives the Evolution of rhino, a Member of the Heterochromatin Protein 1 Family in Drosophila.

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2005-07-01

Full Text Available Heterochromatin comprises a significant component of many eukaryotic genomes. In comparison to euchromatin, heterochromatin is gene poor, transposon rich, and late replicating. It serves many important biological roles, from gene silencing to accurate chromosome segregation, yet little is known about the evolutionary constraints that shape heterochromatin. A complementary approach to the traditional one of directly studying heterochromatic DNA sequence is to study the evolution of proteins that bind and define heterochromatin. One of the best markers for heterochromatin is the heterochromatin protein 1 (HP1, which is an essential, nonhistone chromosomal protein. Here we investigate the molecular evolution of five HP1 paralogs present in Drosophila melanogaster. Three of these paralogs have ubiquitous expression patterns in adult Drosophila tissues, whereas HP1D/rhino and HP1E are expressed predominantly in ovaries and testes respectively. The HP1 paralogs also have distinct localization preferences in Drosophila cells. Thus, Rhino localizes to the heterochromatic compartment in Drosophila tissue culture cells, but in a pattern distinct from HP1A and lysine-9 dimethylated H3. Using molecular evolution and population genetic analyses, we find that rhino has been subject to positive selection in all three domains of the protein: the N-terminal chromo domain, the C-terminal chromo-shadow domain, and the hinge region that connects these two modules. Maximum likelihood analysis of rhino sequences from 20 species of Drosophila reveals that a small number of residues of the chromo and shadow domains have been subject to repeated positive selection. The rapid and positive selection of rhino is highly unusual for a gene encoding a chromosomal protein and suggests that rhino is involved in a genetic conflict that affects the germline, belying the notion that heterochromatin is simply a passive recipient of "junk DNA" in eukaryotic genomes.

Positive selection drives the evolution of rhino, a member of the heterochromatin protein 1 family in Drosophila.

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Danielle Vermaak

2005-07-01

Full Text Available Heterochromatin comprises a significant component of many eukaryotic genomes. In comparison to euchromatin, heterochromatin is gene poor, transposon rich, and late replicating. It serves many important biological roles, from gene silencing to accurate chromosome segregation, yet little is known about the evolutionary constraints that shape heterochromatin. A complementary approach to the traditional one of directly studying heterochromatic DNA sequence is to study the evolution of proteins that bind and define heterochromatin. One of the best markers for heterochromatin is the heterochromatin protein 1 (HP1, which is an essential, nonhistone chromosomal protein. Here we investigate the molecular evolution of five HP1 paralogs present in Drosophila melanogaster. Three of these paralogs have ubiquitous expression patterns in adult Drosophila tissues, whereas HP1D/rhino and HP1E are expressed predominantly in ovaries and testes respectively. The HP1 paralogs also have distinct localization preferences in Drosophila cells. Thus, Rhino localizes to the heterochromatic compartment in Drosophila tissue culture cells, but in a pattern distinct from HP1A and lysine-9 dimethylated H3. Using molecular evolution and population genetic analyses, we find that rhino has been subject to positive selection in all three domains of the protein: the N-terminal chromo domain, the C-terminal chromo-shadow domain, and the hinge region that connects these two modules. Maximum likelihood analysis of rhino sequences from 20 species of Drosophila reveals that a small number of residues of the chromo and shadow domains have been subject to repeated positive selection. The rapid and positive selection of rhino is highly unusual for a gene encoding a chromosomal protein and suggests that rhino is involved in a genetic conflict that affects the germline, belying the notion that heterochromatin is simply a passive recipient of "junk DNA" in eukaryotic genomes.

Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster.

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Johanna Sebald

Full Text Available The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster.

Ras promotes cell survival by antagonizing both JNK and Hid signals in the Drosophila eye.

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Wu, Yue; Zhuang, Yuan; Han, Min; Xu, Tian; Deng, Kejing

2009-10-20

Programmed cell death, or apoptosis, is a fundamental physiological process during normal development or in pathological conditions. The activation of apoptosis can be elicited by numerous signalling pathways. Ras is known to mediate anti-apoptotic signals by inhibiting Hid activity in the Drosophila eye. Here we report the isolation of a new loss-of-function ras allele, rasKP, which causes excessive apoptosis in the Drosophila eye. This new function is likely to be mediated through the JNK pathway since the inhibition of JNK signalling can significantly suppress rasKP-induced apoptosis, whereas the removal of hid only weakly suppresses the phenotype. Furthermore, the reduction of JNK signalling together with the expression of the baculovirus caspase inhibitor p35, which blocks Hid activity, strongly suppresses the rasKP cell death. In addition, we find a strong correlation between rasKP-induced apoptosis in the eye disc and the activation of JNK signalling. In the Drosophila eye, Ras may protect cells from apoptosis by inhibiting both JNK and Hid activities. Surprisingly, reducing Ras activity in the wing, however, does not cause apoptosis but rather affects cell and organ size. Thus, in addition to its requirement for cell viability, Ras appears to mediate different biological roles depending on the developmental context and on the level of its expression.

The influence of sterol metabolism upon radiation-induced aneuploidy of Drosophila melanogaster in the yeast-drosophila system

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Savitsij, V.V.; Luchnikova, E.M.; Inge-Vechtomov, S.I.

1985-01-01

The influence of sterol metabolism upon induced Drosophila melanogaster mutagenesis in an ecology-genetic yeast-drosophila system has been studied. The sterol deficit in fly organism has been created for account of using as food substrate for fremales of biomass of saccharomyces cerevisiae living cells of 9-2-PZ12 train with nyssup(r1) locus mutation which blocks the ergosterol synthesis. It has been found that the Drosophila females content on mutant yeast increases the frequency of losses and non discrepancy of X-chromosomes induced by X-radiation (1000 R). Addition into yeast biomass of 0.1 % cholesterol solution in 10 %-ethanol reduces the oocytes resistance to X-radiation up to control level. Possible hormonal and membrane mechanisms of increasing radiation-induced aneuploidy of Drosophila and the role of sterol metabolism in organism resistance to damaging factors are discussed

Caspase-14 Expression Impairs Retinal Pigment Epithelium Barrier Function: Potential Role in Diabetic Macular Edema

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Selina Beasley

2014-01-01

Full Text Available We recently showed that caspase-14 is a novel molecule in retina with potential role in accelerated vascular cell death during diabetic retinopathy (DR. Here, we evaluated whether caspase-14 is implicated in retinal pigment epithelial cells (RPE dysfunction under hyperglycemia. The impact of high glucose (HG, 30 mM D-glucose on caspase-14 expression in human RPE (ARPE-19 cells was tested, which showed significant increase in caspase-14 expression compared with normal glucose (5 mM D-glucose + 25 mM L-glucose. We also evaluated the impact of modulating caspase-14 expression on RPE cells barrier function, phagocytosis, and activation of other caspases using ARPE-19 cells transfected with caspase-14 plasmid or caspase-14 siRNA. We used FITC-dextran flux assay and electric cell substrate impedance sensing (ECIS to test the changes in RPE cell barrier function. Similar to HG, caspase-14 expression in ARPE-19 cells increased FITC-dextran leakage through the confluent monolayer and decreased the transcellular electrical resistance (TER. These effects of HG were prevented by caspase-14 knockdown. Furthermore, caspase-14 knockdown prevented the HG-induced activation of caspase-1 and caspase-9, the only activated caspases by HG. Phagocytic activity was unaffected by caspase-14 expression. Our results suggest that caspase-14 contributes to RPE cell barrier disruption under hyperglycemic conditions and thus plays a role in the development of diabetic macular edema.

COMPARATIVE STUDY BETWEEN TITANIUM ELASTIC NAILING (TENS AND DYNAMIC COMPRESSION PLATING (DCP IN THE TREATMENT OF FEMORAL DIAPHYSEAL FRACTURES IN CHILDREN

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Ramasubba Reddy

2015-08-01

Full Text Available BACKGROUND : Orthopaedic surgeons have long maintained that all children who have sustained a diaphyseal fracture of femur recover with c onservative treatment, given the excellent remodeling ability of immature bone in children. Angulations, shortenings and malrotations are not always corrected by conservative treatment. Of many surgical options, titanium elastic nailing has been the newer implant which is being used regularly. Although good results have been reported with elastic intramedullary nails, plate fixation continues to be a viable alternative in surgical treatment of femoral shaft fractures. However there are not many studies comp aring the efficiency of titanium elastic nailing and plating for femoral diaphyseal fractures in pediatric age group. AIM : The present study aims to compare the surgical management of diaphyseal fractures of femur in children with Dynamic Compression Plati ng versus Titanium Elastic Nailing. DESIGN : This is a prospective study . MATERIALS AND METHODS : This prospective study was conducted in a tertiary hospital. Patients who presented to the out - patient department and casualty of the hospital with femoral diap hyseal fractures during April 2012 to June 2014 were considered for the study. Subjects fulfilling the predetermined inclusion and exclusion criteria were included in the study. STATISTICAL METHODS : Fisher Exact test, Chi - Square Test, Student t test (Two t ailed, independent . RESULTS : Patients in the age group of 6 - 14 years were considered for the study, Patients were divided into two groups and treated with DCP/TENS. The duration of surgery, hospital stay, and, amount of blood loss was minimal in TENS grou p. Callus was seen early in TENS group. Radiological union was early in TENS group by 2 - 3 weeks. Outcome was better in patients treated with TENS (Excellent - 70%; Satisfactory – 30%; Poor - 0% in comparison to DCP (Excellent - 70%; Satisfactory - 25%; Poor - 5%. CO NCLUSION : TENS

Org-1-dependent lineage reprogramming generates the ventral longitudinal musculature of the Drosophila heart.

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Schaub, Christoph; März, Johannes; Reim, Ingolf; Frasch, Manfred

2015-02-16

Only few examples of transdifferentiation, which denotes the conversion of one differentiated cell type to another, are known to occur during normal development, and more often, it is associated with regeneration processes. With respect to muscles, dedifferentiation/redifferentiation processes have been documented during post-traumatic muscle regeneration in blastema of newts as well as during myocardial regeneration. As shown herein, the ventral longitudinal muscles of the adult Drosophila heart arise from specific larval alary muscles in a process that represents the first known example of syncytial muscle transdifferentiation via dedifferentiation into mononucleate myoblasts during normal development. We demonstrate that this unique process depends on the reinitiation of a transcriptional program previously employed for embryonic alary muscle development, in which the factors Org-1 (Drosophila Tbx1) and Tailup (Drosophila Islet1) are key components. During metamorphosis, the action of these factors is combined with cell-autonomous inputs from the ecdysone steroid and the Hox gene Ultrabithorax, which provide temporal and spatial specificity to the transdifferentiation events. Following muscle dedifferentiation, inductive cues, particularly from the remodeling heart tube, are required for the redifferentiation of myoblasts into ventral longitudinal muscles. Our results provide new insights into mechanisms of lineage commitment and cell-fate plasticity during development. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ire1 mediated mRNA splicing in a C-terminus deletion mutant of Drosophila Xbp1.

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Dina S Coelho

Full Text Available The Unfolded Protein Response is a homeostatic mechanism that permits eukaryotic cells to cope with Endoplasmic Reticulum (ER stress caused by excessive accumulation of misfolded proteins in the ER lumen. The more conserved branch of the UPR relies on an ER transmembrane enzyme, Ire1, which, upon ER stress, promotes the unconventional splicing of a small intron from the mRNA encoding the transcription factor Xbp1. In mammals, two specific regions (the hydrophobic region 2--HR2--and the C-terminal translational pausing site present in the Xbp1unspliced protein mediate the recruitment of the Xbp1 mRNA-ribosome-nascent chain complex to the ER membrane, so that Xbp1 mRNA can be spliced by Ire1. Here, we generated a Drosophila Xbp1 deletion mutant (Excision101 lacking both HR2 and C-terminal region, but not the Ire1 splicing site. We show that Ire1-dependent splicing of Xbp1 mRNA is reduced, but not abolished in Excision101. Our results suggest the existence of additional mechanisms for ER membrane targeting of Xbp1 mRNA that are independent of the C-terminal domain of Drosophila Xbp1unspliced.

Caspase-3 Inhibition Attenuates the Cytopathic Effects of EV71 Infection

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Fengmei Song

2018-04-01

Full Text Available Previous studies demonstrate that human enterovirus 71 (EV71, a primary causative agent for hand, foot, and mouth disease, activates caspase-3 through the non-structural viral 3C protein to induce host cell apoptosis; however, until now it was unclear how 3C activates caspase-3 and how caspase-3 activation affects viral production. Our results demonstrate that 3C binds caspase-8 and caspase-9 but does not directly bind caspase-3 to activate them, and that the proteolytic activity of 3C is required by the activation of caspase-8, caspase-9, and caspase-3. Inhibition of caspase-3 activity attenuates apoptosis in 3C-transfected cells. Furthermore, caspase-3 inhibitor protects host cells from the cytopathic effect of EV71 infection and prevents cell cycle arrest, which is known to be favored for EV71 viral replication. Inhibition of caspase-3 activity decreases EV71 viral protein expression and viral production, but has no effect on viral entry, replication, even polyprotein translation. Therefore, caspase-3 is exploited functionally by EV71 to facilitate its production, which suggests a novel therapeutic approach for the treatment and prevention of hand, foot, and mouth disease.

Molecular dynamics-assisted pharmacophore modeling of caspase-3-isatin sulfonamide complex: Recognizing essential intermolecular contacts and features of sulfonamide inhibitor class for caspase-3 binding.

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Kumar, Sivakumar Prasanth; Patel, Chirag N; Jha, Prakash C; Pandya, Himanshu A

2017-12-01

The identification of isatin sulfonamide as a potent small molecule inhibitor of caspase-3 had fuelled the synthesis and characterization of the numerous sulfonamide class of inhibitors to optimize for potency. Recent works that relied on the ligand-based approaches have successfully shown the regions of optimizations for sulfonamide scaffold. We present here molecular dynamics-based pharmacophore modeling of caspase-3-isatin sulfonamide crystal structure, to elucidate the essential non-covalent contacts and its associated pharmacophore features necessary to ensure caspase-3 optimal binding. We performed 20ns long dynamics of this crystal structure to extract global conformation states and converted into structure-based pharmacophore hypotheses which were rigorously validated using an exclusive focussed library of experimental actives and inactives of sulfonamide class by Receiver Operating Characteristic (ROC) statistic. Eighteen structure-based pharmacophore hypotheses with better sensitivity and specificity measures (>0.6) were chosen which collectively showed the role of pocket residues viz. Cys163 (S 1 sub-site; required for covalent and H bonding with Michael acceptor of inhibitors), His121 (S 1 ; π stack with bicyclic isatin moiety), Gly122 (S 1 ; H bond with carbonyl oxygen) and Tyr204 (S 2 ; π stack with phenyl group of the isatin sulfonamide molecule) as stringent binding entities for enabling caspase-3 optimal binding. The introduction of spatial pharmacophore site points obtained from dynamics-based pharmacophore models in a virtual screening strategy will be helpful to screen and optimize molecules belonging to sulfonamide class of caspase-3 inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interactions between Upf1 and the decapping factors Edc3 and Pat1 in Saccharomyces cerevisiae.

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Kylie D Swisher

Full Text Available In Saccharomyces cerevisiae, mRNA transcripts with premature termination codons are targeted for deadenylation independent decapping and 5' to 3' decay in a quality control pathway termed nonsense-mediated decay (NMD. Critical factors in NMD include Upf1, Upf2, and Upf3, as well as the decapping enzyme, Dcp2/Dcp1. Loss of Upf2 or Upf3 leads to the accumulation of not only Upf1 and Dcp2 in P-bodies, but also of the decapping-activators Pat1, Dhh1, and Lsm1. An interaction between Upf1 and Dcp2 has been identified, which might recruit Dcp2 to the NMD decapping complex. To determine the nature and significance of the Dcp2-Upf1 interaction, we utilized the yeast two-hybrid assay to assess Upf1 interactions with various mRNA decapping factors. We find that although Dcp2 can interact with Upf1, this interaction is indirect and is largely dependent on the Edc3 protein, which interacts with the N-terminal domain of Upf1 at an overlapping, but not identical, site as Upf2. We also found that Pat1 has an independent two-hybrid interaction with the N-terminus of Upf1. Assessment of both reporter and endogenous NMD transcripts suggest that the decapping stimulators, including Edc3 and Pat1, as well as Edc1 and Edc2, are not essential for NMD under normal conditions. This work defines a larger decapping complex involved in NMD, but indicates that components of that complex are not required for general NMD and might either regulate a subset of NMD transcripts or be essential for proper NMD under different environmental conditions.

Definition of a RACK1 Interaction Network in Drosophila melanogaster Using SWATH-MS.

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Kuhn, Lauriane; Majzoub, Karim; Einhorn, Evelyne; Chicher, Johana; Pompon, Julien; Imler, Jean-Luc; Hammann, Philippe; Meignin, Carine

2017-07-05

Receptor for Activated protein C kinase 1 (RACK1) is a scaffold protein that has been found in association with several signaling complexes, and with the 40S subunit of the ribosome. Using the model organism Drosophila melanogaster , we recently showed that RACK1 is required at the ribosome for internal ribosome entry site (IRES)-mediated translation of viruses. Here, we report a proteomic characterization of the interactome of RACK1 in Drosophila S2 cells. We carried out Label-Free quantitation using both Data-Dependent and Data-Independent Acquisition (DDA and DIA, respectively) and observed a significant advantage for the Sequential Window Acquisition of all THeoretical fragment-ion spectra (SWATH) method, both in terms of identification of interactants and quantification of low abundance proteins. These data represent the first SWATH spectral library available for Drosophila and will be a useful resource for the community. A total of 52 interacting proteins were identified, including several molecules involved in translation such as structural components of the ribosome, factors regulating translation initiation or elongation, and RNA binding proteins. Among these 52 proteins, 15 were identified as partners by the SWATH strategy only. Interestingly, these 15 proteins are significantly enriched for the functions translation and nucleic acid binding. This enrichment reflects the engagement of RACK1 at the ribosome and highlights the added value of SWATH analysis. A functional screen did not reveal any protein sharing the interesting properties of RACK1, which is required for IRES-dependent translation and not essential for cell viability. Intriguingly however, 10 of the RACK1 partners identified restrict replication of Cricket paralysis virus (CrPV), an IRES-containing virus. Copyright © 2017 Kuhn et al.

Function of caspase-14 in trophoblast differentiation

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Charles Adrian K

2009-09-01

Full Text Available Abstract Background Within the human placenta, the cytotrophoblast consists of a proliferative pool of progenitor cells which differentiate to replenish the overlying continuous, multi-nucleated syncytiotrophoblast, which forms the barrier between the maternal and fetal tissues. Disruption to trophoblast differentiation and function may result in impaired fetal development and preeclampsia. Caspase-14 expression is limited to barrier forming tissues. It promotes keratinocyte differentiation by cleaving profilaggrin to stabilise keratin intermediate filaments, and indirectly providing hydration and UV protection. However its role in the trophoblast remains unexplored. Methods Using RNA Interference the reaction of control and differentiating trophoblastic BeWo cells to suppressed caspase-14 was examined for genes pertaining to hormonal, cell cycle and cytoskeletal pathways. Results Transcription of hCG, KLF4 and cytokeratin-18 were increased following caspase-14 suppression suggesting a role for caspase-14 in inhibiting their pathways. Furthermore, hCG, KLF4 and cytokeratin-18 protein levels were disrupted. Conclusion Since expression of these molecules is normally increased with trophoblast differentiation, our results imply that caspase-14 inhibits trophoblast differentiation. This is the first functional study of this unusual member of the caspase family in the trophoblast, where it has a different function than in the epidermis. This knowledge of the molecular underpinnings of trophoblast differentiation may instruct future therapies of trophoblast disease.

ENU mutagenesis reveals that Notchless homolog 1 (Drosophila affects Cdkn1a and several members of the Wnt pathway during murine pre-implantation development

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Lossie Amy C

2012-12-01

Full Text Available Abstract Background Our interests lie in determining the genes and genetic pathways that are important for establishing and maintaining maternal-fetal interactions during pregnancy. Mutation analysis targeted to a 34 Mb domain flanked by Trp53 and Wnt3 demonstrates that this region of mouse chromosome 11 contains a large number of essential genes. Two mutant alleles (l11Jus1 and l11Jus4, which fall into the same complementation group, survive through implantation but fail prior to gastrulation. Results Through a positional cloning strategy, we discovered that these homozygous mutant alleles contain non-conservative missense mutations in the Notchless homolog 1 (Drosophila (Nle1 gene. NLE1 is a member of the large WD40-repeat protein family, and is thought to signal via the canonical NOTCH pathway in vertebrates. However, the phenotype of the Nle1 mutant mice is much more severe than single Notch receptor mutations or even in animals in which NOTCH signaling is blocked. To test the hypothesis that NLE1 functions in multiple signaling pathways during pre-implantation development, we examined expression of multiple Notch downstream target genes, as well as select members of the Wnt pathway in wild-type and mutant embryos. We did not detect altered expression of any primary members of the Notch pathway or in Notch downstream target genes. However, our data reveal that Cdkn1a, a NOTCH target, was upregulated in Nle1 mutants, while several members of the Wnt pathway are downregulated. In addition, we found that Nle1 mutant embryos undergo caspase-mediated apoptosis as hatched blastocysts, but not as morulae or blastocysts. Conclusions Taken together, these results uncover potential novel functions for NLE1 in the WNT and CDKN1A pathways during embryonic development in mammals.

Effect of sterol metabolism in the yeast-Drosophila system on the frequency of radiation-induced aneuploidy in the Drosophila melanogaster oocytes

International Nuclear Information System (INIS)

Savitskii, V.V.; Luchnikova, E.M.; Inge-Vechtomov, S.G.

1986-01-01

The effect of sterol metabolism on induced mutagenesis of Drosophila melanogaster was studied in the ecogenetic system of yeast-Drosophila. Sterol deficiency was created in Drosophila by using the biomass of live cells of Saccharomyces cerevisiae strain 9-2-P712 till mutation in locus nys/sup r1/ blocking the synthesis of ergosterol as the food. It was found that rearing of Drosophila females on the mutant yeast increases the frequency of loss and nondisjunction of X chromosomes induced in mature oocytes by X rays (1000 R). Addition of 0.1% of cholesterol solution in 10% ethanol to the yeast biomass restores the resistance of oocyte to X irradiation to the control level. The possible hormonal effect on membrane leading to increased radiation-induced aneuploidy in Drosophila and the role of sterol metabolism in determining the resistance to various damaging factors are discussed

Scavenger receptors mediate the role of SUMO and Ftz-f1 in Drosophila steroidogenesis.

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Ana Talamillo

2013-04-01

Full Text Available SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval-pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor, the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi-mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues.

Hypocapnia induces caspase-3 activation and increases Abeta production.

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Xie, Zhongcong; Moir, Robert D; Romano, Donna M; Tesco, Giuseppina; Kovacs, Dora M; Tanzi, Rudolph E

2004-01-01

At least half of all cases of early onset (<60) familial Alzheimer's disease (FAD) are caused by any of over 150 mutations in three genes: the amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2). Mutant forms of PS1 have been shown to sensitize cells to apoptotic cell death. We investigated the effects of hypocapnia, a risk factor for both cognitive and neurodevelopment deficits, on caspase-3 activation, apoptosis, and amyloid beta-protein (Abeta) production, and assessed the influence of the PS1Delta9 FAD mutation on these effects. For this purpose, we exposed stably transfected H4 human neuroglioma cells to conditions consistent with hypocapnia (PCO2<40 mm Hg) and hypocapnia plus hypoxia (PO2<21%). Hypocapnia (20 mm Hg CO2 for 6 h) induced caspase-3 activation and apoptosis; the PS1Delta9 FAD mutation significantly potentiated these effects. Moreover, the combination of hypocapnia (20 mm Hg CO2) and hypoxia (5%O2) induced caspase-3 activation and apoptosis in a synergistic manner. Hypocapnia (5 and 20 mm Hg CO2 for 6 h) also led to an increased Abeta production. The findings suggest that hypocapnia (e.g. during general anesthesia) could exacerbate AD neuropathogenesis. Copyright (c) 2004 S. Karger AG, Basel.

Oxidative stress, caspase-3 activation and cleavage of ROCK-1 play an essential role in MeHg-induced cell death in primary astroglial cells.

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Dos Santos, Alessandra Antunes; López-Granero, Caridad; Farina, Marcelo; Rocha, João B T; Bowman, Aaron B; Aschner, Michael

2018-03-01

Methylmercury is a toxic environmental contaminant that elicits significant toxicity in humans. The central nervous system is the primary target of toxicity, and is particularly vulnerable during development. Rho-associated protein kinase 1 (ROCK-1) is a major downstream effector of the small GTPase RhoA and a direct substrate of caspase-3. The activation of ROCK-1 is necessary for membrane blebbing during apoptosis. In this work, we examined whether MeHg could affect the RhoA/ROCK-1 signaling pathway in primary cultures of mouse astrocytes. Exposure of cells with 10 μM MeHg decreased cellular viability after 24 h of incubation. This reduction in viability was preceded by a significant increase in intracellular and mitochondrial reactive oxygen species levels, as well as a reduced NAD + /NADH ratio. MeHg also induced an increase in mitochondrial-dependent caspase-9 and caspase-3, while the levels of RhoA protein expression were reduced or unchanged. We further found that MeHg induced ROCK-1 cleavage/activation and promoted LIMK1 and MYPT1 phosphorylation, both of which are the best characterized ROCK-1 downstream targets. Inhibiting ROCK-1 and caspases activation attenuated the MeHg-induced cell death. Collectively, these findings are the first to show that astrocytes exposed to MeHg showed increased cleavage/activation of ROCK-1, which was independent of the small GTPase RhoA. Copyright © 2018. Published by Elsevier Ltd.

A short caspase-3 isoform inhibits chemotherapy-induced apoptosis by blocking apoptosome assembly.

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Frédérique Végran

Full Text Available Alternative splicing of caspase-3 produces a short isoform caspase-3s that antagonizes caspase-3 apoptotic activity. However, the mechanism of apoptosis inhibition by caspase-3s remains unknown. Here we show that exogenous caspase-3 sensitizes MCF-7 and HBL100 breast cancers cells to chemotherapeutic treatments such as etoposide and methotrexate whereas co-transfection with caspase-3s strongly inhibits etoposide and methotrexate-induced apoptosis underlying thus the anti-apoptotic role of caspase-3s. In caspase-3 transfected cells, lamin-A and α-fodrin were cleaved when caspase-3 was activated by etoposide or methotrexate. When caspase-3s was co-transfected, this cleavage was strongly reduced. Depletion of caspase-3 by RNA interference in HBL100 containing endogenous caspase-3s caused reduction in etoposide and methotrexate-induced apoptosis, whereas the depletion of caspase-3s sensitized cells to chemotherapy. In the presence of caspase-3s, a lack of interaction between caspase-3 and caspase-9 was observed. Immunoprecipitation assays showed that caspase-3s binds the pro-forms of caspase-3. This result suggested that the absence of interaction with caspase-9 when both variants of caspase-3 are present contribute to block the apoptosome assembly and inhibit apoptosis. These data support that caspases-3s negatively interferes with caspase-3 activation and apoptosis in breast cancer, and that it can play key roles in the modulation of response to chemotherapeutic treatments.

The silent information regulator 1 (Sirt1) is a positive regulator of the Notch pathway in Drosophila

Czech Academy of Sciences Publication Activity Database

Horváth, Matěj; Mihajlović, Zorana; Slaninová, Věra; Perez-Gomez, Raquel; Moshkin, Y.; Krejčí, Alena

2016-01-01

Roč. 473, č. 22 (2016), s. 4129-4143 ISSN 0264-6021 R&D Projects: GA ČR(CZ) GA14-08583S Institutional support: RVO:60077344 Keywords : Drosophila * silent information regulator 1 * Notch pathway Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.797, year: 2016

Pharmacological caspase inhibitors: Research towards therapeutic perspectives

Czech Academy of Sciences Publication Activity Database

Kudělová, J.; Fleischmannová, Jana; Adamová, Eva; Matalová, Eva

2015-01-01

Roč. 66, č. 4 (2015), s. 473-482 ISSN 0867-5910 R&D Projects: GA ČR GB14-37368G Institutional support: RVO:67985904 Keywords : caspase * caspase inhibitor * apoptosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.804, year: 2015

A novel bicistronic sensor vector for detecting caspase-3 activation.

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Vagner, Tatyana; Mouravlev, Alexandre; Young, Deborah

2015-01-01

Apoptosis is involved in pathological cell death of a wide range of human diseases. One of the most important biochemical markers of apoptosis is activation of caspase-3. Ability to detect caspase-3 activation early in the pathological process is important for determining the timing for interfering with apoptosis initiation and prevention of cell damage. Techniques allowing detection of caspase-3 activity at a single cell level show increased sensitivity, compared to biochemical assays; therefore, we developed a novel bicistronic caspase-3 sensor vector enabling detection of caspase-3 activity in individual cells. We employed green fluorescent protein (GFP) as a reporter for caspase-3 activation in our constructs and assessed the functionality of the generated constructs in transiently transfected Neuro2A and HEK293 cells under basal conditions and following application of okadaic acid (OA) or staurosporine (STS) to induce apoptosis. To ensure responsiveness of the new sensor vector to active caspase-3, we co-transfected the sensor with plasmid(s) overexpressing active caspase-3 and quantified GFP fluorescence using a plate reader. We observed an increase in GFP expression in cells transfected with the new bicistronic caspase-3 sensor in response to both OA and STS. We also showed a significant increase in GFP fluorescence intensity in cells co-expressing the sensor with the plasmid(s) encoding active caspase-3. We generated a novel bicistronic caspase-3 sensor vector which relies on a transcription factor/response element system. The obtained sensor combines high sensitivity of the single cell level detection with the possibility of automated quantification. Copyright © 2015 Elsevier Inc. All rights reserved.

Tumor suppressor roles of CENP-E and Nsl1 in Drosophila epithelial tissues.

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Clemente-Ruiz, Marta; Muzzopappa, Mariana; Milán, Marco

2014-01-01

Depletion of spindle assembly checkpoint (SAC) genes in Drosophila epithelial tissues leads to JNK-dependent programmed cell death and additional blockade of the apoptotic program drives tumorigenesis. A recent report proposes that chromosomal instability (CIN) is not the driving force in the tumorigenic response of the SAC-deficient tissue, and that checkpoint proteins exert a SAC-independent tumor suppressor role. This notion is based on observations that the depletion of CENP-E levels or prevention of Bub3 from binding to the kinetochore in Drosophila tissues unable to activate the apoptotic program induces CIN but does not cause hyperproliferation. Here we re-examined this proposal. In contrast to the previous report, we observed that depletion of CENP-E or Nsl1-the latter mediating kinetochore targeting of Bub3-in epithelial tissues unable to activate the apoptotic program induces significant levels of aneuploidy and drives tumor-like growth. The induction of the JNK transcriptional targets Wingless, a mitogenic molecule, and MMP1, a matrix metaloproteinase 1 involved in basement membrane degradation was also observed in these tumors. An identical response of the tissue was previously detected upon depletion of several SAC genes or genes involved in spindle assembly, chromatin condensation, and cytokinesis, all of which have been described to cause CIN. All together, these results reinforce the role of CIN in driving tumorigenesis in Drosophila epithelial tissues and question the proposed SAC-independent roles of checkpoint proteins in suppressing tumorigenesis. Differences in aneuploidy rates might explain the discrepancy between the previous report and our results.

Pharmacological caspase inhibitors: Research towards therapeutic perspectives

Czech Academy of Sciences Publication Activity Database

Kudělová, J.; Fleischmannová, J.; Adamová, E.; Matalová, Eva

2015-01-01

Roč. 66, č. 4 (2015), s. 473-482 ISSN 0867-5910 R&D Projects: GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 Keywords : caspase * caspase inhibitor * apoptosis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.804, year: 2015

Drosophila atm/telomere fusion is required for telomeric localization of HP1 and telomere position effect.

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Oikemus, Sarah R; McGinnis, Nadine; Queiroz-Machado, Joana; Tukachinsky, Hanna; Takada, Saeko; Sunkel, Claudio E; Brodsky, Michael H

2004-08-01

Terminal deletions of Drosophila chromosomes can be stably protected from end-to-end fusion despite the absence of all telomere-associated sequences. The sequence-independent protection of these telomeres suggests that recognition of chromosome ends might contribute to the epigenetic protection of telomeres. In mammals, Ataxia Telangiectasia Mutated (ATM) is activated by DNA damage and acts through an unknown, telomerase-independent mechanism to regulate telomere length and protection. We demonstrate that the Drosophila homolog of ATM is encoded by the telomere fusion (tefu) gene. In the absence of ATM, telomere fusions occur even though telomere-specific Het-A sequences are still present. High levels of spontaneous apoptosis are observed in ATM-deficient tissues, indicating that telomere dysfunction induces apoptosis in Drosophila. Suppression of this apoptosis by p53 mutations suggests that loss of ATM activates apoptosis through a DNA damage-response mechanism. Loss of ATM reduces the levels of heterochromatin protein 1 (HP1) at telomeres and suppresses telomere position effect. We propose that recognition of chromosome ends by ATM prevents telomere fusion and apoptosis by recruiting chromatin-modifying complexes to telomeres.

Behavioral Teratogenesis in Drosophila melanogaster.

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Mishra, Monalisa; Barik, Bedanta Kumar

2018-01-01

Developmental biology is a fascinating branch of science which helps us to understand the mechanism of development, thus the findings are used in various therapeutic approach. Drosophila melanogaster served as a model to find the key molecules that initiate and regulate the mechanism of development. Various genes, transcription factors, and signaling pathways helping in development are identified in Drosophila. Many toxic compounds, which can affect the development, are also recognized using Drosophila model. These compounds, which can affect the development, are named as a teratogen. Many teratogens identified using Drosophila may also act as a teratogen for a human being since 75% of conservation exist between the disease genes present in Drosophila and human. There are certain teratogens, which do not cause developmental defect if exposed during pregnancy, however; behavioral defect appears in later part of development. Such compounds are named as a behavioral teratogen. Thus, it is worthy to identify the potential behavioral teratogen using Drosophila model. Drosophila behavior is well studied in various developmental stages. This chapter describes various methods which can be employed to test behavioral teratogenesis in Drosophila.

Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2

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Guntur, Ananya R.; Gu, Pengyu; Takle, Kendra; Chen, Jingyi; Xiang, Yang; Yang, Chung-Hui

2015-01-01

The transient receptor potential A1 (TRPA1) channel is an evolutionarily conserved detector of temperature and irritant chemicals. Here, we show that two specific isoforms of TRPA1 in Drosophila are H2O2 sensitive and that they can detect strong UV light via sensing light-induced production of H2O2. We found that ectopic expression of these H2O2-sensitive Drosophila TRPA1 (dTRPA1) isoforms conferred UV sensitivity to light-insensitive HEK293 cells and Drosophila neurons, whereas expressing the H2O2-insensitive isoform did not. Curiously, when expressed in one specific group of motor neurons in adult flies, the H2O2-sensitive dTRPA1 isoforms were as competent as the blue light-gated channelrhodopsin-2 in triggering motor output in response to light. We found that the corpus cardiacum (CC) cells, a group of neuroendocrine cells that produce the adipokinetic hormone (AKH) in the larval ring gland endogenously express these H2O2-sensitive dTRPA1 isoforms and that they are UV sensitive. Sensitivity of CC cells required dTRPA1 and H2O2 production but not conventional phototransduction molecules. Our results suggest that specific isoforms of dTRPA1 can sense UV light via photochemical production of H2O2. We speculate that UV sensitivity conferred by these isoforms in CC cells may allow young larvae to activate stress response—a function of CC cells—when they encounter strong UV, an aversive stimulus for young larvae. PMID:26443856

Distribution of DNA replication proteins in Drosophila cells

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Easwaran, Hariharan P; Leonhardt, Heinrich; Cardoso, M Cristina

2007-01-01

Background DNA replication in higher eukaryotic cells is organized in discrete subnuclear sites called replication foci (RF). During the S phase, most replication proteins assemble at the RF by interacting with PCNA via a PCNA binding domain (PBD). This has been shown to occur for many mammalian replication proteins, but it is not known whether this mechanism is conserved in evolution. Results Fluorescent fusions of mammalian replication proteins, Dnmt1, HsDNA Lig I and HsPCNA were analyzed for their ability to target to RF in Drosophila cells. Except for HsPCNA, none of the other proteins and their deletions showed any accumulation at RF in Drosophila cells. We hypothesized that in Drosophila cells there might be some other peptide sequence responsible for targeting proteins to RF. To test this, we identified the DmDNA Lig I and compared the protein sequence with HsDNA Lig I. The two orthologs shared the PBD suggesting a functionally conserved role for this domain in the Drosophila counterpart. A series of deletions of DmDNA Lig I were analyzed for their ability to accumulate at RF in Drosophila and mammalian cells. Surprisingly, no accumulation at RF was observed in Drosophila cells, while in mammalian cells DmDNA Lig I accumulated at RF via its PBD. Further, GFP fusions with the PBD domains from Dnmt1, HsDNA Lig I and DmDNA Lig I, were able to target to RF only in mammalian cells but not in Drosophila cells. Conclusion We show that S phase in Drosophila cells is characterized by formation of RF marked by PCNA like in mammalian cells. However, other than PCNA none of the replication proteins and their deletions tested here showed accumulation at RF in Drosophila cells while the same proteins and deletions are capable of accumulating at RF in mammalian cells. We hypothesize that unlike mammalian cells, in Drosophila cells, replication proteins do not form long-lasting interactions with the replication machinery, and rather perform their functions via very

Characterization of a Smad motif similar to Drosophila mad in the mouse Msx 1 promoter.

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Alvarez Martinez, Cristina E; Binato, Renata; Gonzalez, Sayonara; Pereira, Monica; Robert, Benoit; Abdelhay, Eliana

2002-03-01

Mouse Msx 1 gene, orthologous of the Drosophila msh, is involved in several developmental processes. BMP family members are major proteins in the regulation of Msx 1 expression. BMP signaling activates Smad 1/5/8 proteins, which associate to Smad 4 before translocating to the nucleus. Analysis of Msx 1 promoter revealed the presence of three elements similar to the consensus established for Mad, the Smad 1 Drosophila counterpart. Notably, such an element was identified in an enhancer important for Msx 1 regulation. Gel shift analysis demonstrated that proteins from 13.5 dpc embryo associate to this enhancer. Remarkably, supershift assays showed that Smad proteins are present in the complex. Purified Smad 1 and 4 also bind to this fragment. We demonstrate that functional binding sites in this enhancer are confined to the Mad motif and flanking region. Our data suggest that this Mad motif may be functional in response to BMP signaling. ©2002 Elsevier Science (USA).

The Drosophila cell adhesion molecule Neuroglian regulates Lissencephaly-1 localisation in circulating immunosurveillance cells

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Williams Michael J

2009-03-01

Full Text Available Abstract Background When the parasitoid wasp Leptopilina boulardi lays its eggs in Drosophila larvae phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. This requires these circulating immunosurveillance cells (haemocytes to change from a non-adhesive to an adhesive state enabling them to bind to the invader. Interestingly, attachment of leukocytes, platelets, and insect haemocytes requires the same adhesion complexes as epithelial and neuronal cells. Results Here evidence is presented showing that the Drosophila L1-type cell adhesion molecule Neuroglian (Nrg is required for haemocytes to encapsulate L. boulardi wasp eggs. The amino acid sequence FIGQY containing a conserved phosphorylated tyrosine is found in the intracellular domain of all L1-type cell adhesion molecules. This conserved tyrosine is phosphorylated at the cell periphery of plasmatocytes and lamellocytes prior to parasitisation, but dephosphorylated after immune activation. Intriguingly, another pool of Nrg located near the nucleus of plasmatocytes remains phosphorylated after parasitisation. In mammalian neuronal cells phosphorylated neurofascin, another L1-type cell adhesion molecule interacts with a nucleokinesis complex containing the microtubule binding protein lissencephaly-1 (Lis1 1. Interestingly in plasmatocytes from Nrg mutants the nucleokinesis regulating protein Lissencephaly-1 (Lis1 fails to localise properly around the nucleus and is instead found diffuse throughout the cytoplasm and at unidentified perinuclear structures. After attaching to the wasp egg control plasmatocytes extend filopodia laterally from their cell periphery; as well as extending lateral filopodia plasmatocytes from Nrg mutants also extend many filopodia from their apical surface. Conclusion The Drosophila cellular adhesion molecule Neuroglian is expressed in haemocytes and its activity is required for the encapsulation of L. boularli eggs. At

Primary structure and functional characterization of a Drosophila dopamine receptor with high homology to human D1/5 receptors.

Science.gov (United States)

Gotzes, F; Balfanz, S; Baumann, A

1994-01-01

Members of the superfamily of G-protein coupled receptors share significant similarities in sequence and transmembrane architecture. We have isolated a Drosophila homologue of the mammalian dopamine receptor family using a low stringency hybridization approach. The deduced amino acid sequence is approximately 70% homologous to the human D1/D5 receptors. When expressed in HEK 293 cells, the Drosophila receptor stimulates cAMP production in response to dopamine application. This effect was mimicked by SKF 38393, a specific D1 receptor agonist, but inhibited by dopaminergic antagonists such as butaclamol and flupentixol. In situ hybridization revealed that the Drosophila dopamine receptor is highly expressed in the somata of the optic lobes. This suggests that the receptor might be involved in the processing of visual information and/or visual learning in invertebrates.

Caspase-9 has a nonapoptotic function in Xenopus embryonic primitive blood formation.

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Tran, Hong Thi; Fransen, Mathias; Dimitrakopoulou, Dionysia; Van Imschoot, Griet; Willemarck, Nicolas; Vleminckx, Kris

2017-07-15

Caspases constitute a family of cysteine proteases centrally involved in programmed cell death, which is an integral part of normal embryonic and fetal development. However, it has become clear that specific caspases also have functions independent of cell death. In order to identify novel apoptotic and nonapoptotic developmental caspase functions, we designed and transgenically integrated novel fluorescent caspase reporter constructs in developing Xenopus embryos and tadpoles. This model organism has an external development, allowing direct and continuous monitoring. These studies uncovered a nonapoptotic role for the initiator caspase-9 in primitive blood formation. Functional experiments further corroborated that caspase-9, but possibly not the executioners caspase-3 and caspase-7, are required for primitive erythropoiesis in the early embryo. These data reveal a novel nonapoptotic function for the initiator caspase-9 and, for the first time, implicate nonapoptotic caspase activity in primitive blood formation. © 2017. Published by The Company of Biologists Ltd.

Staufen- and FMRP-Containing Neuronal RNPs Are Structurally and Functionally Related to Somatic P Bodies

OpenAIRE

Barbee, Scott A.; Estes, Patricia S.; Cziko, Anne-Marie; Hillebrand, Jens; Luedeman, Rene A.; Coller, Jeff M.; Johnson, Nick; Howlett, Iris C.; Geng, Cuiyun; Ueda, Ryu; Brand, Andrea H.; Newbury, Sarah F.; Wilhelm, James E.; Levine, Richard B.; Nakamura, Akira

2006-01-01

Local control of mRNA translation modulates neuronal development, synaptic plasticity, and memory formation. A poorly understood aspect of this control is the role and composition of ribonucleoprotein (RNP) particles that mediate transport and translation of neuronal RNAs. Here, we show that staufen- and FMRPcontaining RNPs in Drosophila neurons contain proteins also present in somatic “P bodies,” including the RNA-degradative enzymes Dcp1p and Xrn1p/Pacman and crucial components of miRNA (ar...

Biological effects of radon in Drosophila

International Nuclear Information System (INIS)

Pimentel P, A.E.; Tavera D, L.; Cruces M, M.P.; Arceo M, C.; Rosa D, M.E. de la

1992-04-01

The main objective of this investigation, is to study the biological effects of the Radon-222 at low dose in 'Drosophila melanogaster'. It is necessary to mention that these effects will analyze from the genetic point of view for: 1) To evaluate in which form the Radon-222 to low dose it influences in some genetic components of the adaptation in Drosophila, such as: fecundity, viability egg-adult and sex proportion. 2) To evaluate which is the genetic effect that induces the Radon to low dose by means of the SMART technique in Drosophila melanogaster, and this way to try of to identify which is the possible mechanism that causes the genetic damage to somatic level. The carried out investigation was divided in three stages: 1. Tests to the vacuum resistance. 2. Test of somatic mutation, and 3. Determination of the presence of radon daughters on the adult of Drosophila. It is necessary to point out that all the experiments were made by triplicate and in each one of them was placed detectors in preset places. Those obtained results are presented inside the 4 charts included in the present work. (Author)

PINK1 is required for timely cell-type specific mitochondrial clearance during Drosophila midgut metamorphosis.

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Liu, Yan; Lin, Jingjing; Zhang, Minjie; Chen, Kai; Yang, Shengxi; Wang, Qun; Yang, Hongqin; Xie, Shusen; Zhou, Yongjian; Zhang, Xi; Chen, Fei; Yang, Yufeng

2016-11-15

Mitophagy is the selective degradation of mitochondria by autophagy, which is an important mitochondrial quality and quantity control process. During Drosophila metamorphosis, the degradation of midgut involves a large change in length and organization, which is mediated by autophagy. Here we noticed a cell-type specific mitochondrial clearance process that occurs in enterocytes (ECs), while most mitochondria remain in intestinal stem cells (ISCs) during metamorphosis. Although PINK1/PARKIN represent the canonical pathway for the elimination of impaired mitochondria in varied pathological conditions, their roles in developmental processes or normal physiological conditions have been less studied. To examine the potential contribution of PINK1 in developmental processes, we monitored the dynamic expression pattern of PINK1 in the midgut development by taking advantage of a newly CRISPR/Cas9 generated knock-in fly strain expressing PINK1-mCherry fusion protein that presumably recapitulates the endogenous expression pattern of PINK1. We disclosed a spatiotemporal correlation between the expression pattern of PINK1 and the mitochondrial clearance or persistence in ECs or ISCs respectively. By mosaic genetic analysis, we then demonstrated that PINK1 and PARKIN function epistatically to mediate the specific timely removal of mitochondria, and are involved in global autophagy in ECs during Drosophila midgut metamorphosis, with kinase-dead PINK1 exerting dominant negative effects. Taken together, our studies concluded that the PINK1/PARKIN is crucial for timely cell-type specific mitophagy under physiological conditions and demonstrated again that Drosophila midgut metamorphosis might serve as an elegant in vivo model to study autophagy. Copyright © 2016 Elsevier Inc. All rights reserved.

Gut-associated microbes of Drosophila melanogaster

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Broderick, Nichole; Lemaitre, Bruno

2012-01-01

There is growing interest in using Drosophila melanogaster to elucidate mechanisms that underlie the complex relationships between a host and its microbiota. In addition to the many genetic resources and tools Drosophila provides, its associated microbiota is relatively simple (1–30 taxa), in contrast to the complex diversity associated with vertebrates (> 500 taxa). These attributes highlight the potential of this system to dissect the complex cellular and molecular interactions that occur between a host and its microbiota. In this review, we summarize what is known regarding the composition of gut-associated microbes of Drosophila and their impact on host physiology. We also discuss these interactions in the context of their natural history and ecology and describe some recent insights into mechanisms by which Drosophila and its gut microbiota interact. “Workers with Drosophila have been considered fortunate in that they deal with the first multicellular invertebrate to be cultured monoxenically (Delcourt and Guyenot, 1910); the first to be handled axenically on a semisynthetic diet (Guyenot, 1917); and the first to be grown on a defined diet (Schultz et al., 1946). This list of advantages is somewhat embarrassing, since it implies an interest in nutrition that, in reality, was only secondary. The very first studies were concerned with the reduction of variability in genetic experiments (Delcourt and Guyenot, 1910) and standardization of the nutritional environment.” -James Sang, 1959 Ann NY Acad 1 PMID:22572876

Osteogenic Potential of Caspases Related to Endochondral Ossification

Czech Academy of Sciences Publication Activity Database

Janečková, E.; Bíliková, P.; Matalová, Eva

2018-01-01

Roč. 66, č. 1 (2018), s. 47-58 ISSN 0022-1554 Institutional support: RVO:67985904 Keywords : caspases * endochronal ossification * growth plate Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Developmental biology Impact factor: 2.511, year: 2016

Application of response surface methodology (RSM) for optimisation of COD, NH3-N and 2,4-DCP removal from recycled paper wastewater in a pilot-scale granular activated carbon sequencing batch biofilm reactor (GAC-SBBR).

Science.gov (United States)

Muhamad, Mohd Hafizuddin; Sheikh Abdullah, Siti Rozaimah; Mohamad, Abu Bakar; Abdul Rahman, Rakmi; Hasan Kadhum, Abdul Amir

2013-05-30

In this study, the potential of a pilot-scale granular activated carbon sequencing batch biofilm reactor (GAC-SBBR) for removing chemical oxygen demand (COD), ammoniacal nitrogen (NH3-N) and 2,4-dichlorophenol (2,4-DCP) from recycled paper wastewater was assessed. For this purpose, the response surface methodology (RSM) was employed, using a central composite face-centred design (CCFD), to optimise three of the most important operating variables, i.e., hydraulic retention time (HRT), aeration rate (AR) and influent feed concentration (IFC), in the pilot-scale GAC-SBBR process for recycled paper wastewater treatment. Quadratic models were developed for the response variables, i.e., COD, NH3-N and 2,4-DCP removal, based on the high value (>0.9) of the coefficient of determination (R(2)) obtained from the analysis of variance (ANOVA). The optimal conditions were established at 750 mg COD/L IFC, 3.2 m(3)/min AR and 1 day HRT, corresponding to predicted COD, NH3-N and 2,4-DCP removal percentages of 94.8, 100 and 80.9%, respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

The GDP-switched GAF domain of DcpA modulates the concerted synthesis/hydrolysis of c-di-GMP in Mycobacterium smegmatis.

Science.gov (United States)

Chen, Hui-Jie; Li, Na; Luo, Ye; Jiang, Yong-Liang; Zhou, Cong-Zhao; Chen, Yuxing; Li, Qiong

2018-04-09

The second messenger c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] plays a key role in bacterial growth, survival and pathogenesis, and thus its intracellular homeostasis should be finely maintained. Mycobacterium smegmatis encodes a GAF (mammalian c G MP-regulated phosphodiesterases, Anabaena a denylyl cyclases and Escherichia coli transcription activator F hlA) domain containing bifunctional enzyme DcpA ( d iguanylate c yclase and p hosphodiesterase A ) that catalyzes the synthesis and hydrolysis of c-di-GMP . Here, we found that M. smegmatis DcpA catalyzes the hydrolysis of c-di-GMP at a higher velocity, compared with synthetic activity, resulting in a sum reaction from the ultimate substrate GTP to the final product pGpG [5'-phosphoguanylyl-(3'-5')-guanosine]. Fusion with the N-terminal GAF domain enables the GGDEF (Gly-Gly-Asp-Glu-Phe) domain of DcpA to dimerize and accordingly gain synthetic activity. Screening of putative metabolites revealed that GDP is the ligand of the GAF domain. Binding of GDP to the GAF domain down-regulates synthetic activity, but up-regulates hydrolytic activity, which, in consequence, might enable a timely response to the transient accumulation of c-di-GMP at the stationary phase or under stresses. Combined with the crystal structure of the EAL (Glu-Ala-Leu) domain and the small-angle X-ray scattering data, we propose a putative regulatory model of the GAF domain finely tuned by the intracellular GTP/GDP ratio. These findings help us to better understand the concerted control of the synthesis and hydrolysis of c-di-GMP in M. smegmatis in various microenvironments. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Feedback regulation of mitochondria by caspase-9 in the B cell receptor-mediated apoptosis.

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Eeva, J; Nuutinen, U; Ropponen, A; Mättö, M; Eray, M; Pellinen, R; Wahlfors, J; Pelkonen, J

2009-12-01

During the germinal centre reaction (GC), B cells with non-functional or self-reactive antigen receptors are negatively selected by apoptosis to generate B cell repertoire with appropriate antigen specificities. We studied the molecular mechanism of Fas/CD95- and B cell receptor (BCR)-induced apoptosis to shed light on the signalling events involved in the negative selection of GC B cells. As an experimental model, we used human follicular lymphoma (FL) cell line HF1A3, which originates from a GC B cell, and transfected HF1A3 cell lines overexpressing Bcl-x(L), c-FLIP(long) or dominant negative (DN) caspase-9. Fas-induced apoptosis was dependent on the caspase-8 activation, since the overexpression of c-FLIP(long), a natural inhibitor of caspase-8 activation, blocked apoptosis induced by Fas. In contrast, caspase-9 activation was not involved in Fas-induced apoptosis. BCR-induced apoptosis showed the typical characteristics of mitochondria-dependent (intrinsic) apoptosis. Firstly, the activation of caspase-9 was involved in BCR-induced DNA fragmentation, while caspase-8 showed only marginal role. Secondly, overexpression of Bcl-x(L) could block all apoptotic changes induced by BCR. As a novel finding, we demonstrate that caspase-9 can enhance the cytochrome-c release and collapse of mitochondrial membrane potential (DeltaPsi(m)) during BCR-induced apoptosis. The requirement of different signalling pathways in apoptosis induced by BCR and Fas may be relevant, since Fas- and BCR-induced apoptosis can thus be regulated independently, and targeted to different subsets of GC B cells.

An Org-1-Tup transcriptional cascade reveals different types of alary muscles connecting internal organs in Drosophila.

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Boukhatmi, Hadi; Schaub, Christoph; Bataillé, Laetitia; Reim, Ingolf; Frendo, Jean-Louis; Frasch, Manfred; Vincent, Alain

2014-10-01

The T-box transcription factor Tbx1 and the LIM-homeodomain transcription factor Islet1 are key components in regulatory circuits that generate myogenic and cardiogenic lineage diversity in chordates. We show here that Org-1 and Tup, the Drosophila orthologs of Tbx1 and Islet1, are co-expressed and required for formation of the heart-associated alary muscles (AMs) in the abdomen. The same holds true for lineage-related muscles in the thorax that have not been described previously, which we name thoracic alary-related muscles (TARMs). Lineage analyses identified the progenitor cell for each AM and TARM. Three-dimensional high-resolution analyses indicate that AMs and TARMs connect the exoskeleton to the aorta/heart and to different regions of the midgut, respectively, and surround-specific tracheal branches, pointing to an architectural role in the internal anatomy of the larva. Org-1 controls tup expression in the AM/TARM lineage by direct binding to two regulatory sites within an AM/TARM-specific cis-regulatory module, tupAME. The contributions of Org-1 and Tup to the specification of Drosophila AMs and TARMs provide new insights into the transcriptional control of Drosophila larval muscle diversification and highlight new parallels with gene regulatory networks involved in the specification of cardiopharyngeal mesodermal derivatives in chordates. © 2014. Published by The Company of Biologists Ltd.

Investigating the effects of nanoparticles on reproduction and development in Drosophila melanogaster and CD-1 mice

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Philbrook, Nicola Anne

Manufactured nanoparticles (NPs) are a class of small (≤ 100 nm) materials that are being used for a variety of purposes, including industrial lubricants, food additives, antibacterial agents, as well as delivery systems for drug and gene therapies. Their unique characteristics due to their small size as well as their parent materials allow them to be exploited in convenience applications; however, some of these properties also allow them to interact with and invade biological systems. Few studies have been performed to determine the potential harm that NPs can inflict on reproductive and developmental processes in organisms. In this study, Drosophila melanogaster and CD-1 mice were orally exposed to varying doses of titanium dioxide (TiO 2) NPs, silver (Ag) NPs, or hydroxyl-functionalized carbon nanotubes (fCNTs) and Drosophila were also exposed to microparticles (MPs) as a control for particle size. The subsequent effect of these materials on reproduction and development were evaluated. Strikingly, each type of NP studied negatively affected either reproduction or development in one or both of the two model systems. TiO2 NPs significantly negative effected both CD-1 mouse development (100 mg/kg or 1000 mg/kg) as well as Drosophila female fecundity (0.005%-0.5% w/v). Ag NPs significantly reduced mouse fetus viability after prenatal exposure to10 mg/kg. Ag NPs also significantly decreased the developmental success of Drosophila when they were directly exposed to these NPs (0.05% - 0.5% w/v) compared to both the vehicle and MP controls. fCNTs significantly increased the presence of morphological defects, resorptions and skeletal abnormalities in CD-1 mice, but had little effect on Drosophila. We speculate that the differences seen in the effects of NP types may be partially due to differences in reproductive physiology as well as each organism's ability to internalize these NPs. Whereas the differing response of each organism to a NP type was likely due in part to

Inner ear dysfunction in caspase-3 deficient mice

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Woo Minna

2011-10-01

Full Text Available Abstract Background Caspase-3 is one of the most downstream enzymes activated in the apoptotic pathway. In caspase-3 deficient mice, loss of cochlear hair cells and spiral ganglion cells coincide closely with hearing loss. In contrast with the auditory system, details of the vestibular phenotype have not been characterized. Here we report the vestibular phenotype and inner ear anatomy in the caspase-3 deficient (Casp3-/- mouse strain. Results Average ABR thresholds of Casp3-/- mice were significantly elevated (P Casp3+/- mice and Casp3+/+ mice at 3 months of age. In DPOAE testing, distortion product 2F1-F2 was significantly decreased (P Casp3-/- mice, whereas Casp3+/- and Casp3+/+ mice showed normal and comparable values to each other. Casp3-/- mice were hyperactive and exhibited circling behavior when excited. In lateral canal VOR testing, Casp3-/- mice had minimal response to any of the stimuli tested, whereas Casp3+/- mice had an intermediate response compared to Casp3+/+ mice. Inner ear anatomical and histological analysis revealed gross hypomorphism of the vestibular organs, in which the main site was the anterior semicircular canal. Hair cell numbers in the anterior- and lateral crista, and utricle were significantly smaller in Casp3-/- mice whereas the Casp3+/- and Casp3+/+ mice had normal hair cell numbers. Conclusions These results indicate that caspase-3 is essential for correct functioning of the cochlea as well as normal development and function of the vestibule.

Apoptotic block in colon cancer cells may be rectified by lentivirus mediated overexpression of caspase-9.

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Xu, D; Wang, C; Shen, X; Yu, Y; Rui, Y; Zhang, D; Zhou, Z

2013-12-01

At present, the inhibition of apoptosis during pathogenesis of colorectal cancer is widely recognized while the role of caspase-9 in this process remains controversial. We aimed to investigate the differential expression of caspase-9 and evaluate the therapeutic potential of expression intervention in this study. We first examined the different expression of caspase-9 in normal colon mucosa, adenoma and cancer, investigating the relationship between its expression and clinico-pathological characteristics. Secondly, overexpression of caspase-9 was established in colon cancer cell lines by lentivirus infection to study the changes in growth, proliferation and apoptosis. Compared with normal colon mucosa, the expression of caspase-9 was higher in adenoma while lower in cancer both at mRNA and protein level (P expression is more common in poorly differentiated cancers (P expression of caspase-9, poorer colony formation and slower cell proliferation. In terms of apoptosis related indicators, caspase-9 overexpression leads to higher apoptosis rate and GO/G1 arrest, while up-regulating the expression of caspase-3 (P expression from colon mucosa, adenoma to cancer suggested it may be involved in the carcinogenesis of colon cancer. The overexpression of caspase-9 exhibits an inhibitory role in cancer growth and proliferation while promoting apoptosis. However, a non-apoptotic role of caspase-9 facilitating differentiation was also implied.

Members of the bcl-2 and caspase families regulate nuclear degeneration during chick lens fibre differentiation.

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Wride, M A; Parker, E; Sanders, E J

1999-09-01

The optical clarity of the lens is ensured by the programmed removal of nuclei and other organelles from the lens fibre cells during development. The morphology of the degenerating nuclei is similar to that observed during apoptosis and is accompanied by DNA fragmentation. Proteins encoded by the bcl-2 proto-oncogene family are important in either promoting or inhibiting apoptosis, and caspases are involved in downstream proteolytic events. Here, the expression of bcl-2 family members (bcl-2, bax, bad, and bcl-x(s/l)) and caspases-1, -2, -3, -4, and -6 was investigated through a range of stages of chick lens development using immunocytochemistry, Western blotting, and affinity labelling for caspases using biotinylated caspase inhibitors. Using differentiating lens epithelial cell cultures, it was demonstrated that the addition to cultures of synthetic peptide inhibitors of caspases -1, -2, -4, -6, and -9 brought about a 50-70% reduction in the number of degenerating nuclei per unit area of culture, as assessed by image analysis. These effects were comparable to those seen when general inhibitors of caspases were added to cultures. On the other hand, inhibitors of caspases-3 and -8 were not effective in significantly reducing the number of TUNEL-labelled nuclei. Expression of the caspase substrates poly(ADP-ribose) polymerase (PARP) and the 45-kDa subunit of DNA fragmentation factor (DFF 45) was also observed in the developing lens. Western blots of cultures to which caspase inhibitors were added revealed alterations in the PARP cleavage pattern, but not in that of DFF. These results demonstrate a role for members of the bcl-2 family and caspases in the degeneration of lens fibre cell nuclei during chick secondary lens fibre development and support the proposal that this process has many characteristics in common with apoptosis. Copyright 1999 Academic Press.

Mejoramiento de las propiedades de tensión en WPC de LDPE: HIPS/fibra natural mediante entrecruzamiento con DCP

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Arnoldo Emilio Delgado Tobón

2014-06-01

Full Text Available Fibra de Retamo Liso (telinemonspessulana fue mezclada en una matriz polimérica formada por: polietileno de baja densidad (LDPE y poliestireno de alto impacto (HIPS en relación 70:30 respectivamente. La fibra natural fue sometida a reducción de tamaño entre 1700 y 850 µm; se le realizó tratamiento superficial de mercerización; el contenido de humedad fue 0,17%. El análisis termo gravimétrico (TGA muestra que la fibra tratada tiene estabilidad de 200,53 °C. La elaboración del compuesto se realizó en mezclador interno y la técnica de obtención de las probetas fue mediante moldeo por compresión. Se evaluó el efecto del agente entrecruzante peróxido de dicumilo (DCP en compuestos con varios porcentajes de fibra: 0; 10; 25; 50 phr. La adición de DCP (1 phr en los compuestos, mejora significativamente las propiedades de tensión con respecto a similares concentraciones de fibra; sin embargo, esta diferencia en los valores de las propiedades disminuye progresivamente cuando la cantidad de fibra se aproxima a 50 phr. El aumento en la concentración de fibra afecta negativamente las propiedades de tensión, con excepción del Módulo de Elasticidad y la Resistencia a la Fluencia. La causa principal es originada por la baja adhesión interfacial fibra natural-matriz polimérica, claramente evidenciada en las imágenes SEM.

Caspase-2 associates with FAN through direct interaction and overlapping functionality.

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Forsberg, Jeremy; Li, Xinge; Zamaraev, Aleksey V; Panaretakis, Theocharis; Zhivotovsky, Boris; Olsson, Magnus

2018-05-23

Caspase-2 has been implicated in diverse cellular processes, and the identification of factors with which it interacts has steadily increased. In the present study, we report a direct interaction between caspase-2 and factor associated with neutral sphingomyelinase activation (FAN) using yeast two-hybrid screening and co-immunoprecipitation. Further, stable suppression of caspase-2 expression in HEK293T and HeLa cells enabled a systematic investigation of putative novel enzyme functionalities, especially with respect to ceramide production, cell migration, IL-6 production and vesicular homeostasis, all of which have been previously reported to be associated with FAN. Lipidomics excluded the involvement of caspase-2 in the generation of ceramide species, but caspase-2-dependent deregulation of IL-6 release, vesicular size and delayed cell relocation supported an association between caspase-2 and FAN. Collectively, these data identify a novel caspase-2-interacting factor, FAN, and expand the role for the enzyme in seemingly non-apoptotic cellular mechanisms. Copyright © 2018 Elsevier Inc. All rights reserved.

Inhibition of CUG-binding protein 1 and activation of caspases are critically involved in piperazine derivative BK10007S induced apoptosis in hepatocellular carcinoma cells.

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Ju-Ha Kim

Full Text Available Though piperazine derivative BK10007S was known to induce apoptosis in pancreatic cancer xenograft model as a T-type CaV3.1 a1G isoform calcium channel blocker, its underlying antitumor mechanism still remains unclear so far. Thus, in the present study, the antitumor mechanism of BK10007S was elucidated in hepatocellular carcinoma cells (HCCs. Herein, BK10007S showed significant cytotoxicity by 3-[4,5-2-yl]-2,5-diphenyltetra-zolium bromide (MTT assay and anti-proliferative effects by colony formation assay in HepG2 and SK-Hep1 cells. Also, apoptotic bodies and terminal deoxynucleotidyl transferase (TdT dUTP Nick End Labeling (TUNEL positive cells were observed in BK10007S treated HepG2 and SK-Hep1 cells by 4',6-diamidino-2-phenylinodole (DAPI staining and TUNEL assay, respectively. Consistently, BK10007S increased sub G1 population in HepG2 and SK-Hep1 cells by cell cycle analysis. Furthermore, Western blotting revealed that BK10007S activated the caspase cascades (caspase 8, 9 and 3, cleaved poly (ADP-ribose polymerase (PARP, and downregulated the expression of cyclin D1, survivin and for CUG-binding protein 1 (CUGBP1 or CELF1 in HepG2 and SK-Hep1 cells. Conversely, overexpression of CUGBP1 reduced cleavages of PARP and caspase 3, cytotoxicity and subG1 population in BK10007S treated HepG2 cells. Overall, these findings provide scientific evidences that BK10007S induces apoptosis via inhibition of CUGBP1 and activation of caspases in hepatocellular carcinomas as a potent anticancer candidate.

LDR reverses DDP resistance in ovarian cancer cells by affecting ERCC-1, Bcl-2, Survivin and Caspase-3 expressions.

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Ju, Xingyan; Yu, Hongsheng; Liang, Donghai; Jiang, Tao; Liu, Yuanwei; Chen, Ling; Dong, Qing; Liu, Xiaoran

2018-06-01

Ovarian cancer is the most frequent cause of death resulting from malignant gynecological tumors. After surgical intervention, cisplatin (DDP) is a major chemotherapy drug for ovarian cancer, but the ovarian cancer cells tend to develop DDP resistance in the clinical setting. Tumor cells are sensitive to low-dose radiation (LDR). However, how the LDR therapy improves the effects of chemotherapy drugs on ovarian cancer is not well understood. This study aimed to explore this issue. The SKOV3/DDP cells were divided into 3 groups, including low-dose group, conventional-dose group, and control group (no radiation). Cell counting kit-8 assay was performed to measure cell proliferation. Flow cytometric analysis was then utilized to quantify the apoptosis with classical Annexin V/propidium iodide co-staining. And Real-time quantitative PCR and western blot were eventually used to analyze the mRNA and protein levels of excision repair cross complementing-group 1 (ERCC1), B-cell lymphoma 2 (Bcl-2), Survivin and Caspase-3, respectively. The IC50 value of DDP in the low-dose group was significantly lower compared with the other two groups. Compared with the conventional-dose group and control group, LDR treatment resulted in significantly more apoptosis. Besides, LDR treatment significantly decreased the mRNA and protein expression of ERCC1, Bcl-2, and Survivin, and enhanced the mRNA and protein expression of Caspase-3 compared with the other two groups. LDR reversed DDP resistance in SKOV3/DDP cells possibly by suppressing ERCC1, Bcl-2, and Survivin expressions, and increasing Caspase-3 expression. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Caspase-3 activation as a bifurcation point between plasticity and cell death

Institute of Scientific and Technical Information of China (English)

Shikha Snigdha; Erica D Smith; G Aleph Prieto; Carl W Cotman

2012-01-01

Death-mediating proteases such as caspases and caspase-3 in particular,have been implicated in neurodegenerative processes,aging and Alzheimer's disease.However,emerging evidence suggests that in addition to their classical role in cell death,caspases play a key role in modulating synaptic function.It is remarkable that active caspases-3,which can trigger widespread damage and degeneration,aggregates in structures as delicate as synapses and persists in neurons without causing acute cell death.Here,we evaluate this dichotomy,and discuss the hypothesis that caspase-3 may be a bifurcation point in cellular signaling,able to orient the neuronal response to stress down either pathological/apoptotic pathways or towards physiological cellular remodeling.We propose that temporal,spatial and other regulators of caspase activity are key determinants of the ultimate effect of caspase-3 activation in neurons.This concept has implications for differential roles of caspase-3 activation across the lifespan.Specifically,we propose that limited caspase-3 activation is critical for synaptic function in the healthy adult brain while chronic activation is involved in degenerative processes in the aging brain.

Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl-1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation.

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Ge, Yan; Yoshiie, Kiyotaka; Kuribayashi, Futoshi; Lin, Mingqun; Rikihisa, Yasuko

2005-01-01

The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signalling pathways through which the obligatory intracellular bacterium Anaplasma phagocytophilum inhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated. bfl-1 mRNA levels in uninfected neutrophils after 12 h in culture were reduced to approximately 5-25% of 0 h levels, but remained high in infected neutrophils. The eukaryotic RNA synthesis inhibitor, actinomycin D, prevented the maintenance of bfl-1 mRNA levels by A. phagocytophilum. Differences in mcl-1, bax, bcl-w, bad or bak mRNA levels in infected versus uninfected neutrophils were not remarkable. By using mitochondrial fluorescent dyes, Mitotracker Red and JC-1, it was found that most uninfected neutrophils lost mitochondrial membrane potential after 10-12 h incubation, whereas A. phagocytophilum-infected neutrophils maintained high membrane potential. Caspase 3 activity and the degree of apoptosis were lower in dose-dependent manner in A. phagocytophilum-infected neutrophils at 16 h post infection, as compared to uninfected neutrophils. Anti-active caspase 3 antibody labelling showed less positively stained population in infected neutrophils compared to those in uninfected neutrophils after 12 h incubation. These results suggest that A. phagocytophilum inhibits human neutrophil apoptosis via transcriptional upregulation of bfl-1 and inhibition of mitochondria-mediated activation of caspase 3.

Modeling Human Cancers in Drosophila.

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Sonoshita, M; Cagan, R L

2017-01-01

Cancer is a complex disease that affects multiple organs. Whole-body animal models provide important insights into oncology that can lead to clinical impact. Here, we review novel concepts that Drosophila studies have established for cancer biology, drug discovery, and patient therapy. Genetic studies using Drosophila have explored the roles of oncogenes and tumor-suppressor genes that when dysregulated promote cancer formation, making Drosophila a useful model to study multiple aspects of transformation. Not limited to mechanism analyses, Drosophila has recently been showing its value in facilitating drug development. Flies offer rapid, efficient platforms by which novel classes of drugs can be identified as candidate anticancer leads. Further, we discuss the use of Drosophila as a platform to develop therapies for individual patients by modeling the tumor's genetic complexity. Drosophila provides both a classical and a novel tool to identify new therapeutics, complementing other more traditional cancer tools. © 2017 Elsevier Inc. All rights reserved.

Semi-automated quantitative Drosophila wings measurements.

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Loh, Sheng Yang Michael; Ogawa, Yoshitaka; Kawana, Sara; Tamura, Koichiro; Lee, Hwee Kuan

2017-06-28

Drosophila melanogaster is an important organism used in many fields of biological research such as genetics and developmental biology. Drosophila wings have been widely used to study the genetics of development, morphometrics and evolution. Therefore there is much interest in quantifying wing structures of Drosophila. Advancement in technology has increased the ease in which images of Drosophila can be acquired. However such studies have been limited by the slow and tedious process of acquiring phenotypic data. We have developed a system that automatically detects and measures key points and vein segments on a Drosophila wing. Key points are detected by performing image transformations and template matching on Drosophila wing images while vein segments are detected using an Active Contour algorithm. The accuracy of our key point detection was compared against key point annotations of users. We also performed key point detection using different training data sets of Drosophila wing images. We compared our software with an existing automated image analysis system for Drosophila wings and showed that our system performs better than the state of the art. Vein segments were manually measured and compared against the measurements obtained from our system. Our system was able to detect specific key points and vein segments from Drosophila wing images with high accuracy.

Differential splicing of the apoptosis-associated speck like protein containing a caspase recruitment domain (ASC regulates inflammasomes

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Rojanasakul Yon

2010-05-01

Full Text Available Abstract Background The apoptotic speck-like protein containing a caspase recruitment domain (ASC is the essential adaptor protein for caspase 1 mediated interleukin (IL-1β and IL-18 processing in inflammasomes. It bridges activated Nod like receptors (NLRs, which are a family of cytosolic pattern recognition receptors of the innate immune system, with caspase 1, resulting in caspase 1 activation and subsequent processing of caspase 1 substrates. Hence, macrophages from ASC deficient mice are impaired in their ability to produce bioactive IL-1β. Furthermore, we recently showed that ASC translocates from the nucleus to the cytosol in response to inflammatory stimulation in order to promote an inflammasome response, which triggers IL-1β processing and secretion. However, the precise regulation of inflammasomes at the level of ASC is still not completely understood. In this study we identified and characterized three novel ASC isoforms for their ability to function as an inflammasome adaptor. Methods To establish the ability of ASC and ASC isoforms as functional inflammasome adaptors, IL-1β processing and secretion was investigated by ELISA in inflammasome reconstitution assays, stable expression in THP-1 and J774A1 cells, and by restoring the lack of endogenous ASC in mouse RAW264.7 macrophages. In addition, the localization of ASC and ASC isoforms was determined by immunofluorescence staining. Results The three novel ASC isoforms, ASC-b, ASC-c and ASC-d display unique and distinct capabilities to each other and to full length ASC in respect to their function as an inflammasome adaptor, with one of the isoforms even showing an inhibitory effect. Consistently, only the activating isoforms of ASC, ASC and ASC-b, co-localized with NLRP3 and caspase 1, while the inhibitory isoform ASC-c, co-localized only with caspase 1, but not with NLRP3. ASC-d did not co-localize with NLRP3 or with caspase 1 and consistently lacked the ability to function as an

Caspases in retinal ganglion cell death and axon regeneration

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Thomas, Chloe N; Berry, Martin; Logan, Ann; Blanch, Richard J; Ahmed, Zubair

2017-01-01

Retinal ganglion cells (RGC) are terminally differentiated CNS neurons that possess limited endogenous regenerative capacity after injury and thus RGC death causes permanent visual loss. RGC die by caspase-dependent mechanisms, including apoptosis, during development, after ocular injury and in progressive degenerative diseases of the eye and optic nerve, such as glaucoma, anterior ischemic optic neuropathy, diabetic retinopathy and multiple sclerosis. Inhibition of caspases through genetic or pharmacological approaches can arrest the apoptotic cascade and protect a proportion of RGC. Novel findings have also highlighted a pyroptotic role of inflammatory caspases in RGC death. In this review, we discuss the molecular signalling mechanisms of apoptotic and inflammatory caspase responses in RGC specifically, their involvement in RGC degeneration and explore their potential as therapeutic targets. PMID:29675270

Neuronal Cbl Controls Biosynthesis of Insulin-Like Peptides in Drosophila melanogaster

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Yu, Yue; Sun, Ying; He, Shengqi; Yan, Cheng; Rui, Liangyou; Li, Wenjun; Liu, Yong

2012-01-01

The Cbl family proteins function as both E3 ubiquitin ligases and adaptor proteins to regulate various cellular signaling events, including the insulin/insulin-like growth factor 1 (IGF1) and epidermal growth factor (EGF) pathways. These pathways play essential roles in growth, development, metabolism, and survival. Here we show that in Drosophila melanogaster, Drosophila Cbl (dCbl) regulates longevity and carbohydrate metabolism through downregulating the production of Drosophila insulin-lik...

Impacts of Bone Marrow Stem Cells on Caspase-3 Levels after Spinal Cord Injury in Mice

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Noushin Gashmardi

2017-11-01

Full Text Available Spinal cord injury (SCI is a drastic disability that leads to spinal cord impairment. This study sought to determine the effects of bone marrow stem cells (BMSCs on caspase-3 levels after acute SCI in mice. Forty-two mice were randomly divided into 3 groups: control (2 subcategories, subjected to no intervention; sham (3 subcategories, subjected to acute SCI; and experimental (2 subcategories, subjected to SCI and cell transplantation. In the experimental group, 2×105 BMSCs were injected intravenously 1 day after SCI. The mesenchymal property of the cells was assessed. The animals in the 3 groups were sacrificed 1, 21, and 35 days after the induction of injury and caspase-3 levels were evaluated using a caspase-3 assay kit. The obtained values were analyzed with ANOVA and Tukey tests using GraphPad and SPSS. Based on the assessments, the transplanted cells were spindle-shaped and were negative for the hematopoietic markers of CD34 and CD45 and positive for the expression of the mesenchymal marker of CD90 and osteogenic induction. The caspase-3 levels showed a significant increase in the sham and experimental groups in comparison to the control group. One day after SCI, the caspase-3 level was significantly higher in the sham group (1.157±0.117 than in the other groups (P<0.000. Twenty-one days after SCI, the caspase-3 level was significantly lower in the experimental group than in the sham group (0.4±0.095 vs. 0.793±0.076; P˂0.000. Thirty-five days following SCI, the caspase-3 level was lower in the experimental group than in the sham group (0.223±0.027 vs. 0.643±0.058; P˂0.000. We conclude that BMSC transplantation was able to downregulate the caspase-3 level after acute SCI, underscoring the role of caspase-3 as a marker for the assessment of treatment efficacy in acute SCI.

Fate and origin of 1,2-dichloropropane in an unconfined shallow aquifer.

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Tesoriero, A J; Löffler, F E; Liebscher, H

2001-02-01

A shallow aquifer with different redox zones overlain by intensive agricultural activity was monitored for the occurrence of 1,2-dichloropropane (DCP) to assess the fate and origin of this pollutant. DCP was detected more frequently in groundwater samples collected in aerobic and nitrate-reducing zones than those collected from iron-reducing zones. Simulated DCP concentrations for groundwater entering an iron-reducing zone were calculated from a fate and transport model that included dispersion, sorption, and hydrolysis but not degradation. Simulated concentrations were well in excess of measured values, suggesting that microbial degradation occurred in the iron-reducing zone. Microcosm experiments were conducted using aquifer samples collected from iron-reducing and aerobic zones to evaluate the potential for microbial degradation of DCP and to explain field observations. Hydrogenolysis of DCP and production of monochlorinated propanes in microcosm experiments occurred only with aquifer materials collected from the iron-reducing zone, and no dechlorination was observed in microcosms established with aquifer materials collected from the aerobic zones. Careful analyses of the DCP/1,2,2-trichloropropane ratios in groundwater indicated that older fumigant formulations were responsible for the high levels of DCP present in this aquifer.

The death effector domains of caspase-8 induce terminal differentiation.

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Ainhoa Mielgo

2009-11-01

Full Text Available The differentiation and senescence programs of metazoans play key roles in regulating normal development and preventing aberrant cell proliferation, such as cancer. These programs are intimately associated with both the mitotic and apoptotic pathways. Caspase-8 is an apical apoptotic initiator that has recently been appreciated to coordinate non-apoptotic roles in the cell. Most of these functions are attributed to the catalytic domain, however, the amino-terminal death effector domains (DEDs, which belong to the death domain superfamily of proteins, can also play key roles during development. Here we describe a novel role for caspase-8 DEDs in regulating cell differentiation and senescence. Caspase-8 DEDs accumulate during terminal differentiation and senescence of epithelial, endothelial and myeloid cells; genetic deletion or shRNA suppression of caspase-8 disrupts cell differentiation, while re-expression of DEDs rescues this phenotype. Among caspase-8 deficient neuroblastoma cells, DED expression attenuated tumor growth in vivo and proliferation in vitro via disruption of mitosis and cytokinesis, resulting in upregulation of p53 and induction of differentiation markers. These events occur independent of caspase-8 catalytic activity, but require a critical lysine (K156 in a microtubule-binding motif in the second DED domain. The results demonstrate a new function for the DEDs of caspase-8, and describe an unexpected mechanism that contributes to cell differentiation and senescence.

Evolutionary changes in gene expression, coding sequence and copy-number at the Cyp6g1 locus contribute to resistance to multiple insecticides in Drosophila.

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Thomas W R Harrop

Full Text Available Widespread use of insecticides has led to insecticide resistance in many populations of insects. In some populations, resistance has evolved to multiple pesticides. In Drosophila melanogaster, resistance to multiple classes of insecticide is due to the overexpression of a single cytochrome P450 gene, Cyp6g1. Overexpression of Cyp6g1 appears to have evolved in parallel in Drosophila simulans, a sibling species of D. melanogaster, where it is also associated with insecticide resistance. However, it is not known whether the ability of the CYP6G1 enzyme to provide resistance to multiple insecticides evolved recently in D. melanogaster or if this function is present in all Drosophila species. Here we show that duplication of the Cyp6g1 gene occurred at least four times during the evolution of different Drosophila species, and the ability of CYP6G1 to confer resistance to multiple insecticides exists in D. melanogaster and D. simulans but not in Drosophila willistoni or Drosophila virilis. In D. virilis, which has multiple copies of Cyp6g1, one copy confers resistance to DDT and another to nitenpyram, suggesting that the divergence of protein sequence between copies subsequent to the duplication affected the activity of the enzyme. All orthologs tested conferred resistance to one or more insecticides, suggesting that CYP6G1 had the capacity to provide resistance to anthropogenic chemicals before they existed. Finally, we show that expression of Cyp6g1 in the Malpighian tubules, which contributes to DDT resistance in D. melanogaster, is specific to the D. melanogaster-D. simulans lineage. Our results suggest that a combination of gene duplication, regulatory changes and protein coding changes has taken place at the Cyp6g1 locus during evolution and this locus may play a role in providing resistance to different environmental toxins in different Drosophila species.

Peroxynitrite induces apoptosis of mouse cochlear hair cells via a Caspase-independent pathway in vitro.

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Cao, Zhixin; Yang, Qianqian; Yin, Haiyan; Qi, Qi; Li, Hongrui; Sun, Gaoying; Wang, Hongliang; Liu, Wenwen; Li, Jianfeng

2017-11-01

Peroxynitrite (ONOO - ) is a potent and versatile oxidant implicated in a number of pathophysiological processes. The present study was designed to investigate the effect of ONOO - on the cultured cochlear hair cells (HCs) of C57BL/6 mice in vitro as well as the possible mechanism underlying the action of such an oxidative stress. The in vitro primary cultured cochlear HCs were subjected to different concentrations of ONOO - , then, the cell survival and morphological changes were examined by immunofluorescence and transmission electron microscopy (TEM), the apoptosis was determined by Terminal deoxynucleotidyl transferase dUNT nick end labeling (TUNEL) assay, the mRNA expressions of Caspase-3, Caspase-8, Caspase-9, Apaf1, Bcl-2, and Bax were analyzed by RT-PCR, and the protein expressions of Caspase-3 and AIF were assessed by immunofluorescence. This work demonstrated that direct exposure of primary cultured cochlear HCs to ONOO - could result in a base-to-apex gradient injury of HCs in a concentration-dependent manner. Furthermore, ONOO - led to much more losses of outer hair cells than inner hair cells mainly through the induction of apoptosis of HCs as evidenced by TEM and TUNEL assays. The mRNA expressions of Caspase-8, Caspase-9, Apaf1, and Bax were increased and, meanwhile, the mRNA expression of Bcl-2 was decreased in response to ONOO - treatment. Of interesting, the expression of Caspase-3 had no significant change, whereas, the expression alteration of AIF was observed. These results suggested that ONOO - can effectively damage the survival of cochlear HCs via triggering the apoptotic pathway. The findings from this work suggest that ONOO - -induced apoptosis is mediated, at least in part, via a Caspase-independent pathway in cochlear HCs.

Drosophila's contribution to stem cell research [v1; ref status: indexed, http://f1000r.es/5h7

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Gyanesh Singh

2015-06-01

Full Text Available The discovery of Drosophila stem cells with striking similarities to mammalian stem cells has brought new hope for stem cell research. A recent development in Drosophila stem cell research is bringing wider opportunities for contemporary stem cell biologists. In this regard, Drosophila germ cells are becoming a popular model of stem cell research. In several cases, genes that controlled Drosophila stem cells were later discovered to have functional homologs in mammalian stem cells. Like mammals, Drosophila germline stem cells (GSCs are controlled by both intrinsic as well as external signals. Inside the Drosophila testes, germline and somatic stem cells form a cluster of cells (the hub. Hub cells depend on JAK-STAT signaling, and, in absence of this signal, they do not self-renew. In Drosophila, significant changes occur within the stem cell niche that contributes to a decline in stem cell number over time. In case of aging Drosophila, somatic niche cells show reduced DE-cadherin and unpaired (Upd proteins. Unpaired proteins are known to directly decrease stem cell number within the niches, and, overexpression of upd within niche cells restored GSCs in older males also . Stem cells in the midgut of Drosophila are also very promising. Reduced Notch signaling was found to increase the number of midgut progenitor cells. On the other hand, activation of the Notch pathway decreased proliferation of these cells. Further research in this area should lead to the discovery of additional factors that regulate stem and progenitor cells in Drosophila.

Substrate interactions in dehalogenation of 1,2-dichloroethane, 1,2-dichloropropane, and 1,1,2-trichloroethane mixtures by Dehalogenimonas spp.

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Dillehay, Jacob L; Bowman, Kimberly S; Yan, Jun; Rainey, Fred A; Moe, William M

2014-04-01

When chlorinated alkanes are present as soil or groundwater pollutants, they often occur in mixtures. This study evaluated substrate interactions during the anaerobic reductive dehalogenation of chlorinated alkanes by the type strains of two Dehalogenimonas species, D. lykanthroporepellens and D. alkenigignens. Four contaminant mixtures comprised of combinations of the chlorinated solvents 1,2-dichloroethane (1,2-DCA), 1,2-dichloropropane (1,2-DCP), and 1,1,2-trichloroethane (1,1,2-TCA) were assessed for each species. Chlorinated solvent depletion and daughter product formation determined as a function of time following inoculation into anaerobic media revealed preferential dechlorination of 1,1,2-TCA over both 1,2-DCA and 1,2-DCP for both species. 1,2-DCA in particular was not dechlorinated until 1,1,2-TCA reached low concentrations. In contrast, both species concurrently dechlorinated 1,2-DCA and 1,2-DCP over a comparably large concentration range. This is the first report of substrate interactions during chlorinated alkane dehalogenation by pure cultures, and the results provide insights into the chlorinated alkane transformation processes that may be expected for contaminant mixtures in environments where Dehalogenimonas spp. are present.

Metabolic Regulation of CaMKII Protein and Caspases in Xenopus laevis Egg Extracts*

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McCoy, Francis; Darbandi, Rashid; Chen, Si-Ing; Eckard, Laura; Dodd, Keela; Jones, Kelly; Baucum, Anthony J.; Gibbons, Jennifer A.; Lin, Sue-Hwa; Colbran, Roger J.; Nutt, Leta K.

2013-01-01

The metabolism of the Xenopus laevis egg provides a cell survival signal. We found previously that increased carbon flux from glucose-6-phosphate (G6P) through the pentose phosphate pathway in egg extracts maintains NADPH levels and calcium/calmodulin regulated protein kinase II (CaMKII) activity to phosphorylate caspase 2 and suppress cell death pathways. Here we show that the addition of G6P to oocyte extracts inhibits the dephosphorylation/inactivation of CaMKII bound to caspase 2 by protein phosphatase 1. Thus, G6P sustains the phosphorylation of caspase 2 by CaMKII at Ser-135, preventing the induction of caspase 2-mediated apoptotic pathways. These findings expand our understanding of oocyte biology and clarify mechanisms underlying the metabolic regulation of CaMKII and apoptosis. Furthermore, these findings suggest novel approaches to disrupt the suppressive effects of the abnormal metabolism on cell death pathways. PMID:23400775

Cytotoxicity of diacetoxyscirpenol is associated with apoptosis by activation of caspase-8 and interruption of cell cycle progression by down-regulation of cdk4 and cyclin B1 in human Jurkat T cells

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Jun, Do Youn; Kim, Jun Seok; Park, Hae Sun; Song, Woo Sun; Bae, Young Seuk; Kim, Young Ho

2007-01-01

To understand the mechanism underlying T-cell toxicity of diacetoxyscirpenol (DAS) from Fusarium sambucinum, its apoptogenic as well as growth retardation activity was investigated in human Jurkat T cells. Exposure to DAS (0.01-0.15 μM) caused apoptotic DNA fragmentation along with caspase-8 activation, Bid cleavage, mitochondrial cytochrome c release, activation of caspase-9 and caspase-3, and PARP degradation, without any alteration in the levels of Fas or FasL. Under these conditions, necrosis was not accompanied. The cytotoxicity of DAS was not blocked by the anti-Fas neutralizing antibody ZB-4. Although the DAS-induced apoptotic events were completely prevented by overexpression of Bcl-xL, the cells overexpressing Bcl-xL were unable to divide in the presence of DAS, resulting from the failure of cell cycle progression possibly due to down-regulation in the protein levels of cdk4 and cyclin B1. The DAS-mediated apoptosis and activation of caspase-8, -9, and -3 were abrogated by either pan-caspase inhibitor (z-VAD-fmk) or caspase-8 inhibitor (z-IETD-fmk). While the DAS-mediated apoptosis and activation of caspase-9 and caspase-3 were slightly suppressed by the mitochondrial permeability transition pore inhibitor (CsA), both caspase-8 activation and Bid cleavage were not affected by CsA. The activated normal peripheral T cells possessed a similar susceptibility to the cytotoxicity of DAS. These results demonstrate that the T-cell toxicity of DAS is attributable to not only apoptosis initiated by caspase-8 activation and subsequent mitochondrion-dependent or -independent activation of caspase cascades, which can be regulated by Bcl-xL, but also interruption of cell cycle progression caused by down-regulation of cdk4 and cyclin B1 proteins

Recognition and Detoxification of the Insecticide DDT by Drosophila melanogaster Glutathione S-Transferase D1

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Low, Wai Yee; Feil, Susanne C.; Ng, Hooi Ling; Gorman, Michael A.; Morton, Craig J.; Pyke, James; McConville, Malcolm J.; Bieri, Michael; Mok, Yee-Foong; Robin, Charles; Gooley, Paul R.; Parker, Michael W.; Batterham, Philip (SVIMR-A); (Melbourne)

2010-06-14

GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 {angstrom} resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model of the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional {sup 1}H,{sup 15}N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity.

BmDredd is an initiator caspase and participates in Emodin-induced apoptosis in the silkworm, Bombyx mori.

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Wang, La; Song, Juan; Bao, Xi-Yan; Chen, Peng; Yi, Hua-Shan; Pan, Min-Hui; Lu, Cheng

2016-10-15

The identification and analysis of the caspases is essential to research into apoptosis in lepidoptera insects. The domesticated silkworm, Bombyx mori, is the model system for lepidopterans. In this study, we cloned and characterized a B. mori Dredd gene, BmDredd, the proposed insect homologue of human caspase-8, which encoded a polypeptide of 543 amino acids. BmDredd possesses a long N-terminal prodomain, a p20 domain, and a p10 domain. When transiently expressed in Escherichia coli cells, BmDredd underwent spontaneous cleavage and exhibited high proteolytic activity for caspase-8 substrate but relatively low for caspase-3 or -9 substrate. In addition, BmDredd induced apoptosis when transiently expressed in BmN-SWU1 cells, an ovarian cell line of B. mori. Moreover, after the treatment of Emodin, a novel apoptosis inducer, endogenous BmDredd expression level, the caspase-8 activity and the apoptotic rate increased notably in BmN-SWU1 cells. When BmDredd was subjected to interference in BmN-SWU1 cells and Emodin treatment, BmDredd expression levels decreased and the apoptotic rate also decreased significantly. These results suggest BmDredd is the homologue of human caspase-8 and plays a role in Emodin-induced apoptosis in BmN-SWU1 cells of B. mori. Copyright © 2016 Elsevier B.V. All rights reserved.

Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress.

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Zhang, Qiang; Liu, Jianing; Chen, Shulan; Liu, Jing; Liu, Lijuan; Liu, Guirong; Wang, Fang; Jiang, Wenxin; Zhang, Caixia; Wang, Shuangyu; Yuan, Xiao

2016-04-01

It is well recognized that mandibular growth, which is caused by a variety of functional appliances, is considered to be the result of both neuromuscular and skeletal adaptations. Accumulating evidence has demonstrated that apoptosis plays an important role in the adaptation of skeletal muscle function. However, the underlying mechanism of apoptosis that is induced by stretch continues to be incompletely understood. Endoplasmic reticulum stress (ERS), a newly defined signaling pathway, initiates apoptosis. This study seeks to determine if caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress in myoblast and its underlying mechanism. Apoptosis was assessed by Hochest staining, DAPI staining and annexin V binding and PI staining. ER chaperones, such as GRP78, CHOP and caspase-12, were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Furthermore, caspase-12 inhibitor was used to value the mechanism of the caspase-12 pathway. Apoptosis of myoblast, which is subjected to cyclic stretch, was observed in a time-dependent manner. We found that GRP78 mRNA and protein were significantly increased and CHOP and caspase-12 were activated in myoblast that was exposed to cyclic stretch. Caspase-12 inhibition reduced stretch-induced apoptosis, and caspase-12 activated caspase-3 to induce apoptosis. We concluded that caspase-12 played an important role in stretch-induced apoptosis that is associated by endoplasmic reticulum stress by activating caspase-3.

Amyloid β42 peptide is toxic to non-neural cells in Drosophila yielding a characteristic metabolite profile and the effect can be suppressed by PI3K

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Mercedes Arnés

2017-11-01

Full Text Available The human Aβ42 peptide is associated with Alzheimer's disease through its deleterious effects in neurons. Expressing the human peptide in adult Drosophila in a tissue- and time-controlled manner, we show that Aβ42 is also toxic in non-neural cells, neurosecretory and epithelial cell types in particular. This form of toxicity includes the aberrant signaling by Wingless morphogen leading to the eventual activation of Caspase 3. Preventing Caspase 3 activation by means of p53 keeps epithelial cells from elimination but maintains the Aβ42 toxicity yielding more severe deleterious effects to the organism. Metabolic profiling by nuclear magnetic resonance (NMR of adult flies at selected ages post Aβ42 expression onset reveals characteristic changes in metabolites as early markers of the pathological process. All morphological and most metabolic features of Aβ42 toxicity can be suppressed by the joint overexpression of PI3K.

Dscam1-mediated self-avoidance counters netrin-dependent targeting of dendrites in Drosophila.

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Matthews, Benjamin J; Grueber, Wesley B

2011-09-13

Dendrites and axons show precise targeting and spacing patterns for proper reception and transmission of information in the nervous system. Self-avoidance promotes complete territory coverage and nonoverlapping spacing between processes from the same cell [1, 2]. Neurons that lack Drosophila Down syndrome cell adhesion molecule 1 (Dscam1) show aberrant overlap, fasciculation, and accumulation of dendrites and axons, demonstrating a role in self-recognition and repulsion leading to self-avoidance [3-11]. Fasciculation and accumulation of processes suggested that Dscam1 might promote process spacing by counterbalancing developmental signals that otherwise promote self-association [9, 12]. Here we show that Dscam1 functions to counter Drosophila sensory neuron dendritic targeting signals provided by secreted Netrin-B and Frazzled, a netrin receptor. Loss of Dscam1 function resulted in aberrant dendrite accumulation at a Netrin-B-expressing target, whereas concomitant loss of Frazzled prevented accumulation and caused severe deficits in dendritic territory coverage. Netrin misexpression was sufficient to induce ectopic dendritic targeting in a Frazzled-dependent manner, whereas Dscam1 was required to prevent ectopic accumulation, consistent with separable roles for these receptors. Our results suggest that Dscam1-mediated self-avoidance counters extrinsic signals that are required for normal dendritic patterning, but whose action would otherwise favor neurite accumulation. Counterbalancing roles for Dscam1 may be deployed in diverse contexts during neural circuit formation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Ofloxacin induces apoptosis in microencapsulated juvenile rabbit chondrocytes by caspase-8-dependent mitochondrial pathway

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Sheng Zhiguo; Cao Xiaojuan; Peng Shuangqing; Wang Changyong; Li Qianqian; Wang Yimei; Liu Mifeng

2008-01-01

Quinolones (QNs)-induced arthropathy is an important toxic effect in immature animals leading to restriction of their therapeutic use in pediatrics. However, the exact mechanism still remains unclear. Recently, we have demonstrated that ofloxacin, a typical QN, induces apoptosis of alginate microencapsulated juvenile rabbit joint chondrocytes by disturbing the β 1 integrin functions and inactivating the ERK/MAPK signaling pathway. In this study, we extend our initial observations to further elucidate the mechanism(s) of ofloxacin-induced apoptosis by utilizing specific caspase inhibitors. Pretreatment with both caspase-9-specific inhibitor zLEHD-fmk and caspase-8 inhibitor zIETD-fmk attenuated ofloxacin-induced apoptosis and activation of caspase-3 of chondrocyte in a concentration-dependent manner, as determined by fluorescent dye staining, enzyme activity assay and immunoblotting. Furthermore, the activation of caspase-9, -8 and -3 stimulated by ofloxacin was significantly inhibited in the presence of zIETD-fmk while pretreatment with zLEHD-fmk only blocked the activation of caspase-9 and -3. Ofloxacin also stimulated a concentration-dependent translocation of cytochrome c from mitochondria into the cytosol and a decrease of mitochondrial transmembrane potential, which was completely inhibited by zIETD-fmk. In addition, ofloxacin was found to increase the level of Bax, tBid, p53 in a concentration- and time-dependent manner. Taken together, The current results indicate that the caspase-8-dependent mitochondrial pathway is primarily involved in the ofloxacin-induced apoptosis of microencapsulated juvenile rabbit joint chondrocytes

Functional PAK-2 knockout and replacement with a caspase cleavage-deficient mutant in mice reveals differential requirements of full-length PAK-2 and caspase-activated PAK-2p34.

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Marlin, Jerry W; Chang, Yu-Wen E; Ober, Margaret; Handy, Amy; Xu, Wenhao; Jakobi, Rolf

2011-06-01

p21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.

The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

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Schatten, H.; Hedrick, J.; Chakrabarti, A.

1998-01-01

Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

Fludarabine inhibits STAT1-mediated up-regulation of caspase-3 expression in dexamethasone-induced osteoblasts apoptosis and slows the progression of steroid-induced avascular necrosis of the femoral head in rats.

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Feng, Zhenhua; Zheng, Wenhao; Tang, Qian; Cheng, Liang; Li, Hang; Ni, Wenfei; Pan, Xiaoyun

2017-08-01

Steroid-induced avascular necrosis of the femoral head (SANFH) is a major limitation of long-term or excessive clinical administration of glucocorticoids. Fludarabine, which is a compound used to treat various hematological malignancies, such as chronic lymphocytic leukemia, acts by down-regulating signal transducer and activator of transcription 1 (STAT1) by inhibiting STAT1 phosphorylation in both normal and cancer cells. This study assessed the effects of fludarabine in vitro (primary murine osteoblasts) and in vivo (rat SANFH model). In vitro, pretreatment with fludarabine significantly inhibited Dexamethasone (Dex)-induced apoptosis in osteoblasts, which was examined by TUNEL staining. Treatment with Dex caused a remarkable decrease in the expression of Bcl-2; an increase in cytochrome c release; activation of BAX, caspase-9, and caspase-3; and an obvious enhancement in STAT1 phosphorylation. However, treatment resulted in the up-regulation of caspase-3 expression. Enhanced P-STAT1 activity and up-regulation of caspase-3 expression were also observed in osteoblasts. In vivo, the subchondral trabeculae in fludarabine-treated rats exhibited less bone loss and a lower ratio of empty lacunae. Taken together, our results suggest that STAT1-mediated up-regulation of caspase-3 is involved in osteoblast apoptosis induced by Dex and indicates that fludarabine may serve as a potential agent for the treatment of SANFH.

SAG/ROC-SCFβ-TrCP E3 Ubiquitin Ligase Promotes Pro-Caspase-3 Degradation as a Mechanism of Apoptosis Protection

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Mingjia Tan

2006-12-01

Full Text Available Skp1-cullin-F-box protein (SCF is a multicomponent E3 ubiquitin (Ub ligase that ubiquitinates a number of important biologic molecules such as p27, β-catenin, and lκB for proteasomal degradation, thus regulating cell proliferation and survival. One SCF component, SAG/ROC2/Rbx2/Hrt2, a RING finger protein, was first identified as a redox-inducible protein, which, when overexpressed, inhibited apoptosis both in vitro and in vivo. We report here that sensitive to apoptosis gene (SAG, as well as its family member ROC1/Rbxi, bound to the proinactive form of caspase-3 (pro-caspase-3. Binding was likely mediated through F-box protein, β-transducin repeat-containing protein (β-TrCP, which binds to the first 38 amino acids of pro-caspase-3. Importantly, β-TrCP1 expression significantly shortened the protein half-life of pro-caspase-3, whereas expression of a dominant-negative β-TrCP1 mutant with the F-box domain deleted extended it. An in vitro ubiquitination assay showed that SAG/ROC-SCF -Trcp promoted ubiquitination of pro-caspase-3. Furthermore, endogenous levels of pro-caspase-3 were decreased by overexpression of SAG/ROC-SCFβ-TrCP E3 Ub ligases, but increased on siRNA silencing of SAG, regulator of cullin-1 (ROC1, or β-TrCPs, leading to increased apoptosis by etoposide and TNF-related apoptosis-inducing ligand through increased activation of caspase-3. Thus, pro-caspase-3 appears to be a substrate of SAG/ROC-SCFβ-TrCP E3 Ub ligase, which protects cells from apoptosis through increased apoptosis threshold by reducing the basal level of pro-caspase-3.

Caspase-dependent inhibition of store-operated Ca2+ entry into apoptosis-committed Jurkat cells

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Onopiuk, Marta; Wierzbicka, Katarzyna; Brutkowski, Wojciech; Szczepanowska, Joanna; Zablocki, Krzysztof

2010-01-01

Activation of T-cells triggers store-operated Ca 2+ entry, which begins a signaling cascade leading to induction of appropriate gene expression and eventually lymphocyte proliferation and differentiation. The simultaneous enhancement of Fas ligand gene expression in activated cells allows the immune response to be limited by committing the activated cells to apoptosis. In apoptotic cells the store-operated calcium entry is significantly inhibited. It has been documented that moderate activation of Fas receptor may cause reversible inhibition of store-operated channels by ceramide released from hydrolyzed sphingomyelin. Here we show that activation of Fas receptor in T-cells results in caspase-dependent decrease of cellular STIM1 and Orai1 protein content. This effect may be responsible for the substantial inhibition of Ca 2+ entry into Jurkat cells undergoing apoptosis. In turn, this inhibition might prevent overloading of cells with calcium and protect them against necrosis. -- Research highlights: → Fas activation reduces STIM1 and Orai1 protein content in caspase dependent manner. → Fas activation partially reduces mitochondrial potential in caspase dependent manner. → Fas stimulation inhibits of store-operated Ca 2+ entry in caspase dependent manner. → Inhibition of Ca 2+ entry in apoptotic cells may protect them from secondary necrosis.

Cytosolic and nuclear caspase-8 have opposite impact on survival after liver resection for hepatocellular carcinoma

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Koschny, Ronald; Schemmer, Peter; Schirmacher, Peter; Ganten, Tom M; Brost, Sylvia; Hinz, Ulf; Sykora, Jaromir; Batke, Emanuela M; Singer, Stephan; Breuhahn, Kai; Stremmel, Wolfgang; Walczak, Henning

2013-01-01

An imbalance between proliferation and apoptosis is one of the main features of carcinogenesis. TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis upon binding to the TRAIL death receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2, whereas binding to TRAIL-R3 and TRAIL-R4 might promote cell survival and proliferation. The anti-tumor activity of TRAIL-R1 and TRAIL-R2 agonists is currently investigated in clinical trials. To gain further insight into the regulation of apoptosis in hepatocellular carcinoma (HCC), we investigated the TRAIL pathway and the regulators of apoptosis caspase-8, Bcl-xL and Mcl-1 in patients with HCC regarding patient survival. We analyzed 157 hepatocellular carcinoma patients who underwent partial liver resection or orthotopic liver transplantation and healthy control liver tissue using immunohistochemistry on tissue microarrays for the expression of TRAIL-R1 to TRAIL-R4, caspase-8, Bcl-xL and Mcl-1. Immunohistochemical data were evaluated for potential associations with clinico-pathological parameters and survival. Whereas TRAIL-R1 was downregulated in HCC in comparison to normal liver tissue, TRAIL-R2 and –R4 were upregulated in HCC, especially in G2 and G3 tumors. TRAIL-R1 downregulation and upregulation of TRAIL-R2 and TRAIL-R4 correlated with tumor dedifferentiation (G2/G3). TRAIL-R3, Bcl-xL and Mcl-1 showed no differential expression in tumor tissue compared to normal tissue. The expression levels of TRAIL receptors did not correlate with patient survival after partial hepatectomy. Interestingly, in tumor tissue, but not in normal hepatocytes, caspase-8 showed a strong nuclear staining. Low cytosolic and high nuclear staining intensity of caspase-8 significantly correlated with impaired survival after partial hepatectomy, which, for cytosolic caspase-8, was independent from tumor grade. Assessment of TRAIL-receptor expression patterns may have therapeutic implications for the use of TRAIL receptor agonists in HCC therapy

The Drosophila melanogaster host model

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Igboin, Christina O.; Griffen, Ann L.; Leys, Eugene J.

2012-01-01

The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen–host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial–host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis–host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed. PMID:22368770

The Drosophila melanogaster host model

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Christina O. Igboin

2012-02-01

Full Text Available The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen–host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial–host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis–host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed.

The Drosophila melanogaster host model.

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Igboin, Christina O; Griffen, Ann L; Leys, Eugene J

2012-01-01

The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen-host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial-host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis-host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed.

The Drosophila homolog of the mammalian imprint regulator, CTCF, maintains the maternal genomic imprint in Drosophila melanogaster

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Rasheva Vanya

2010-07-01

Full Text Available Abstract Background CTCF is a versatile zinc finger DNA-binding protein that functions as a highly conserved epigenetic transcriptional regulator. CTCF is known to act as a chromosomal insulator, bind promoter regions, and facilitate long-range chromatin interactions. In mammals, CTCF is active in the regulatory regions of some genes that exhibit genomic imprinting, acting as insulator on only one parental allele to facilitate parent-specific expression. In Drosophila, CTCF acts as a chromatin insulator and is thought to be actively involved in the global organization of the genome. Results To determine whether CTCF regulates imprinting in Drosophila, we generated CTCF mutant alleles and assayed gene expression from the imprinted Dp(1;fLJ9 mini-X chromosome in the presence of reduced CTCF expression. We observed disruption of the maternal imprint when CTCF levels were reduced, but no effect was observed on the paternal imprint. The effect was restricted to maintenance of the imprint and was specific for the Dp(1;fLJ9 mini-X chromosome. Conclusions CTCF in Drosophila functions in maintaining parent-specific expression from an imprinted domain as it does in mammals. We propose that Drosophila CTCF maintains an insulator boundary on the maternal X chromosome, shielding genes from the imprint-induced silencing that occurs on the paternally inherited X chromosome. See commentary: http://www.biomedcentral.com/1741-7007/8/104

Study of radioadaptive response in Drosophila melanogaster at different oogenesis stages

International Nuclear Information System (INIS)

Glushkova, I.V.; Aksyutik, T.V.

2005-01-01

We study radioadaptive response in the Canton-S strain of Drosophila melanogaster at different oogenesis stages using the test of dominant lethal mutations (DLM). AR was not revealed at the stages of 14-7 and 7--1 oocytes in the studied Drosophila stock. It is likely to be associated with a genetic constitution of the Drosophila strain under study. (authors)

N,N-dimethyl phytosphingosine induces caspase-8-dependent cytochrome c release and apoptosis through ROS generation in human leukemia cells

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Kim, Byeong Mo; Choi, Yun Jung; Han, Youngsoo; Yun, Yeon-Sook; Hong, Sung Hee

2009-01-01

N,N-dimethyl phytosphingosine (DMPS) blocks the conversion of sphingosine to sphingosine-1-phosphate (S1P) by the enzyme sphingosine kinase (SK). In this study, we elucidated the apoptotic mechanisms of DMPS action on a human leukemia cell line using functional pharmacologic and genetic approaches. First, we demonstrated that DMPS-induced apoptosis is evidenced by nuclear morphological change, distinct internucleosomal DNA fragmentation, and an increased sub-G1 cell population. DMPS treatment led to the activation of caspase-9 and caspase-3, accompanied by the cleavage of poly(ADP-ribose) polymerase (PARP) and led to cytochrome c release, depolarization of the mitochondrial membrane potential, and downregulation of the anti-apoptotic members of the bcl-2 family. Ectopic expression of bcl-2 and bcl-xL conferred resistance of HL-60 cells to DMPS-induced cell death, suggesting that DMPS-induced apoptosis occurs predominantly through the activation of the intrinsic mitochondrial pathway. We also observed that DMPS activated the caspase-8-Bid-Bax pathway and that the inhibition of caspase-8 by z-IETD-fmk or small interfering RNA suppressed the cleavage of Bid, cytochrome c release, caspase-3 activation, and apoptotic cell death. In addition, cells subjected to DMPS exhibited significantly increased reactive oxygen species (ROS) generation, and ROS scavengers, such as quercetin and Tiron, but not N-acetylcysteine (NAC), inhibited DMPS-induced activations of caspase-8, -3 and subsequent apoptotic cell death, indicating the role of ROS in caspase-8-mediated apoptosis. Taken together, these results indicate that caspase-8 acts upstream of caspase-3, and that the caspase-8-mediated mitochondrial pathway is important in DMPS-induced apoptosis. Our results also suggest that ROS are critical regulators of caspase-8-mediated apoptosis in DMPS-treated leukemia cells.

Caspase-12 ablation preserves muscle function in the mdx mouse

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Moorwood, Catherine; Barton, Elisabeth R.

2014-01-01

Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin. Several downstream consequences of dystrophin deficiency are triggers of endoplasmic reticulum (ER) stress, including loss of calcium homeostasis, hypoxia and oxidative stress. During ER stress, misfolded proteins accumulate in the ER lumen and the unfolded protein response (UPR) is triggered, leading to adaptation or apoptosis. We hypothesized that ER stress is heightened in dystrophic muscles and contributes to the pathology of DMD. We observed increases in the ER stress markers BiP and cleaved caspase-4 in DMD patient biopsies, compared with controls, and an increase in multiple UPR pathways in muscles of the dystrophin-deficient mdx mouse. We then crossed mdx mice with mice null for caspase-12, the murine equivalent of human caspase-4, which are resistant to ER stress. We found that deleting caspase-12 preserved mdx muscle function, resulting in a 75% recovery of both specific force generation and resistance to eccentric contractions. The compensatory hypertrophy normally found in mdx muscles was normalized in the absence of caspase-12; this was found to be due to decreased fibre sizes, and not to a fibre type shift or a decrease in fibrosis. Fibre central nucleation was not significantly altered in the absence of caspase-12, but muscle fibre degeneration found in the mdx mouse was reduced almost to wild-type levels. In conclusion, we have identified heightened ER stress and abnormal UPR signalling as novel contributors to the dystrophic phenotype. Caspase-4 is therefore a potential therapeutic target for DMD. PMID:24879640

Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

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Ding, L.; Wu, J.P.; Xu, G.; Zhu, B.; Zeng, Q.M.; Li, D.F.; Lu, W.

2014-01-01

Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis

Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

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Ding, L.; Wu, J.P. [Fudan University, Jinshan Hospital, Department of Orthopaedics, Shanghai, China, Department of Orthopaedics, Jinshan Hospital, Fudan University, Shanghai (China); Xu, G. [Fudan University, Jinshan Hospital, Center Laboratory, Shanghai, China, Center Laboratory, Jinshan Hospital, Fudan University, Shanghai (China); Zhu, B.; Zeng, Q.M.; Li, D.F.; Lu, W. [Fudan University, Jinshan Hospital, Department of Orthopaedics, Shanghai, China, Department of Orthopaedics, Jinshan Hospital, Fudan University, Shanghai (China)

2014-05-09

Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

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L. Ding

2014-06-01

Full Text Available Current studies find that degenerated cartilage endplates (CEP of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

Involvement of caspase-dependent and -independent apoptotic pathways in cisplatin-induced apoptosis

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Liu, Lei; Zhang, Yingjie; Wang, Xianwang

2009-02-01

Cisplatin, an efficient anticancer agent, can trigger multiple apoptotic pathways in cancer cells. However, the signal transduction pathways in response to cisplatin-based chemotherapy are complicated, and the mechanism is not fully understood. In current study, we showed that, during cisplatin-induced apoptosis of human lung adenocarcinoma cells, both the caspase-dependent and -independent pathways were activated. Herein, we reported that after cisplatin treatment, the activities of caspase-9/-3 were sharply increased; pre-treatment with Z-LEHD-fmk (inhibitor of caspase-9), Z-DEVD-fmk (inhibitor of caspase-3), and Z-VAD-fmk (a pan-caspase inhibitor) increased cell viability and decreased apoptosis, suggesting that caspase-mediated apoptotic pathway was activated following cisplatin treatment. Confocal imaging of the cells transfected with AIF-GFP demonstrated that AIF release occurred about 9 h after cisplatin treatment. The event proceeded progressively over time, coinciding with a nuclear translocation and lasting for more than 2 hours. Down-regulation of AIF by siRNA also significantly increased cell viability and decreased apoptosis, these results suggested that AIF-mediated caspase-independent apoptotic pathway was involved in cispatin-induced apoptosis. In conclusion, the current study demonstrated that both caspase-dependent and -independent apoptotic pathways were involved in cisplatin-induced apoptosis in human lung adenocarcinoma cells.

THE EXPRESSION OF Bcl-2 AND PRO-CASPASE 3 IN HEAD AND NECK SQUAMOUS CELL CARCINOMA

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Andrej Cör

2002-12-01

Full Text Available Background. Head and neck squamous cell carcinoma (HNSCC is the sixth most common cancer and accounts for 6% of cancers worldwide. A better understanding of its biology could lead to improved treatment options. Generally, the goal of cancer treatment is to abolish cell proliferation and to induce necrotic or aptoptotic cell death. Apoptosis has been recognized as a key mechanism of tumour cell elimination. Different apoptotic signals converge to induce caspase cascade activation. Caspase 3 is the central executioner caspase and is necessary for effective apoptotic cell death. Bcl-2 protein family regulates apoptosis. The Bcl-2 protein itself is a product of a proto-oncogene and has an antiapoptotic action.Methods. In our study, the expression of Bcl-2 and pro-caspase 3 by immunohistochemistry in 28 HNSCC graded into well, moderately and poorly differentiated cancers were investigated.Results. Our results of Bcl-2 expression confirm and extend previous reports in which Bcl-2 over-expression has been recognised as an important parameter in HNSCC biological behaviour. Three of 28 tumours (11% showed significant Bcl-2 expression. Two of them were poorly and one was moderately differentiated. Pro-caspase 3 immunoreactivity was confined mainly to the cytoplasm. Absent or low pro-caspase 3 immunoreactivity was found only in 1 of 6 well differentiated and in 1of 10 moderately differentiated tumours in contrast to 5 of 12 poorly differentiated tumours. In six of 12 poorly differentiated tumours procasapse 3 immunoreactivity was strongly positive. In two cases hyperplastic epithelium was strongly positive in contrast to adjacent HNSCC in the same slide which was completely negative for pro-caspase 3.Conclusions. Our results indicate downregulation of pro-caspase 3 expression, especially in poorly differentiated HNSCC. Further studies are needed to test whether this is related to HNSCC behaviour and predict treatment outcome.

Negative regulation of MAP kinase signaling in Drosophila by Ptp61F/PTP1B.

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Tchankouo-Nguetcheu, Stéphane; Udinotti, Mario; Durand, Marjorie; Meng, Tzu-Ching; Taouis, Mohammed; Rabinow, Leonard

2014-10-01

PTP1B is an important negative regulator of insulin and other signaling pathways in mammals. However, the role of PTP1B in the regulation of RAS-MAPK signaling remains open to deliberation, due to conflicting evidence from different experimental systems. The Drosophila orthologue of mammalian PTP1B, PTP61F, has until recently remained largely uncharacterized. To establish the potential role of PTP61F in the regulation of signaling pathways in Drosophila and particularly to help resolve its fundamental function in RAS-MAPK signaling, we generated a new allele of Ptp61F as well as employed both RNA interference and overexpression alleles. Our results validate recent data showing that the activity of insulin and Abl kinase signaling is increased in Ptp61F mutants and RNA interference lines. Importantly, we establish negative regulation of the RAS/MAPK pathway by Ptp61F activity in whole animals. Of particular interest, our results document the modulation of hyperactive MAP kinase activity by Ptp61F alleles, showing that the phosphatase intervenes to directly or indirectly regulate MAP kinase itself.

[Studies of the repair of radiation-induced genetic damage in Drosophila]. Annual progress report, February 1, 1993 - November 1, 1994

International Nuclear Information System (INIS)

Hawley, R.S.

1998-01-01

This research focuses on two repair deficient mutations in Drosophila melanogaster, namely mei-9, mei-41. In addition, the authors propose to extend this study to include the mus-312 mutation. They expect these studies to provide substantial insights into both the molecular mechanisms of DNA repair in Drosophila and the role these genes play in normal biological processes

Hermann Muller and Mutations in Drosophila

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dropdown arrow Site Map A-Z Index Menu Synopsis Hermann Muller and Mutations in Drosophila Resources with University of Texas. In Austin his experiments on fruit flies (Drosophila) first showed that exposure to September to spend a year at the only Drosophila laboratory in Europe which was doing parallel work

Inhibiting HIF-1α Decreases Expression of TNF-α and Caspase-3 in Specific Brain Regions Exposed Kainic Acid-Induced Status Epilepticus

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Jixue Yang

2016-01-01

Full Text Available Background/Aims: A recent study demonstrates that pro-inflammatory cytokines (PICs, i.e., IL-1β, IL-6 and TNF-α in specific brain regions of rats play a role in regulating kainic acid (KA-induced status epilepticus (SE via a GABAergic mechanism. The purposes of this report were to examine contributions of hypoxia inducible factor subtype 1α (HIF-1α to expression of PICs in these specific brain regions in epileptic rats. Particularly, we investigated the parietal cortex, hippocampus and amygdala. In addition, we further examined expression of Caspase-3 indicating cell apoptosis in those brain regions of epileptic rats after infusing 2-methoxyestradiol (2-MET, inhibitor of HIF-1α and etanercept (TNF-α receptor antagonist. Methods: ELISA was used to determine the levels of HIF-1α and PICs and western blot analysis was used to examine Caspase-3 expression. Results: Our data show that HIF-1α was significantly increased in the parietal cortex, hippocampus and amygdala 1, 3 and 7 days after induction of SE (Pvs. control rats. Our results also show that inhibiting HIF-1α by central infusion of 2-MET significantly decreased the amplified TNF-α expression in these brain regions evoked by SE (Pvs. vehicle control, but did not modify IL-1β and IL-6. Our results demonstrate that 2-MET and etanercept attenuated an increase in Caspase-3 evoked by SE. Conclusion: Overall, we suggest that HIF-1α activated by SE is likely to contribute to epileptic activity via a TNF-α pathway, which has pharmacological implications to target specific HIF-1α and TNF-α pathways for neuronal dysfunction and vulnerability related to epilepsy.

Contribution of caspase-3 differs by p53 status in apoptosis induced by X-irradiation

International Nuclear Information System (INIS)

Kobayashi, Daisuke; Tokino, Takashi; Watanabe, Naoki

2001-01-01

We investigated the effect of p53 status on involvement of caspase-3 activation in cell death induced by X-irradiation, using rat embryonic fibroblasts (REFs) transduced with a temperature-sensitive mutant (mt) p53 gene. Cells with wild-type (wt) p53 showed greater resistance to X-irradiation than cells with mt p53. In cells with wt p53, X-irradiation-induced apoptosis was not inhibited by the caspase-3 inhibitor acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspartyl-aldehyde (Ac-DMQD-CHO) and caspase-3 activity was not elevated following X-irradiation, although induction of p53 and p21/WAF-1 protein was observed. In contrast, irradiated cells with mt p53 showed 89% inhibition of cell death with Ac-DMQD-CHO and 98% inhibition with the antioxidant N-acetyl-L-cysteine (NAC). In cells with mt p53, caspase-3 activity was increased approximately 5 times beyond baseline activity at 24 h after irradiation. This increase was almost completely inhibited by NAC. However, inhibition of caspase-3 by Ac-DMQD-CHO failed to decrease production of reactive oxygen species by cells with mt p53. Differential involvement of caspase-3 is a reason for differences in sensitivity to X-irradiation in cells with different p53 status. Caspase-3 activation appears to occur downstream from generation of reactive oxygen species occurring independently of wt p53 during X-irradiation-induced cell death. (author)

The neurotoxicant PCB-95 by increasing the neuronal transcriptional repressor REST down-regulates caspase-8 and increases Ripk1, Ripk3 and MLKL expression determining necroptotic neuronal death.

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Guida, Natascia; Laudati, Giusy; Serani, Angelo; Mascolo, Luigi; Molinaro, Pasquale; Montuori, Paolo; Di Renzo, Gianfranco; Canzoniero, Lorella M T; Formisano, Luigi

2017-10-15

Our previous study showed that the environmental neurotoxicant non-dioxin-like polychlorinated biphenyl (PCB)-95 increases RE1-silencing transcription factor (REST) expression, which is related to necrosis, but not apoptosis, of neurons. Meanwhile, necroptosis is a type of a programmed necrosis that is positively regulated by receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL) and negatively regulated by caspase-8. Here we evaluated whether necroptosis contributes to PCB-95-induced neuronal death through REST up-regulation. Our results demonstrated that in cortical neurons PCB-95 increased RIPK1, RIPK3, and MLKL expression and decreased caspase-8 at the gene and protein level. Furthermore, the RIPK1 inhibitor necrostatin-1 or siRNA-mediated RIPK1, RIPK3 and MLKL expression knockdown significantly reduced PCB-95-induced neuronal death. Intriguingly, PCB-95-induced increases in RIPK1, RIPK3, MLKL expression and decreases in caspase-8 expression were reversed by knockdown of REST expression with a REST-specific siRNA (siREST). Notably, in silico analysis of the rat genome identified a REST consensus sequence in the caspase-8 gene promoter (Casp8-RE1), but not the RIPK1, RIPK3 and MLKL promoters. Interestingly, in PCB-95-treated neurons, REST binding to the Casp8-RE1 sequence increased in parallel with a reduction in its promoter activity, whereas under the same experimental conditions, transfection of siREST or mutation of the Casp8-RE1 sequence blocked PCB-95-induced caspase-8 reduction. Since RIPK1, RIPK3 and MLKL rat genes showed no putative REST binding site, we assessed whether the transcription factor cAMP Responsive Element Binding Protein (CREB), which has a consensus sequence in all three genes, affected neuronal death. In neurons treated with PCB-95, CREB protein expression decreased in parallel with a reduction in binding to the RIPK1, RIPK3 and MLKL gene promoter sequence. Furthermore, CREB overexpression was

The Predominant Pathway of Apoptosis in THP-1 Macrophage-Derived Foam Cells Induced by 5-Aminolevulinic Acid-Mediated Sonodynamic Therapy is the Mitochondria-Caspase Pathway Despite the Participation of Endoplasmic Reticulum Stress

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Huan Wang

2014-05-01

Full Text Available Background: In advanced atherosclerosis, chronic endoplasmic reticulum (ER stress induces foam cells apoptosis and generates inflammatory reactions. Methods: THP-1 macrophage-derived foam cells (FC were incubated with 1 mM 5-aminolevulinic acid (ALA. After ALA mediated sonodynamic therapy (ALA-SDT, apoptosis of FC was assayed by Annexin V-PI staining. Intracellular reactive oxygen species (ROS and mitochondrial membrane potential were detected by staining with CellROX® Green Reagent and jc-1. Pretreatment of FC with N-acetylcysteine (NAC, Z-VAD-FMK or 4-phenylbutyrate (4-PBA, mitochondria apoptotic pathway associated proteins and C/EBP-homologous (CHOP expressions were assayed by wertern blotting. Results: Burst of apoptosis of FC was observed at 5-hour after ALA-SDT with 6-hour incubation of ALA and 0.4 W/cm2 ultrasound. After ALA-SDT, intracellular ROS level increased and mitochondrial membrane potential collapsed. Translocations of cytochrome c from mitochondria into cytosol and Bax from cytosol into mitochondria, cleaved caspase 9, cleaved caspase 3, upregulation of CHOP, as well as downregulation of Bcl-2 after ALA-SDT were detected, which could be suppressed by NAC. Activation of mitochondria-caspase pathway could not be inhibited by 4-PBA. Cleaved caspase 9 and caspase 3 as well as apoptosis induced by ALA-SDT could be inhibited by Z-VAD-FMK. Conclusion: The mitochondria-caspase pathway is predominant in the apoptosis of FC induced by ALA-SDT though ER stress participates in.

BID is cleaved by caspase-8 within a native complex on the mitochondrial membrane

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Schug, Z. T.; Gonzalvez, F.; Houtkooper, R. H.; Vaz, F. M.; Gottlieb, E.

2011-01-01

Caspase-8 stably inserts into the mitochondrial outer membrane during extrinsic apoptosis. Inhibition of caspase-8 enrichment on the mitochondria impairs caspase-8 activation and prevents apoptosis. However, the function of active caspase-8 on the mitochondrial membrane remains unknown. In this

Memantine Can Reduce Ethanol-Induced Caspase-3 Activity and Apoptosis in H4 Cells by Decreasing Intracellular Calcium.

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Wang, Xiaolong; Chen, Jiajun; Wang, Hongbo; Yu, Hao; Wang, Changliang; You, Jiabin; Wang, Pengfei; Feng, Chunmei; Xu, Guohui; Wu, Xu; Zhao, Rui; Zhang, Guohua

2017-08-01

Caspase-3 activation and apoptosis are associated with various neurodegenerative disorders. Calcium activation is an important factor in promoting apoptosis. We, therefore, assessed the role of intracellular calcium in ethanol-induced activation of caspase-3 in H4 human neuroglioma cells and the protective effect of the NMDA receptor antagonist, memantine, on ethanol-induced apoptosis in H4 cells. H4 cells were treated with 100 mM EtOH (in culture medium) for 2 days. For interaction studies, cells were treated with memantine (4 μM), EDTA (1 mM), or BAPTA-AM (10 μM) before treatment with EtOH. Knockdown of the gene encoding the NR1 subunit of the NMDA receptor was performed using RNAi. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. Cell viability was detected using an MTS cell proliferation kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration. The levels of NR1, caspase-3, IP3R1, and SERCA1 proteins were detected by western blotting. NR1, IP3R1, and SERCA1 mRNA levels were detected by qPCR. We observed increased expression of NR1, IP3R1, SERCA1, and increased intracellular levels of calcium ions in H4 cells exposed to ethanol. In addition, the calcium chelators, EDTA and BAPTA, and RNAi disruption of the NMDA receptor reduced ethanol-induced caspase-3 activation in H4 cells. Memantine treatment reduced the ethanol-induced increase of intracellular calcium, caspase-3 activation, apoptosis, and the ethanol-induced decrease in cell viability. Our results indicate that ethanol-induced caspase-3 activation and apoptosis are likely to be dependent on cytosolic calcium levels and that they can be reduced by memantine treatment.

Expression and activation of Daphnia pulex Caspase-3 are involved in regulation of aging.

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Tong, Qiaoqiong; Zhang, Mengmeng; Cao, Xiao; Xu, Shanliang; Wang, Danli; Zhao, Yunlong

2017-11-15

Death-mediating proteases such as Caspases have been implicated in aging. Remarkably, active Caspase-3 can trigger widespread damage and degeneration, playing a key role in causing cell death. In order to explore the relationship between Caspase-3 and aging in Daphnia pulex, we cloned and analyzed the full-length cDNA sequence of its Caspase-3 gene. Both mRNA expression and activity of D. pulex Caspase-3 increased with age. Moreover, different forms of Caspase-3 appeared with aging. The expression of casp3-L was higher and decreased with age, while that of casp3-S was weak and increased with age, consistent with the trend in Caspase-3 activity. Mhc mRNA expression declined over time and was negatively correlated with age and Caspase-3. In situ hybridization results showed that Caspase-3 mRNA was expressed in different growth and reproduction stages, and its expression levels in embryos and larva were lower than that in adult D. pulex. Western blot analysis revealed the presence of Caspase-3 in the form of zymogens with a molecular weight of ~36kDa. Overall, this study explored age-associated gene regulation to provide a basis for the molecular mechanism of D. pulex reproductive conversion. Copyright © 2017 Elsevier B.V. All rights reserved.

Long-term fluorescence lifetime imaging of a genetically encoded sensor for caspase-3 activity in mouse tumor xenografts

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Zherdeva, Victoria; Kazachkina, Natalia I.; Shcheslavskiy, Vladislav; Savitsky, Alexander P.

2018-03-01

Caspase-3 is known for its role in apoptosis and programmed cell death regulation. We detected caspase-3 activation in vivo in tumor xenografts via shift of mean fluorescence lifetimes of a caspase-3 sensor. We used the genetically encoded sensor TR23K based on the red fluorescent protein TagRFP and chromoprotein KFP linked by 23 amino acid residues (TagRFP-23-KFP) containing a specific caspase cleavage DEVD motif to monitor the activity of caspase-3 in tumor xenografts by means of fluorescence lifetime imaging-Forster resonance energy transfer. Apoptosis was induced by injection of paclitaxel for A549 lung adenocarcinoma and etoposide and cisplatin for HEp-2 pharynx adenocarcinoma. We observed a shift in lifetime distribution from 1.6 to 1.9 ns to 2.1 to 2.4 ns, which indicated the activation of caspase-3. Even within the same tumor, the lifetime varied presumably due to the tumor heterogeneity and the different depth of tumor invasion. Thus, processing time-resolved fluorescence images allows detection of both the cleaved and noncleaved states of the TR23K sensor in real-time mode during the course of several weeks noninvasively. This approach can be used in drug screening, facilitating the development of new anticancer agents as well as improvement of chemotherapy efficiency and its adaptation for personal treatment.

The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8

Energy Technology Data Exchange (ETDEWEB)

Manzo-Merino, Joaquin [Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología, México/Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. Av. San Fernando No. 22, Col. Sección XVI, Tlalpan 14080 (Mexico); Massimi, Paola [International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34149 Trieste (Italy); Lizano, Marcela, E-mail: lizanosoberon@gmail.com [Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología, México/Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. Av. San Fernando No. 22, Col. Sección XVI, Tlalpan 14080 (Mexico); Banks, Lawrence, E-mail: banks@icgeb.org [International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34149 Trieste (Italy)

2014-02-15

The HPV-16 E6 and E6{sup ⁎} proteins have been shown previously to be capable of regulating caspase 8 activity. We now show that the capacity of E6 to interact with caspase 8 is common to diverse HPV types, being also seen with HPV-11 E6, HPV-18 E6 and HPV-18 E6{sup ⁎}. Unlike most E6-interacting partners, caspase 8 does not appear to be a major proteasomal target of E6, but instead E6 appears able to stimulate caspase 8 activation, without affecting the overall apoptotic activity. This would appear to be mediated in part by the ability of the HPV E6 oncoproteins to recruit active caspase 8 to the nucleus. - Highlights: • Multiple HPV E6 oncoproteins interact with the caspase 8 DED domain. • HPV E6 stimulates activation of caspase 8. • HPV E6 promotes nuclear accumulation of caspase 8.

The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8

International Nuclear Information System (INIS)

Manzo-Merino, Joaquin; Massimi, Paola; Lizano, Marcela; Banks, Lawrence

2014-01-01

The HPV-16 E6 and E6 ⁎ proteins have been shown previously to be capable of regulating caspase 8 activity. We now show that the capacity of E6 to interact with caspase 8 is common to diverse HPV types, being also seen with HPV-11 E6, HPV-18 E6 and HPV-18 E6 ⁎ . Unlike most E6-interacting partners, caspase 8 does not appear to be a major proteasomal target of E6, but instead E6 appears able to stimulate caspase 8 activation, without affecting the overall apoptotic activity. This would appear to be mediated in part by the ability of the HPV E6 oncoproteins to recruit active caspase 8 to the nucleus. - Highlights: • Multiple HPV E6 oncoproteins interact with the caspase 8 DED domain. • HPV E6 stimulates activation of caspase 8. • HPV E6 promotes nuclear accumulation of caspase 8

BmICE-2 is a novel pro-apoptotic caspase involved in apoptosis in the silkworm, Bombyx mori.

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Yi, Hua-Shan; Pan, Cai-Xia; Pan, Chun; Song, Juan; Hu, Yan-Fen; Wang, La; Pan, Min-Hui; Lu, Cheng

2014-02-28

In this study we identified a potential pro-apoptotic caspase gene, Bombyx mori(B. mori)ICE-2 (BmICE-2) which encoded a polypeptide of 284 amino acid residues, including a (169)QACRG(173) sequence which surrounded the catalytic site and contained a p20 and a p10 domain. BmICE-2 expressed in Escherichia coli (E. coli) exhibited high proteolytic activity for the synthetic human initiator caspase-9 substrates Ac-LEHD-pNA, but little activity towards the effector caspase-3 substrates Ac-DEVD-pNA. When BmICE-2 was transiently expressed in BmN-SWU1 silkworm B. mori cells, we found that the high proteolytic activity for Ac-LEHD-pNA triggered caspase-3-like protease activity resulting in spontaneous cleavage and apoptosis in these cells. This effect was not replicated in Spodoptera frugiperda 9 cells. In addition, spontaneous cleavage of endogenous BmICE-2 in BmN-SWU1 cells could be induced by actinomycin D. These results suggest that BmICE-2 may be a novel pro-apoptotic gene with caspase-9 activity which is involved apoptotic processes in BmN-SWU1 silkworm B. mori cells. Copyright © 2014 Elsevier Inc. All rights reserved.

A novel Drosophila injury model reveals severed axons are cleared through a Draper/MMP-1 signaling cascade

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Purice, Maria D; Ray, Arpita; Münzel, Eva Jolanda; Pope, Bernard J; Park, Daniel J; Speese, Sean D; Logan, Mary A

2017-01-01

Neural injury triggers swift responses from glia, including glial migration and phagocytic clearance of damaged neurons. The transcriptional programs governing these complex innate glial immune responses are still unclear. Here, we describe a novel injury assay in adult Drosophila that elicits widespread glial responses in the ventral nerve cord (VNC). We profiled injury-induced changes in VNC gene expression by RNA sequencing (RNA-seq) and found that responsive genes fall into diverse signaling classes. One factor, matrix metalloproteinase-1 (MMP-1), is induced in Drosophila ensheathing glia responding to severed axons. Interestingly, glial induction of MMP-1 requires the highly conserved engulfment receptor Draper, as well as AP-1 and STAT92E. In MMP-1 depleted flies, glia do not properly infiltrate neuropil regions after axotomy and, as a consequence, fail to clear degenerating axonal debris. This work identifies Draper-dependent activation of MMP-1 as a novel cascade required for proper glial clearance of severed axons. DOI: http://dx.doi.org/10.7554/eLife.23611.001 PMID:28825401

The Roles of ROS and Caspases in TRAIL-Induced Apoptosis and Necroptosis in Human Pancreatic Cancer Cells.

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Min Zhang

Full Text Available Death signaling provided by tumor necrosis factor (TNF-related apoptosis-inducing ligand (TRAIL can induce death in cancer cells with little cytotoxicity to normal cells; this cell death has been thought to involve caspase-dependent apoptosis. Reactive oxygen species (ROS are also mediators that induce cell death, but their roles in TRAIL-induced apoptosis have not been elucidated fully. In the current study, we investigated ROS and caspases in human pancreatic cancer cells undergoing two different types of TRAIL-induced cell death, apoptosis and necroptosis. TRAIL treatment increased ROS in two TRAIL-sensitive pancreatic cancer cell lines, MiaPaCa-2 and BxPC-3, but ROS were involved in TRAIL-induced apoptosis only in MiaPaCa-2 cells. Unexpectedly, inhibition of ROS by either N-acetyl-L-cysteine (NAC, a peroxide inhibitor, or Tempol, a superoxide inhibitor, increased the annexin V-/propidium iodide (PI+ early necrotic population in TRAIL-treated cells. Additionally, both necrostatin-1, an inhibitor of receptor-interacting protein kinase 1 (RIP1, and siRNA-mediated knockdown of RIP3 decreased the annexin V-/PI+ early necrotic population after TRAIL treatment. Furthermore, an increase in early apoptosis was induced in TRAIL-treated cancer cells under inhibition of either caspase-2 or -9. Caspase-2 worked upstream of caspase-9, and no crosstalk was observed between ROS and caspase-2/-9 in TRAIL-treated cells. Together, these results indicate that ROS contribute to TRAIL-induced apoptosis in MiaPaCa-2 cells, and that ROS play an inhibitory role in TRAIL-induced necroptosis of MiaPaCa-2 and BxPC-3 cells, with caspase-2 and -9 playing regulatory roles in this process.

Smac Mimetic Bypasses Apoptosis Resistance in FADD- or Caspase-8-Deficient Cells by Priming for Tumor Necrosis Factor α-Induced Necroptosis

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Bram Laukens

2011-10-01

Full Text Available Searching for new strategies to bypass apoptosis resistance, we investigated the potential of the Smac mimetic BV6 in Jurkat leukemia cells deficient in key molecules of the death receptor pathway. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant, FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis in a synergistic manner. In contrast to TNFα, Smac mimetic significantly enhances CD95-induced apoptosis in wild-type but not in FADD-deficient cells. Interestingly, Smac mimetic- and TNFα-mediated cell death occurs without characteristic features of apoptosis (i.e., caspase activation, DNA fragmentation in FADD-deficient cells. By comparison, Smac mimetic and TNFα trigger activation of caspase-8, -9, and -3 and DNA fragmentation in wild-type cells. Consistently, the caspase inhibitor zVAD.fmk fails to block Smac mimetic- and TNFα-triggered cell death in FADD- or caspase-8-deficient cells, while it confers protection in wild-type cells. By comparison, necrostatin-1, an RIP1 kinase inhibitor, abolishes Smac mimetic- and TNFα-induced cell death in FADD- or caspase-8-deficient. Thus, Smac mimetic enhances TNFα-induced cell death in leukemia cells via two distinct pathways in a context-dependent manner: it primes apoptosis-resistant cells lacking FADD or caspase-8 to TNFα-induced, RIP1-dependent and caspase-independent necroptosis, whereas it sensitizes apoptosis-proficient cells to TNFα-mediated, caspase-dependent apoptosis. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance.

A non-redundant role for Drosophila Mkk4 and hemipterous/Mkk7 in TAK1-mediated activation of JNK.

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Peter Geuking

Full Text Available BACKGROUND: The JNK pathway is a mitogen-activated protein (MAP kinase pathway involved in the regulation of numerous physiological processes during development and in response to environmental stress. JNK activity is controlled by two MAPK kinases (MAPKK, Mkk4 and Mkk7. Mkk7 plays a prominent role upon Tumor Necrosis Factor (TNF stimulation. Eiger, the unique TNF-superfamily ligand in Drosophila, potently activates JNK signaling through the activation of the MAPKKK Tak1. METHODOLOGY/PRINCIPAL FINDINGS: In a dominant suppressor screen for new components of the Eiger/JNK-pathway in Drosophila, we have identified an allelic series of the Mkk4 gene. Our genetic and biochemical results demonstrate that Mkk4 is dispensable for normal development and host resistance to systemic bacterial infection but plays a non-redundant role as a MAPKK acting in parallel to Hemipterous/Mkk7 in dTAK1-mediated JNK activation upon Eiger and Imd pathway activation. CONCLUSIONS/SIGNIFICANCE: In contrast to mammals, it seems that in Drosophila both MAPKKs, Hep/Mkk7 and Mkk4, are required to induce JNK upon TNF or pro-inflammatory stimulation.

The D1 family dopamine receptor, DopR, potentiates hind leg grooming behavior in Drosophila.

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Pitmon, E; Stephens, G; Parkhurst, S J; Wolf, F W; Kehne, G; Taylor, M; Lebestky, T

2016-03-01

Drosophila groom away debris and pathogens from the body using their legs in a stereotyped sequence of innate motor behaviors. Here, we investigated one aspect of the grooming repertoire by characterizing the D1 family dopamine receptor, DopR. Removal of DopR results in decreased hind leg grooming, as substantiated by quantitation of dye remaining on mutant and RNAi animals vs. controls and direct scoring of behavioral events. These data are also supported by pharmacological results that D1 receptor agonists fail to potentiate grooming behaviors in headless DopR flies. DopR protein is broadly expressed in the neuropil of the thoracic ganglion and overlaps with TH-positive dopaminergic neurons. Broad neuronal expression of dopamine receptor in mutant animals restored normal grooming behaviors. These data provide evidence for the role of DopR in potentiating hind leg grooming behaviors in the thoracic ganglion of adult Drosophila. This is a remarkable juxtaposition to the considerable role of D1 family dopamine receptors in rodent grooming, and future investigations of evolutionary relationships of circuitry may be warranted. © 2016 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.

The role of the Drosophila LAMMER protein kinase DOA in somatic ...

Indian Academy of Sciences (India)

2010-09-06

Sep 6, 2010 ... Somatic sexual identity in Drosophila melanogaster is under the control of a ... between stages 12 and 14, in response to an X to autosome .... MER kinases used were Drosophila DOA, mouse CLK1, human CLK2 and.

Metabolomic Studies in Drosophila.

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Cox, James E; Thummel, Carl S; Tennessen, Jason M

2017-07-01

Metabolomic analysis provides a powerful new tool for studies of Drosophila physiology. This approach allows investigators to detect thousands of chemical compounds in a single sample, representing the combined contributions of gene expression, enzyme activity, and environmental context. Metabolomics has been used for a wide range of studies in Drosophila , often providing new insights into gene function and metabolic state that could not be obtained using any other approach. In this review, we survey the uses of metabolomic analysis since its entry into the field. We also cover the major methods used for metabolomic studies in Drosophila and highlight new directions for future research. Copyright © 2017 by the Genetics Society of America.

Evaluation of Off-season Potential Breeding Sources for Spotted Wing Drosophila (Drosophila suzukii Matsumura) in Michigan.

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Bal, Harit K; Adams, Christopher; Grieshop, Matthew

2017-12-05

It has been suggested that fruit wastes including dropped and unharvested fruits, and fruit byproducts (i.e., pomace) found in fruit plantings and cideries or wine-making facilities could serve as potential off-season breeding sites for spotted wing Drosophila (Drosophila suzukii Matsumura (Diptera: Drosophilidae)). This idea, however, has yet to be widely tested. The goal of our study was to determine the potential of dropped fruit and fruit wastes as Fall spotted wing Drosophila breeding resources in Michigan, USA. Fruit waste samples were collected from 15 farms across the lower peninsula of Michigan and were evaluated for spotted wing Drosophila and other drosophilid emergence and used in host suitability bioassays. All of the dropped apples, pears, grapes, and raspberries and 40% of apple and 100% of grape fruit pomace evaluated were found to contain spotted wing Drosophila with the highest numbers collected from dropped grapes and pears. Greater spotted wing Drosophila recovery was found in fruit wastes at sites attached with cideries and wine-making facilities and with multiple cultivated fruit crops than sites with no cideries and only one crop. Females oviposited in raspberry, pear, apple, grape, apple pomace and grape pomace samples with the highest rates of reproduction in raspberries. Our results demonstrate that fruit wastes including dropped berry, pomme and stone fruits, as well as fruit compost may be important late season reproductive resources for spotted wing Drosophila. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Requirement of matrix metalloproteinase-1 for intestinal homeostasis in the adult Drosophila midgut

International Nuclear Information System (INIS)

Lee, Shin-Hae; Park, Joung-Sun; Kim, Young-Shin; Chung, Hae-Young; Yoo, Mi-Ae

2012-01-01

Stem cells are tightly regulated by both intrinsic and extrinsic signals as well as the extracellular matrix (ECM) for tissue homeostasis and regenerative capacity. Matrix metalloproteinases (MMPs), proteolytic enzymes, modulate the turnover of numerous substrates, including cytokine precursors, growth factors, and ECM molecules. However, the roles of MMPs in the regulation of adult stem cells are poorly understood. In the present study, we utilize the Drosophila midgut, which is an excellent model system for studying stem cell biology, to show that Mmp1 is involved in the regulation of intestinal stem cells (ISCs). The results showed that Mmp1 is expressed in the adult midgut and that its expression increases with age and with exposure to oxidative stress. Mmp1 knockdown or Timp-overexpressing flies and flies heterozygous for a viable, hypomorphic Mmp1 allele increased ISC proliferation in the gut, as shown by staining with an anti-phospho-histone H3 antibody and BrdU incorporation assays. Reduced Mmp1 levels induced intestinal hyperplasia, and the Mmp1depletion-induced ISC proliferation was rescued by the suppression of the EGFR signaling pathway, suggesting that Mmp1 regulates ISC proliferation through the EGFR signaling pathway. Furthermore, adult gut-specific knockdown and whole-animal heterozygotes of Mmp1 increased additively sensitivity to paraquat-induced oxidative stress and shortened lifespan. Our data suggest that Drosophila Mmp1 is involved in the regulation of ISC proliferation for maintenance of gut homeostasis. -- Highlights: ► Mmp1 is expressed in the adult midgut. ► Mmp1 is involved in the regulation of ISC proliferation activity. ► Mmp1-related ISC proliferation is associated with EGFR signaling. ► Mmp1 in the gut is required for the intestinal homeostasis and longevity.

Requirement of matrix metalloproteinase-1 for intestinal homeostasis in the adult Drosophila midgut

Energy Technology Data Exchange (ETDEWEB)

Lee, Shin-Hae; Park, Joung-Sun [Department of Molecular Biology, College of Natural Science, Pusan National University, Busan 609-735 (Korea, Republic of); Kim, Young-Shin [Research Institute of Genetic Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Chung, Hae-Young [Molecular Inflammation Research Center for Aging Intervention (MRCA), College of Pharmacy, Pusan National University, Busan 609-735 (Korea, Republic of); Yoo, Mi-Ae, E-mail: mayoo@pusan.ac.kr [Department of Molecular Biology, College of Natural Science, Pusan National University, Busan 609-735 (Korea, Republic of)

2012-03-10

Stem cells are tightly regulated by both intrinsic and extrinsic signals as well as the extracellular matrix (ECM) for tissue homeostasis and regenerative capacity. Matrix metalloproteinases (MMPs), proteolytic enzymes, modulate the turnover of numerous substrates, including cytokine precursors, growth factors, and ECM molecules. However, the roles of MMPs in the regulation of adult stem cells are poorly understood. In the present study, we utilize the Drosophila midgut, which is an excellent model system for studying stem cell biology, to show that Mmp1 is involved in the regulation of intestinal stem cells (ISCs). The results showed that Mmp1 is expressed in the adult midgut and that its expression increases with age and with exposure to oxidative stress. Mmp1 knockdown or Timp-overexpressing flies and flies heterozygous for a viable, hypomorphic Mmp1 allele increased ISC proliferation in the gut, as shown by staining with an anti-phospho-histone H3 antibody and BrdU incorporation assays. Reduced Mmp1 levels induced intestinal hyperplasia, and the Mmp1depletion-induced ISC proliferation was rescued by the suppression of the EGFR signaling pathway, suggesting that Mmp1 regulates ISC proliferation through the EGFR signaling pathway. Furthermore, adult gut-specific knockdown and whole-animal heterozygotes of Mmp1 increased additively sensitivity to paraquat-induced oxidative stress and shortened lifespan. Our data suggest that Drosophila Mmp1 is involved in the regulation of ISC proliferation for maintenance of gut homeostasis. -- Highlights: Black-Right-Pointing-Pointer Mmp1 is expressed in the adult midgut. Black-Right-Pointing-Pointer Mmp1 is involved in the regulation of ISC proliferation activity. Black-Right-Pointing-Pointer Mmp1-related ISC proliferation is associated with EGFR signaling. Black-Right-Pointing-Pointer Mmp1 in the gut is required for the intestinal homeostasis and longevity.

A Drosophila Model to Image Phagosome Maturation

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Douglas A. Brooks

2013-03-01

Full Text Available Phagocytosis involves the internalization of extracellular material by invagination of the plasma membrane to form intracellular vesicles called phagosomes, which have functions that include pathogen degradation. The degradative properties of phagosomes are thought to be conferred by sequential fusion with endosomes and lysosomes; however, this maturation process has not been studied in vivo. We employed Drosophila hemocytes, which are similar to mammalian professional macrophages, to establish a model of phagosome maturation. Adult Drosophila females, carrying transgenic Rab7-GFP endosome and Lamp1-GFP lysosome markers, were injected with E. coli DH5α and the hemocytes were collected at 15, 30, 45 and 60 minutes after infection. In wild-type females, E. coli were detected within enlarged Rab7-GFP positive phagosomes at 15 to 45 minutes after infection; and were also observed in enlarged Lamp1-GFP positive phagolysosomes at 45 minutes. Two-photon imaging of hemocytes in vivo confirmed this vesicle morphology, including enlargement of Rab7-GFP and Lamp1-GFP structures that often appeared to protrude from hemocytes. The interaction of endosomes and lysosomes with E. coli phagosomes observed in Drosophila hemocytes was consistent with that previously described for phagosome maturation in human ex vivo macrophages. We also tested our model as a tool for genetic analysis using 14-3-3e mutants, and demonstrated altered phagosome maturation with delayed E. coli internalization, trafficking and/or degradation. These findings demonstrate that Drosophila hemocytes provide an appropriate, genetically amenable, model for analyzing phagosome maturation ex vivo and in vivo.

Use of Drosophila to study DNA repair

International Nuclear Information System (INIS)

Boyd, J.B.; Harris, P.V.; Sakaguchi, K.

1988-01-01

This paper discusses Drosophila, the premier metazoan organism for analyzing many fundamental features of eukaryotic gene regulation. The authors present adaptations of several approaches for studying DNA repair to an analysis of repair-defective mutants in Drosophila. A current understanding of Drosophila DNA repair is described

Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

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Park Sung

2008-12-01

Full Text Available Abstract Background Caspase-3, a principal apoptotic effector that cleaves the majority of cellular substrates, is an important medicinal target for the treatment of cancers and neurodegenerative diseases. Large amounts of the protein are required for drug discovery research. However, previous efforts to express the full-length caspase-3 gene in E. coli have been unsuccessful. Results Overproducers of thrombin-activatable full-length caspase-3 precursors were prepared by engineering the auto-activation sites of caspase-3 precursor into a sequence susceptible to thrombin hydrolysis. The engineered precursors were highly expressed as soluble proteins in E. coli and easily purified by affinity chromatography, to levels of 10–15 mg from 1 L of E. coli culture, and readily activated by thrombin digestion. Kinetic evaluation disclosed that thrombin digestion enhanced catalytic activity (kcat/KM of the precursor proteins by two orders of magnitude. Conclusion A novel method for a large-scale preparation of active caspase-3 was developed by a strategic engineering to lack auto-activation during expression with amino acid sequences susceptible to thrombin, facilitating high-level expression in E. coli. The precursor protein was easily purified and activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3 in high yields.

[Changes in Ca(2+)concentration and caspase-3 expression and their relationship in Raji cells exposed to electromagnetic radiation].

Science.gov (United States)

Wang, Wei; Liu, Huan-xin; Wang, De-wen; Zuo, Hong-yan; Peng, Rui-yun

2013-02-01

To study the effects of electromagnetic pulse (EMP), S-band high power microwave (S-HPM), and X-band high power microwave (X-HPM) on the Ca(2+) concentration and caspase-3 expression in Raji cells and the relationship between Ca(2+) concentration and caspase-3 expression, and to investigate the regulatory mechanism of electromagnetic radiation damage. Raji cells were cultured conventionally. Some cells were irradiated by EMP, S-HPM, and X-HPM in the logarithmic growth phase for 6 hours and then collected; others received sham irradiation as a control. The Ca(2+) concentration in the cells was measured by laser scanning confocal microscope; the caspase-3 expression in the cells was evaluated by Western blot. Compared with the control group (Ca(2+) fluorescence intensity = 43.08 ± 2.08; caspase-3 expression level = 0.444 ± 0.13), the EMP,S-HPM, and X-HPM groups had significantly increased Ca(2+) concentrations, with Ca(2+) fluorescence intensities of 69.56 ± 1.71, 50.06 ± 1.89, and 70.68 ± 1.59, respectively (P < 0.01), and had upregulated caspase-3 expression, with expression levels of 0.964 ± 0.12, 0.586 ± 0.16, and 0.970 ± 0.07, respectively (P < 0.01). Each of the EMP and X-HPM groups had significantly higher Ca(2+) fluorescence intensity and caspase-3 expression level than the S-HPM group (P < 0.01), but there were no significant differences between the EMP and X-HPM groups. The linear regression analysis showed that the caspase-3 expression was upregulated as the Ca(2+) concentration increased, with a positive correlation between them (P < 0.01). EMP, S-HPM, and X-HPM cause damage probably by increasing the Ca(2+) concentration in cells and in turn inducing caspase-3 overexpression.

Retinal Cell Death Caused by Sodium Iodate Involves Multiple Caspase-Dependent and Caspase-Independent Cell-Death Pathways

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Jasmin Balmer

2015-07-01

Full Text Available Herein, we have investigated retinal cell-death pathways in response to the retina toxin sodium iodate (NaIO3 both in vivo and in vitro. C57/BL6 mice were treated with a single intravenous injection of NaIO3 (35 mg/kg. Morphological changes in the retina post NaIO3 injection in comparison to untreated controls were assessed using electron microscopy. Cell death was determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL staining. The activation of caspases and calpain was measured using immunohistochemistry. Additionally, cytotoxicity and apoptosis in retinal pigment epithelial (RPE cells, primary retinal cells, and the cone photoreceptor (PRC cell line 661W were assessed in vitro after NaIO3 treatment using the ApoToxGlo™ assay. The 7-AAD/Annexin-V staining was performed and necrostatin (Nec-1 was administered to the NaIO3-treated cells to confirm the results. In vivo, degenerating RPE cells displayed a rounded shape and retracted microvilli, whereas PRCs featured apoptotic nuclei. Caspase and calpain activity was significantly upregulated in retinal sections and protein samples from NaIO3-treated animals. In vitro, NaIO3 induced necrosis in RPE cells and apoptosis in PRCs. Furthermore, Nec-1 significantly decreased NaIO3-induced RPE cell death, but had no rescue effect on treated PRCs. In summary, several different cell-death pathways are activated in retinal cells as a result of NaIO3.

1,3-Dichloro-2-propanol inhibits progesterone production through the expression of steroidogenic enzymes and cAMP concentration in Leydig cells.

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Sun, Jianxia; Bai, Shun; Bai, Weibin; Zou, Feiyan; Zhang, Lei; Li, Guoqiang; Hu, Yunfeng; Li, Mingwei; Yan, Rian; Su, Zhijian; Huang, Yadong

2014-07-01

1,3-Dichloro-2-propanol (1,3-DCP) is a well-known food processing contaminant that has been shown to impede male reproductive function. However, its mechanism of action remains elusive. In this study, the effects of 1,3-DCP on progesterone production were investigated using the R2C Leydig cell model. 1,3-DCP significantly reduced cell viability from 7.48% to 97.4% at doses comprised between 0.5 and 6mM. Single cell gel/comet assays and atomic force microscopy assays showed that 1,3-DCP induced early phase cell apoptosis. In addition, 1,3-DCP significantly reduced progesterone production detected by radioimmunoassay (RIA). The results from quantitative polymerase chain reaction and western blotting demonstrated that the mRNA expression levels of steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase were significantly down-regulated in R2C cells. Particularly, the change rhythm of Star expression was highly consistent with progesterone production. Furthermore, the cyclic adenosine monophosphate (cAMP) and the mitochondrial membrane potential mediated by ROS, which are involved in regulating progesterone synthesis were also decreased in response to the 1,3-DCP treatment. Overall, the data presented here suggested that 1,3-DCP interferes with the male steroidogenic capacity mainly by down-regulating the level of cAMP and the key enzymes involved in the androgen synthesis pathway. Copyright © 2014 Elsevier Ltd. All rights reserved.

TORC1 Inhibition by Rapamycin Promotes Antioxidant Defences in a Drosophila Model of Friedreich's Ataxia.

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Pablo Calap-Quintana

Full Text Available Friedreich's ataxia (FRDA, the most common inherited ataxia in the Caucasian population, is a multisystemic disease caused by a significant decrease in the frataxin level. To identify genes capable of modifying the severity of the symptoms of frataxin depletion, we performed a candidate genetic screen in a Drosophila RNAi-based model of FRDA. We found that genetic reduction in TOR Complex 1 (TORC1 signalling improves the impaired motor performance phenotype of FRDA model flies. Pharmacologic inhibition of TORC1 signalling by rapamycin also restored this phenotype and increased the lifespan and ATP levels. Furthermore, rapamycin reduced the altered levels of malondialdehyde + 4-hydroxyalkenals and total glutathione of the model flies. The rapamycin-mediated protection against oxidative stress is due in part to an increase in the transcription of antioxidant genes mediated by cap-n-collar (Drosophila ortholog of Nrf2. Our results suggest that autophagy is indeed necessary for the protective effect of rapamycin in hyperoxia. Rapamycin increased the survival and aconitase activity of model flies subjected to high oxidative insult, and this improvement was abolished by the autophagy inhibitor 3-methyladenine. These results point to the TORC1 pathway as a new potential therapeutic target for FRDA and as a guide to finding new promising molecules for disease treatment.

Mouse strain-dependent caspase activation during acetaminophen hepatotoxicity does not result in apoptosis or modulation of inflammation

Energy Technology Data Exchange (ETDEWEB)

Williams, C. David [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Koerner, Michael R., E-mail: mkoern2@illinois.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Lampe, Jed N. [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Farhood, Anwar [Department of Pathology, Brackenridge Hospital, Austin, TX 78701 (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States)

2011-12-15

The mechanisms of acetaminophen (APAP)-mediated hepatic oncotic necrosis have been extensively characterized. However, it was recently demonstrated that fed CD-1 mice have a transient caspase activation which initiates apoptosis. To evaluate these findings in more detail, outbred (Swiss Webster, SW) and inbred (C57BL/6) mice were treated with APAP with or without pan-caspase inhibitor and compared to the apoptosis model of galactosamine (GalN)/endotoxin (ET). Fasted or fed APAP-treated C57BL/6 mice showed no evidence of caspase-3 processing or activity. Interestingly, a minor, temporary increase in caspase-3 processing and activity (150% above baseline) was observed after APAP treatment only in fed SW mice. The degree of caspase-3 activation in SW mice after APAP was minor compared to that observed in GalN/ET-treated mice (1600% above baseline). The pancaspase inhibitor attenuated caspase activation and resulted in increased APAP-induced injury (plasma ALT, necrosis scoring). The caspase inhibitor did not affect apoptosis because regardless of treatment only < 0.5% of hepatocytes showed consistent apoptotic morphology after APAP. In contrast, > 20% apoptotic cells were observed in GalN/ET-treated mice. Presence of the caspase inhibitor altered hepatic glutathione levels in SW mice, which could explain the exacerbation of injury. Additionally, the infiltration of hepatic neutrophils was not altered by the fed state of either mouse strain. Conclusion: Minor caspase-3 activation without apoptotic cell death can be observed only in fed mice of some outbred strains. These findings suggest that although the severity of APAP-induced liver injury varies between fed and fasted animals, the mechanism of cell death does not fundamentally change. -- Highlights: Black-Right-Pointing-Pointer During acetaminophen overdose caspase-3 can be activated in fed mice of certain outbred strains. Black-Right-Pointing-Pointer Hepatic ATP levels are not the determining factor for caspase

Acute and long-term outcomes in a Drosophila melanogaster model of classic galactosemia occur independently of galactose-1-phosphate accumulation

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Jennifer M. I. Daenzer

2016-11-01

Full Text Available Classic galactosemia (CG is a potentially lethal inborn error of metabolism that results from the profound loss of galactose-1-phosphate uridylyltransferase (GALT, the second enzyme in the Leloir pathway of galactose metabolism. Neonatal detection and dietary restriction of galactose minimizes or resolves the acute sequelae of CG, but fails to prevent the long-term complications experienced by a majority of patients. One of the substrates of GALT, galactose-1-phosphate (Gal-1P, accumulates to high levels in affected infants, especially following milk exposure, and has been proposed as the key mediator of acute and long-term pathophysiology in CG. However, studies of treated patients demonstrate no association between red blood cell Gal-1P level and long-term outcome severity. Here, we used genetic, epigenetic and environmental manipulations of a Drosophila melanogaster model of CG to test the role of Gal-1P as a candidate mediator of outcome in GALT deficiency. Specifically, we both deleted and knocked down the gene encoding galactokinase (GALK in control and GALT-null Drosophila, and assessed the acute and long-term outcomes of the resulting animals in the presence and absence of dietary galactose. GALK is the first enzyme in the Leloir pathway of galactose metabolism and is responsible for generating Gal-1P in humans and Drosophila. Our data confirmed that, as expected, loss of GALK lowered or eliminated Gal-1P accumulation in GALT-null animals. However, we saw no concomitant rescue of larval survival or adult climbing or fecundity phenotypes. Instead, we saw that loss of GALK itself was not benign and in some cases phenocopied or exacerbated the outcome seen in GALT-null animals. These findings strongly contradict the long-standing hypothesis that Gal-1P alone underlies pathophysiology of acute and long-term outcomes in GALT-null Drosophila and suggests that other metabolite(s of galactose, and/or other pathogenic factors, might be involved.

Transcripts of mobile element MDG1 during ontogenesis of Drosophila melanogaster

International Nuclear Information System (INIS)

Kuvakina, A.I.; Nurminskii, D.I.; Kogan, G.L.; Gvozdev, V.A.

1989-01-01

It has been demonstrated by Northern hybridization using a single-stranded labeled probes that the number of MDG1 transcripts as well as their size change during ontogenesis of Drosophila. The transcripts of MDG1 were not found in unfertilized eggs. The full-length transcript of MDG1 (about 7 kb long) appears in the embryonic and larval cells, and its quantity sharply increases in pupae and adults. A transcript of about 5 kb length is also found in the pupae and adults. Another, about 2 kb long transcript forms in the embryos, pupae and adults, which is absent in larvae. The main transcript in the larval cells, complementary to the inner part of the body of MDG1, is about 1 kb long. The transcription level of MDG1 and the mobile element copia do not change under heat shock at adult stage

Detecting novel low-abundant transcripts in Drosophila

DEFF Research Database (Denmark)

Lee, Sanggyu; Bao, Jingyue; Zhou, Guolin

2005-01-01

Increasing evidence suggests that low-abundant transcripts may play fundamental roles in biological processes. In an attempt to estimate the prevalence of low-abundant transcripts in eukaryotic genomes, we performed a transcriptome analysis in Drosophila using the SAGE technique. We collected 244......,313 SAGE tags from transcripts expressed in Drosophila embryonic, larval, pupae, adult, and testicular tissue. From these SAGE tags, we identified 40,823 unique SAGE tags. Our analysis showed that 55% of the 40,823 unique SAGE tags are novel without matches in currently known Drosophila transcripts...... in the Drosophila genome. Our study reveals the presence of a significant number of novel low-abundant transcripts in Drosophila, and highlights the need to isolate these novel low-abundant transcripts for further biological studies. Udgivelsesdato: 2005-Jun...

Evaluation of a Diabetes Coach Program Aimed to Improve the Care of Children and Youth With Type 1 Diabetes and With Compromised Control.

Science.gov (United States)

Luca, Paola; Haugrud, Bodiel; Husband, Allison; Dawrant, Jonathan; Pacaud, Danièle

2018-01-31

To evaluate the impact of the Diabetes Coach Program (DCP) on glycated hemoglobin (A1C) levels in youth with type 1 diabetes. The youth were referred to and participated in the DCP between October 2011 and May 2016. The Diabetes Coach visited families in their homes every 1 to 2 weeks and updated patients' diabetes teams regularly. A1C levels before the DCP were compared with A1C levels during and after discharge from the DCP. Six participating families completed satisfaction surveys via telephone. The DCP included 23 participants (43% male; median age, 11 years; range, 8.8 to 14.5 years); median duration of type 1 diabetes, 1.7 years (range, 0.1 to 6.3 years); median time in the DCP, 1.5 years (range, 0.2 to 2.5 years). During involvement in the program, median A1C levels decreased from baselines of 11.1% (range, 8.9% to 15.3%) to 10.2% (range, 7.6% to 12.4%) (p=0.0028). For 11 of 13 patients discharged from the DCP, the most recent median A1C levels, 11.2% (range, 9.1% to 13.6%), an average of 2.4 years later, were not different from the initial A1C levels (p=0.85). Family feedback was overwhelmingly positive. Participation in the DCP decreased A1C values in pediatric patients; however, the levels were not sustained after visits stopped. Pediatric health coaches may play an important role in the management of type 1 diabetes, but further research is needed to explore their benefits and how positive effects can be sustained. Copyright © 2018 Diabetes Canada. Published by Elsevier Inc. All rights reserved.

Novel Triazole linked 2-phenyl benzoxazole derivatives induce apoptosis by inhibiting miR-2, miR-13 and miR-14 function in Drosophila melanogaster.

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Mondal, Tanmoy; Lavanya, A V S; Mallick, Akash; Dadmala, Tulshiram L; Kumbhare, Ravindra M; Bhadra, Utpal; Bhadra, Manika Pal

2017-06-01

Apoptosis is an important phenomenon in multi cellular organisms for maintaining tissue homeostasis and embryonic development. Defect in apoptosis leads to a number of disorders like- autoimmune disorder, immunodeficiency and cancer. 21-22 nucleotides containing micro RNAs (miRNAs/miRs) function as a crucial regulator of apoptosis alike other cellular pathways. Recently, small molecules have been identified as a potent inducer of apoptosis. In this study, we have identified novel Triazole linked 2-phenyl benzoxazole derivatives (13j and 13h) as a negative regulator of apoptosis inhibiting micro RNAs (miR-2, miR-13 and miR-14) in a well established in vivo model Drosophila melanogaster where the process of apoptosis is very similar to human apoptosis. These compounds inhibit miR-2, miR-13 and miR-14 activity at their target sites, which induce an increased caspase activity, and in turn influence the caspase dependent apoptotic pathway. These two compounds also increase the mitochondrial reactive oxygen species (ROS) level to trigger apoptotic cell death.

TSA-induced cell death in prostate cancer cell lines is caspase-2 dependent and involves the PIDDosome.

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Taghiyev, Agshin F; Guseva, Natalya V; Glover, Rebecca A; Rokhlin, Oskar W; Cohen, Michael B

2006-09-01

The histone deacetylase inhibitor Trichostatin A (TSA) has previously been found to induce caspase activity in the human prostate cancer cell lines DU145 and LNCaP. TSA treatment resulted in the release of cytochrome c and Smac/DIABLO from mitochondria in DU145, and activation of caspase-9 in both cell lines. We concluded that TSA mediated its effect via the mitochondrial pathway. The aim of the current study was to determine how TSA initiated the caspase cascade. The results revealed that caspase-2 plays an important role in TSA-induced apoptosis. Inhibition of caspase-2 by siRNA or expression of caspase-2dn substantially decreased caspase activity after TSA treatment in both cell lines, siRNA caspase-2 also inhibited TSA-induced cell death. Caspase-2 acts upstream of caspase-8 and -9 and mediates mitochondrial cytochrome c release. Coimmunoprecipitation experiments show that caspase-2 formed protein complexes with RADD/RAIDD and PIDD. Together, these data indicate that caspase-2 initiates caspase cascade after TSA treatment and involves the formation of the PIDDosome.

The molecular mechanisms of OPA1-mediated optic atrophy in Drosophila model and prospects for antioxidant treatment.

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Will Yarosh

2008-01-01

Full Text Available Mutations in optic atrophy 1 (OPA1, a nuclear gene encoding a mitochondrial protein, is the most common cause for autosomal dominant optic atrophy (DOA. The condition is characterized by gradual loss of vision, color vision defects, and temporal optic pallor. To understand the molecular mechanism by which OPA1 mutations cause optic atrophy and to facilitate the development of an effective therapeutic agent for optic atrophies, we analyzed phenotypes in the developing and adult Drosophila eyes produced by mutant dOpa1 (CG8479, a Drosophila ortholog of human OPA1. Heterozygous mutation of dOpa1 by a P-element or transposon insertions causes no discernable eye phenotype, whereas the homozygous mutation results in embryonic lethality. Using powerful Drosophila genetic techniques, we created eye-specific somatic clones. The somatic homozygous mutation of dOpa1 in the eyes caused rough (mispatterning and glossy (decreased lens and pigment deposition eye phenotypes in adult flies; this phenotype was reversible by precise excision of the inserted P-element. Furthermore, we show the rough eye phenotype is caused by the loss of hexagonal lattice cells in developing eyes, suggesting an increase in lattice cell apoptosis. In adult flies, the dOpa1 mutation caused an increase in reactive oxygen species (ROS production as well as mitochondrial fragmentation associated with loss and damage of the cone and pigment cells. We show that superoxide dismutase 1 (SOD1, Vitamin E, and genetically overexpressed human SOD1 (hSOD1 is able to reverse the glossy eye phenotype of dOPA1 mutant large clones, further suggesting that ROS play an important role in cone and pigment cell death. Our results show dOpa1 mutations cause cell loss by two distinct pathogenic pathways. This study provides novel insights into the pathogenesis of optic atrophy and demonstrates the promise of antioxidants as therapeutic agents for this condition.

Caspase-mediated apoptosis induction in zebrafish cerebellar Purkinje neurons.

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Weber, Thomas; Namikawa, Kazuhiko; Winter, Barbara; Müller-Brown, Karina; Kühn, Ralf; Wurst, Wolfgang; Köster, Reinhard W

2016-11-15

The zebrafish is a well-established model organism in which to study in vivo mechanisms of cell communication, differentiation and function. Existing cell ablation methods are either invasive or they rely on the cellular expression of prokaryotic enzymes and the use of antibiotic drugs as cell death-inducing compounds. We have recently established a novel inducible genetic cell ablation system based on tamoxifen-inducible Caspase 8 activity, thereby exploiting mechanisms of cell death intrinsic to most cell types. Here, we prove its suitability in vivo by monitoring the ablation of cerebellar Purkinje cells (PCs) in transgenic zebrafish that co-express the inducible caspase and a fluorescent reporter. Incubation of larvae in tamoxifen for 8 h activated endogenous Caspase 3 and cell death, whereas incubation for 16 h led to the near-complete loss of PCs by apoptosis. We observed synchronous cell death autonomous to the PC population and phagocytosing microglia in the cerebellum, reminiscent of developmental apoptosis in the forebrain. Thus, induction of apoptosis through targeted activation of caspase by tamoxifen (ATTAC TM ) further expands the repertoire of genetic tools for conditional interrogation of cellular functions. © 2016. Published by The Company of Biologists Ltd.

Ebi/AP-1 suppresses pro-apoptotic genes expression and permits long-term survival of Drosophila sensory neurons.

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Young-Mi Lim

Full Text Available Sensory organs are constantly exposed to physical and chemical stresses that collectively threaten the survival of sensory neurons. Failure to protect stressed neurons leads to age-related loss of neurons and sensory dysfunction in organs in which the supply of new sensory neurons is limited, such as the human auditory system. Transducin β-like protein 1 (TBL1 is a candidate gene for ocular albinism with late-onset sensorineural deafness, a form of X-linked age-related hearing loss. TBL1 encodes an evolutionarily conserved F-box-like and WD40 repeats-containing subunit of the nuclear receptor co-repressor/silencing mediator for retinoid and thyroid hormone receptor and other transcriptional co-repressor complexes. Here we report that a Drosophila homologue of TBL1, Ebi, is required for maintenance of photoreceptor neurons. Loss of ebi function caused late-onset neuronal apoptosis in the retina and increased sensitivity to oxidative stress. Ebi formed a complex with activator protein 1 (AP-1 and was required for repression of Drosophila pro-apoptotic and anti-apoptotic genes expression. These results suggest that Ebi/AP-1 suppresses basal transcription levels of apoptotic genes and thereby protects sensory neurons from degeneration.

Hearing regulates Drosophila aggression.

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Versteven, Marijke; Vanden Broeck, Lies; Geurten, Bart; Zwarts, Liesbeth; Decraecker, Lisse; Beelen, Melissa; Göpfert, Martin C; Heinrich, Ralf; Callaerts, Patrick

2017-02-21

Aggression is a universal social behavior important for the acquisition of food, mates, territory, and social status. Aggression in Drosophila is context-dependent and can thus be expected to involve inputs from multiple sensory modalities. Here, we use mechanical disruption and genetic approaches in Drosophila melanogaster to identify hearing as an important sensory modality in the context of intermale aggressive behavior. We demonstrate that neuronal silencing and targeted knockdown of hearing genes in the fly's auditory organ elicit abnormal aggression. Further, we show that exposure to courtship or aggression song has opposite effects on aggression. Our data define the importance of hearing in the control of Drosophila intermale aggression and open perspectives to decipher how hearing and other sensory modalities are integrated at the neural circuit level.

The Significance of Caspase-Cleaved Cytokeratin 18 in Pleural Effusion

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Lee, Keu Sung; Chung, Joo Yang; Jung, Yun Jung; Chung, Wou Young; Park, Joo Hun; Sheen, Seung Soo; Lee, Kyi Beom

2014-01-01

Background Apoptosis plays a role in the development of pleural effusion. Caspase-cleaved cytokeratin 18, a marker for epithelial cell apoptosis, was evaluated in pleural effusion. Methods A total of 79 patients with pleural effusion were enrolled. The underlying causes were lung cancer (n=24), parapneumonic effusion (n=15), tuberculous effusion (n=28), and transudates (n=12). The levels of M30, an epitope of caspase-cleaved cytokeratin 18, were measured in blood and pleural fluids using enzyme-linked immunosorbent assay along with routine cellular and biochemical parameters. The expression of M30 was evaluated in the pleural tissues using immunohistochemistry for M30. Results The M30 levels in pleural fluid were significantly higher in patients with tuberculosis (2,632.1±1,467.3 U/mL) than in patients with lung cancer (956.5±618.5 U/mL), parapneumonic effusion (689.9±413.6 U/mL), and transudates (273.6±144.5 U/mL; all peffusion from all other effusions was 0.93. In the immunohistochemical analysis of M30, all pathologic types of cancer cells showed moderate to high expression, and the epithelioid cells in granulomas showed high expression in tuberculous pleural tissues. Conclusion Caspase-cleaved cytokeratin 18 was most prominently observed in tuberculous pleural effusion and showed utility as a clinical marker. The main source of M30 was found to be the epithelioid cells of granulomas in tuberculous pleural tissues. PMID:24523813

Lack of caspase-3 attenuates immobilization-induced muscle atrophy and loss of tension generation along with mitigation of apoptosis and inflammation

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Zhu, Shimei; Nagashima, Michio; Khan, Mahammad A.S; Yasuhara, Shingo; Kaneki, Masao; Jeevendra Martyn, J. A.

2012-01-01

Introduction Immobilization by casting induces disuse muscle atrophy (DMA). Methods Using wild type (WT) and caspase-3 knockout (KO) mice, we evaluated the effect of caspase-3 on muscle mass, apoptosis and inflammation during DMA. Results Caspase-3 deficiency significantly attenuated muscle mass decrease [gastrocnemius: 28 ± 1% in KO vs. 41 ± 3% in WT; soleus: 47 ± 2% in KO vs. 56 ± 2% in WT; (P immobilized versus contralateral hindlimb. Lack of caspase-3 decreased immobilization-induced increased apoptotic myonuclei (3.2-fold) and macrophage infiltration (2.2-fold) in soleus muscle and attenuated increased monocyte chemoattractant protein-1 mRNA expression (2-fold in KO vs. 18-fold in WT) in gastrocnemius. Conclusion Caspase-3 plays a key role in DMA and associated decreased tension, presumably by acting on the apoptosis and inflammation pathways. PMID:23401051

Snipper, an Eri1 homologue, affects histone mRNA abundance and is crucial for normal Drosophila melanogaster development.

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Alexiadis, Anastasios; Delidakis, Christos; Kalantidis, Kriton

2017-07-01

The conserved 3'-5' RNA exonuclease ERI1 is implicated in RNA interference inhibition, 5.8S rRNA maturation and histone mRNA maturation and turnover. The single ERI1 homologue in Drosophila melanogaster Snipper (Snp) is a 3'-5' exonuclease, but its in vivo function remains elusive. Here, we report Snp requirement for normal Drosophila development, since its perturbation leads to larval arrest and tissue-specific downregulation results in abnormal tissue development. Additionally, Snp directly interacts with histone mRNA, and its depletion results in drastic reduction in histone transcript levels. We propose that Snp protects the 3'-ends of histone mRNAs and upon its absence, histone transcripts are readily degraded. This in turn may lead to cell cycle delay or arrest, causing growth arrest and developmental perturbations. © 2017 Federation of European Biochemical Societies.

Programmed Cell Death and Caspase Functions During Neural Development.

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Yamaguchi, Yoshifumi; Miura, Masayuki

2015-01-01

Programmed cell death (PCD) is a fundamental component of nervous system development. PCD serves as the mechanism for quantitative matching of the number of projecting neurons and their target cells through direct competition for neurotrophic factors in the vertebrate peripheral nervous system. In addition, PCD plays roles in regulating neural cell numbers, canceling developmental errors or noise, and tissue remodeling processes. These findings are mainly derived from genetic studies that prevent cells from dying by apoptosis, which is a major form of PCD and is executed by activation of evolutionarily conserved cysteine protease caspases. Recent studies suggest that caspase activation can be coordinated in time and space at multiple levels, which might underlie nonapoptotic roles of caspases in neural development in addition to apoptotic roles. © 2015 Elsevier Inc. All rights reserved.

Chronic Obstructive Pulmonary Disease-Derived Circulating Cells Release IL-18 and IL-33 under Ultrafine Particulate Matter Exposure in a Caspase-1/8-Independent Manner

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Gianluigi De Falco

2017-10-01

Full Text Available Chronic obstructive pulmonary disease (COPD is considered the fourth-leading causes of death worldwide; COPD is caused by inhalation of noxious indoor and outdoor particles, especially cigarette smoke that represents the first risk factor for this respiratory disorder. To mimic the effects of particulate matter on COPD, we isolated peripheral blood mononuclear cells (PBMCs and treated them with combustion-generated ultrafine particles (UFPs obtained from two different fuel mixtures, namely, pure ethylene and a mixture of ethylene and dimethylfuran (the latter mimicking the combustion of biofuels. UFPs were separated in two fractions: (1 sub-10 nm particles, named nano organic carbon (NOC particles and (2 primarily soot particles of 20–40 nm and their agglomerates (200 nm. We found that both NOC and soot UFPs induced the release of IL-18 and IL-33 from unstable/exacerbated COPD-derived PBMCs. This effect was associated with higher levels of mitochondrial dysfunction and derived reactive oxygen species, which were higher in PBMCs from unstable COPD patients after combustion-generated UFP exposure. Moreover, lower mRNA expression of the repairing enzyme OGG1 was associated with the higher levels of 8-OH-dG compared with non-smoker and smokers. It was interesting that IL-18 and IL-33 release from PBMCs of unstable COPD patients was not NOD-like receptor 3/caspase-1 or caspase-8-dependent, but rather correlated to caspase-4 release. This effect was not evident in stable COPD-derived PBMCs. Our data suggest that combustion-generated UFPs induce the release of caspase-4-dependent inflammasome from PBMCs of COPD patients compared with healthy subjects, shedding new light into the biology of this key complex in COPD.

Anticancer Effect of Ursodeoxycholic Acid in Human Oral Squamous Carcinoma HSC-3 Cells through the Caspases

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Pang, Liang; Zhao, Xin; Liu, Weiwei; Deng, Jiang; Tan, Xiaotong; Qiu, Lihua

2015-01-01

Bear bile was used as a traditional medicine or tonic in East Asia, and ursodeoxycholic acid (UDCA) is the most important compound in bear bile. Further, synthetic UDCA is also used in modern medicine and nutrition; therefore, its further functional effects warrant research, in vitro methods could be used for the fundamental research of its anticancer effects. In this study, the apoptotic effects of UDCA in human oral squamous carcinoma HSC-3 cells through the activation of caspases were observed by the experimental methods of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay, DAPI (4’,6-diamidino-2-phenylindole) staining, flow cytometry analysis, RT-PCR (reverse transcription-polymerase chain reaction) assay and Western blot assay after HSC-3 cells were treated by different concentrations of UDCA. With 0 to 400 μg/mL UDCA treatment, UDCA had strong growth inhibitory effects in HSC-3 cells, but had almost no effect in HOK normal oral cells. At concentrations of 100, 200 and 400 μg/mL, UDCA could induce apoptosis compared to untreated control HSC-3 cells. Treatment of 400 μg/mL UDCA could induce more apoptotic cancer cells than 100 and 200 μg/mL treatment; the sub-G1 DNA content of 400 μg/mL UDCA treated cancer cells was 41.3% versus 10.6% (100 μg/mL) and 22.4% (200 μg/mL). After different concentrations of UDCA treatment, the mRNA and protein expressions of caspase-3, caspase-8, caspase-9, Bax, Fas/FasL (Fas ligand), TRAIL (TNF-related apoptosis-inducing ligand), DR4 (death receptor 4) and DR5 (death receptor 5) were increased in HSC-3 cells, and mRNA and protein expressions of Bcl-2 (B-cell lymphoma 2), Bcl-xL (B-cell lymphoma-extra large), XIAP (X-linked inhibitor of apoptosis protein), cIAP-1 (cellular inhibitor of apoptosis 1), cIAP-2 (cellular inhibitor of apoptosis 2) and survival were decreased. Meanwhile, at the highest concentration of 400 μg/mL, caspase-3, caspase-8, caspase-9, Bax, Fas/FasL, TRAIL, DR4, DR5, and Iκ

Anticancer Effect of Ursodeoxycholic Acid in Human Oral Squamous Carcinoma HSC-3 Cells through the Caspases

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Liang Pang

2015-05-01

Full Text Available Bear bile was used as a traditional medicine or tonic in East Asia, and ursodeoxycholic acid (UDCA is the most important compound in bear bile. Further, synthetic UDCA is also used in modern medicine and nutrition; therefore, its further functional effects warrant research, in vitro methods could be used for the fundamental research of its anticancer effects. In this study, the apoptotic effects of UDCA in human oral squamous carcinoma HSC-3 cells through the activation of caspases were observed by the experimental methods of MTT (3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide assay, DAPI (4’,6-diamidino-2-phenylindole staining, flow cytometry analysis, RT-PCR (reverse transcription-polymerase chain reaction assay and Western blot assay after HSC-3 cells were treated by different concentrations of UDCA. With 0 to 400 μg/mL UDCA treatment, UDCA had strong growth inhibitory effects in HSC-3 cells, but had almost no effect in HOK normal oral cells. At concentrations of 100, 200 and 400 μg/mL, UDCA could induce apoptosis compared to untreated control HSC-3 cells. Treatment of 400 μg/mL UDCA could induce more apoptotic cancer cells than 100 and 200 μg/mL treatment; the sub-G1 DNA content of 400 μg/mL UDCA treated cancer cells was 41.3% versus 10.6% (100 μg/mL and 22.4% (200 μg/mL. After different concentrations of UDCA treatment, the mRNA and protein expressions of caspase-3, caspase-8, caspase-9, Bax, Fas/FasL (Fas ligand, TRAIL (TNF-related apoptosis-inducing ligand, DR4 (death receptor 4 and DR5 (death receptor 5 were increased in HSC-3 cells, and mRNA and protein expressions of Bcl-2 (B-cell lymphoma 2, Bcl-xL (B-cell lymphoma-extra large, XIAP (X-linked inhibitor of apoptosis protein, cIAP-1 (cellular inhibitor of apoptosis 1, cIAP-2 (cellular inhibitor of apoptosis 2 and survival were decreased. Meanwhile, at the highest concentration of 400 μg/mL, caspase-3, caspase-8, caspase-9, Bax, Fas/FasL, TRAIL, DR4, DR5, and

A comparison of Frost expression among species and life stages of Drosophila.

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Bing, X; Zhang, J; Sinclair, Brent J

2012-02-01

Frost (Fst) is a gene associated with cold exposure in Drosophila melanogaster. We used real-time PCR to assess whether cold exposure induces expression of Fst in 10 different life stages of D. melanogaster, and adults of seven other Drosophila species. We exposed groups of individuals to 0 °C (2 h), followed by 1 h recovery (22 °C). Frost was significantly upregulated in response to cold in eggs, third instar larvae, and 2- and 5-day-old male and female adults in D. melanogaster. Life stages in which cold did not upregulate Fst had high constitutive expression. Frost is located on the opposite strand of an intron of Diuretic hormone (DH), but cold exposure did not upregulate DH. Frost orthologues were identified in six other species within the Melanogaster group (Drosophila sechellia, Drosophila simulans, Drosophila yakuba, Drosophila erecta, Drosophila ananassae and Drosophila mauritiana). Frost orthologues were upregulated in response to cold exposure in both sexes in adults of all of these species. The predicted structure of a putative Frost consensus protein shows highly conserved tandem repeats of motifs involved in cell signalling (PEST and TRAF2), suggesting that Fst might encode an adaptor protein involved in acute stress or apoptosis signalling in vivo. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

A Drosophila wing spot test

International Nuclear Information System (INIS)

Ayaki, Toshikazu; Yoshikawa, Isao; Niikawa, Norio; Hoshi, Masaharu.

1986-01-01

A Drosophila wing spot test system was used to investigate the effects of low doses of X-rays, gamma rays, and both 2.3 and 14.1 MeV neutrons on somatic chromosome mutation (SCM) induction. The incidence of SCM was significantly increased with any type of radiation, with evident linear dose-response relationship within the range of 3 to 20 cGy. It was estimated that relative biological effectiveness value for SCM induction of 2.3 MeV neutrons to X-rays and gamma rays is much higher than that of 14.1 MeV neutrons to those photons (2.4 vs 8.0). The Drosophila wing spot test system seems to become a promising in vivo experimental method for higher animals in terms of the lack of necessity for a marvelously large number of materials required in conventional test system. (Namekawa, K.)

Effect of non-nutritive sugars to decrease the survivorship of spotted wing drosophila, Drosophila suzukii

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In this study, we investigated the effects of non-nutritive sugars and sugar alcohols on the survivorship of spotted wing drosophila, Drosophila suzukii, and found erythritol and erythrose as potentially toxic to the fly. In a dose-dependent study, erythritol and erythrose significantly reduced fly ...

Flying Drosophila orient to sky polarization.

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Weir, Peter T; Dickinson, Michael H

2012-01-10

Insects maintain a constant bearing across a wide range of spatial scales. Monarch butterflies and locusts traverse continents [1, 2], and foraging bees and ants travel hundreds of meters to return to their nests [1, 3, 4], whereas many other insects fly straight for only a few centimeters before changing direction. Despite this variation in spatial scale, the brain region thought to underlie long-distance navigation is remarkably conserved [5, 6], suggesting that the use of a celestial compass is a general and perhaps ancient capability of insects. Laboratory studies of Drosophila have identified a local search mode in which short, straight segments are interspersed with rapid turns [7, 8]. However, this flight mode is inconsistent with measured gene flow between geographically separated populations [9-11], and individual Drosophila can travel 10 km across desert terrain in a single night [9, 12, 13]-a feat that would be impossible without prolonged periods of straight flight. To directly examine orientation behavior under outdoor conditions, we built a portable flight arena in which a fly viewed the natural sky through a liquid crystal device that could experimentally rotate the polarization angle. Our findings indicate that Drosophila actively orient using the sky's natural polarization pattern. Copyright © 2012 Elsevier Ltd. All rights reserved.

Detection of Mitochondrial Caspase Activity in Real Time In Situ in Live Cells

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Zhang, Yingpei; Haskins, Catherine; Lopez-Cruzan, Marisa; Zhang, Jianhua; Centonze, Victoria E.; Herman, Brian

2004-08-01

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP caspase 3 substrate YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.

Org-1, the Drosophila ortholog of Tbx1, is a direct activator of known identity genes during muscle specification.

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Schaub, Christoph; Nagaso, Hideyuki; Jin, Hong; Frasch, Manfred

2012-03-01

Members of the T-Box gene family of transcription factors are important players in regulatory circuits that generate myogenic and cardiogenic lineage diversities in vertebrates. We show that during somatic myogenesis in Drosophila, the single ortholog of vertebrate Tbx1, optomotor-blind-related-gene-1 (org-1), is expressed in a small subset of muscle progenitors, founder cells and adult muscle precursors, where it overlaps with the products of the muscle identity genes ladybird (lb) and slouch (slou). In addition, org-1 is expressed in the lineage of the heart-associated alary muscles. org-1 null mutant embryos lack Lb and Slou expression within the muscle lineages that normally co-express org-1. As a consequence, the respective muscle fibers and adult muscle precursors are either severely malformed or missing, as are the alary muscles. To address the mechanisms that mediate these regulatory interactions between Org-1, Lb and Slou, we characterized distinct enhancers associated with somatic muscle expression of lb and slou. We demonstrate that these lineage- and stage-specific cis-regulatory modules (CRMs) bind Org-1 in vivo, respond to org-1 genetically and require T-box domain binding sites for their activation. In summary, we propose that org-1 is a common and direct upstream regulator of slou and lb in the developmental pathway of these two neighboring muscle lineages. Cross-repression between slou and lb and combinatorial activation of lineage-specific targets by Org-1-Slou and Org-1-Lb, respectively, then leads to the distinction between the two lineages. These findings provide new insights into the regulatory circuits that control the proper pattering of the larval somatic musculature in Drosophila.

A Quantitative RNAi Screen for JNK Modifiers Identifies Pvr as a Novel Regulator of Drosophila Immune Signaling

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Bond, David; Foley, Edan

2009-01-01

Drosophila melanogaster responds to gram-negative bacterial challenges through the IMD pathway, a signal transduction cassette that is driven by the coordinated activities of JNK, NF-κB and caspase modules. While many modifiers of NF-κB activity were identified in cell culture and in vivo assays, the regulatory apparatus that determines JNK inputs into the IMD pathway is relatively unexplored. In this manuscript, we present the first quantitative screen of the entire genome of Drosophila for novel regulators of JNK activity in the IMD pathway. We identified a large number of gene products that negatively or positively impact on JNK activation in the IMD pathway. In particular, we identified the Pvr receptor tyrosine kinase as a potent inhibitor of JNK activation. In a series of in vivo and cell culture assays, we demonstrated that activation of the IMD pathway drives JNK-dependent expression of the Pvr ligands, Pvf2 and Pvf3, which in turn act through the Pvr/ERK MAP kinase pathway to attenuate the JNK and NF-κB arms of the IMD pathway. Our data illuminate a poorly understood arm of a critical and evolutionarily conserved innate immune response. Furthermore, given the pleiotropic involvement of JNK in eukaryotic cell biology, we believe that many of the novel regulators identified in this screen are of interest beyond immune signaling. PMID:19893628

Control of G1 in the developing Drosophila eye: rca1 regulates Cyclin A.

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Dong, X; Zavitz, K H; Thomas, B J; Lin, M; Campbell, S; Zipursky, S L

1997-01-01

In the developing eye of Drosophila melanogaster, cells become synchronized in the G1 phase of the cell cycle just prior to the onset of cellular differentiation and morphogenesis. In roughex (rux) mutants, cells enter S phase precociously because of ectopic activation of a Cyclin A/Cdk complex in early G1. This leads to defects in cell fate and pattern formation, and results in abnormalities in the morphology of the adult eye. A screen for dominant suppressors of the rux eye phenotype led to the identification of mutations in cyclin A, string (cdc25), and new cell cycle genes. One of these genes, regulator of cyclin A (rca1), encodes a novel protein required for both mitotic and meiotic cell cycle progression. rca1 mutants arrest in G2 of embryonic cell cycle 16 with a phenotype very similar to cyclin A loss of function mutants. Expression of rca1 transgenes in G1 or in postmitotic neurons promotes Cyclin A protein accumulation and drives cells into S phase in a Cyclin A-dependent fashion.

Proteases and caspase-like activity in the yeast Saccharomyces cerevisiae.

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Wilkinson, Derek; Ramsdale, Mark

2011-10-01

A variety of proteases have been implicated in yeast PCD (programmed cell death) including the metacaspase Mca1 and the separase Esp1, the HtrA-like serine protease Nma111, the cathepsin-like serine carboxypeptideases and a range of vacuolar proteases. Proteasomal activity is also shown to have an important role in determining cell fate, with both pro- and anti-apoptotic roles. Caspase 3-, 6- and 8-like activities are detected upon stimulation of yeast PCD, but not all of this activity is associated with Mca1, implicating other proteases with caspase-like activity in the yeast cell death response. Global proteolytic events that accompany PCD are discussed alongside a consideration of the conservation of the death-related degradome (both at the level of substrate choice and cleavage site). The importance of both gain-of-function changes in the degradome as well as loss-of-function changes are highlighted. Better understanding of both death-related proteases and their substrates may facilitate the design of future antifungal drugs or the manipulation of industrial yeasts for commercial exploitation.

Paradoxical sleep deprivation changes testicular malondialdehyde and caspase-3 expression in male rats

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Fitranto Arjadi

2015-08-01

Full Text Available BACKGROUND Sleep deprivation is a significant problem among adult men and is considered as a risk factor for several diseases. Paradoxical sleep deprivation (PSD induces Leydig cell apoptosis through elevation of corticosterone, with testicular malondialdehyde (MDA and Leydig cell caspase-3 expression as parameters. The aim of this study was to observe testicular MDA level and caspase-3 expression treated with paradoxical sleep deprivation (PSD, immobilization, and footshock stress and to determine the stress model with a significant effect in white male rats (Rattus norvegicus . METHODS This experimental randomized study of posttest only with control group design was conducted on 24 white male Wistar strain rats, randomly allocated into four treatment groups, i.e. control (K1 without any stress treatment, PSD (KII, immobilization (KIII, and footshock stress (KIV. Treatments were given for 25 days to produce chronic stress. Testicular MDA concentration was examined by the ELISA method while caspase-3 was examined by the TUNEL method. RESULTS Mean testicular MDA concentration with one-way ANOVA test showed differences in means between the groups (p=0.000 and post hoc Tukey-HSD test showed significant results between PSD stress group versus control, immobilization and footshock stress groups. One-way ANOVA test showed a significant difference in caspase-3 expression in at least two treatment groups (p=0.008 and post-hoc Tuckey-LSD test showed significant differences between controls and all stress groups. CONCLUSION Sleep deprivation is a type of stress inducing changes in testicular MDA concentration and caspase-3 expression in male rat testes.

Glucuronylated core 1 glycans are required for precise localization of neuromuscular junctions and normal formation of basement membranes on Drosophila muscles.

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Itoh, Kazuyoshi; Akimoto, Yoshihiro; Kondo, Shu; Ichimiya, Tomomi; Aoki, Kazuhiro; Tiemeyer, Michael; Nishihara, Shoko

2018-04-15

T antigen (Galβ1-3GalNAcα1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 β1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including decreased number of circulating hemocytes, hyper-differentiation of hematopoietic stem cells in lymph glands, malformation of the central nervous system, mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAβ1-3Galβ1-3GalNAcα1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. In this study, for the first time, we unraveled biological roles of glucuronylated T antigen. Our data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila β1,3-glucuronyltransferase-P (dGlcAT-P). We created dGlcAT-P null mutants and found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. We also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, we showed a genetic interaction between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization

The Drosophila HEM-2/NAP1 homolog KETTE controls axonal pathfinding and cytoskeletal organization.

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Hummel, T; Leifker, K; Klämbt, C

2000-04-01

In Drosophila, the correct formation of the segmental commissures depends on neuron-glial interactions at the midline. The VUM midline neurons extend axons along which glial cells migrate in between anterior and posterior commissures. Here, we show that the gene kette is required for the normal projection of the VUM axons and subsequently disrupts glial migration. Axonal projection defects are also found for many other moto- and interneurons. In addition, kette affects the cell morphology of mesodermal and epidermal derivatives, which show an abnormal actin cytoskeleton. The KETTE protein is homologous to the transmembrane protein HEM-2/NAP1 evolutionary conserved from worms to vertebrates. In vitro analysis has shown a specific interaction of the vertebrate HEM-2/NAP1 with the SH2-SH3 adapter protein NCK and the small GTPase RAC1, which both have been implicated in regulating cytoskeleton organization and axonal growth. Hypomorphic kette mutations lead to axonal defects similar to mutations in the Drosophila NCK homolog dreadlocks. Furthermore, we show that kette and dock mutants genetically interact. NCK is thought to interact with the small G proteins RAC1 and CDC42, which play a role in axonal growth. In line with these observations, a kette phenocopy can be obtained following directed expression of mutant DCDC42 or DRAC1 in the CNS midline. In addition, the kette mutant phenotype can be partially rescued by expression of an activated DRAC1 transgene. Our data suggest an important role of the HEM-2 protein in cytoskeletal organization during axonal pathfinding.

highroad Is a Carboxypetidase Induced by Retinoids to Clear Mutant Rhodopsin-1 in Drosophila Retinitis Pigmentosa Models

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Huai-Wei Huang

2018-02-01

Full Text Available Rhodopsins require retinoid chromophores for their function. In vertebrates, retinoids also serve as signaling molecules, but whether these molecules similarly regulate gene expression in Drosophila remains unclear. Here, we report the identification of a retinoid-inducible gene in Drosophila, highroad, which is required for photoreceptors to clear folding-defective mutant Rhodopsin-1 proteins. Specifically, knockdown or genetic deletion of highroad blocks the degradation of folding-defective Rhodopsin-1 mutant, ninaEG69D. Moreover, loss of highroad accelerates the age-related retinal degeneration phenotype of ninaEG69D mutants. Elevated highroad transcript levels are detected in ninaEG69D flies, and interestingly, deprivation of retinoids in the fly diet blocks this effect. Consistently, mutations in the retinoid transporter, santa maria, impairs the induction of highroad in ninaEG69D flies. In cultured S2 cells, highroad expression is induced by retinoic acid treatment. These results indicate that cellular quality-control mechanisms against misfolded Rhodopsin-1 involve regulation of gene expression by retinoids.

Simple route of caspase-3 FRET sensor synthesis using “click chemistry”

OpenAIRE

Lišková, M. (Marcela); Křenková, J. (Jana); Klepárník, K. (Karel); Pazdera, P.; Foret, F. (František)

2015-01-01

Programmed cell death or apoptosis is regulated process of cell suicide. The central role in apoptosis play cysteine proteases called caspases. Caspases recognize tetra-peptide sequences Asp-Glu-Val-Asp (DEVD) on their substrates and hydrolyze peptide bonds after aspartic acid residues. Various techniques for the determination of caspase-3 are commercially available e.g. Enzyme Linked Immuno-Sorbent Assay (ELISA), Western blotting or flow cytometric analysis. The products of the cleavage can ...

Analysis of the interaction between human RITA and Drosophila Suppressor of Hairless.

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Brockmann, Birgit; Mastel, Helena; Oswald, Franz; Maier, Dieter

2014-12-01

Notch signalling mediates intercellular communication, which is effected by the transcription factor CSL, an acronym for vertebrate CBF1/RBP-J, Drosophila Suppressor of Hairless [Su(H)] and C. elegans Lag1. Nuclear import of CBF1/RBP-J depends on co-activators and co-repressors, whereas the export relies on RITA. RITA is a tubulin and CBF1/RBP-J binding protein acting as a negative regulator of Notch signalling in vertebrates. RITA protein is highly conserved in eumatazoa, but no Drosophila homologue was yet identified. In this work, the activity of human RITA in the fly was addressed. To this end, we generated transgenic flies that allow a tissue specific induction of human RITA, which was demonstrated by Western blotting and in fly tissues. Unexpectedly, overexpression of RITA during fly development had little phenotypic consequences, even when overexpressed simultaneously with either Su(H) or the Notch antagonist Hairless. We demonstrate the in vivo binding of human RITA to Su(H) and to tubulin by co-immune precipitation. Moreover, RITA and tubulin co-localized to some degree in several Drosophila tissues. Overall our data show that human RITA, albeit binding to Drosophila Su(H) and tubulin, cannot influence the Notch signalling pathway in the fly, suggesting that a nuclear export mechanism of Su(H), if existent in Drosophila, does not depend on RITA. © 2015 The Authors.

Metabolic Enhancer Piracetam Attenuates the Translocation of Mitochondrion-Specific Proteins of Caspase-Independent Pathway, Poly [ADP-Ribose] Polymerase 1 Up-regulation and Oxidative DNA Fragmentation.

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Verma, Dinesh Kumar; Gupta, Sonam; Biswas, Joyshree; Joshi, Neeraj; Sivarama Raju, K; Wahajuddin, Mu; Singh, Sarika

2018-03-12

Piracetam, a nootropic drug, has been clinically used for decades; however, its mechanism of action still remains enigmatic. The present study was undertaken to evaluate the role of mitochondrion-specific factors of caspase-independent pathway like apoptotic-inducing factor (AIF) and endonuclease-G (endo-G) in piracetam-induced neuroprotection. N2A cells treated with lipopolysaccharide (LPS) exhibited significant cytotoxicity, impaired mitochondrial activity, and reactive oxygen species generation which was significantly attenuated with piracetam co-treatment. Cells co-treated with LPS and piracetam exhibited significant uptake of piracetam in comparison to only piracetam-treated cells as estimated by liquid chromatography-mass spectrometry (LC-MSMS). LPS treatment caused significant translocation of AIF and endonuclease-G in neuronal N2A cells which were significantly attenuated with piracetam co-treatment. Significant over-expression of proinflammatory cytokines was also observed after treatment of LPS to cells which was inhibited with piracetam co-treatment demonstrating its anti-inflammatory property. LPS-treated cells exhibited significant oxidative DNA fragmentation and poly [ADP-ribose] polymerase-1 (PARP-1) up-regulation in nucleus, both of which were attenuated with piracetam treatment. Antioxidant melatonin but not z-VAD offered the inhibited LPS-induced DNA fragmentation indicating the involvement of oxidative DNA fragmentation. Further, we did not observe the altered caspase-3 level after LPS treatment initially while at a later time point, significantly augmented level of caspase-3 was observed which was not inhibited with piracetam treatment. In total, our findings indicate the interference of piracetam in mitochondrion-mediated caspase-independent pathway, as well as its anti-inflammatory and antioxidative properties. Graphical Abstract Graphical abstract indicating the novel interference of metabolic enhancer piracetam (P) in neuronal death

A caspase-2-RFXANK interaction and its implication for MHC class II expression.

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Forsberg, Jeremy; Li, Xinge; Akpinar, Birce; Salvatori, Roger; Ott, Martin; Zhivotovsky, Boris; Olsson, Magnus

2018-01-23

Despite recent achievements implicating caspase-2 in tumor suppression, the enzyme stands out from the apoptotic caspase family as a factor whose function requires further clarification. To specify enzyme characteristics through the definition of interacting proteins in apoptotic or non-apoptotic settings, a yeast 2-hybrid (Y2H) screen was performed using the full-length protein as bait. The current report describes the analysis of a captured prey and putative novel caspase-2 interacting factor, the regulatory factor X-associated ankyrin-containing protein (RFXANK), previously associated with CIITA, the transactivator regulating cell-type specificity and inducibility of MHC class II gene expression. The interaction between caspase-2 and RFXANK was verified by co-immunoprecipitations using both exogenous and endogenous proteins, where the latter approach suggested that binding of the components occurs in the cytoplasm. Cellular co-localization was confirmed by transfection of fluorescently conjugated proteins. Enhanced caspase-2 processing in RFXANK-overexpressing HEK293T cells treated with chemotherapeutic agents further supported Y2H data. Yet, no distinct differences with respect to MHC class II expression were observed in plasma membranes of antigen-presenting cells derived from wild type and caspase-2 -/- mice. In contrast, increased levels of the total MHC class II protein was evident in protein lysates from caspase-2 RNAi-silenced leukemia cell lines and B-cells isolated from gene-targeted mice. Together, these data identify a novel caspase-2-interacting factor, RFXANK, and indicate a potential non-apoptotic role for the enzyme in the control of MHC class II gene regulation.

Fenretinide-induced caspase-8 activation and apoptosis in an established model of metastatic neuroblastoma

International Nuclear Information System (INIS)

Raguénez, Gilda; Mühlethaler-Mottet, Annick; Meier, Roland; Duros, Caroline; Bénard, Jean; Gross, Nicole

2009-01-01

Resistance of high-risk metastatic neuroblastoma (HR-NB) to high dose chemotherapy (HD-CT) raises a major therapeutic challenge in pediatric oncology. Patients are treated by maintenance CT. For some patients, an adjuvant retinoid therapy is proposed, such as the synthetic retinoid fenretinide (4-HPR), an apoptotic inducer. Recent studies demonstrated that NB metastasis process is enhanced by the loss of caspase-8 involved in the Integrin-Mediated Death (IMD) process. As the role of caspase-8 appears to be critical in preventing metastasis, we aimed at studying the effect of 4-HPR on caspase-8 expression in metastatic neuroblasts. We used the human IGR-N-91 MYCN-amplified NB experimental model, able to disseminate in vivo from the primary nude mouse tumor xenograft (PTX) into myocardium (Myoc) and bone marrow (BM) of the animal. NB cell lines, i.e., IGR-N-91 and SH-EP, were treated with various doses of Fenretinide (4-HPR), then cytotoxicity was analyzed by MTS proliferation assay, apoptosis by the propidium staining method, gene or protein expressions by RT-PCR and immunoblotting and caspases activity by colorimetric protease assays. The IGR-N-91 parental cells do not express detectable caspase-8. However the PTX cells established from the primary tumor in the mouse, are caspase-8 positive. In contrast, metastatic BM and Myoc cells show a clear down-regulation of the caspase-8 expression. In parallel, the caspases -3, -9, -10, Bcl-2, or Bax expressions were unchanged. Our data show that in BM, compared to PTX cells, 4-HPR up-regulates caspase-8 expression that parallels a higher sensitivity to apoptotic cell death. Stable caspase-8-silenced SH-EP cells appear more resistant to 4-HPR-induced cell death compared to control SH-EP cells. Moreover, 4-HPR synergizes with drugs since apoptosis is restored in VP16- or TRAIL-resistant-BM cells. These results demonstrate that 4-HPR in up-regulating caspase-8 expression, restores and induces apoptotic cell death in

Anti-apoptotic effect of caspase inhibitors on H₂O₂-treated HeLa cells through early suppression of its oxidative stress.

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Park, Woo Hyun

2014-05-01

Oxidative stress-induced cytotoxicity in cervical cancer cells may be of toxicological interest. In the present study, the effects of exogenous H2O2 on cell growth and death in HeLa cervical cancer cells were investigated, and the anti-apoptotic effects of various caspase (pan-caspase, caspase-3, -8 or -9) inhibitors on H2O2-treated HeLa cells were also evaluated with regard to reactive oxygen species (ROS) and glutathione (GSH) levels. Based on MTT assays, H2O2 inhibited the growth of HeLa cells with an IC50 value of ~75 µM at 24 h. H2O2 increased the number of dead cells and Annexin V-FITC-positive cells in the HeLa cells, which was accompanied by the activation of caspase-3 and the loss of mitochondrial membrane potential (MMP; ΔΨm). However, relatively higher doses of H2O2 induced necrosis in HeLa cells. Caspase inhibitors significantly prevented H2O2-induced HeLa cell death. H2O2 increased ROS including O2•- at 24 h and increased the activity of catalase in HeLa cells. H2O2 also increased the ROS level at 1 h, and several caspase inhibitors attenuated the increased level at 1 h but not at 6, 12 and 24 h. H2O2 decreased the GSH level in HeLa cells at 1 h, and several caspase inhibitors attenuated the decreased level of GSH at this time. H2O2 induced GSH depletion at 24 h. In conclusion, H2O2 inhibited the growth of HeLa cells via apoptosis and/or necrosis, which was accompanied by intracellular increases in ROS levels and GSH depletion. Caspase inhibitors are suggested to suppress H2O2-induced oxidative stress to rescue HeLa cells at the early time point of 1 h.

Differential effects of phytotherapic preparations in the hSOD1 Drosophila melanogaster model of ALS

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De Rose, Francescaelena; Marotta, Roberto; Talani, Giuseppe; Catelani, Tiziano; Solari, Paolo; Poddighe, Simone; Borghero, Giuseppe; Marrosu, Francesco; Sanna, Enrico; Kasture, Sanjay; Acquas, Elio; Liscia, Anna

2017-01-01

The present study was aimed at characterizing the effects of Withania somnifera (Wse) and Mucuna pruriens (Mpe) on a Drosophila melanogaster model for Amyotrophic Lateral Sclerosis (ALS). In particular, the effects of Wse and Mpe were assessed following feeding the flies selectively overexpressing the wild human copper, zinc-superoxide dismutase (hSOD1-gain-of-function) in Drosophila motoneurons. Although ALS-hSOD1 mutants showed no impairment in life span, with respect to GAL4 controls, the results revealed impairment of climbing behaviour, muscle electrophysiological parameters (latency and amplitude of ePSPs) as well as thoracic ganglia mitochondrial functions. Interestingly, Wse treatment significantly increased lifespan of hSDO1 while Mpe had not effect. Conversely, both Wse and Mpe significantly rescued climbing impairment, and also latency and amplitude of ePSPs as well as failure responses to high frequency DLM stimulation. Finally, mitochondrial alterations were any more present in Wse- but not in Mpe-treated hSOD1 mutants. Hence, given the role of inflammation in the development of ALS, the high translational impact of the model, the known anti-inflammatory properties of these extracts, and the viability of their clinical use, these results suggest that the application of Wse and Mpe might represent a valuable pharmacological strategy to counteract the progression of ALS and related symptoms. PMID:28102336

First record of spotted wing drosophila Drosophila suzukii (Diptera: Drosophilidae in Montenegro

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Snježana Hrnčić

2015-01-01

Full Text Available The spotted wing drosophila Drosophila suzukii Matsumura (Diptera: Drosophilidae is an invasive pest originating from Southeast Asia. It was detected for the first time in Europe in 2008 (Spain and Italy and subsequently in other European countries. It is a highly polyphagous pest that infests healthy, ripening fruit and presents a serious threat to fruit production, particularly of soft skinned fruit. In the first half of October 2013, a new fruit fly species was unexpectedly detected in Tephri traps baited with the three-component female-biased attractant BioLure that is regularly used for monitoring the Mediterranean fruit fly Ceratitis capitata Wiedem. (Diptera: Tephritidae in Montenegro. Brief visual inspection identified the new species as the spotted wing drosophila D. suzukii. The pest was first recorded in several localities on the Montenegrin seacoast around Boka Kotor Bay. After the finding, all Drosophila specimens were collected from traps for further laboratory observation. A quick follow-up monitoring of other Tephri traps was carried out within the next few days on the rest of the seacoast (localities from Tivat to Ulcinj. Additionally, Tephri traps were set up around Lake Skadar and in the city of Podgorica, as well as on fresh fruit markets in Podgorica. The results of this preliminary study showed that D. suzukii was present in all surveyed locations and adults were captured until late December. Both sexes were found in traps with BioLure. Our data show that D. suzukii is present in southern parts of Montenegro and there is a serious threat of its further spreading, particularly towards northern parts of the country where the main raspberry and blueberry production is placed. The results also show that Tephri traps baited with BioLure can be used for detection and monitoring of spotted wing drosophila.

Motor Control of Drosophila Courtship Song

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Troy R. Shirangi

2013-11-01

Full Text Available Many animals utilize acoustic signals—or songs—to attract mates. During courtship, Drosophila melanogaster males vibrate a wing to produce trains of pulses and extended tone, called pulse and sine song, respectively. Courtship songs in the genus Drosophila are exceedingly diverse, and different song features appear to have evolved independently of each other. How the nervous system allows such diversity to evolve is not understood. Here, we identify a wing muscle in D. melanogaster (hg1 that is uniquely male-enlarged. The hg1 motoneuron and the sexually dimorphic development of the hg1 muscle are required specifically for the sine component of the male song. In contrast, the motoneuron innervating a sexually monomorphic wing muscle, ps1, is required specifically for a feature of pulse song. Thus, individual wing motor pathways can control separate aspects of courtship song and may provide a “modular” anatomical substrate for the evolution of diverse songs.

Metabolome analysis of Drosophila melanogaster during embryogenesis.

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An, Phan Nguyen Thuy; Yamaguchi, Masamitsu; Bamba, Takeshi; Fukusaki, Eiichiro

2014-01-01

The Drosophila melanogaster embryo has been widely utilized as a model for genetics and developmental biology due to its small size, short generation time, and large brood size. Information on embryonic metabolism during developmental progression is important for further understanding the mechanisms of Drosophila embryogenesis. Therefore, the aim of this study is to assess the changes in embryos' metabolome that occur at different stages of the Drosophila embryonic development. Time course samples of Drosophila embryos were subjected to GC/MS-based metabolome analysis for profiling of low molecular weight hydrophilic metabolites, including sugars, amino acids, and organic acids. The results showed that the metabolic profiles of Drosophila embryo varied during the course of development and there was a strong correlation between the metabolome and different embryonic stages. Using the metabolome information, we were able to establish a prediction model for developmental stages of embryos starting from their high-resolution quantitative metabolite composition. Among the important metabolites revealed from our model, we suggest that different amino acids appear to play distinct roles in different developmental stages and an appropriate balance in trehalose-glucose ratio is crucial to supply the carbohydrate source for the development of Drosophila embryo.

Uncovering a Dual Regulatory Role for Caspases During Endoplasmic Reticulum Stress-induced Cell Death.

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Anania, Veronica G; Yu, Kebing; Gnad, Florian; Pferdehirt, Rebecca R; Li, Han; Ma, Taylur P; Jeon, Diana; Fortelny, Nikolaus; Forrest, William; Ashkenazi, Avi; Overall, Christopher M; Lill, Jennie R

2016-07-01

Many diseases are associated with endoplasmic reticulum (ER) stress, which results from an accumulation of misfolded proteins. This triggers an adaptive response called the "unfolded protein response" (UPR), and prolonged exposure to ER stress leads to cell death. Caspases are reported to play a critical role in ER stress-induced cell death but the underlying mechanisms by which they exert their effect continue to remain elusive. To understand the role caspases play during ER stress, a systems level approach integrating analysis of the transcriptome, proteome, and proteolytic substrate profile was employed. This quantitative analysis revealed transcriptional profiles for most human genes, provided information on protein abundance for 4476 proteins, and identified 445 caspase substrates. Based on these data sets many caspase substrates were shown to be downregulated at the protein level during ER stress suggesting caspase activity inhibits their cellular function. Additionally, RNA sequencing revealed a role for caspases in regulation of ER stress-induced transcriptional pathways and gene set enrichment analysis showed expression of multiple gene targets of essential transcription factors to be upregulated during ER stress upon inhibition of caspases. Furthermore, these transcription factors were degraded in a caspase-dependent manner during ER stress. These results indicate that caspases play a dual role in regulating the cellular response to ER stress through both post-translational and transcriptional regulatory mechanisms. Moreover, this study provides unique insight into progression of the unfolded protein response into cell death, which may help identify therapeutic strategies to treat ER stress-related diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Optogenetic pacing in Drosophila melanogaster

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Alex, Aneesh; Li, Airong; Tanzi, Rudolph E.; Zhou, Chao

2015-01-01

Electrical stimulation is currently the gold standard for cardiac pacing. However, it is invasive and nonspecific for cardiac tissues. We recently developed a noninvasive cardiac pacing technique using optogenetic tools, which are widely used in neuroscience. Optogenetic pacing of the heart provides high spatial and temporal precisions, is specific for cardiac tissues, avoids artifacts associated with electrical stimulation, and therefore promises to be a powerful tool in basic cardiac research. We demonstrated optogenetic control of heart rhythm in a well-established model organism, Drosophila melanogaster. We developed transgenic flies expressing a light-gated cation channel, channelrhodopsin-2 (ChR2), specifically in their hearts and demonstrated successful optogenetic pacing of ChR2-expressing Drosophila at different developmental stages, including the larva, pupa, and adult stages. A high-speed and ultrahigh-resolution optical coherence microscopy imaging system that is capable of providing images at a rate of 130 frames/s with axial and transverse resolutions of 1.5 and 3.9 μm, respectively, was used to noninvasively monitor Drosophila cardiac function and its response to pacing stimulation. The development of a noninvasive integrated optical pacing and imaging system provides a novel platform for performing research studies in developmental cardiology. PMID:26601299

Xbp1-Independent Ire1 Signaling Is Required for Photoreceptor Differentiation and Rhabdomere Morphogenesis in Drosophila

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Dina S. Coelho

2013-11-01

Full Text Available The unfolded protein response (UPR is composed by homeostatic signaling pathways that are activated by excessive protein misfolding in the endoplasmic reticulum. Ire1 signaling is an important mediator of the UPR, leading to the activation of the transcription factor Xbp1. Here, we show that Drosophila Ire1 mutant photoreceptors have defects in the delivery of rhodopsin-1 to the rhabdomere and in the secretion of Spacemaker/Eyes Shut into the interrhabdomeral space. However, these defects are not observed in Xbp1 mutant photoreceptors. Ire1 mutant retinas have higher mRNA levels for targets of regulated Ire1-dependent decay (RIDD, including for the fatty acid transport protein (fatp. Importantly, the downregulation of fatp by RNAi rescues the rhodopsin-1 delivery defects observed in Ire1 mutant photoreceptors. Our results show that the role of Ire1 during photoreceptor differentiation is independent of Xbp1 function and demonstrate the physiological relevance of the RIDD mechanism in this specific paradigm.

Caspase-2 cleavage of tau reversibly impairs memory.

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Zhao, Xiaohui; Kotilinek, Linda A; Smith, Benjamin; Hlynialuk, Chris; Zahs, Kathleen; Ramsden, Martin; Cleary, James; Ashe, Karen H

2016-11-01

In Alzheimer's disease (AD) and other tauopathies, the tau protein forms fibrils, which are believed to be neurotoxic. However, fibrillar tau has been dissociated from neuron death and network dysfunction, suggesting the involvement of nonfibrillar species. Here we describe a novel pathological process in which caspase-2 cleavage of tau at Asp314 impairs cognitive and synaptic function in animal and cellular models of tauopathies by promoting the missorting of tau to dendritic spines. The truncation product, Δtau314, resists fibrillation and is present at higher levels in brains from cognitively impaired mice and humans with AD. The expression of tau mutants that resisted caspase-2 cleavage prevented tau from infiltrating spines, dislocating glutamate receptors and impairing synaptic function in cultured neurons, and it prevented memory deficits and neurodegeneration in mice. Decreasing the levels of caspase-2 restored long-term memory in mice that had existing deficits. Our results suggest an overall treatment strategy for re-establishing synaptic function and restoring memory in patients with AD by preventing tau from accumulating in dendritic spines.

Sucrose Improves Insecticide Activity Against Drosophila suzukii (Diptera: Drosophilidae).

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Cowles, Richard S; Rodriguez-Saona, Cesar; Holdcraft, Robert; Loeb, Gregory M; Elsensohn, Johanna E; Hesler, Steven P

2015-04-01

The addition of sucrose to insecticides targeting spotted wing drosophila, Drosophila suzukii (Matsumura), enhanced lethality in laboratory, semifield, and field tests. In the laboratory, 0.1% sucrose added to a spray solution enhanced spotted wing drosophila feeding. Flies died 120 min earlier when exposed to spinosad residues at label rates enhanced with sucrose. Added sucrose reduced the LC50 for dried acetamiprid residues from 82 to 41 ppm in the spray solution. Laboratory bioassays of spotted wing drosophila mortality followed exposure to grape and blueberry foliage and/or fruit sprayed and aged in the field. On grape foliage, the addition of 2.4 g/liter of sugar with insecticide sprays resulted in an 11 and 6% increase of spotted wing drosophila mortality at 1 and 2 d exposures to residues, respectively, averaged over seven insecticides with three concentrations. In a separate experiment, spinetoram and cyantraniliprole reduced by 95-100% the larval infestation of blueberries, relative to the untreated control, 7 d after application at labeled rates when applied with 1.2 g/liter sucrose in a spray mixture, irrespective of rainfall; without sucrose infestation was reduced by 46-91%. Adding sugar to the organically acceptable spinosyn, Entrust, reduced larval infestation of strawberries by >50% relative to without sugar for five of the six sample dates during a season-long field trial. In a small-plot field test with blueberries, weekly applications in alternating sprays of sucrose plus reduced-risk insecticides, spinetoram or acetamiprid, reduced larval infestation relative to the untreated control by 76%; alternating bifenthrin and phosmet (without sucrose) reduced infestation by 65%. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Caspases and osteogenic markers-in vitro screening of inhibition impact

Czech Academy of Sciences Publication Activity Database

Adamová, Eva; Janečková, Eva; Klepárník, Karel; Matalová, Eva

2016-01-01

Roč. 52, č. 2 (2016), s. 144-148 ISSN 1071-2690 R&D Projects: GA ČR(CZ) GB14-37368G; GA ČR(CZ) GA14-28254S Institutional support: RVO:67985904 ; RVO:68081715 Keywords : osteogenesis * chondrogenesis * caspases * caspase-3 * gene expression Subject RIV: EA - Cell Biology; CB - Analytical Chemistry, Separation (UIACH-O) Impact factor: 0.897, year: 2016

Progesterone production requires activation of caspase-3 in preovulatory granulosa cells in a serum starvation model.

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An, Li-Sha; Yuan, Xiao-Hua; Hu, Ying; Shi, Zi-Yun; Liu, Xiao-Qin; Qin, Li; Wu, Gui-Qing; Han, Wei; Wang, Ya-Qin; Ma, Xu

2012-11-01

Granulosa cells proliferate, differentiate, and undergo apoptosis throughout follicular development. Previous studies have demonstrated that stimulation of progesterone production is accompanied by caspase-3 activation. Moreover, we previously reported that arsenic enhanced caspase-3 activity coupled with progesterone production. Inhibition of caspase-3 activity can significantly inhibit progesterone production induced by arsenic or follicle-stimulating hormone (FSH). Here, we report that serum starvation induces caspase-3 activation coupled with augmentation of progesterone production. Serum starvation also increased the levels of cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein, both of which may contribute to progesterone synthesis in preovulatory granulosa cells. Inhibition of caspase-3 activity resulted in a decrease in progesterone production. Deactivation of caspase-3 activity by caspase-3 specific inhibitor also resulted in decreases in P450scc and StAR expression, which may partly contribute to the observed decrease in progesterone production. Our study demonstrates for the first time that progesterone production in preovulatory granulosa cells is required for caspase-3 activation in a serum starvation model. Inhibition of caspase-3 activity can result in decreased expression of the steroidogenic proteins P450scc and StAR. Our work provides further details on the relationship between caspase-3 activation and steroidogenesis and indicates that caspase-3 plays a critical role in progesterone production by granulosa cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Hypergravity-induced altered behavior in Drosophila

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Hosamani, Ravikumar; Wan, Judy; Marcu, Oana; Bhattacharya, Sharmila

2012-07-01

Microgravity and mechanical stress are important factors of the spaceflight environment, and affect astronaut health and behavior. Structural, functional, and behavioral mechanisms of all cells and organisms are adapted to Earth's gravitational force, 1G, while altered gravity can pose challenges to their adaptability to this new environment. On ground, hypergravity paradigms have been used to predict and complement studies on microgravity. Even small changes that take place at a molecular and genetic level during altered gravity may result in changes in phenotypic behavior. Drosophila provides a robust and simple, yet very reliable model system to understand the complexity of hypergravity-induced altered behavior, due to availability of a plethora of genetic tools. Locomotor behavior is a sensitive parameter that reflects the array of molecular adaptive mechanisms recruited during exposure to altered gravity. Thus, understanding the genetic basis of this behavior in a hypergravity environment could potentially extend our understanding of mechanisms of adaptation in microgravity. In our laboratory we are trying to dissect out the cellular and molecular mechanisms underlying hypergravity-induced oxidative stress, and its potential consequences on behavioral alterations by using Drosophila as a model system. In the present study, we employed pan-neuronal and mushroom body specific knock-down adult flies by using Gal4/UAS system to express inverted repeat transgenes (RNAi) to monitor and quantify the hypergravity-induced behavior in Drosophila. We established that acute hypergravity (3G for 60 min) causes a significant and robust decrease in the locomotor behavior in adult Drosophila, and that this change is dependent on genes related to Parkinson's disease, such as DJ-1α , DJ-1β , and parkin. In addition, we also showed that anatomically the control of this behavior is significantly processed in the mushroom body region of the fly brain. This work links a molecular

Neuronal Cbl Controls Biosynthesis of Insulin-Like Peptides in Drosophila melanogaster

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Yu, Yue; Sun, Ying; He, Shengqi; Yan, Cheng; Rui, Liangyou; Li, Wenjun

2012-01-01

The Cbl family proteins function as both E3 ubiquitin ligases and adaptor proteins to regulate various cellular signaling events, including the insulin/insulin-like growth factor 1 (IGF1) and epidermal growth factor (EGF) pathways. These pathways play essential roles in growth, development, metabolism, and survival. Here we show that in Drosophila melanogaster, Drosophila Cbl (dCbl) regulates longevity and carbohydrate metabolism through downregulating the production of Drosophila insulin-like peptides (dILPs) in the brain. We found that dCbl was highly expressed in the brain and knockdown of the expression of dCbl specifically in neurons by RNA interference increased sensitivity to oxidative stress or starvation, decreased carbohydrate levels, and shortened life span. Insulin-producing neuron-specific knockdown of dCbl resulted in similar phenotypes. dCbl deficiency in either the brain or insulin-producing cells upregulated the expression of dilp genes, resulting in elevated activation of the dILP pathway, including phosphorylation of Drosophila Akt and Drosophila extracellular signal-regulated kinase (dERK). Genetic interaction analyses revealed that blocking Drosophila epidermal growth factor receptor (dEGFR)-dERK signaling in pan-neurons or insulin-producing cells by overexpressing a dominant-negative form of dEGFR abolished the effect of dCbl deficiency on the upregulation of dilp genes. Furthermore, knockdown of c-Cbl in INS-1 cells, a rat β-cell line, also increased insulin biosynthesis and glucose-stimulated secretion in an ERK-dependent manner. Collectively, these results suggest that neuronal dCbl regulates life span, stress responses, and metabolism by suppressing dILP production and the EGFR-ERK pathway mediates the dCbl action. Cbl suppression of insulin biosynthesis is evolutionarily conserved, raising the possibility that Cbl may similarly exert its physiological actions through regulating insulin production in β cells. PMID:22778134

Caspase-Independent Apoptosis Induced by Reperfusion Following Ischemia without Bile Duct Occlusion in Rat Liver.

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Matsui, Nobuaki; Yoshioka, Rie; Nozawa, Asako; Kobayashi, Naonobu; Shichijo, Yukari; Yoshikawa, Tadatoshi; Akagi, Masaaki

2017-01-01

The contribution of caspases to hepatic ischemia/reperfusion (I/R)-induced apoptosis has not been completely understood yet. Several studies have demonstrated increased caspase activity during I/R and the protective effect of caspase inhibitors against I/R injuries. However, reports with opposing results also exist. Herein, we examined the contribution of caspases to the I/R-induced hepatic apoptosis in rats using caspase inhibitors and specific substrates of caspases. Hepatic I/R was induced via a 2-h occlusion of the portal vein and the hepatic artery, without conducting bile duct occlusion. DNA laddering and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL)-positive cells were increased at 3 h after reperfusion. Pretreatment with caspase inhibitors (Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-cmk) 2 or 10 mg/kg intravenously (i.v.), 20 mg/kg intraperitoneally (i.p.), Z-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VAD-fmk) 3 mg/kg i.v.) failed to reduce apoptosis induced by I/R. Interestingly, apoptosis induced by the portal triad (hepatic artery, portal vein, and bile duct) occlusion/reperfusion could be marginally attenuated using Z-Asp-cmk (2 mg/kg i.v.). The cleavage activity for Ac-DEVD-α-(4-methylcoumaryl-7-amide) (MCA), a caspase-3/7/8/9 substrate, was significantly increased by I/R. Conversely, the cleavage activities for Ac-DNLD-MCA and MCA-VDQVDGW[K-DNP]-NH 2 , specific substrates for caspase-3 and -7 respectively, were decreased by I/R. Protein expression of the cellular inhibitor of apoptosis protein 2 (c-IAP2), an endogenous caspase inhibitor, was increased by ischemia. Nuclear translocation of the apoptosis-inducing factor (AIF), an initiator protein of caspase-independent apoptosis, was also increased during I/R. These results suggest that caspases are inhibited by c-IAP2 induced during ischemia and that AIF may be involved in initiation of apoptosis induced by hepatic I/R without

PIDDosome Expression and the Role of Caspase-2 Activation for Chemotherapy-Induced Apoptosis in RCCs

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Sebastian Heikaus

2010-01-01

Full Text Available Background: The importance of caspase-2 activation for mediating apoptosis in cancer is not clear and seems to differ between different tumour types. Furthermore, only few data have been obtained concerning the expression of caspase-2, which can be alternatively spliced into caspase-2L and caspase-2S, and the other PIDDosome members PIDD and RAIDD in human tumours in vivo. We, therefore, investigated their expression in renal cell carcinomas (RCCs of the clear cell type in vivo and analysed the role of caspase-2 in chemotherapy-induced apoptosis in RCCs in vitro.

Essential roles of caspases and their upstream regulators in rotenone-induced apoptosis

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Lee Jihjong; Huang, M.-S.; Yang, I-C.; Lai, T.-C.; Wang, J.-L.; Pang, V.F.; Hsiao, M.; Kuo, M.Y.P.

2008-01-01

In the present study, we examined whether caspases and their upstream regulators are involved in rotenone-induced cytotoxicity. Rotenone significantly inhibited the proliferation of oral cancer cell lines in a dose-dependent manner compared to normal oral mucosal fibroblasts. Flow cytometric analysis of DNA content showed that rotenone treatment induced apoptosis following G2/M arrest. Western blotting showed activation of both the caspase-8 and caspase-9 pathways, which differed from previous studies conducted in other cell types. Furthermore, p53 protein and its downstream pro-apoptotic target, Bax, were induced in SAS cells after treatment with rotenone. Rotenone-induced apoptosis was inhibited by antioxidants (glutathione, N-acetylcysteine, and tiron). In conclusion, our results demonstrate significant involvement of caspases and their upstream regulators in rotenone-induced cytotoxicity

High rate of translocation-based gene birth on the Drosophila Y chromosome.

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Tobler, Ray; Nolte, Viola; Schlötterer, Christian

2017-10-31

The Y chromosome is a unique genetic environment defined by a lack of recombination and male-limited inheritance. The Drosophila Y chromosome has been gradually acquiring genes from the rest of the genome, with only seven Y-linked genes being gained over the past 63 million years (0.12 gene gains per million years). Using a next-generation sequencing (NGS)-powered genomic scan, we show that gene transfers to the Y chromosome are much more common than previously suspected: at least 25 have arisen across three Drosophila species over the past 5.4 million years (1.67 per million years for each lineage). The gene transfer rate is significantly lower in Drosophila melanogaster than in the Drosophila simulans clade, primarily due to Y-linked retrotranspositions being significantly more common in the latter. Despite all Y-linked gene transfers being evolutionarily recent (Drosophila Y chromosome to be more dynamic than previously appreciated. Our analytical method provides a powerful means to identify Y-linked gene transfers and will help illuminate the evolutionary dynamics of the Y chromosome in Drosophila and other species. Copyright © 2017 the Author(s). Published by PNAS.

Genomic Signatures of Speciation in Sympatric and Allopatric Hawaiian Picture-Winged Drosophila.

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Kang, Lin; Settlage, Robert; McMahon, Wyatt; Michalak, Katarzyna; Tae, Hongseok; Garner, Harold R; Stacy, Elizabeth A; Price, Donald K; Michalak, Pawel

2016-05-30

The Hawaiian archipelago provides a natural arena for understanding adaptive radiation and speciation. The Hawaiian Drosophila are one of the most diverse endemic groups in Hawaiì with up to 1,000 species. We sequenced and analyzed entire genomes of recently diverged species of Hawaiian picture-winged Drosophila, Drosophila silvestris and Drosophila heteroneura from Hawaiì Island, in comparison with Drosophila planitibia, their sister species from Maui, a neighboring island where a common ancestor of all three had likely occurred. Genome-wide single nucleotide polymorphism patterns suggest the more recent origin of D. silvestris and D. heteroneura, as well as a pervasive influence of positive selection on divergence of the three species, with the signatures of positive selection more prominent in sympatry than allopatry. Positively selected genes were significantly enriched for functional terms related to sensory detection and mating, suggesting that sexual selection played an important role in speciation of these species. In particular, sequence variation in Olfactory receptor and Gustatory receptor genes seems to play a major role in adaptive radiation in Hawaiian pictured-winged Drosophila. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

big bang gene modulates gut immune tolerance in Drosophila.

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Bonnay, François; Cohen-Berros, Eva; Hoffmann, Martine; Kim, Sabrina Y; Boulianne, Gabrielle L; Hoffmann, Jules A; Matt, Nicolas; Reichhart, Jean-Marc

2013-02-19

Chronic inflammation of the intestine is detrimental to mammals. Similarly, constant activation of the immune response in the gut by the endogenous flora is suspected to be harmful to Drosophila. Therefore, the innate immune response in the gut of Drosophila melanogaster is tightly balanced to simultaneously prevent infections by pathogenic microorganisms and tolerate the endogenous flora. Here we describe the role of the big bang (bbg) gene, encoding multiple membrane-associated PDZ (PSD-95, Discs-large, ZO-1) domain-containing protein isoforms, in the modulation of the gut immune response. We show that in the adult Drosophila midgut, BBG is present at the level of the septate junctions, on the apical side of the enterocytes. In the absence of BBG, these junctions become loose, enabling the intestinal flora to trigger a constitutive activation of the anterior midgut immune response. This chronic epithelial inflammation leads to a reduced lifespan of bbg mutant flies. Clearing the commensal flora by antibiotics prevents the abnormal activation of the gut immune response and restores a normal lifespan. We now provide genetic evidence that Drosophila septate junctions are part of the gut immune barrier, a function that is evolutionarily conserved in mammals. Collectively, our data suggest that septate junctions are required to maintain the subtle balance between immune tolerance and immune response in the Drosophila gut, which represents a powerful model to study inflammatory bowel diseases.

Imaging of activated caspase-3 in living cell by fluorescence resonance energy transfer during photosensitization-induced apoptosis

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Wu, Yunxia; Xing, Da; Chen, Qun; Tang, Yonghong

2005-01-01

Photodynamic therapy (PDT) is a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in cell, and activation of caspase-3 is considered to be the final step in many apoptosis pathways. The changes of caspase-3 activation in cell during TNFα- and photodynamic therapy-induced apoptosis was measured by fluorescence resonance energy transfer (FRET) analysis. FRET probe consisting of fusions of an enhanced cyan fluorescent protein (ECFP), Venus and a linker peptide containing the caspase-3 cleavage sequence DEVD was utilized. Therefore, activated caspase-3 cleaved the linker peptide of FRET probe and disrupted the FRET signal. Human lung adenocarcinoma cell line (ASTC-a-1) were stably transfected with the plasmid (ECFP-DEVD-Venus) and then were treated by TNF-α and PDT, respectively. Experimental results indicated that caspase-3 activation resulted in cleavage of linker peptide and subsequent disruption of the FRET signal during TNFα- and photodynamic therapy-induced apoptosis, and that the activation of caspase-3 induced by photodynamic therapy was faster than that induce by TNF-α. The study supports that using FRET technique and different recombinant substrates as FRET probes could be used to detect the process of PDT-induced apoptosis and provide a new means to investigate apoptotic mechanism of PDT.

New record for the invasive Spotted Wing Drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae) in Anillaco, Argentina

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The invasive Spotted Wing Drosophila (SWD), Drosophila suzukii Matsumura, is reported for the first time in La Rioja, Argentina. This represents a major range expansion for this species. The natural enemies of SWD, Leptopilina clavipes and Ganaspis hookeri were also collected with the SWD at the s...

Drosophila atonal fully rescues the phenotype of Math1 null mice: new functions evolve in new cellular contexts

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Wang, Vincent Y.; Hassan, Bassem A.; Bellen, Hugo J.; Zoghbi, Huda Y.

2002-01-01

Many genes share sequence similarity between species, but their properties often change significantly during evolution. For example, the Drosophila genes engrailed and orthodenticle and the onychophoran gene Ultrabithorax only partially substitute for their mouse or Drosophila homologs. We have been analyzing the relationship between atonal (ato) in the fruit fly and its mouse homolog, Math1. In flies, ato acts as a proneural gene that governs the development of chordotonal organs (CHOs), which serve as stretch receptors in the body wall and joints and as auditory organs in the antennae. In the fly CNS, ato is important not for specification but for axonal arborization. Math1, in contrast, is required for the specification of cells in both the CNS and the PNS. Furthermore, Math1 serves a role in the development of secretory lineage cells in the gut, a function that does not parallel any known to be served by ato. We wondered whether ato and Math1 might be more functionally homologous than they appear, so we expressed Math1 in ato mutant flies and ato in Math1 null mice. To our surprise, the two proteins are functionally interchangeable.

Real-time monitoring of caspase cascade activation in living cells.

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Zhu, Lei; Huang, Xinglu; Choi, Ki Young; Ma, Ying; Zhang, Fan; Liu, Gang; Lee, Seulki; Chen, Xiaoyuan

2012-10-10

We introduce a simple, versatile and robust one-step technique that enables real-time imaging of multiple intracellular caspase activities in living cells without the need for complicated synthetic protocols. Conventional fluorogenic probes or recently reported activatable probes have been designed to target various proteases but are limited to extracellular molecules. Only a few have been applied to image intracellular proteases in living cells because most of these probes have limited cell-permeability. Our platform does not need complicated synthetic processes; instead it involves a straightforward peptide synthesis and a simple mixing step with a commercial transfection agent. The transfection agent efficiently delivered the highly quenched fluorogenic probes, comprised of distinctive pairs of dyes and quenchers, to the initiator caspase-8 and the effector caspase-3 in MDA-MB-435 cells, allowing dual-imaging of the activities of both caspases during the apoptotic process induced by TNF-related apoptosis induced ligand (TRAIL). With the combination of multiple fluorogenic probes, this simple platform can be applied to multiplexed imaging of selected intracellular proteases to study apoptotic processes in pathologies or for cell-based high throughput screening systems for drug discovery. Published by Elsevier B.V.

Drosophila-Cdh1 (Rap/Fzr) a regulatory subunit of APC/C is required for synaptic morphology, synaptic transmission and locomotion.

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