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Sample records for apoptotic dna degradation

  1. The roles and acting mechanism of Caenorhabditis elegans DNase II genes in apoptotic dna degradation and development.

    Directory of Open Access Journals (Sweden)

    Huey-Jen Lai

    Full Text Available DNase II enzymes are acidic endonucleases that have been implicated in mediating apoptotic DNA degradation, a critical cell death execution event. C. elegans genome contains three DNase II homologues, NUC-1, CRN-6, and CRN-7, but their expression patterns, acting sites, and roles in apoptotic DNA degradation and development are unclear. We have conducted a comprehensive analysis of three C. elegans DNase II genes and found that nuc-1 plays a major role, crn-6 plays an auxiliary role, and crn-7 plays a negligible role in resolving 3' OH DNA breaks generated in apoptotic cells. Promoter swapping experiments suggest that crn-6 but not crn-7 can partially substitute for nuc-1 in mediating apoptotic DNA degradation and both fail to replace nuc-1 in degrading bacterial DNA in intestine. Despite of their restricted and largely non-overlapping expression patterns, both CRN-6 and NUC-1 can mediate apoptotic DNA degradation in many cells, suggesting that they are likely secreted nucleases that are retaken up by other cells to exert DNA degradation functions. Removal or disruption of NUC-1 secretion signal eliminates NUC-1's ability to mediate DNA degradation across its expression border. Furthermore, blocking cell corpse engulfment does not affect apoptotic DNA degradation mediated by nuc-1, suggesting that NUC-1 acts in apoptotic cells rather than in phagocytes to resolve 3' OH DNA breaks. Our study illustrates how multiple DNase II nucleases play differential roles in apoptotic DNA degradation and development and reveals an unexpected mode of DNase II action in mediating DNA degradation.

  2. Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation.

    Science.gov (United States)

    Fath, A; Bethke, P C; Jones, R L

    1999-11-01

    Barley aleurone cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevent PCD. We tested the hypothesis that PCD in aleurone cells occurs by apoptosis, and show that the hallmark of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when aleurone cells die. We show that endogenous barley aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from aleurone layers or protoplasts. Barley aleurone cells contain three nucleases whose activities are regulated by GA and ABA. CA induction and ABA repression of nuclease activities correlate with PCD in aleurone cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living aleurone cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during aleurone PCD.

  3. Evidence of apoptotic effects of 2,4-D and butachlor on walking catfish, Clarias batrachus, by transmission electron microscopy and DNA degradation studies.

    Science.gov (United States)

    Ateeq, Bushra; Farah, M Abul; Ahmad, Waseem

    2006-01-25

    Apoptosis or programmed cell death is characterized morphologically by chromatin condensation, cell shrinkage, fragmentation of the nucleus and cytoplasm, and consequently formation of apoptotic bodies. It has also been best characterized by the cleavage of DNA into nucleosomal size fragments of 180-200 bp or multiples of the same. Contrary to this, under extreme conditions, the cells were found to show adaptive response to apoptosis and unable to regulate their own death; necrosis is therefore predominantly observed. In the present study, we showed induction of apoptosis in Clarias batrachus due to sublethal concentration of 2,4-D and butachlor at multiple exposure time. The first phase of the study involved light microscopy (LM) and transmission electron microscopy (TEM) for ultrastructural abnormalities of the germinal tissues. While, in the second phase of the study, DNA degradation of blood and hepatic tissue was resolved on agarose gel electrophoresis. In histopathological studies, large numbers of stage II oocytes were noted for nuclear blebbing irrespective of the test chemical. Some of the butachlor-exposed oocytes showed vacuolation and electron dense cytoplasm along with thickened nuclear envelope, having close association with the lysosomes on the cytoplasmic side. Some oocytes undergo nuclear blebbing having inner dense core and translucent cytoplasm. Leydig cells were slightly hypertrophied and few appeared pycnotic, a process involving necrotic changes in which the cell nuclei were characterized by rounding up and condensation resulting in hyperchromatic staining or pycnosis. In testicular tissue, spermatogonial nuclei had irregular large clumps of heterochromatin adjoining the nuclear membrane indicating initial stage of apoptotic cell death. Electrophoretic separation resulted in a ladder pattern of blood DNA and smear like pattern of hepatic DNA. These results indicate that the above herbicides are able to induce apoptosis both at molecular as

  4. C1q protein binds to the apoptotic nucleolus and causes C1 protease degradation of nucleolar proteins.

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    Cai, Yitian; Teo, Boon Heng Dennis; Yeo, Joo Guan; Lu, Jinhua

    2015-09-11

    In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus.

  5. Single gene retrieval from thermally degraded DNA

    Indian Academy of Sciences (India)

    Lianwen Zhang; Lianwen Zhang

    2005-12-01

    To simulate single gene retrieval from ancient DNA, several related factors have been investigated. By monitoring a 889 bp polymerase chain reaction (PCR) product and genomic DNA degradation, we find that heat and oxygen (especially heat) are both crucial factors influencing DNA degradation. The heat influence, mainly represented by temperature and heating time, affects the DNA degradation via DNA depurination followed by cleavage of nearby phosphodiesters. The heating time influence is temperature-dependent. By reactive oxygen species (ROS) scavenging and 1,3-diphenyl-isobenzofuran (DPBF) bleaching experiments the influence of oxygen on DNA thermal degradation was shown to occur via a singlet oxygen pathway. A comparative study of the thermal degradation of cellular DNA and isolated DNA showed that cellular lipids can aggravate DNA thermal degradation. These results confirm the possibility of gene amplification from thermally degraded DNA. They can be used to evaluate the feasibility of the retrieval of single gene from ancient remains.

  6. NUC-1, a caenorhabditis elegans DNase II homolog, functions in an intermediate step of DNA degradation during apoptosis.

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    Wu, Y C; Stanfield, G M; Horvitz, H R

    2000-03-01

    One hallmark of apoptosis is the degradation of chromosomal DNA. We cloned the Caenorhabditis elegans gene nuc-1, which is involved in the degradation of the DNA of apoptotic cells, and found that nuc-1 encodes a homolog of mammalian DNase II. We used the TUNEL technique to assay DNA degradation in nuc-1 and other mutants defective in programmed cell death and discovered that TUNEL labels apoptotic cells only during a transient intermediate stage. Mutations in nuc-1 allowed the generation of TUNEL-reactive DNA but blocked the conversion of TUNEL-reactive DNA to a subsequent TUNEL-unreactive state. Completion of DNA degradation did not occur in the absence of cell-corpse engulfment. Our data suggest that the process of degradation of the DNA of a cell corpse occurs in at least three distinct steps and requires activities provided by both the dying and the engulfing cell.

  7. Cloning and sequencing of a DNA fragment encoding N37 apoptotic peptide derived from p53

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective It was reported that p53 apoptotic peptide (N37) could inhibit p73 gene through being bound with iASPP,which could induce tumor cell apoptosis. To further explore the function of N37,we constructed the cloning plasmid of DNA fragment encoding p53 (N37) apoptotic peptide by using DNA synthesis and molecular biology methods. Methods According to human p53 sequence from the GenBank database,the primer of p53(N37) gene was designed using Primer V7.0 software. The DNA fragment encoding p53 (N37) apopto...

  8. DNA repair protein degradation mediated by helicase-like transcription factor in radiation-induced apoptotic cells%人类解旋酶样转录因子介导辐射凋亡细胞DNA修复蛋白的降解

    Institute of Scientific and Technical Information of China (English)

    朱应葆; 韩云; 孙艳; 梁莉; 贾廷珍

    2010-01-01

    目的 探讨人类解旋酶样转录因子(HLTF)转染对辐射诱导细胞凋亡时DNA修复相关蛋白水平的影响.方法 将野生型和RING结构域突变型HLTF分别转染人肺癌细胞A549,60Coγ射线15 Gy照射诱导细胞凋亡,用Western blot检测HRAD17和HRAD52蛋白水平的变化,免疫共沉淀检测特定复合物的存在.结果 γ射线诱导细胞凋亡时,野生型HLTF转染组与对照组相比,DNA修复蛋白HRAD17和HRAD52水平明显降低,而RING结构域突变组与对照组相比则没有明显变化;辐射诱导细胞凋亡时,HLTF可与HRAD17和HRAD52形成复合物.结论 HLTF可介导辐射诱导的凋亡细胞中HRAD17和HRAD52的降解,其机制可能是通过蛋白质复合物的相互作用使DNA修复蛋白泛素化而降解.%Objective To study the effects of helicase-like transcription factor (HLTF)transfection on DNA repair protein level in radiation-induced apoptotic cells.Methods Human lung carcinoma A549 cells were cultured and transfected with FLAG-tagged wild type HLTF (wild type HLTF transfection group),RING structure domain (ubiquitin conjugating region) mutatation HLTF expressing plasmid (mutant transfection group),empty plasmid (congtrol group) respectively.And the other cells were used as mock transfection group.All cells were irradiated with 15 Gy of 60Co γ-rays to induce apoptosis.Western blotting was used to detect the protein levels of the DNA repair proteins HRAD17 and HRAD52 in the transfected cells.Results The levels of HRAD17 and HRAD52 in the wild type HLTF transfection group was significantly lower than that of the control group.There was no significant difference in HRAD17 and HRAD52 levels between the mock transfection group and ubiquity in conjugating region mutation group.complexes of HLTF and HRAD17 and HRAD52 could be found in the irradiation-induced cells.Conclusions HLTF mediates the degradation of HRAD17 and HRAD52 in the irradiation-induced apoptotic cells possibly by the interaction of the

  9. The anti-apoptotic factor Che-1/AATF links transcriptional regulation, cell cycle control, and DNA damage response

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    Fanciulli Maurizio

    2007-07-01

    Full Text Available Abstract Che-1 is a RNA polymerase II binding protein involved in the transcriptional regulation of E2F target genes and in cell proliferation. Recently, it has been shown that Che-1 accumulates in cells responding to genotoxic agents such as Doxorubicin and ionizing radiation. The DNA damage-activated checkpoint kinases ATM and Chk2 interact with and phosphorylate Che-1, enhancing its accumulation and stability, and promoting Che-1-mediated transcription of p53-responsive genes and of p53 itself, as evidenced by microarray analysis. This transcriptional response is suppressed by expression of a Che-1 mutant lacking ATM and Chk2 phosphorylation amino acid residues, or by depletion of Che-1 by RNA silencing. In addition, chromatin immunoprecipitation analysis has shown that Che-1 is released from E2F target genes and recruited to the p21 and p53 promoters after DNA damage. Che-1 contributes to the maintenance of the G2/M checkpoint in response to genotoxic stress. These findings identify a new mechanism by which the checkpoint kinases regulate, via the novel effector Che-1, the p53 pathway. Lastly, increasing evidence suggests that Che-1 may be involved in apoptotic signaling in neural tissues. In cortical neurons, Che-1 exhibits anti-apoptotic activity, protecting cells from neuronal damage induced by amyloid β-peptide. In cerebellar granule neurons, Che-1 interacts with Tau in the cytoplasmic compartment and this interaction is modulated during neuronal apoptosis. Finally, Che-1 directly interacts with the neuronal cell-death inducer "NRAGE" which downregulates endogenous Che-1 by targeting it for proteasome-dependent degradation. These findings identify Che-1 as a novel cytoprotective factor against apoptotic insults and suggest that Che-1 may represent a potential target for therapeutic application.

  10. Apoptotic susceptibility to DNA damage of pluripotent stem cells facilitates pharmacologic purging of teratoma risk.

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    Smith, Alyson J; Nelson, Natalie G; Oommen, Saji; Hartjes, Katherine A; Folmes, Clifford D; Terzic, Andre; Nelson, Timothy J

    2012-10-01

    Pluripotent stem cells have been the focus of bioengineering efforts designed to generate regenerative products, yet harnessing therapeutic capacity while minimizing risk of dysregulated growth remains a challenge. The risk of residual undifferentiated stem cells within a differentiated progenitor population requires a targeted approach to eliminate contaminating cells prior to delivery. In this study we aimed to validate a toxicity strategy that could selectively purge pluripotent stem cells in response to DNA damage and avoid risk of uncontrolled cell growth upon transplantation. Compared with somatic cell types, embryonic stem cells and induced pluripotent stem cells displayed hypersensitivity to apoptotic induction by genotoxic agents. Notably, hypersensitivity in pluripotent stem cells was stage-specific and consistently lost upon in vitro differentiation, with the mean half-maximal inhibitory concentration increasing nearly 2 orders of magnitude with tissue specification. Quantitative polymerase chain reaction and Western blotting demonstrated that the innate response was mediated through upregulation of the BH3-only protein Puma in both natural and induced pluripotent stem cells. Pretreatment with genotoxic etoposide purged hypersensitive pluripotent stem cells to yield a progenitor population refractory to teratoma formation upon transplantation. Collectively, this study exploits a hypersensitive apoptotic response to DNA damage within pluripotent stem cells to decrease risk of dysregulated growth and augment the safety profile of transplant-ready, bioengineered progenitor cells.

  11. In vivo DNA damaging and apoptotic potential of silver nanoparticles in Swiss albino mice

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    Al Gurabi MA

    2015-01-01

    Full Text Available Mohammed A Al Gurabi, Daoud Ali, Saad Alkahtani, Saud Alarifi Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia Abstract: Nanoparticles can potentially cause adverse effects on organs, tissue, cell levels, and protein levels because of their physicochemical properties. Silver nanoparticles (AgNPs are being used on a wide scale in world consumer markets; their potential hazards for humans remain largely unknown. This study aimed to investigate the intraperitoneal toxicity of AgNPs (26 mg per kg of body weight, 52 mg per kg of body weight, and 78 mg per kg of body weight over 72 hours in Swiss albino mice. AgNPs induced a significant increase in serum liver injury markers including alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase. Induction of DNA damage was also studied in mice injected with AgNPs. Apoptosis (detected by using the terminal deoxynucleotidyl transferase deoxyuridine triphosphatase nick end labeling assay method in liver tissue and DNA strand breaks (detected by using the comet assay method in lymphocytes revealed that a concentration of 78 mg of AgNPs per kg body weight can cause significant apoptosis and DNA damage. The DNA damage and apoptosis raise the concern about the safety associated with application of the AgNPs. Significantly more alterations were induced in the hepatocytes of animals exposed to AgNP doses than in the control animals. The induced histological and apoptotic changes may be due to AgNP toxicity. immunohistochemical and ultrastructural of AgNP. Keywords: silver nanoparticles, liver tissue, histology, apoptosis, DNA damage, Swiss albino mice

  12. Next-generation sequencing offers new insights into DNA degradation

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren; Orlando, Ludovic Antoine Alexandre; Willerslev, Eske

    2012-01-01

    The processes underlying DNA degradation are central to various disciplines, including cancer research, forensics and archaeology. The sequencing of ancient DNA molecules on next-generation sequencing platforms provides direct measurements of cytosine deamination, depurination and fragmentation r...

  13. Relation between serum xenobiotic induced receptor activities and sperm DNA damage and sperm apoptotic markers in European and Inuit populations

    DEFF Research Database (Denmark)

    Long, Manhai; Stronati, Alessanda; Bizzaro, Davide;

    2007-01-01

    -mediated luciferase reporter gene expression. Sperm DNA damage was measured using terminal deoxynucleotidyl transferase-driven dUTP nick labeling assay (TUNEL) and pro- (Fas) and anti-apoptotic (Bcl-xL) markers were determined by immune methods. Different features of xenobiotic-induced receptor activity in serum...

  14. Fatal outcome in bacteremia is characterized by high plasma cell free DNA concentration and apoptotic DNA fragmentation: a prospective cohort study.

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    Reetta Huttunen

    Full Text Available INTRODUCTION: Recent studies have shown that apoptosis plays a critical role in the pathogenesis of sepsis. High plasma cell free DNA (cf-DNA concentrations have been shown to be associated with sepsis outcome. The origin of cf-DNA is unclear. METHODS: Total plasma cf-DNA was quantified directly in plasma and the amplifiable cf-DNA assessed using quantitative PCR in 132 patients with bacteremia caused by Staphylococcus aureus, Streptococcus pneumoniae, ß-hemolytic streptococcae or Escherichia coli. The quality of cf-DNA was analyzed with a DNA Chip assay performed on 8 survivors and 8 nonsurvivors. Values were measured on days 1-4 after positive blood culture, on day 5-17 and on recovery. RESULTS: The maximum cf-DNA values on days 1-4 (n = 132 were markedly higher in nonsurvivors compared to survivors (2.03 vs 1.26 ug/ml, p1.52 ug/ml remained an independent risk factor for case fatality in a logistic regression model. Qualitative analysis of cf-DNA showed that cf-DNA displayed a predominating low-molecular-weight cf-DNA band (150-200 bp in nonsurvivors, corresponding to the size of the apoptotic nucleosomal DNA. cf-DNA concentration showed a significant positive correlation with visually graded apoptotic band intensity (R = 0.822, p<0.001. CONCLUSIONS: Plasma cf-DNA concentration proved to be a specific independent prognostic biomarker in bacteremia. cf-DNA displayed a predominating low-molecular-weight cf-DNA band in nonsurvivors corresponding to the size of apoptotic nucleosomal DNA.

  15. Compromised proteasome degradation elevates neuronal nitric oxide synthase levels and induces apoptotic cell death.

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    Lam, Philip Y; Cadenas, Enrique

    2008-10-15

    The significance of impairment of proteasome activity in PC12 cells was examined in connection with nitrative/nitrosative stress and apoptotic cell death. Treatment of differentiated PC12 cells with MG132, a proteasome inhibitor, elicited a dose- and time-dependent increase in neuronal nitric oxide synthase (nNOS) protein levels, decreased cell viability, and increased cytotoxicity. Viability and cytotoxicity were ameliorated by L-NAME (a broad NOS inhibitor). Nitric oxide/peroxynitrite formation was increased upon treatment of PC12 cells with MG132 and decreased upon treatment with the combination of MG132 and 7-NI (a specific inhibitor of nNOS). The decreases in cell viability appeared to be effected by an activation of JNK and its effect on mitochondrial Bcl-x(L) phosphorylation. These effects are strengthened by the activation of caspase-9 along with increased caspase-3 activity upon treatment of PC12 cells with MG132. These results suggest that impairment of proteasome activity and consequent increases in nNOS levels lead to a nitrative stress that involves the coordinated response of JNK cytosolic signaling and mitochondrion-driven apoptotic pathways.

  16. Identification of a nuclear-localized nuclease from wheat cells undergoing programmed cell death that is able to trigger DNA fragmentation and apoptotic morphology on nuclei from human cells.

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    Domínguez, Fernando; Cejudo, Francisco J

    2006-08-01

    PCD (programmed cell death) in plants presents important morphological and biochemical differences compared with apoptosis in animal cells. This raises the question of whether PCD arose independently or from a common ancestor in plants and animals. In the present study we describe a cell-free system, using wheat grain nucellar cells undergoing PCD, to analyse nucleus dismantling, the final stage of PCD. We have identified a Ca2+/Mg2+ nuclease and a serine protease localized to the nucleus of dying nucellar cells. Nuclear extracts from nucellar cells undergoing PCD triggered DNA fragmentation and other apoptotic morphology in nuclei from different plant tissues. Inhibition of the serine protease did not affect DNA laddering. Furthermore, we show that the nuclear extracts from plant cells triggered DNA fragmentation and apoptotic morphology in nuclei from human cells. The inhibition of the nucleolytic activity with Zn2+ or EDTA blocked the morphological changes of the nucleus. Moreover, nuclear extracts from apoptotic human cells triggered DNA fragmentation and apoptotic morphology in nuclei from plant cells. These results show that degradation of the nucleus is morphologically and biochemically similar in plant and animal cells. The implication of this finding on the origin of PCD in plants and animals is discussed.

  17. Early apoptotic changes in human spermatozoa and their relationships with conventional semen parameters and sperm DNA fragmentation

    Institute of Scientific and Technical Information of China (English)

    Hao-Bo Zhang; Shao-Ming Lu; Chun-Yan Ma; Li Wang; Xiao Li; Zi-Jiang Chen

    2008-01-01

    Aim: To investigate whether early apoptotic changes in spermatozoa can be significant markers for sperm quality.Methods: Two early apoptotic changes in the semen of 56 men were assessed using Annexin V (AN)/propidium iodide (PI) staining for phosphatidylserine externalization and JC-1 staining for mitochondrial membrane potential (MMP). The results were compared with conventional semen parameters and DNA fragmentation identified using the TUNEL assay. Results: The different labeling patterns in the bivariate Annexin V/PI analysis identified four distinc-tive spermatozoa populations. The percentage of AN-/PI- spermatozoa positively correlated with conventional semen parameters and MMP, but negatively correlated with TUNEL (+) spermatozoa. As for the AN-/PI+ fraction, we found an opposite result in comparison to AN-/PI- spermatozoa. The level of early apoptotic AN+/PI- spermatozoa negatively correlated with MMP and sperm motility. The level of late apoptotic AN+/PI+ spermatozoa negatively correlated with conventional semen parameters and MMP, and positively correlated with TUNEL (+) spermatozoa. MMP positively correlated with conventional semen parameters, but negatively correlated with TUNEL (+) spermatozoa. Conclusion:Although early apoptotic AN+/PI- spermatozoa only negatively correlates with sperm motility, the differences in proportion of each subpopulation of spermatozoa (especially, the percentage of AN-/PI-spermatozoa), and decreased MMP might be significant markers for diagnosing male infertility. They possibly bring additional information to predict the outcome of in vitro fertilization.

  18. The role of antigen specificity in the binding of murine monoclonal anti-DNA antibodies to microparticles from apoptotic cells.

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    Ullal, Anirudh J; Marion, Tony N; Pisetsky, David S

    2014-10-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and markers of underlying immune system disturbances. These antibodies bind to both single-stranded and double-stranded DNA, mediating pathogenesis by forming immune complexes. As shown recently, DNA in blood exists in both free and particulate forms, with DNA representing an important component of microparticles. Microparticles are membrane-bound vesicles containing nuclear molecules, released by membrane blebbing during cell death and activation. A panel of monoclonal NZB/NZW F1 anti-DNA antibodies was tested for binding to microparticles generated from apoptotic THP-1 and Jurkat cells. These studies showed that only certain anti-DNA antibodies in the panel, specific for double-stranded DNA, bound to microparticles. Binding to particles was reduced by soluble DNA or DNase treatment. Together, these results indicate that particle binding is a feature of only certain anti-DNA antibodies, reflecting immunochemical properties of the antibodies and the nature of the exposed DNA antigens.

  19. Differential cDNA cloning by enzymatic degrading subtraction (EDS).

    OpenAIRE

    1994-01-01

    We describe a new method, called enzymatic degrading subtraction (EDS), for the construction of subtractive libraries from PCR amplified cDNA. The novel features of this method are that i) the tester DNA is blocked by thionucleotide incorporation; ii) the rate of hybridization is accelerated by phenol-emulsion reassociation; and iii) the driver cDNA and hybrid molecules are enzymatically removed by digestion with exonucleases III and VII rather than by physical partitioning. We demonstrate th...

  20. [DNA degradation during standard alkaline of thermal denaturation].

    Science.gov (United States)

    Drozhdeniuk, A P; Sulimova, G E; Vaniushin, B F

    1976-01-01

    Essential degradation 8 DNA (up to 10 per cent) with liberation of acid-soluble fragments takes place on the standard alkaline (0,01 M sodium phosphate, pH 12, 60 degrees, 15 min) or thermal (0.06 M sodium phosphate buffer, pH 6.8, 102 degrees C, 15 min) denaturation. This degradation is more or less selective: fraction of low molecular weight fragments, isolated by hydroxyapatite cromatography and eluted by 0.06 M sodium phosphate buffer, pH 6.8 is rich in adenine and thymine and contains about 2 times less 5-methylcytosine than the total wheat germ DNA. The degree of degradation of DNA on thermal denaturation is higher than on alkaline degradation. Therefore while studying reassociation of various DNA, one and the same standard method of DNA denaturation should be used. Besides, both the level of DNA degradation and the nature of the resulting products (fragments) should be taken into account.

  1. DNA binding and apoptotic induction ability of harmalol in HepG2: Biophysical and biochemical approaches.

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    Sarkar, Sarita; Bhattacharjee, Paromita; Bhadra, Kakali

    2016-10-25

    Harmalol administration caused remarkable reduction in proliferation of HepG2 cells with GI50 of 14.2 μM, without showing much cytotoxicity in embryonic liver cell line, WRL-68. Data from circular dichroism (CD) and differential scanning calorimetric (DSC) analysis of harmalol-CT DNA complex shows conformational changes with prominent CD perturbation and stabilization of CT DNA by 8 °C. Binding constant and stoichiometry was calculated using the above biophysical techniques. The Scatchard plot constructed from CD data showed cooperative binding, from which the cooperative binding affinity (K'ω) of 4.65 ± 0.7 × 10(5) M(-1), and n value of 4.16 were deduced. The binding parameter obtained from DSC melting data was in good agreement with the above CD data. Furthermore, dose dependent apoptotic induction ability of harmalol was studied in HepG2 cells using different biochemical assays. Generation of ROS, DNA damage, changes in cellular external and ultramorphology, alteration of membrane, formation of comet tail, decreased mitochondrial membrane potential and a significant increase in Sub Go/G1 population made the cancer cell, HepG2, prone to apoptosis. Up regulation of p53 and caspase 3 further indicated the apoptotic role of harmalol.

  2. Proteolysis of the human DNA polymerase delta smallest subunit p12 by μ-calpain in calcium-triggered apoptotic HeLa cells.

    Directory of Open Access Journals (Sweden)

    Xiaoting Fan

    Full Text Available Degradation of p12 subunit of human DNA polymerase delta (Pol δ that results in an interconversion between Pol δ4 and Pol δ3 forms plays a significant role in response to replication stress or genotoxic agents triggered DNA damage. Also, the p12 is readily degraded by human calpain in vitro. However, little has been done for the investigation of its degree of participation in any of the more common apoptosis. Here, we first report that the p12 subunit is a substrate of μ-calpain. In calcium-triggered apoptotic HeLa cells, the p12 is degraded at 12 hours post-induction (hpi, restored thereafter by 24 hpi, and then depleted again after 36 hpi in a time-dependent manner while the other three subunits are not affected. It suggests a dual function of Pol δ by its interconversion between Pol δ4 and Pol δ3 that is involved in a novel unknown apoptosis mechanism. The proteolysis of p12 could be efficiently blocked by both calpain inhibitor ALLN and proteasome inhibitor MG132. In vitro pull down and co-immunoprecipitation assays show that the μ-calpain binds to p12 through the interaction of μ-calpain with Pol δ other three subunits, not p12 itself, and PCNA, implying that the proteolysis of p12 by μ-calpain might be through a Pol δ4/PCNA complex. The p12 cleavage sites by μ-calpain are further determined as the location within a 16-amino acids peptide 28-43 by in vitro cleavage assays. Thus, the p12/Pol δ is a target as a nuclear substrate of μ-calpain in a calcium-triggered apoptosis and appears to be a potential marker in the study of the chemotherapy of cancer therapies.

  3. Investigation of DNA damage response and apoptotic gene methylation pattern in sporadic breast tumors using high throughput quantitative DNA methylation analysis technology

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    Prakash Neeraj

    2010-11-01

    Full Text Available Abstract Background- Sporadic breast cancer like many other cancers is proposed to be a manifestation of abnormal genetic and epigenetic changes. For the past decade our laboratory has identified genes involved in DNA damage response (DDR, apoptosis and immunesurvelliance pathways to influence sporadic breast cancer risk in north Indian population. Further to enhance our knowledge at the epigenetic level, we performed DNA methylation study involving 17 gene promoter regions belonging to DNA damage response (DDR and death receptor apoptotic pathway in 162 paired normal and cancerous breast tissues from 81 sporadic breast cancer patients, using a high throughput quantitative DNA methylation analysis technology. Results- The study identified five genes with statistically significant difference between normal and tumor tissues. Hypermethylation of DR5 (P = 0.001, DCR1 (P = 0.00001, DCR2 (P = 0.0000000005 and BRCA2 (P = 0.007 and hypomethylation of DR4 (P = 0.011 in sporadic breast tumor tissues suggested a weak/aberrant activation of the DDR/apoptotic pathway in breast tumorigenesis. Negative correlation was observed between methylation status and transcript expression levels for TRAIL, DR4, CASP8, ATM, CHEK2, BRCA1 and BRCA2 CpG sites. Categorization of the gene methylation with respect to the clinicopathological parameters showed an increase in aberrant methylation pattern in advanced tumors. These uncharacteristic methylation patterns corresponded with decreased death receptor apoptosis (P = 0.047 and DNA damage repair potential (P = 0.004 in advanced tumors. The observation of BRCA2 -26 G/A 5'UTR polymorphism concomitant with the presence of methylation in the promoter region was novel and emerged as a strong candidate for susceptibility to sporadic breast tumors. Conclusion- Our study indicates that methylation of DDR-apoptotic gene promoters in sporadic breast cancer is not a random phenomenon. Progressive epigenetic alterations in advancing

  4. DNA Profiling Success Rates from Degraded Skeletal Remains in Guatemala.

    Science.gov (United States)

    Johnston, Emma; Stephenson, Mishel

    2016-07-01

    No data are available regarding the success of DNA Short Tandem Repeat (STR) profiling from degraded skeletal remains in Guatemala. Therefore, DNA profiling success rates relating to 2595 skeletons from eleven cases at the Forensic Anthropology Foundation of Guatemala (FAFG) are presented. The typical postmortem interval was 30 years. DNA was extracted from bone powder and amplified using Identifiler and Minifler. DNA profiling success rates differed between cases, ranging from 50.8% to 7.0%, the overall success rate for samples was 36.3%. The best DNA profiling success rates were obtained from femur (36.2%) and tooth (33.7%) samples. DNA profiles were significantly better from lower body bones than upper body bones (p = forensic DNA sampling strategies in future victim recovery investigations.

  5. A putative role of Drep1 in apoptotic DNA fragmentation system in fly is mediated by direct interaction with Drep2 and Drep4.

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    Park, Ok Kyung; Park, Hyun Ho

    2013-04-01

    DNA fragmentation is common phenomenon for apoptotic cell death. DNA fragmentation factor, called DFF40 (CAD: mouse homologue), is a main nuclease for apoptotic DNA fragmentation. Nuclease activity of DFF40 is normally inhibited by DFF45 by tight interaction via CIDE domain without apoptotic stimuli. Once effector caspase is activated during apoptosis signaling, it cleave DFF45, allowing DFF40 to enter the nucleus and cleave chromosomal DNA. Unlike mammalian system, apoptotic DNA fragmentation in the fly might be controlled by four DFF-related proteins, known as Drep1, Drep2, Drep3 and Drep4. Although the function of Drep1 and Drep4 is well known as DFF45 and DFF40 homologues, respectively, the function of Drep2 and Drep3 is still unclear. DFF-related proteins contain a conserved CIDE domain of ~90 amino acid residues that is involved in protein-protein interaction. Here, we showed that Drep1 directly bind to Drep2 as well as Drep4 via CIDE domain. In addition, we found that the interaction of Drep2 and Drep4 to Drep1 was not competitive indicating that Drep2 and Drep4 bind different place of Drep1. All together, we suggest that Drep1 might be involved in apoptotic DNA fragmentation of fly system by direct interaction with Drep2 as well as Drep4.

  6. Dual apoptotic DNA fragmentation system in the fly: Drep2 is a novel nuclease of which activity is inhibited by Drep3.

    Science.gov (United States)

    Park, Ok Kyeung; Park, Hyun Ho

    2012-09-21

    DNA fragmentation is the hallmark of apoptotic cells and mainly mediated by the DNA fragmentation factor DFF40(CAD)/DFF45(ICAD). DFF40 is a novel nuclease, whereas DFF45 is an inhibitor that can suppress the nuclease activity. Apoptotic DNA fragmentation in the fly is controlled by four DFF-related proteins, known as Drep1, 2, 3 and 4. However, the functions of Drep2 and Drep3 are totally unknown. Here, we found that Drep2 is a novel nuclease whose activity is inhibited by Drep3 through a tight interaction with the CIDE domain. Our results suggest that the fly has dual apoptotic DNA fragmentation systems: Drep1: Drep4 and Drep2: Drep3 complexes.

  7. Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq).

    Science.gov (United States)

    Graham, Carly F; Glenn, Travis C; McArthur, Andrew G; Boreham, Douglas R; Kieran, Troy; Lance, Stacey; Manzon, Richard G; Martino, Jessica A; Pierson, Todd; Rogers, Sean M; Wilson, Joanna Y; Somers, Christopher M

    2015-11-01

    Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next-generation DNA sequencing (RADSeq, GBS) is unknown. We experimentally examined the effects of in situDNA degradation on data generation for a modified double-digest RADSeq approach (3RAD). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish (Coregonus clupeaformis) following 0-, 12-, 48- and 96-h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12-48 h) performed well. In contrast, the number of RADtags per individual, number of variable sites, and percentage of identical RADtags retained were all dramatically reduced when libraries were made using highly degraded DNA (96-h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2- to 16-fold), and additional read-length trimming. We conclude that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded.

  8. [Cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy].

    Science.gov (United States)

    Tan, Jiao; Wang, Ya-Ping; Wang, Hui-Xin; Liang, Jian-Ming; Zhang, Meng; Sun, Xun; Huang, Yong-Zhuo

    2014-12-01

    To develop a cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy, we prepared the AVPI-LMWP/pTRAIL self-assembled complexes containing a therapeutic combination of peptide drug AVPI and DNA drug TRAIL. The chimeric apoptotic peptide AVPI-LMWP was synthesized using the standard solid-phase synthesis. The cationic AVPI-LMWP could condense pTRAIL by electrostatic interaction. The physical-chemical properties of the AVPI-LMWP/pTRAIL complexes were characterized. The cellular uptake efficiency and the inhibitory activity of the AVPI-LMWP/pTRAIL complexes on tumor cell were also performed. The results showed that the AVPI-LMWP/pTRAIL complexes were successfully prepared by co-incubation. With the increase of mass ratio (AVPI-LMWP/DNA), the particle size was decreased and the zeta potential had few change. Agarose gel electrophoresis showed that AVPI-LMWP could fully bind and condense pTRAIL at a mass ratio above 15:1. Cellular uptake efficiency was improved along with the increased ratio of W(AVPI-LMWP)/WpTRAIL. The in vitro cytotoxicity experiments demonstrated that the AVPI-LMWP/pTRAIL (W:W = 20:1) complexes was significantly more effective than the pTRAIL, AVPI-LMWP alone or LMWP/pTRAIL complexes on inhibition of HeLa cell growth. Our studies indicated that the AVPI-LMWP/pTRAIL co-delivery system could deliver plasmid into HeLa cell and induce tumor cell apoptosis efficiently, which showed its potential in cancer therapy using combination of apoptoic peptide and gene drugs.

  9. LDFF, the large molecular weight DNA fragmentation factor, is responsible for the large molecular weight DNA degradation during apoptosis in Xenopus egg extracts

    Institute of Scientific and Technical Information of China (English)

    Zhi Gang LU; Chuan Mao ZHANG; Zhong He ZHAI

    2004-01-01

    DNA degradation is a biochemical hallmark in apoptosis. It has been demonstrated in many cell types that there are two stages of DNA fragmentation during the apoptotic execution. In the early stage, chromatin DNA is cut into large molecular weight DNA fragments, although the responsible nuclease(s) has not been recognized. In the late stage, the chromatin DNA is cleaved further into short oligonucleosomal fragments by a well-characterized nuclease in apoptosis,the caspase-activated DNase (CAD/DFF40). In this study, we demonstrate that large molecular weight DNA fragmentation also occurs in Xenopus egg extracts in apoptosis. We show that the large molecular weight DNA fragmentation factor (LDFF) is not the Xenopus CAD homolog XCAD. LDFF is activated by caspase-3. The large molecular weight DNA fragmentation activity of LDFF is Mg2+-dependent and Ca2+-independent, can occur in both acidic and neutral pH conditions and can tolerate 45℃ treatment. These results indicate that LDFF in Xenopus egg extracts might be a new DNase (or DNases) responsible for the large DNA fragmentation.

  10. Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells.

    Science.gov (United States)

    Krüger, Katharina; Ziegler, Verena; Hartmann, Christina; Henninger, Christian; Thomale, Jürgen; Schupp, Nicole; Fritz, Gerhard

    2016-02-01

    The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury.

  11. DNase I induced DNA degradation is inhibited by neomycin.

    Science.gov (United States)

    Woegerbauer, M; Burgmann, H; Davies, J; Graninger, W

    2000-03-01

    Preparations of antimicrobials from biotechnological sources containing nucleic acids may serve as vector for the dissemination of resistance genes. An essential prerequisite for the acquisition of a new resistance phenotype in a transformational scenario is the availability of physically intact DNA molecules capable of transforming competent microorganisms. DNA is thought to be an easy target for catabolic processes when present in the natural habitat of bacteria (e.g. gastrointestinal tract, soil) due to the overall presence of nucleolytic enzymes. Aminoglycoside antibiotics are known to display a strong affinity to nucleic acids rendering these compounds to be primary candidates for exerting DNA protective functions in the gastrointestinal tract when applied orally during antibiotic chemotherapy. Using a DNase I protection assay it could be demonstrated that neomycin B at a concentration of 2 mM completely inhibited degradation of plasmid DNA in vitro. No inhibition of degradation was observed with streptomycin and kanamycin and the non-aminoglycoside antibiotics oxytetracycline and ampicillin under identical assay conditions. Thus, neomycin preparations may be able to promote structural integrity of contaminating DNA-fragments in DNase-rich environments.

  12. Interaction of DNA-lesions induced by sodium fluoride and radiation and its influence in apoptotic induction in cancer cell lines

    Directory of Open Access Journals (Sweden)

    Santosh Podder

    2015-01-01

    Full Text Available Fluoride is an essential trace element but also an environmental contaminant with major sources of exposure being drinking water, food and pesticides. Previous studies showed that sodium fluoride (NaF at 5 mM or more is required to induce apoptosis and chromosome aberrations and proposed that DNA damage and apoptosis play an important role in toxicity of excessive fluoride. The aim of this study is directed to understand the nature of DNA-lesions induced by NaF by allowing its interaction with radiation induced DNA-lesions. NaF 5 mM was used after observing inability to induce DNA damages and apoptosis by single exposure with 50 μM or 1 mM NaF. Co-exposure to NaF and radiation significantly increased the frequency of aberrant metaphases and exchange aberrations in human lymphocytes and arrested the cells in G1 stage instead of apoptotic death. Flow cytometric analysis, DNA fragmentation and PARP-cleavage analysis clearly indicated that 5 mM NaF together with radiation (1 Gy induced apoptosis in both U87 and K562 cells due to down regulation of expression of anti-apoptotic proteins, like Bcl2 in U87 and inhibitors of apoptotic proteins like survivin and cIAP in K562 cells. This study herein suggested that single exposure with extremely low concentration of NaF unable to induce DNA lesions whereas higher concentration induced DNA lesions interact with the radiation-induced DNA lesions. Both are probably repaired rapidly thus showed increased interactive effect. Coexposure to NaF and radiation induces more apoptosis in cancer cell lines which could be due to increased exchange aberrations through lesions interaction and downregulating anti-apoptotic genes.

  13. Degradable starch nanoparticle assisted ethanol precipitation of DNA.

    Science.gov (United States)

    Ip, Alexander C-F; Tsai, Tsung Hao; Khimji, Imran; Huang, Po-Jung Jimmy; Liu, Juewen

    2014-09-22

    Precipitation of DNA from a large volume of aqueous solution is an important step in many molecular biology and analytical chemistry experiments. Currently, this is mainly achieved by ethanol precipitation, where a long-term incubation (usually overnight) at low temperature of -20 to -80°C with high salt concentration is required. This method also requires a large quantity of DNA to form a visible pellet and was tested mainly for double-stranded DNA. To improve DNA precipitation, co-precipitating polymers such as linear polyacrylamide has been used. In this work, we report that starch nanoparticles (SNPs) can achieve convenient DNA precipitation at room temperature with a low salt concentration and short incubation time. This method requires as low as 0.01-0.1% SNPs and can precipitate both single- and double-stranded DNA of various lengths. The effect of salt concentration, pH and the crosslinking density of SNPs has been systematically studied. Compared to other types of precipitating agents, SNPs are highly biocompatible and can be degraded by a common enzyme (amylase). This work suggests a novel application of a bio-based material that is prepared in mass production.

  14. Simultaneous detection and semiquantification of DNA damage in normal and apoptotic cells: triple-immunofluorescent labeling using DAPI, antibodies, and TUNEL.

    Science.gov (United States)

    Agrawal, Anant; Godar, Dianne E

    2012-07-01

    We developed a triple-labeling immunofluorescence technique that simultaneously identifies total DNA (DAPI), DNA damage (antibodies), and dead cells [terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells] and a method that semiquantifies DNA damage in paraffin-embedded tissues. Using this technique in combination with our analysis method, scientists can now simultaneously detect and compare the relative amounts of DNA damage of almost any kind (except single-strand and double-strand breaks), using indirect fluorescent antibody labeling, in both normal and dying cells of different tissues. Simultaneous labeling of DNA damage and dead or TUNEL-positive cells can reduce processing costs and analysis time, and can lead to discoveries concerning how cells die from different DNA damages. We used increasing doses of UV (290 to 400 nm) radiation to create DNA damage in the form of cyclobutane pyrimidine dimers and 6-4 photoproducts that kill some of the cells in 3-dimensional tissue-engineered skin and vaginal samples. We describe a protocol that reliably detects and semiquantifies DNA damage in both normal and apoptotic cells. We show this triple-labeling immunofluorescence technique and analysis method yields linear UV dose response curves for damage to DNA bases that allows semiquantification of cyclobutane pyrimidine dimers and calculation of its repair rate (T=1 and 24 h), whereas TUNEL allows quantification of the number of apoptotic cells. Scientists can now create beautiful fluorescent pictures that simultaneously detect DNA damage in both normal and apoptotic cells to assess and semiquantify the damage to understand better how different insults lead to the cell's demise.

  15. A functional yeast survival screen of tumor-derived cDNA libraries designed to identify anti-apoptotic mammalian oncogenes.

    Directory of Open Access Journals (Sweden)

    Moritz Eißmann

    Full Text Available Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems.

  16. Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil)

    NARCIS (Netherlands)

    Yamada, T.; Takatsu, Y.; Kasumi, K.; Ichimura, K.; Doorn, van W.G.

    2006-01-01

    We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the num

  17. DNA fragmentation in apoptosis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo. Recent work also suggests the existence of additional endonucleases for DNA degradation. Understanding the roles of individual endonucleases in apoptosis, and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis, as well as in various diseased states, will be a major research focus in the near future.

  18. Statistical model for degraded DNA samples and adjusted probabilities for allelic drop-out

    DEFF Research Database (Denmark)

    Tvedebrink, Torben; Eriksen, Poul Svante; Mogensen, Helle Smidt;

    2012-01-01

    Abstract DNA samples found at a scene of crime or obtained from the debris of a mass disaster accident are often subject to degradation. When using the STR DNA technology, the DNA profile is observed via a so-called electropherogram (EPG), where the alleles are identified as signal peaks above a ...

  19. Statistical model for degraded DNA samples and adjusted probabilities for allelic drop-out

    DEFF Research Database (Denmark)

    Tvedebrink, Torben; Eriksen, Poul Svante; Mogensen, Helle Smidt

    2012-01-01

    DNA samples found at a scene of crime or obtained from the debris of a mass disaster accident are often subject to degradation. When using the STR DNA technology, the DNA profile is observed via a so-called electropherogram (EPG), where the alleles are identified as signal peaks above a certain l...

  20. Human Noxin is an anti-apoptotic protein in response to DNA damage of A549 non-small cell lung carcinoma.

    Science.gov (United States)

    Won, Kyoung-Jae; Im, Joo-Young; Yun, Chae-Ok; Chung, Kyung-Sook; Kim, Young Joo; Lee, Jung-Sun; Jung, Young-Jin; Kim, Bo-Kyung; Song, Kyung Bin; Kim, Young-Ho; Chun, Ho-Kyung; Jung, Kyeong Eun; Kim, Moon-Hee; Won, Misun

    2014-06-01

    Human Noxin (hNoxin, C11Orf82), a homolog of mouse noxin, is highly expressed in colorectal and lung cancer tissues. hNoxin contains a DNA-binding C-domain in RPA1, which mediates DNA metabolic processes, such as DNA replication and DNA repair. Expression of hNoxin is associated with S phase in cancer cells and in normal cells. Expression of hNoxin was induced by ultraviolet (UV) irradiation. Knockdown of hNoxin caused growth inhibition of colorectal and lung cancer cells. The comet assay and western blot analysis revealed that hNoxin knockdown induced apoptosis through activation of p38 mitogen-activated protein kinase (MAPK)/p53 in non-small cell lung carcinoma A549 cells. Furthermore, simultaneous hNoxin knockdown and treatment with DNA-damaging agents, such as camptothecin (CPT) and UV irradiation, enhanced apoptosis, whereas Trichostatin A (TSA) did not. However, transient overexpression of hNoxin rescued cells from DNA damage-induced apoptosis but did not block apoptosis in the absence of DNA damage. These results suggest that hNoxin may be associated with inhibition of apoptosis in response to DNA damage. An adenovirus expressing a short hairpin RNA against hNoxin transcripts significantly suppressed the growth of A549 tumor xenografts, indicating that hNoxin knockdown has in vivo anti-tumor efficacy. Thus, hNoxin is a DNA damage-induced anti-apoptotic protein and potential therapeutic target in cancer.

  1. Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review.

    Science.gov (United States)

    Gryson, Nicolas

    2010-03-01

    The applicability of a DNA-based method for GMO detection and quantification depends on the quality and quantity of the DNA. Important food-processing conditions, for example temperature and pH, may lead to degradation of the DNA, rendering PCR analysis impossible or GMO quantification unreliable. This review discusses the effect of several food processes on DNA degradation and subsequent GMO detection and quantification. The data show that, although many of these processes do indeed lead to the fragmentation of DNA, amplification of the DNA may still be possible. Length and composition of the amplicon may, however, affect the result, as also may the method of extraction used. Also, many techniques are used to describe the behaviour of DNA in food processing, which occasionally makes it difficult to compare research results. Further research should be aimed at defining ingredients in terms of their DNA quality and PCR amplification ability, and elaboration of matrix-specific certified reference materials.

  2. Human identification & forensic analyses of degraded or low level DNA

    NARCIS (Netherlands)

    Westen, Antoinette-Andrea

    2013-01-01

    DNA-based human identification is employed in varying situations, such as disaster victim identification, relationship testing and forensic analyses. When DNA is of low quality and/or quantity, standard methods for DNA profiling may not suffice. The research described in this thesis is aimed at the

  3. Two PI 3-kinases and one PI 3-phosphatase together establish the cyclic waves of phagosomal PtdIns(3P critical for the degradation of apoptotic cells.

    Directory of Open Access Journals (Sweden)

    Nan Lu

    2012-01-01

    Full Text Available Phosphatidylinositol 3-phosphate (PtdIns(3P is a signaling molecule important for many membrane trafficking events, including phagosome maturation. The level of PtdIns(3P on phagosomes oscillates in two waves during phagosome maturation. However, the physiological significance of such oscillation remains unknown. Currently, the Class III PI 3-kinase (PI3K Vps34 is regarded as the only kinase that produces PtdIns(3P in phagosomal membranes. We report here that, in the nematode C. elegans, the Class II PI3K PIKI-1 plays a novel and crucial role in producing phagosomal PtdIns(3P. PIKI-1 is recruited to extending pseudopods and nascent phagosomes prior to the appearance of PtdIns(3P in a manner dependent on the large GTPase dynamin (DYN-1. PIKI-1 and VPS-34 act in sequence to provide overlapping pools of PtdIns(3P on phagosomes. Inactivating both piki-1 and vps-34 completely abolishes the production of phagosomal PtdIns(3P and disables phagosomes from recruiting multiple essential maturation factors, resulting in a complete arrest of apoptotic-cell degradation. We have further identified MTM-1, a PI 3-phosphatase that antagonizes the activities of PIKI-1 and VPS-34 by down-regulating PtdIns(3P on phagosomes. Remarkably, persistent appearance of phagosomal PtdIns(3P, as a result of inactivating mtm-1, blocks phagosome maturation. Our findings demonstrate that the proper oscillation pattern of PtdIns(3P on phagosomes, programmed by the coordinated activities of two PI3Ks and one PI 3-phosphatase, is critical for phagosome maturation. They further shed light on how the temporally controlled reversible phosphorylation of phosphoinositides regulates the progression of multi-step cellular events.

  4. Regulation of Apoptotic Endonucleases by EndoG

    Science.gov (United States)

    Zhdanov, Dmitry D.; Fahmi, Tariq; Wang, Xiaoying; Apostolov, Eugene O.; Sokolov, Nikolai N.; Javadov, Sabzali

    2015-01-01

    Cells contain several apoptotic endonucleases, which appear to act simultaneously before and after cell death by destroying the host cell DNA. It is largely unknown how the endonucleases are being induced and whether they can regulate each other. This study was performed to determine whether apoptotic mitochondrial endonuclease G (EndoG) can regulate expression of other apoptotic endonucleases. The study showed that overexpression of mature EndoG in kidney tubular epithelial NRK-52E cells can increase expression of caspase-activated DNase (CAD) and four endonucleases that belong to DNase I group including DNase I, DNase X, DNase IL2, and DNase γ, but not endonucleases of the DNase 2 group. The induction of DNase I-type endonucleases was associated with DNA degradation in promoter/exon 1 regions of the endonuclease genes. These results together with findings on colocalization of immunostained endonucleases and TUNEL suggest that DNA fragmentation after EndoG overexpression was caused by DNase I endonucleases and CAD in addition to EndoG itself. Overall, these data provide first evidence for the existence of the integral network of apoptotic endonucleases regulated by EndoG. PMID:25849439

  5. Comparison of three methods of DNA extraction from human bones with different degrees of degradation.

    Science.gov (United States)

    Jakubowska, Joanna; Maciejewska, Agnieszka; Pawłowski, Ryszard

    2012-01-01

    There is a necessity for deceased identification as a result of many accidents and sometimes bones are the only accessible source of DNA. So far, a universal method that allows for extraction of DNA from materials at different stages of degradation does not exist. The aims of this study were: the comparison of three methods of DNA extraction from bones with different degree of degradation and an evaluation of the usefulness of these methods in forensic genetics. The efficiency of DNA extraction, the degree of extract contamination by polymerase chain reaction (PCR) inhibitors and the possibility of determining the STR loci profile were especially being compared. Nuclear DNA from bones at different states of degradation was isolated using three methods: classical, organic phenol-chloroform extraction, DNA extraction from crystal aggregates and extraction by total demineralisation. Total demineralisation is the best method for most cases of DNA extraction from bones, although it does not provide pure DNA. DNA extraction from aggregates removes inhibitors much better and is also a good method of choice when identity determination of exhumed remains is necessary. In the case of not buried bones (remains found outside) total demineralisation or phenol-chloroform protocols are more efficient for successful DNA extraction.

  6. Exonuclease VII is involved in "reckless" DNA degradation in UV-irradiated Escherichia coli.

    Science.gov (United States)

    Repar, Jelena; Briški, Nina; Buljubašić, Maja; Zahradka, Ksenija; Zahradka, Davor

    2013-01-20

    The recA mutants of Escherichia coli exhibit an abnormal DNA degradation that starts at sites of double-strand DNA breaks (DSBs), and is mediated by RecBCD exonuclease (ExoV). This "reckless" DNA degradation occurs spontaneously in exponentially growing recA cells, and is stimulated by DNA-damaging agents. We have previously found that the xonA and sbcD mutations, which inactivate exonuclease I (ExoI) and SbcCD nuclease, respectively, markedly suppress "reckless" DNA degradation in UV-irradiated recA cells. In the present work, we show that inactivation of exonuclease VII (ExoVII) by an xseA mutation contributes to attenuation of DNA degradation in UV-irradiated recA mutants. The xseA mutation itself has only a weak effect, however, it acts synergistically with the xonA or sbcD mutations in suppressing "reckless" DNA degradation. The quadruple xseA xonA sbcD recA mutants show no sign of DNA degradation during post-irradiation incubation, suggesting that ExoVII, together with ExoI and SbcCD, plays a crucial role in regulating RecBCD-catalyzed chromosome degradation. We propose that these nucleases act on DSBs to create blunt DNA ends, the preferred substrates for the RecBCD enzyme. In addition, our results show that in UV-irradiated recF recA(+) cells, the xseA, xonA, and sbcD mutations do not affect RecBCD-mediated DNA repair, suggesting that ExoVII, ExoI and SbcCD nucleases are not essential for the initial targeting of RecBCD to DSBs. It is possible that the DNA-blunting activity provided by ExoVII, ExoI and SbcCD is required for an exchange of RecBCD molecules on dsDNA ends during ongoing "reckless" DNA degradation.

  7. Serum induced degradation of 3D DNA box origami observed by high speed atomic force microscope

    DEFF Research Database (Denmark)

    Jiang, Zaixing; Zhang, Shuai; Yang, Chuanxu;

    2015-01-01

    3D DNA origami holds tremendous potential to encapsulate and selectively release therapeutic drugs. Observations of real-time performance of 3D DNA origami structures in physiological environment will contribute much to its further applications. Here, we investigate the degradation kinetics of 3D...... DNA box origami in serum using high-speed atomic force microscope optimized for imaging 3D DNA origami in real time. The time resolution allows characterizing the stages of serum effects on individual 3D DNA box origami with nanometer resolution. Our results indicate that the whole digest process...... is a combination of a rapid collapse phase and a slow degradation phase. The damages of box origami mainly happen in the collapse phase. Thus, the structure stability of 3D DNA box origami should be further improved, especially in the collapse phase, before clinical applications...

  8. TEMPORAL MODELING OF DNA DEGRADATION IN BONE REMAINS

    Directory of Open Access Journals (Sweden)

    Andrei Stefan

    2012-06-01

    Full Text Available The aim of this study is to follow the changes that occur, in time, at DNA level and to establish an efficient and reliable protocol for ancestral DNA extraction from bones found in archaeological sites. To test whether the protocol is efficient and capable of yielding good quality DNA, extraction was first performed on fresh bones. The material consists of fresh pig (Sus scrofa and cow (Bos taurus bones that were grounded by using a drill operating at low speed. The bone powder was then incubated in lysis buffer in the presence of proteinase K. DNA isolation and purification were done by using the phenol:chloroform protocol and DNA was precipitated with absolute ethanol stored at -20oC. The extractions were carried out once every month for a total of four extractions

  9. HIC1 (hypermethylated in cancer 1) SUMOylation is dispensable for DNA repair but is essential for the apoptotic DNA damage response (DDR) to irreparable DNA double-strand breaks (DSBs).

    Science.gov (United States)

    Paget, Sonia; Dubuissez, Marion; Dehennaut, Vanessa; Nassour, Joe; Harmon, Brennan T; Spruyt, Nathalie; Loison, Ingrid; Abbadie, Corinne; Rood, Brian R; Leprince, Dominique

    2017-01-10

    The tumor suppressor gene HIC1 (Hypermethylated In Cancer 1) encodes a transcriptional repressor mediating the p53-dependent apoptotic response to irreparable DNA double-strand breaks (DSBs) through direct transcriptional repression of SIRT1. HIC1 is also essential for DSB repair as silencing of endogenous HIC1 in BJ-hTERT fibroblasts significantly delays DNA repair in functional Comet assays. HIC1 SUMOylation favours its interaction with MTA1, a component of NuRD complexes. In contrast with irreparable DSBs induced by 16-hours of etoposide treatment, we show that repairable DSBs induced by 1 h etoposide treatment do not increase HIC1 SUMOylation or its interaction with MTA1. Furthermore, HIC1 SUMOylation is dispensable for DNA repair since the non-SUMOylatable E316A mutant is as efficient as wt HIC1 in Comet assays. Upon induction of irreparable DSBs, the ATM-mediated increase of HIC1 SUMOylation is independent of its effector kinase Chk2. Moreover, irreparable DSBs strongly increase both the interaction of HIC1 with MTA1 and MTA3 and their binding to the SIRT1 promoter. To characterize the molecular mechanisms sustained by this increased repression potential, we established global expression profiles of BJ-hTERT fibroblasts transfected with HIC1-siRNA or control siRNA and treated or not with etoposide. We identified 475 genes potentially repressed by HIC1 with cell death and cell cycle as the main cellular functions identified by pathway analysis. Among them, CXCL12, EPHA4, TGFβR3 and TRIB2, also known as MTA1 target-genes, were validated by qRT-PCR analyses. Thus, our data demonstrate that HIC1 SUMOylation is important for the transcriptional response to non-repairable DSBs but dispensable for DNA repair.

  10. Singlet oxygen mediated DNA degradation by copper nanoparticles: potential towards cytotoxic effect on cancer cells

    Directory of Open Access Journals (Sweden)

    Sengupta Tapas K

    2011-03-01

    Full Text Available Abstract The DNA degradation potential and anti-cancer activities of copper nanoparticles of 4-5 nm size are reported. A dose dependent degradation of isolated DNA molecules by copper nanoparticles through generation of singlet oxygen was observed. Singlet oxygen scavengers such as sodium azide and Tris [hydroxyl methyl] amino methane were able to prevent the DNA degradation action of copper nanoparticles confirming the involvement of activated oxygen species in the degradation process. Additionally, it was observed that the copper nanoparticles are able to exert cytotoxic effect towards U937 and Hela cells of human histiocytic lymphoma and human cervical cancer origins, respectively by inducing apoptosis. The growth characteristics of U937 and Hela cells were studied applying various concentrations of the copper nanoparticles.

  11. Effect of inhibitors of cellular metabolism on postradiation repair and degradation of DNA in rat thymocytes

    Energy Technology Data Exchange (ETDEWEB)

    Ivannik, B.P.; Golubeva, R.V.; Proskuryakov, S.Ya.; Murzaev, V.I.; Ryabchenko, N.I.

    1979-10-01

    The viscosimetric method was used to determine the molecular weight of high polymer single-stranded DNA in alkaline nuclear lysates for the study of the effects of a number of inhibitors of synthesis of DNA (hydroxyurea), macroergic elements (2,4-dinitrophenol, EDTA) and DNAase (Na/sup +/ citrate, Ca/sup + +/ ions) on the process of repair and secondary post-radiation degradation of DNA of rat thymocytes exposed to radiation in a dosage of 3 kR.

  12. Degradation of transgene DNA in genetically modified herbicide-tolerant rice during food processing.

    Science.gov (United States)

    Song, Shangxin; Zhou, Guanghong; Gao, Feng; Zhang, Wei; Qiu, Liangyan; Dai, Sifa; Xu, Xinglian; Xiao, Hongmei

    2011-12-01

    In order to assess the effect of food processing on the degradation of exogenous DNA components in sweet rice wine and rice crackers made from genetically modified (GM) rice (Oryza sativa L.), we developed genomic DNA extraction methods and compared the effect of different food processing procedures on DNA degradation. It was found that the purity, quantity and quality of DNA by alkaline lysis method were higher than by CTAB (cetyltrimethylammonium bromide) method. For sweet rice wine, CAMV35S (cauliflower mosaic virus 35S) promoter and NOS (nopaline synthase) terminator were degraded by the third day, whereas the exogenous gene Bar (bialaphos resistance) remained unaffected. For rice crackers, boiling, drying and microwaving contributed to the initial degradations of DNA. Baking resulted in further degradations, and frying led to the most severe changes. These results indicated that the stability of DNA in GM rice was different under different processing conditions. For sweet rice wine, Bar was most stable, followed by NOS, CAMV35S, and SPS. For rice crackers, CAMV35S was most stable, followed by SPS, NOS, and Bar.

  13. Action of hydrogen peroxide on degradation of DNA after irradiation in Escherichia coli.

    Science.gov (United States)

    Keller, K M; Pollard, E C

    1977-05-01

    Hydrogen peroxide (H2O2), which produces breaks in cellular DNA, has not hitherto been shown to cause degradation of DNA. In this investigation it is shown that if transcription is blocked with rifampin, treatment with H2O2 causes degradation of DNA to nearly the same extent as does gamma-radiation. Further, if cells are given a treatment with H2O2 and incubated for 50 min, the amount of degradation in a second treatment is markedly less. This is attributed to the induction of the inhibitor of post-irradiation degradation of DNA (prd) by the first treatment. There is thus a double action of H2O2: first, to induce inhibition, and second, to cause degradation of DNA to begin in non-induced cells. The genetic dependence of induction by H2O2 mimics that of ionizing radiation. Accordingly, the induction process does not occur in recA- and lex- cells, because they are not inducible and is absent in recB- cells because they lack exonuclease V, the major component of prd. Potassium iodide (KI), an OH radical scavenger, negates the action of peroxide on DNA. The results obtained in this study suggest a possible theory for the evolution of radiation response systems

  14. Therapeutic targeting of liver cancer with a recombinant DNA vaccine containing the hemagglutinin-neuraminidase gene of Newcastle disease virus via apoptotic-dependent pathways.

    Science.gov (United States)

    Chen, Li-Gang; Liu, Yuan-Sheng; Zheng, Tang-Hui; Chen, Xu; Li, Ping; Xiao, Chuan-Xing; Ren, Jian-Lin

    2016-11-01

    A total of ~38.6 million mortalities occur due to liver cancer annually, worldwide. Although a variety of therapeutic methods are available, the efficacy of treatment at present is extremely limited due to an increased risk of malignancy and inherently poor prognosis of liver cancer. Gene therapy is considered a promising option, and has shown notable potential for the comprehensive therapy of liver cancer, in keeping with advances that have been made in the development of cancer molecular biology. The present study aimed to investigate the synergistic effects of the abilities of the hemagglutinin neuraminidase protein of Newcastle disease virus (NDV), the pro-apoptotic factor apoptin from chicken anaemia virus, and the interferon-γ inducer interleukin-18 (IL-18) in antagonizing liver cancer. Therefore, a recombinant DNA plasmid expressing the three exogenous genes, VP3, IL-18 and hemagglutinin neuraminidase (HN), was constructed. Flow cytometry, acridine orange/ethidium bromide staining and analysis of caspase-3 activity were performed in H22 cell lines transfected with the recombinant DNA plasmid. In addition, 6-week-old C57BL/6 mice were used to establish a H22 hepatoma-bearing mouse model. Mice tumor tissue was analyzed by immunohistochemistry and scanning electron microscopy. The results of the present study revealed that the recombinant DNA vaccine containing the VP3, IL-18 and HN genes inhibited cell proliferation and induced autophagy via the mitochondrial pathway in vivo and in vitro.

  15. Maternal inheritance of mitochondrial DNA: degradation of paternal mitochondria by allogeneic organelle autophagy, allophagy.

    Science.gov (United States)

    Sato, Miyuki; Sato, Ken

    2012-03-01

    Maternal inheritance of mitochondrial DNA (mtDNA) is generally observed in many eukaryotes. Sperm-derived paternal mitochondria and their mtDNA enter the oocyte cytoplasm upon fertilization and then normally disappear during early embryogenesis. However, the mechanism underlying this clearance of paternal mitochondria has remained largely unknown. Recently, we showed that autophagy is required for the elimination of paternal mitochondria in Caenorhabditis elegans embryos. Shortly after fertilization, autophagosomes are induced locally around the penetrated sperm components. These autophagosomes engulf paternal mitochondria, resulting in their lysosomal degradation during early embryogenesis. In autophagy-defective zygotes, paternal mitochondria and their genomes remain even in the larval stage. Therefore, maternal inheritance of mtDNA is accomplished by autophagic degradation of paternal mitochondria. We also found that another kind of sperm-derived structure, called the membranous organelle, is degraded by zygotic autophagy as well. We thus propose to term this allogeneic (nonself) organelle autophagy as allophagy.

  16. Proteasome-dependent degradation of replisome components regulates faithful DNA replication.

    Science.gov (United States)

    Roseaulin, Laura C; Noguchi, Chiaki; Noguchi, Eishi

    2013-08-15

    The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF(Pof3) (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.

  17. The development of miniplex primer sets for the analysis of degraded DNA

    Science.gov (United States)

    McCord, Bruce; Opel, Kerry; Chung, Denise; Drabek, Jiri; Tatarek, Nancy; Meadows Jantz, Lee; Butler, John

    2005-05-01

    In this project, a new set of multiplexed PCR reactions has been developed for the analysis of degraded DNA. These DNA markers, known as Miniplexes, utilize primers that have shorter amplicons for use in short tandem repeat (STR) analysis of degraded DNA. In our work we have defined six of these new STR multiplexes, each of which consists of 3 to 4 reduced size STR loci, and each labeled with a different fluorescent dye. When compared to commercially available STR systems, reductions in size of up to 300 basepairs are possible. In addition, these newly designed amplicons consist of loci that are fully compatible with the the national computer DNA database known as CODIS. To demonstrate compatibility with commercial STR kits, a concordance study of 532 DNA samples of Caucasian, African American, and Hispanic origin was undertaken There was 99.77% concordance between allele calls with the two methods. Of these 532 samples, only 15 samples showed discrepancies at one of 12 loci. These occurred predominantly at 2 loci, vWA and D13S317. DNA sequencing revealed that these locations had deletions between the two primer binding sites. Uncommon deletions like these can be expected in certain samples and will not affect the utility of the Miniplexes as tools for degraded DNA analysis. The Miniplexes were also applied to enzymatically digested DNA to assess their potential in degraded DNA analysis. The results demonstrated a greatly improved efficiency in the analysis of degraded DNA when compared to commercial STR genotyping kits. A series of human skeletal remains that had been exposed to a variety of environmental conditions were also examined. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only sixteen percent of the samples tested generated full profiles with a commercial kit. In addition, complete profiles were obtained for eleven of the twelve Miniplex loci which had amplicon size ranges less than 200 base pairs

  18. Modulation of eDNA release and degradation affects Staphylococcus aureus biofilm maturation.

    Directory of Open Access Journals (Sweden)

    Ethan E Mann

    Full Text Available Recent studies have demonstrated a role for Staphylococcus aureus cidA-mediated cell lysis and genomic DNA release in biofilm adherence. The current study extends these findings by examining both temporal and additional genetic factors involved in the control of genomic DNA release and degradation during biofilm maturation. Cell lysis and DNA release were found to be critical for biofilm attachment during the initial stages of development and the released DNA (eDNA remained an important matrix component during biofilm maturation. This study also revealed that an lrgAB mutant exhibits increased biofilm adherence and matrix-associated eDNA consistent with its proposed role as an inhibitor of cidA-mediated lysis. In flow-cell assays, both cid and lrg mutations had dramatic effects on biofilm maturation and tower formation. Finally, staphylococcal thermonuclease was shown to be involved in biofilm development as a nuc mutant formed a thicker biofilm containing increased levels of matrix-associated eDNA. Together, these findings suggest a model in which the opposing activities of the cid and lrg gene products control cell lysis and genomic DNA release during biofilm development, while staphylococcal thermonuclease functions to degrade the eDNA, possibly as a means to promote biofilm dispersal.

  19. DNA degradation within mouse brain and dental pulp cells 72 hours postmortem

    Institute of Scientific and Technical Information of China (English)

    Jilong Zheng; Xiaona Li; Di Shan; Han Zhang; Dawei Guan

    2012-01-01

    In this study, we sought to elucidate the process of DNA degradation in brain and dental pulp cells of mice, within postmortem 0-72 hours, by using the single cell gel electrophoresis assay and professional comet image analysis and processing techniques. The frequency of comet-like cells, the percentage of tail DNA, tail length, tail moment, Olive moment and tail area increased in tandem with increasing postmortem interval. In contrast, the head radius, the percentage of head DNA and head area showed a decreasing trend. Linear regression analysis revealed a high correlation between these parameters and the postmortem interval. The findings suggest that the single cell gel electrophoresis assay is a quick and sensitive method to detect DNA degradation in brain and dental pulp cells, providing an objective and accurate new way to estimate postmortem interval.

  20. An enzyme-based DNA preparation method for application to forensic biological samples and degraded stains.

    Science.gov (United States)

    Lounsbury, Jenny A; Coult, Natalie; Miranian, Daniel C; Cronk, Stephen M; Haverstick, Doris M; Kinnon, Paul; Saul, David J; Landers, James P

    2012-09-01

    Extraction of DNA from forensic samples typically uses either an organic extraction protocol or solid phase extraction (SPE) and these methods generally involve numerous sample transfer, wash and centrifugation steps. Although SPE has been successfully adapted to the microdevice, it can be problematic because of lengthy load times and uneven packing of the solid phase. A closed-tube enzyme-based DNA preparation method has recently been developed which uses a neutral proteinase to lyse cells and degrade proteins and nucleases [14]. Following a 20 min incubation of the buccal or whole blood sample with this proteinase, DNA is polymerase chain reaction (PCR)-ready. This paper describes the optimization and quantitation of DNA yield using this method, and application to forensic biological samples, including UV- and heat-degraded whole blood samples on cotton or blue denim substrates. Results demonstrate that DNA yield can be increased from 1.42 (±0.21)ng/μL to 7.78 (±1.40)ng/μL by increasing the quantity of enzyme per reaction by 3-fold. Additionally, there is a linear relationship between the amount of starting cellular material added and the concentration of DNA in the solution, thereby allowing DNA yield estimations to be made. In addition, short tandem repeat (STR) profile results obtained using DNA prepared with the enzyme method were comparable to those obtained with a conventional SPE method, resulting in full STR profiles (16 of 16 loci) from liquid samples (buccal swab eluate and whole blood), dried buccal swabs and bloodstains and partial profiles from UV or heat-degraded bloodstains on cotton or blue denim substrates. Finally, the DNA preparation method is shown to be adaptable to glass or poly(methyl methacrylate) (PMMA) microdevices with little impact on STR peak height but providing a 20-fold reduction in incubation time (as little as 60 s), leading to a ≥1 h reduction in DNA preparation time.

  1. p16(INK4A) enhances the transcriptional and the apoptotic functions of p53 through DNA-dependent interaction.

    Science.gov (United States)

    Al-Khalaf, Huda H; Nallar, Shreeram C; Kalvakolanu, Dhananjaya V; Aboussekhra, Abdelilah

    2017-02-20

    p16(INK4A) and p53 are two important tumor suppressor proteins that play essential roles during cell proliferation and aging through regulating the expression of several genes. Here, we report that p16(INK4A) and p53 co-regulate a plethora of transcripts. Furthermore, both proteins colocalize in the nucleus of human primary skin fibroblasts and breast luminal cells, and form a heteromer whose level increases in response to genotoxic stress as well as aging of human fibroblasts and various mouse organs. CDK4 is also present in this heteromeric complex, which is formed only in the presence of DNA both in vitro using pure recombinant proteins and in vivo. We have also shown that p16(INK4A) enhances the binding efficiency of p53 to its cognate sequence presents in the CDKN1A promoter in vitro, and both proteins are present at the promoters of CDKN1A and BAX in vivo. Importantly, the fourth ankyrin repeat of p16(INK4A) and the C-terminal domain of p53 were necessary for the physical association between these two proteins. The physiologic importance of this association was revealed by the inability of cancer-associated p16(INK4A) mutants to interact with p53 and to transactivate the expression of its major targets CDKN1A and BAX in the p16-defective U2OS cells expressing either wild-type or mutated p16(INK4A) . Furthermore, the association between p16(INK4A) and p53 was capital for their nuclear colocalization, the X-ray-dependent induction of p21 and Bax proteins as well as the induction of apoptosis in various types of cells. Together, these results show DNA-dependent physical interaction between p16(INK4A) and p53.

  2. Determination of the Early Time of Death by Computerized Image Analysis of DNA Degradation: Which Is the Best Quantitative Indicator of DNA Degradation?

    Institute of Scientific and Technical Information of China (English)

    LIU Lijiang; SHU Xiji; REN Liang; ZHOU Hongyan; LI Yan; LIU Wei; ZHU Cheng; LIU Liang

    2007-01-01

    This study evaluated the correlation between DNA degradation of the splenic lymphocytes and the early time of death, examined the early time of death by computerized image analysis technique (CIAT) and identified the best parameter that quantitatively reflects the DNA degradation.The spleen tissues from 34 SD rats were collected, subjected to cell smearing every 2 h within the first 36 h after death, stained by Feulgen-Van's staining, three indices reflecting DNA content in splenic lymphocytes, including integral optical density (IOD), average optical density (AOD), average gray scale (AG) were measured by the image analysis. Our results showed that IOD and AOD decreased and AG increased over time within the first 36 h. A stepwise linear regression analysis showed that only AG was fitted. A correlation between the postmortem interval (PMI) and AG was identified and the corresponding regression equation was obtained. Our study suggests that CIAT is a useful and promising tool for the estimation of early PMI with good objectivity and reproducibility,and AG is a more effective and better quantitative indicator for the estimation of PMI within the first 36 h after death in rats.

  3. Apoptotic markers and DNA damage are related to late phase of stroke: Involvement of dyslipidemia and inflammation.

    Science.gov (United States)

    Pascotini, Eduardo Tanuri; Flores, Ariane Ethur; Kegler, Aline; Gabbi, Patricia; Bochi, Guilherme Vargas; Algarve, Thais Doeler; Prado, Ana Lucia Cervi; Duarte, Marta M M F; da Cruz, Ivana B M; Moresco, Rafael Noal; Royes, Luiz Fernando Freire; Fighera, Michele Rechia

    2015-11-01

    Oxidative stress and brain inflammation are thought to contribute to the pathophysiology of cerebral injury in acute stroke, leading to apoptosis and cell death. Lipid accumulation may lead to progression of carotid plaques and inflammation, contributing to increased acute stroke risk. However, little is known about these events and markers in the late stroke (>6 months) and if dyslipidemia could contribute to disease's pathophysiology in a later phase. In this case-control study, we recruited patients in the late stroke phase (n=40) and health subjects (control group; n = 40). Dichlorodihydrofluorescein (DCFH), nitrite/nitrate (NOx), Tumor necrosis factor-alpha (TNF-α), Acetylcholinesterase (AChE), Caspase 8 (CASP 8), Caspase 3 (CASP 3) and Picogreen (PG) were measured in periphery blood samples. Furthermore, a correlation among all measured markers (DCFH, NOx, TNF-α, AChE, CASP 8, CASP 3 and PG) was realized. The marker levels were also compared to triglycerides (TG), total (CHO), LDL and HDL cholesterol levels and medications used. Statistical analyses showed that stroke patients presented an increase of DCFH, NOx, TNF-α and AChE levels when compared to control subjects. In addition, we observed that stroke patients had significantly higher CASP 8, CASP 3 and PG levels than control group. A significant correlation between TNF-α with CASP 8 (r = 0.4) and CASP 3 (r = 0.4) levels was observed, but not with oxidative/nitrosative markers. Moreover, we observed that stroke patients with dyslipidemia had significantly higher TNF-α, CASP 8 and CASP 3 levels than stroke without dyslipidemia and control groups. Our findings suggest that oxidative and inflammatory markers may be still increased and lead to caspase activation and DNA damage even after 6 months to cerebral injury. Furthermore, it is plausible to propose that dyslipidemia may contribute to worsen proinflammatory state in a later phase of stroke and an increased risk to new neurovascular events.

  4. Post mortem DNA degradation of human tissue experimentally mummified in salt.

    Science.gov (United States)

    Shved, Natallia; Haas, Cordula; Papageorgopoulou, Christina; Akguel, Guelfirde; Paulsen, Katja; Bouwman, Abigail; Warinner, Christina; Rühli, Frank

    2014-01-01

    Mummified human tissues are of great interest in forensics and biomolecular archaeology. The aim of this study was to analyse post mortem DNA alterations in soft tissues in order to improve our knowledge of the patterns of DNA degradation that occur during salt mummification. In this study, the lower limb of a female human donor was amputated within 24 h post mortem and mummified using a process designed to simulate the salt dehydration phase of natural or artificial mummification. Skin and skeletal muscle were sampled at multiple time points over a period of 322 days and subjected to genetic analysis. Patterns of genomic fragmentation, miscoding lesions, and overall DNA degradation in both nuclear and mitochondrial DNA was assessed by different methods: gel electrophoresis, multiplex comparative autosomal STR length amplification, cloning and sequence analysis, and PCR amplification of different fragment sizes using a damage sensitive recombinant polymerase. The study outcome reveals a very good level of DNA preservation in salt mummified tissues over the course of the experiment, with an overall slower rate of DNA fragmentation in skin compared to muscle.

  5. An investigation of the effects of Cinnamomum cassia bark extracts on oxidative DNA damage and possible cytotoxic and apoptotic activities in transformed/untransformed cell lines from Type 1 diabetic patients, in vitro.

    Directory of Open Access Journals (Sweden)

    Ferzan Lermioglu Erciyas

    2015-05-01

    Full Text Available It was shown that patients with Type 1 diabetes mellitus (T1DM had increased level of oxidative DNA damage and decreased efficacy of DNA repair. These changes were implicated in the increased cancer risk in patients with diabetes mellitus. Cinnamon bark extracts have diverse biological activities including antidiabetic and anti-tumor properties. Cinnamomum cassia (C. cassia is a common used cinnamon species present in commercial cinnamon preparations. We aimed to investigate the effects of cinnamon extracts prepared from C. cassia bark on endogenous and hydrogen peroxide (H2O2-induced oxidative DNA damage, as well as cytotoxic and apoptotic activities in this study. Type 1 diabetic (T1DM lymphocytes (GM02765, GM01838 and fibroblasts (GM01837 were obtained from NIGMS Human Genetic Cell Repository of Coriell Institute, New Jersey, USA. Cytotoxicity analysis were performed by using a tetrazolium salt, 4-[3-(4-iodophenyl 2-(4-nitrophenyl 2H-5-tetrazolio] 1,3-benzene disulfonate (WST-1. The effects of extracts on endogenous and H2O2-induced oxidative DNA damage were studied using the single cell gel electrophoresis (SCGE; Comet Assay, a technique allowing DNA damage in a single cell. Apoptotic activities of extracts were investigated by TUNEL and Annexin V/PI assays. using flow cytometry. IC50 and IC20 values of the extracts varied and the effects on endogenous and H2O2-induced DNA damage were different regarding cell lines and extracts. Although their protective effects at some doses against to H2O2-induced oxidative damage, our results suggested DNA damaging and apoptotic potential of cinnamon bark extracts on Type 1 diabetic cell lines, in vitro.

  6. Novel Phenanthrene-Degrading Bacteria Identified by DNA-Stable Isotope Probing.

    Directory of Open Access Journals (Sweden)

    Longfei Jiang

    Full Text Available Microorganisms responsible for the degradation of phenanthrene in a clean forest soil sample were identified by DNA-based stable isotope probing (SIP. The soil was artificially amended with either 12C- or 13C-labeled phenanthrene, and soil DNA was extracted on days 3, 6 and 9. Terminal restriction fragment length polymorphism (TRFLP results revealed that the fragments of 219- and 241-bp in HaeIII digests were distributed throughout the gradient profile at three different sampling time points, and both fragments were more dominant in the heavy fractions of the samples exposed to the 13C-labeled contaminant. 16S rRNA sequencing of the 13C-enriched fraction suggested that Acidobacterium spp. within the class Acidobacteria, and Collimonas spp. within the class Betaproteobacteria, were directly involved in the uptake and degradation of phenanthrene at different times. To our knowledge, this is the first report that the genus Collimonas has the ability to degrade PAHs. Two PAH-RHDα genes were identified in 13C-labeled DNA. However, isolation of pure cultures indicated that strains of Staphylococcus sp. PHE-3, Pseudomonas sp. PHE-1, and Pseudomonas sp. PHE-2 in the soil had high phenanthrene-degrading ability. This emphasizes the role of a culture-independent method in the functional understanding of microbial communities in situ.

  7. Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif.

    Science.gov (United States)

    Land, Allison M; Wang, Jiayi; Law, Emily K; Aberle, Ryan; Kirmaier, Andrea; Krupp, Annabel; Johnson, Welkin E; Harris, Reuben S

    2015-11-24

    APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy.

  8. Dual-degradable disulfide-containing PEI–Pluronic/DNA polyplexes: transfection efficiency and balancing protection and DNA release

    Directory of Open Access Journals (Sweden)

    Zhang L

    2013-09-01

    Full Text Available Lifen Zhang,* Zhenzhen Chen,* Yanfeng LiState Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Institute of Biochemical Engineering and Environmental Technology, Lanzhou University, Lanzhou, People's Republic of China*These authors contributed equally to this workAbstract: Polymeric gene-delivery vectors to achieve lack of toxicity and a balance between protection and DNA release remains a formidable challenge. Incorporating intracellular environment-responsive degradable bonds is an appreciable step toward developing safer transfection agents. In this study, novel, dual-degradable polycation copolymers (Pluronic-diacrylate [PA]–polyethyleneimine [PEI]–SS were synthesized through the addition of low molecular weight (800 Da PEI cross-linked with SS (PEI-SS to PA. Three PA-PEI-SS copolymers (PA-PEI-SS1, 2, and 3 with different PEI-SS to Pluronic molar ratios were investigated and found to strongly condense plasmid DNA into positively charged nanoparticles with an average particle size of approximately 200 nm and to possess higher stability against DNase I digestion and sodium heparin. Disulfide and ester bonds of the copolymers were susceptible to intracellular redox conditions. In vitro experiments demonstrated that the PA-PEI-SS copolymers had significantly lower cytotoxicity and higher transfection efficiency in both BGC-823 and 293T cell lines than the controls of degradable PEI-SS and nondegradable 25 kDa PEI. Transfection activity was influenced by the PEI-SS content in the polymers and PA-PEI-SS1 showed the highest efficiency of the three copolymers. These studies suggest that these dual-degradable copolymers could be used as potential biocompatible gene delivery carriers.Keywords: Pluronic, PEI, gene vector, dual-degradable, disulfide-containing linker

  9. The development of reduced size STR amplicons as tools for analysis of degraded DNA.

    Science.gov (United States)

    Butler, John M; Shen, Yin; McCord, Bruce R

    2003-09-01

    New multiplex PCR sets of commonly used short tandem repeat (STR) markers have been developed to produce PCR products that are reduced in size when compared to standard commercial STR kits. The reduction in size of these amplicons can facilitate the examination and analysis of degraded DNA evidence by improving amplification efficiency. This "miniSTR" approach will permit current forensic practitioners to use STR markers and instrumentation already present in their laboratories and to generate genotyping data that is directly comparable to reference samples and searchable through the FBI's Combined DNA Index System (CODIS) databases. This paper discusses the development of these new primer sets and presents some initial results in the analysis of degraded and aged DNA samples. A method for removal of problematic fluorescent dye artifacts is also described. Comparison studies in over 100 samples have verified that these miniSTR primers can provide fully concordant results to commercial STR kits and can provide improved signal from degraded DNA specimens. These miniplex sets should prove valuable in the analysis of samples where allele dropout and reduced sensitivity of larger STR alleles occurs.

  10. Heat degradation of eukaryotic and bacterial DNA: an experimental model for paleomicrobiology

    Directory of Open Access Journals (Sweden)

    Nguyen-Hieu Tung

    2012-09-01

    Full Text Available Abstract Background Theoretical models suggest that DNA degradation would sharply limit the PCR-based detection of both eukaryotic and prokaryotic DNA within ancient specimens. However, the relative extent of decay of eukaryote and prokaryote DNA over time is a matter of debate. In this study, the murine macrophage cell line J774, alone or infected with Mycobacterium smegmatis bacteria, were killed after exposure to 90°C dry heat for intervals ranging from 1 to 48 h in order to compare eukaryotic cells, extracellular bacteria and intracellular bacteria. The sizes of the resulting mycobacterial rpoB and murine rpb2 homologous gene fragments were then determined by real-time PCR and fluorescent probing. Findings The cycle threshold (Ct values of PCR-amplified DNA fragments from J774 cells and the M. smegmatis negative controls (without heat exposure varied from 26–33 for the J774 rpb2 gene fragments and from 24–29 for M. smegmatis rpoB fragments. After 90°C dry heat incubation for up to 48 h, the Ct values of test samples increased relative to those of the controls for each amplicon size. For each dry heat exposure time, the Ct values of the 146-149-bp fragments were lower than those of 746-747-bp fragments. During the 4- to 24-h dry heat incubation, the non-infected J774 cell DNA was degraded into 597-bp rpb2 fragments. After 48 h, however, only 450-bp rpb2 fragments of both non-infected and infected J774 cells could be amplified. In contrast, the 746-bp rpoB fragments of M. smegmatis DNA could be amplified after the 48-h dry heat exposure in all experiments. Infected and non-infected J774 cell DNA was degraded more rapidly than M. smegmatis DNA after dry heat exposure (ANOVA test, p  Conclusion In this study, mycobacterial DNA was more resistant to dry-heat stress than eukaryotic DNA. Therefore, the detection of large, experimental, ancient mycobacterial DNA fragments is a suitable approach for paleomicrobiological studies.

  11. Capillary electrophoresis of miniSTR markers to genotype highly degraded DNA samples.

    Science.gov (United States)

    Coble, Michael D

    2012-01-01

    The amplification of short tandem repeat (STR) markers throughout the human nuclear DNA genome are used to associate crime scene evidence to the perpetrator's profile in criminal investigations. For highly challenged or compromised materials such as stains exposed to the elements, skeletal remains from missing persons cases, or fragmented and degraded samples from mass disasters, obtaining a full STR profile may be difficult if not impossible. With the introduction of short amplicon STR or "miniSTR" typing, it is possible to obtain STR genetic information from highly challenged samples without the need to sequence the hypervariable regions of the mitochondrial DNA (mtDNA) genome. Non-Combined DNA Index System (CODIS) STR markers have been developed to obtain information beyond the core CODIS loci. This chapter will focus on the steps necessary to prepare and use one of the non-CODIS (NC) multiplexes, NC01 (Coble and Butler 2005), for analysis on capillary electrophoresis instrumentation.

  12. Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR

    Directory of Open Access Journals (Sweden)

    Kleiboeker Steven B

    2005-04-01

    Full Text Available Abstract Although PCR and RT-PCR provided a valuable approach for detection of pathogens, the high level of sensitivity of these assays also makes them prone to false positive results. In addition to cross-contamination with true positive samples, false positive results are also possible due to "carry-over" contamination of samples with amplicon DNA generated by previous reactions. To reduce this source of false positives, amplicon generated by reactions in which dUTP was substituted for dTTP can be degraded by uracil DNA glycosylase (UNG. UNG does not degrade RNA but will cleave contaminating uracil-containing DNA while leaving thymine-containing DNA intact. The availability of heat-labile UNG makes use of this approach feasible for RT-PCR. In this study, real-time RT-PCR was used to quantify UNG degradation of amplicon DNA and the effect of UNG on RNA detection. Using the manufacturers' recommended conditions, complete degradation of DNA was not observed for samples containing 250 copies of amplicon DNA. Doubling the UNG concentration resulted in degradation of the two lowest concentrations of DNA tested, but also resulted in an increase of 1.94 cycles in the CT for RNA detection. To improve DNA degradation while minimizing the effect on RNA detection, a series of time, temperature and enzyme concentrations were evaluated. Optimal conditions were found to be 0.25 U UNG per 25 μl reaction with a 20 min, 30°C incubation prior to RT-PCR. Under these conditions, high concentrations of amplicon DNA could be degraded while the CT for RNA detection was increased by 1.2 cycles.

  13. DDB2 association with PCNA is required for its degradation after UV-induced DNA damage.

    Science.gov (United States)

    Cazzalini, Ornella; Perucca, Paola; Mocchi, Roberto; Sommatis, Sabrina; Prosperi, Ennio; Stivala, Lucia Anna

    2014-01-01

    DDB2 is a protein playing an essential role in the lesion recognition step of the global genome sub-pathway of nucleotide excision repair (GG-NER) process. Among the proteins involved in the DNA damage response, p21(CDKN1A) (p21) has been reported to participate in NER, but also to be removed by proteolytic degradation, thanks to its association with PCNA. DDB2 is involved in the CUL4-DDB1 complex mediating p21 degradation; however, the direct interaction between DDB2, p21 and PCNA has been never investigated. Here, we show that DDB2 co-localizes with PCNA and p21 at local UV-induced DNA-damage sites, and these proteins co-immunoprecipitate in the same complex. In addition, we provide evidence that p21 is not able to bind directly DDB2, but, to this end, the presence of PCNA is required. Direct physical association of recombinant DDB2 protein with PCNA is mediated by a conserved PIP-box present in the N-terminal region of DDB2. Mutation of the PIP-box resulted in the loss of protein interaction. Interestingly, the same mutation, or depletion of PCNA by RNA interference, greatly impaired DDB2 degradation induced by UV irradiation. These results indicate that DDB2 is a PCNA-binding protein, and that this association is required for DDB2 proteolytic degradation.

  14. Strain dependent UV degradation of Escherichia coli DNA monitored by Fourier transform infrared spectroscopy.

    Science.gov (United States)

    Muntean, Cristina M; Lapusan, Alexandra; Mihaiu, Liora; Stefan, Razvan

    2014-01-05

    In this work we present a method for detection of DNA isolated from nonpathogenic Escherichia coli strains, respectively. Untreated and UV irradiated bacterial DNAs were analyzed by FT-IR spectroscopy, to investigate their screening characteristic features and their structural radiotolerance at 253.7nm. FT-IR spectra, providing a high molecular structural information, have been analyzed in the wavenumber range 800-1800cm(-1). FT-IR signatures, spectroscopic band assignments and structural interpretations of these DNAs are reported. Also, UV damage at the DNA molecular level is of interest. Strain dependent UV degradation of DNA from E. coli has been observed. Particularly, alterations in nucleic acid bases, base pairing and base stacking have been found. Also changes in the DNA conformation and deoxyribose were detected. Based on this work, specific E. coli DNA-ligand interactions, drug development and vaccine design for a better understanding of the infection mechanism caused by an interference between pathogenic and nonpathogenic bacteria and for a better control of disease, respectively, might be further investigated using Fourier transform infrared spectroscopy. Besides, understanding the pathways for UV damaged DNA response, like nucleic acids repair mechanisms is appreciated.

  15. Difficulties of sex determination from forensic bone degraded DNA: A comparison of three methods.

    Science.gov (United States)

    Quincey, Danielle; Carle, Georges; Alunni, Véronique; Quatrehomme, Gérald

    2013-09-01

    Sex determination is of paramount importance in forensic anthropology. Numerous anthropological methods have been described, including visual assessments and various measurements of bones. Nevertheless, whatever the method used, the percentage of correct classification of a single bone usually varies between 80% and 95%, due to significant intra- and inter-population variations, and sometimes variations coming from secular trends. DNA is increasingly used in a forensic context. But forensic DNA extraction from bone raises several issues, because the samples are very often badly altered and/or in very small quantity. Nuclear DNA is difficult to get from degraded samples, according to low copy number, at least in comparison with mitochondrial DNA. In a forensic context (as in a paeleoanthropological context) DNA sex determination is usually complicated by the weak amount of DNA, the degraded nature of nucleic acids, the presence of enzymatic inhibitors in DNA extracts, the possible faint amplification of Y band and the risk of contamination during either excavation or manipulation of samples. The aim of this work was to compare three methods of DNA sex determination from bones: procedure #1 using a single PCR amplification, procedure #2 using a double PCR amplification, and procedure #3 adding bleaching for decontamination of the bone, instead of simply rubbing the bone. These processes were applied to samples of bones (49 samples coming from 39 individuals) that were in various states of post mortem alteration. The main results are the following. (i) No DNA could be extracted from three skulls (parietal bones, mastoid process), the compact bone of one rib, and the diaphysis of one femur; (ii) there was a contamination in three skulls; and (iii) the Y band did not appear in two male cases, with one of the three procedures (male tibia, procedure #2) and with procedures #2 and #3 (male femur). This study emphasises the main issue while working with altered bones: the

  16. Turmeric toxicity in A431 epidermoid cancer cells associates with autophagy degradation of anti-apoptotic and anti-autophagic p53 mutant.

    Science.gov (United States)

    Thongrakard, Visa; Titone, Rossella; Follo, Carlo; Morani, Federica; Suksamrarn, Apichart; Tencomnao, Tewin; Isidoro, Ciro

    2014-12-01

    The keratinocyte-derived A431 Squamous Cell Carcinoma cells express the p53R273H mutant, which has been reported to inhibit apoptosis and autophagy. Here, we show that the crude extract of turmeric (Curcuma longa), similarly to its bioactive component Curcumin, could induce both apoptosis and autophagy in A431 cells, and these effects were concomitant with degradation of p53. Turmeric and curcumin also stimulated the activity of mTOR, which notoriously promotes cell growth and acts negatively on basal autophagy. Rapamycin-mediated inhibition of mTOR synergized with turmeric and curcumin in causing p53 degradation, increased the production of autophagosomes and exacerbated cell toxicity leading to cell necrosis. Small-interference mediated silencing of the autophagy proteins BECLIN 1 or ATG7 abrogated the induction of autophagy and largely rescued p53 stability in Turmeric-treated or Curcumin-treated cells, indicating that macroautophagy was mainly responsible for mutant p53 degradation. These data uncover a novel mechanism of turmeric and curcumin toxicity in chemoresistant cancer cells bearing mutant p53.

  17. DNA damage accumulation and TRF2 degradation in atypical Werner syndrome fibroblasts with LMNA mutations.

    Science.gov (United States)

    Saha, Bidisha; Zitnik, Galynn; Johnson, Simon; Nguyen, Quyen; Risques, Rosa A; Martin, George M; Oshima, Junko

    2013-01-01

    Segmental progeroid syndromes are groups of disorders with multiple features suggestive of accelerated aging. One subset of adult-onset progeroid syndromes, referred to as atypical Werner syndrome, is caused by mutations in the LMNA gene, which encodes a class of nuclear intermediate filaments, lamin A/C. We previously described rapid telomere attrition and accelerated replicative senescence in cultured fibroblasts overexpressing mutant lamin A. In this study, we investigated the cellular phenotypes associated with accelerated telomere shortening in LMNA mutant primary fibroblasts. In early passage primary fibroblasts with R133L or L140R LMNA mutations, shelterin protein components were already reduced while cells still retained telomere lengths comparable to those of controls. There was a significant inverse correlation between the degree of abnormal nuclear morphology and the level of TRF2, a shelterin subunit, suggesting a potential causal relationship. Stabilization of the telomeres via the introduction of the catalytic subunit of human telomerase, hTERT (human telomerase reverse transcriptase), did not prevent degradation of shelterin components, indicating that reduced TRF2 in LMNA mutants is not mediated by short telomeres. Interestingly, γ-H2AX foci (reflecting double strand DNA damage) in early passage LMNA mutant primary fibroblasts and LMNA mutant hTERT fibroblasts were markedly increased in non-telomeric regions of DNA. Our results raise the possibility that mutant lamin A/C causes global genomic instability with accumulation of non-telomeric DNA damage as an early event, followed by TRF2 degradation and telomere shortening.

  18. Degradation of DNA in Haemophilus influenzae cells after x-ray irradiation. II. Comparison with theoretical models

    Energy Technology Data Exchange (ETDEWEB)

    Randolph, M.L.

    1976-01-01

    Models of the kinetics of degradation of DNA in bacterial cells following exposure to x-rays are developed and tested using data obtained with various strains of Haemophilus influenzae cells. Different mathematical models allow for different initial distributions of DNA lengths, depending on cell phase and assumed replication model, and for unilateral or bilateral degradation from initiation points, which are taken as single-strand breaks. In order to explain the observed magnitude and time course of degradation an interruption-of-degradation probability, which may be interpreted as the result of an x-ray inducible inhibitor, is introduced. Interruption of degradation is interpreted as an early and essential, but not necessarily sufficient, step for repair of DNA. Empirical best fits to the kinetic data were obtained by iterative calculation methods based on varying the rate parameters. Depending somewhat on the strain and cell phase, the data seem best described by assuming bilateral degradation, perhaps at different rates, whose total for log phase wild-type cells is roughly 5 x 10/sup 3/ nucleotides/min and an interruption rate of about 0.03/min. For stationary phase the total degradation rate is greater and the probability of degradation unchanged. Differences in the kinetics between strains are discussed briefly.

  19. Assessment of DNA degradation induced by thermal and UV radiation processing: implications for quantification of genetically modified organisms.

    Science.gov (United States)

    Ballari, Rajashekhar V; Martin, Asha

    2013-12-01

    DNA quality is an important parameter for the detection and quantification of genetically modified organisms (GMO's) using the polymerase chain reaction (PCR). Food processing leads to degradation of DNA, which may impair GMO detection and quantification. This study evaluated the effect of various processing treatments such as heating, baking, microwaving, autoclaving and ultraviolet (UV) irradiation on the relative transgenic content of MON 810 maize using pRSETMON-02, a dual target plasmid as a model system. Amongst all the processing treatments examined, autoclaving and UV irradiation resulted in the least recovery of the transgenic (CaMV 35S promoter) and taxon-specific (zein) target DNA sequences. Although a profound impact on DNA degradation was seen during the processing, DNA could still be reliably quantified by Real-time PCR. The measured mean DNA copy number ratios of the processed samples were in agreement with the expected values. Our study confirms the premise that the final analytical value assigned to a particular sample is independent of the degree of DNA degradation since the transgenic and the taxon-specific target sequences possessing approximately similar lengths degrade in parallel. The results of our study demonstrate that food processing does not alter the relative quantification of the transgenic content provided the quantitative assays target shorter amplicons and the difference in the amplicon size between the transgenic and taxon-specific genes is minimal.

  20. Combination of TRAIL with bortezomib shifted apoptotic signaling from DR4 to DR5 death receptor by selective internalization and degradation of DR4.

    Directory of Open Access Journals (Sweden)

    Maxim L Bychkov

    Full Text Available TRAIL (tumor necrosis factor-related apoptosis-inducing ligand mediates apoptosis in cancer cells through death receptors DR4 and DR5 preferring often one receptor over another in the cells expressing both receptors. Receptor selective mutant variants of TRAIL and agonistic antibodies against DR4 and DR5 are highly promising anticancer agents. Here using DR5 specific mutant variant of TRAIL--DR5-B we have demonstrated for the first time that the sensitivity of cancer cells can be shifted from one TRAIL death receptor to another during co-treatment with anticancer drugs. First we have studied the contribution of DR4 and DR5 in HCT116 p53+/+ and HCT116 p53-/- cells and demonstrated that in HCT116 p53+/+ cells the both death receptors are involved in TRAIL-induced cell death while in HCT116 p53-/- cells prevailed DR4 signaling. The expression of death (DR4 and DR5 as well as decoy (DcR1 and DcR2 receptors was upregulated in the both cell lines either by TRAIL or by bortezomib. However, combined treatment of cells with two drugs induced strong time-dependent and p53-independent internalization and further lysosomal degradation of DR4 receptor. Interestingly DR5-B variant of TRAIL which do not bind with DR4 receptor also induced elimination of DR4 from cell surface in combination with bortezomib indicating the ligand-independent mechanism of the receptor internalization. Eliminatory internalization of DR4 resulted in activation of DR5 receptor thus DR4-dependent HCT116 p53-/- cells became highly sensitive to DR5-B in time-dependent manner. Internalization and degradation of DR4 receptor depended on activation of caspases as well as of lysosomal activity as it was completely inhibited by Z-VAD-FMK, E-64 and Baf-A1. In light of our findings, it is important to explore carefully which of the death receptors is active, when sensitizing drugs are combined with agonistic antibodies to the death receptors or receptor selective variants of TRAIL to enhance

  1. Combination of TRAIL with bortezomib shifted apoptotic signaling from DR4 to DR5 death receptor by selective internalization and degradation of DR4.

    Science.gov (United States)

    Bychkov, Maxim L; Gasparian, Marine E; Dolgikh, Dmitry A; Kirpichnikov, Mikhail P

    2014-01-01

    TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) mediates apoptosis in cancer cells through death receptors DR4 and DR5 preferring often one receptor over another in the cells expressing both receptors. Receptor selective mutant variants of TRAIL and agonistic antibodies against DR4 and DR5 are highly promising anticancer agents. Here using DR5 specific mutant variant of TRAIL--DR5-B we have demonstrated for the first time that the sensitivity of cancer cells can be shifted from one TRAIL death receptor to another during co-treatment with anticancer drugs. First we have studied the contribution of DR4 and DR5 in HCT116 p53+/+ and HCT116 p53-/- cells and demonstrated that in HCT116 p53+/+ cells the both death receptors are involved in TRAIL-induced cell death while in HCT116 p53-/- cells prevailed DR4 signaling. The expression of death (DR4 and DR5) as well as decoy (DcR1 and DcR2) receptors was upregulated in the both cell lines either by TRAIL or by bortezomib. However, combined treatment of cells with two drugs induced strong time-dependent and p53-independent internalization and further lysosomal degradation of DR4 receptor. Interestingly DR5-B variant of TRAIL which do not bind with DR4 receptor also induced elimination of DR4 from cell surface in combination with bortezomib indicating the ligand-independent mechanism of the receptor internalization. Eliminatory internalization of DR4 resulted in activation of DR5 receptor thus DR4-dependent HCT116 p53-/- cells became highly sensitive to DR5-B in time-dependent manner. Internalization and degradation of DR4 receptor depended on activation of caspases as well as of lysosomal activity as it was completely inhibited by Z-VAD-FMK, E-64 and Baf-A1. In light of our findings, it is important to explore carefully which of the death receptors is active, when sensitizing drugs are combined with agonistic antibodies to the death receptors or receptor selective variants of TRAIL to enhance cancer treatment

  2. Degradable polymer-coated gold nanoparticles for co-delivery of DNA and siRNA.

    Science.gov (United States)

    Bishop, Corey J; Tzeng, Stephany Y; Green, Jordan J

    2015-01-01

    Gold nanoparticles have utility for in vitro, ex vivo and in vivo imaging applications as well as for serving as a scaffold for therapeutic delivery and theranostic applications. Starting with gold nanoparticles as a core, layer-by-layer degradable polymer coatings enable the simultaneous co-delivery of DNA and short interfering RNA (siRNA). To engineer release kinetics, polymers which degrade through two different mechanisms can be utilized to construct hybrid inorganic/polymeric particles. During fabrication of the nanoparticles, the zeta potential reverses upon the addition of each oppositely charged polyelectrolyte layer and the final nanoparticle size reaches approximately 200nm in diameter. When the hybrid gold/polymer/nucleic acid nanoparticles are added to human primary brain cancer cells in vitro, they are internalizable by cells and reach the cytoplasm and nucleus as visualized by transmission electron microscopy and observed through exogenous gene expression. This nanoparticle delivery leads to both exogenous DNA expression and siRNA-mediated knockdown, with the knockdown efficacy superior to that of Lipofectamine® 2000, a commercially available transfection reagent. These gold/polymer/nucleic acid hybrid nanoparticles are an enabling theranostic platform technology capable of delivering combinations of genetic therapies to human cells.

  3. Hyperthermia enhances mapatumumab-induced apoptotic death through ubiquitin-mediated degradation of cellular FLIP(long) in human colon cancer cells.

    Science.gov (United States)

    Song, X; Kim, S-Y; Zhou, Z; Lagasse, E; Kwon, Y T; Lee, Y J

    2013-04-04

    Colorectal cancer is the third leading cause of cancer-related mortality in the world; the main cause of death of colorectal cancer is hepatic metastases, which can be treated with hyperthermia using isolated hepatic perfusion (IHP). In this study, we report that mild hyperthermia potently reduced cellular FLIP(long), (c-FLIP(L)), a major regulator of the death receptor (DR) pathway of apoptosis, thereby enhancing humanized anti-DR4 antibody mapatumumab (Mapa)-mediated mitochondria-independent apoptosis. We observed that overexpression of c-FLIP(L) in CX-1 cells abrogated the synergistic effect of Mapa and hyperthermia, whereas silencing of c-FLIP in CX-1 cells enhanced Mapa-induced apoptosis. Hyperthermia altered c-FLIP(L) protein stability without concomitant reductions in FLIP mRNA. Ubiquitination of c-FLIP(L) was increased by hyperthermia, and proteasome inhibitor MG132 prevented heat-induced downregulation of c-FLIP(L). These results suggest the involvement of the ubiquitin-proteasome system in this process. We also found lysine residue 195 (K195) to be essential for c-FLIP(L) ubiquitination and proteolysis, as mutant c-FLIP(L) lysine 195 arginine (arginine replacing lysine) was left virtually un-ubiquitinated and was refractory to hyperthermia-triggered degradation, and thus partially blocked the synergistic effect of Mapa and hyperthermia. Our observations reveal that hyperthermia transiently reduced c-FLIP(L) by proteolysis linked to K195 ubiquitination, which contributed to the synergistic effect between Mapa and hyperthermia. This study supports the application of hyperthermia combined with other regimens to treat colorectal hepatic metastases.

  4. DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment

    Energy Technology Data Exchange (ETDEWEB)

    Winderl, C.; Penning, H.; von Netzer, F.; Meckenstock, R.U.; Lueders, T. [Helmholtz Zentrum Munchen, Neuherberg (Germany)

    2010-10-15

    Global groundwater resources are constantly challenged by a multitude of contaminants such as aromatic hydrocarbons. Especially in anaerobic habitats, a large diversity of unrecognized microbial populations may be responsible for their degradation. Still, our present understanding of the respective microbiota and their ecophysiology is almost exclusively based on a small number of cultured organisms, mostly within the Proteobacteria. Here, by DNA-based stable isotope probing (SIP), we directly identified the most active sulfate-reducing toluene degraders in a diverse sedimentary microbial community originating from a tar-oil-contaminated aquifer at a former coal gasification plant. On incubation of fresh sediments with {sup 13}C{sub 7}-toluene, the production of both sulfide and (CS{sub 2}){sup 13}CO{sub 2} was clearly coupled to the {sup 13}Clabeling of DNA of microbes related to Desulfosporosinus spp. within the Peptococcaceae (Clostridia). The screening of labeled DNA fractions also suggested a novel benzylsuccinate synthase alpha-subunit (bssA) sequence type previously only detected in the environment to be tentatively affiliated with these degraders. However, carbon flow from the contaminant into degrader DNA was only similar to 50%, pointing toward high ratios of heterotrophic CS{sub 2}-fixation during assimilation of acetyl-CoA originating from the contaminant by these degraders. These findings demonstrate that the importance of non-proteobacterial populations in anaerobic aromatics degradation, as well as their specific ecophysiology in the subsurface may still be largely ungrasped.

  5. Serum-induced degradation of 3D DNA box origami observed with high-speed atomic force microscopy

    Institute of Scientific and Technical Information of China (English)

    Zaixing Jiang[1,2,3; Shuai Zhang[2,3; Chuanxu Yang[2; Jorgen Kjems[2; Yudong Huang[1; Flemming Besenbacher[2; Mingdong Dong[2

    2015-01-01

    3D DNA origami holds tremendous potential for the encapsulation and selective release of therapeutic drugs. Observations of the real-time performance of these structures in physiological environments will contribute to the development of future applications. We investigated the degradation kinetics of 3D DNA box origami in serum by using high-speed atomic force microscope optimized for imaging 3D DNA origami in real time. The time resolution allowed to characterize the stages of serum effects on individual 3D DNA boxes origami with nanometer resolution. Our results indicate that the digestion process is a combination of rapid collapse and slow degradation phases. Damage to box origami occurs mainly in the collapse phase. Thus, the structural stability of 3D DNA box origami should be improved, especially in the collapse phase, before these structures are used in clinical applications.

  6. Inhibition of proteasomal degradation of rpn4 impairs nonhomologous end-joining repair of DNA double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Donghong Ju

    Full Text Available BACKGROUND: The proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the transcription factor Rpn4 induces the proteasome genes and is rapidly degraded by the assembled proteasome. The integrity of the Rpn4-proteasome feedback loop is critical for cell viability under stressed conditions. We have demonstrated that inhibition of Rpn4 degradation sensitizes cells to DNA damage, particularly in response to high doses of DNA damaging agents. The underlying mechanism, however, remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using yeast genetics and biochemical approach we show that inhibition of Rpn4 degradation displays a synthetic growth defect with deletion of the MEC1 checkpoint gene and sensitizes several checkpoint mutants to DNA damage. In addition, inhibition of Rpn4 degradation leads to a defect in repair of double-strand breaks (DSBs by nonhomologous end-joining (NHEJ. The expression levels of several key NHEJ genes are downregulated and the recruitment of Yku70 to a DSB is reduced by inhibition of Rpn4 degradation. We find that Rpn4 and the proteasome are recruited to a DSB, suggesting their direct participation in NHEJ. Inhibition of Rpn4 degradation may result in a concomitant delay of release of Rpn4 and the proteasome from a DSB. CONCLUSION/SIGNIFICANCE: This study provides the first evidence for the role of proteasomal degradation of Rpn4 in NHEJ.

  7. Multiple DNA extractions coupled with stable-isotope probing of anthracene-degrading bacteria in contaminated soil.

    Science.gov (United States)

    Jones, Maiysha D; Singleton, David R; Sun, Wei; Aitken, Michael D

    2011-05-01

    In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the (13)C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-(13)C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from (13)C-enriched DNA and were designated "anthracene group 1." Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP.

  8. DNA degradation by aqueous extract of Aloe vera in the presence of copper ions.

    Science.gov (United States)

    Naqvi, Shoa; Ullah, M F; Hadi, S M

    2010-06-01

    The plant Aloe vera has long been used in medicine, as dietary supplements and for cosmetic purposes. Aloe vera extracts are a rich source of polyphenols, such as aloin and aloe emodin and have shown a wide range of pharmacological properties, including anti-inflammatory and anti-cancer properties. The bioactive component aloe emodin has been reported to induce apoptosis in various cancer cell lines. Many of the biological activities of Aloe vera have been attributed to its antioxidant properties. However, most plant-derived polyphenols that are also present in Aloe vera may exhibit pro-oxidant properties either alone or in the presence of transition metals, such as copper. Previous reports from this laboratory have implicated the pro-oxidant action as one of the mechanisms for their anti-cancer properties. In the present paper, we show that aqueous extract of Aloe vera is also able to cause DNA degradation in the presence of copper ions. Further, the extract is also able to reduce Cu(II) to Cu(I) and generate reactive oxygen species, such as superoxide anion and hydroxyl radicals in a dose-dependent manner, which correlates with ability of the extract to cause DNA breakage. Thus, the study shows that in addition to antioxidant activity, Aloe vera extract also possess pro-oxidant properties, leading to oxidative DNA breakage.

  9. Fluorescence formation from the interaction of DNA with lipid oxidation degradation products.

    Science.gov (United States)

    Frankel, E N; Neff, W E; Brooks, D D; Fujimoto, K

    1987-06-23

    To clarify the mechanism of fluorescence formation between DNA and lipid degradation products in the presence of ferric chloride and ascorbic acid, a number of carbonyl compounds and decomposition products of pure methyl linolenate hydroperoxides were examined. Keto derivatives of methyl ricinoleate, linoleate, and oleate, alkanals and 2-alkenals produced little or no fluorescence with DNA in the presence of ferric chloride-ascorbic acid. 2,4-Alkadienals were more active and 2,4,7-decatrienal was the most active. Mixtures of volatile aldehydes prepared from linolenate hydroperoxide decomposed either thermally or with iron and ascorbate had the same activity as 2,4,7-decatrienal. Higher molecular-weight products from the decomposition of methyl linolenate hydroperoxides showed relatively low activity. beta-Carotene, alpha-tocopherol and other antioxidants effectively reduced the amount of fluorescence formed by linolenate hydroperoxides. The results suggest that, in addition to hydroperoxide decomposition products, singlet oxygen and/or free radical species contribute significantly to the fluorescence formed from the interaction of methyl linolenate hydroperoxides with DNA in the presence of ferric chloride and ascorbic acid.

  10. Nanobiosensor for Detection and Quantification of DNA Sequences in Degraded Mixed Meats

    Directory of Open Access Journals (Sweden)

    M. E. Ali

    2011-01-01

    Full Text Available A novel class of nanobiosensor was developed by integrating a 27-nucleotide AluI fragment of swine cytochrome b (cytb gene to a 3-nm diameter citrate-tannate coated gold nanoparticle (GNP. The biosensor detected 0.5% and 1% pork in raw and 2.5-h autoclaved pork-beef binary admixtures in a single step without any separation or washing. The hybridization kinetics of the hybrid sensor was studied with synthetic and AluI digested real pork targets from moderate to extreme target concentrations and a sigmoidal relationship was found. Using the kinetic curve, a convenient method for quantifying and counting target DNA copy number was developed. The accuracy of the method was over 90% and 80% for raw and autoclaved pork-beef binary admixtures in the range of 5–100% pork adulteration. The biosensor probe identified a target DNA sequence that was several-folds shorter than a typical PCR-template. This offered the detection and quantitation of potential targets in highly processed or degraded samples where PCR amplification was not possible due to template crisis. The assay was a viable alternative approach of qPCR for detecting, quantifying and counting copy number of shorter size DNA sequences to address a wide ranging biological problem in food industry, diagnostic laboratories and forensic medicine.

  11. Early-stage apoptosis is associated with DNA-damage-independent ATM phosphorylation and chromatin decondensation in NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Schou, Kenneth Bødtker; Schneider, Linda; Christensen, Søren Tvorup

    2008-01-01

    Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used...

  12. Degradation of paternal mitochondria after fertilization: implications for heteroplasmy, assisted reproductive technologies and mtDNA inheritance.

    Science.gov (United States)

    Sutovsky, Peter; Van Leyen, Klaus; McCauley, Tod; Day, Billy N; Sutovsky, Miriam

    2004-01-01

    Maternal inheritance of mitochondrial DNA has long been regarded as a major paradox in developmental biology. While some confusion may still persist in popular science, research data clearly document that the paternal sperm-borne mitochondria of most mammalian species enter the ooplasm at fertilization and are specifically targeted for degradation by the resident ubiquitin system. Ubiquitin is a proteolytic chaperone that forms covalently linked polyubiquitin chains on the targeted proteinaceous substrates. The polyubiquitin tag redirects the substrate proteins to a 26-S proteasome, a multi-subunit proteolytic organelle. Thus, specific proteasomal inhibitors reversibly block sperm mitochondrial degradation in ooplasm. Lysosomal degradation and the activity of membrane-lipoperoxidating enzyme 15-lipoxygenase (15-LOX) may also contribute to sperm mitochondrial degradation in the ooplasm, but probably is not crucial. Prohibitin, the major protein of the inner mitochondrial membrane, appears to be ubiquitinated in the sperm mitochondria. Occasional occurrence of paternal inheritance of mtDNA has been suggested in mammals including humans. While most such evidence has been widely disputed, it warrants further examination. Of particular concern is the documented heteroplasmy, i.e. mixed mtDNA inheritance after ooplasmic transplantation. Intracytoplasmic sperm injection (ICSI) has inherent potential for delaying the degradation of sperm mitochondria. However, paternal mtDNA inheritance after ICSI has not been documented so far.

  13. DNA degradation and genetic analysis of empty puparia: genetic identification limits in forensic entomology.

    Science.gov (United States)

    Mazzanti, Morena; Alessandrini, Federica; Tagliabracci, Adriano; Wells, Jeffrey D; Campobasso, Carlo P

    2010-02-25

    Puparial cases are common remnants of necrophagous flies in crime investigations. They usually represent the longest developmental time and, therefore, they can be very useful for the estimation of the post-mortem interval (PMI). However, before any PMI estimate, it is crucial to identify the species of fly eclosed from each puparium associated with the corpse. Morphological characteristics of the puparium are often distinctive enough to permit a species identification. But, even an accurate morphological analysis of empty puparia cannot discriminate among different species of closely related flies. Furthermore, morphological identification may be impossible if the fly puparia are poorly preserved or in fragments. This study explores the applicability of biomolecular techniques on empty puparia and their fragments for identification purposes. A total of 63 empty puparia of necrophagous Diptera resulting from forensic casework were examined. Samples were divided into three groups according to size, type and time of eclosion in order to verify whether the physical characteristics and puparia weathering can influence the amount of DNA extraction. The results suggest that a reliable genetic identification of forensically important flies may also be performed from empty puparia and/or their fragments. However, DNA degradation can deeply compromise the genetic analysis since the older the fly puparia, the smaller are the amplified fragments.

  14. Antigenotoxic and Apoptotic Activity of Green Tea Polyphenol Extracts on Hexavalent Chromium-Induced DNA Damage in Peripheral Blood of CD-1 Mice: Analysis with Differential Acridine Orange/Ethidium Bromide Staining

    Directory of Open Access Journals (Sweden)

    María del Carmen García-Rodríguez

    2013-01-01

    Full Text Available This study was conducted to investigate the modulating effects of green tea polyphenols on genotoxic damage and apoptotic activity induced by hexavalent chromium [Cr (VI] in CD-1 mice. Animals were divided into the following groups: (i injected with vehicle; (ii treated with green tea polyphenols (30 mg/kg via gavage; (iii injected with CrO3 (20 mg/kg intraperitoneally; (iv treated with green tea polyphenols in addition to CrO3. Genotoxic damage was evaluated by examining micronucleated polychromatic erythrocytes (MN-PCEs obtained from peripheral blood at 0, 24, 48, and 72 h after treatment. Induction of apoptosis and cell viability were assessed by differential acridine orange/ethidium bromide (AO/EB staining. Treatment of green tea polyphenols led to no significant changes in the MN-PCEs. However, CrO3 treatment significantly increased MN-PCEs at 24 and 48 h after injection. Green tea polyphenols treatment prior to CrO3 injection led to a decrease in MN-PCEs compared to the group treated with CrO3 only. The average of apoptotic cells was increased at 48 h after treatment compared to control mice, suggesting that apoptosis could contribute to eliminate the DNA damaged cells induced by Cr (VI. Our findings support the proposed protective effects of green tea polyphenols against the genotoxic damage induced by Cr (VI.

  15. Insights into the processes behind the contamination of degraded human teeth and bone samples with exogenous sources of DNA

    DEFF Research Database (Denmark)

    Gilbert, M. T. P.; Hansen, Anders J.; Willerslev, E.

    2006-01-01

    A principal problem facing human DNA studies that use old and degraded remains is contamination from other sources of human DNA. In this study we have attempted to contaminate deliberately bones and teeth sampled from a medieval collection excavated in Trondheim, Norway, in order to investigate...... and are difficult to decontaminate using the tested protocol. We believe that this is largely due to the porous nature of bone and teeth facilitating the deep penetration of the contaminant DNA. To simulate a more realistic handling situation, 27 further teeth were directly handled and washed, then decontaminated...

  16. Nucleo-cytoplasmic communication in apoptotic response to genotoxic and inflammatory stress

    Institute of Scientific and Technical Information of China (English)

    Jean Y. J. WANG

    2005-01-01

    Genotoxic agents or inflammatory cytokines activate cellular stress responses and trigger programmed cell death.We have identified a signal transduction module, including three nuclear proteins that participate in the regulation of cell death induced by chemotherapeutic agents and tumor necrosis factor (TNF). In this nuclear signaling module, retinoblastoma protein (Rb) functions as an inhibitor of apoptotic signal transduction. Inactivation of Rb by phosphorylation or caspase-dependent cleavage/degradation is required for cell death to occur. Rb inhibits the Abl tyrosine kinase. Thus,Rb inactivation is a pre-requisite for Abl activation by DNA damage or TNF. Activation of nuclear Abl and its downstream effector p73 induces mitochondriadependent cell death. The involvement of these nuclear signal transducers in TNF induced apoptosis, which does not require new gene expression, indicates that nuclear events other than transcription can contribute to extrinsic apoptotic signal transduction.

  17. Investigating bacterial populations in styrene-degrading biofilters by 16S rDNA tag pyrosequencing.

    Science.gov (United States)

    Portune, Kevin J; Pérez, M Carmen; Álvarez-Hornos, F Javier; Gabaldón, Carmen

    2015-01-01

    Microbial biofilms are essential components in the elimination of pollutants within biofilters, yet still little is known regarding the complex relationships between microbial community structure and biodegradation function within these engineered ecosystems. To further explore this relationship, 16S rDNA tag pyrosequencing was applied to samples taken at four time points from a styrene-degrading biofilter undergoing variable operating conditions. Changes in microbial structure were observed between different stages of biofilter operation, and the level of styrene concentration was revealed to be a critical factor affecting these changes. Bacterial genera Azoarcus and Pseudomonas were among the dominant classified genera in the biofilter. Canonical correspondence analysis (CCA) and correlation analysis revealed that the genera Brevundimonas, Hydrogenophaga, and Achromobacter may play important roles in styrene degradation under increasing styrene concentrations. No significant correlations (P > 0.05) could be detected between biofilter operational/functional parameters and biodiversity measurements, although biological heterogeneity within biofilms and/or technical variability within pyrosequencing may have considerably affected these results. Percentages of selected bacterial taxonomic groups detected by fluorescence in situ hybridization (FISH) were compared to results from pyrosequencing in order to assess the effectiveness and limitations of each method for identifying each microbial taxon. Comparison of results revealed discrepancies between the two methods in the detected percentages of numerous taxonomic groups. Biases and technical limitations of both FISH and pyrosequencing, such as the binding of FISH probes to non-target microbial groups and lack of classification of sequences for defined taxonomic groups from pyrosequencing, may partially explain some differences between the two methods.

  18. Quantitative analysis of genomic DNA degradation in whole blood under various storage conditions for molecular diagnostic testing.

    Science.gov (United States)

    Permenter, Jessalyn; Ishwar, Arjun; Rounsavall, Angie; Smith, Maddie; Faske, Jennifer; Sailey, Charles J; Alfaro, Maria P

    2015-12-01

    Proper storage of whole blood is crucial for isolating nucleic acids from leukocytes and to ensure adequate performance of downstream assays in the molecular diagnostic laboratory. Short-term and long-term storage recommendations are lacking for successful isolation of genomic DNA (gDNA). Container type (EDTA or heparin), temperature (4 °C and room temperature) and time (1-130 days) were assessed as criterion for sample acceptance policies. The percentage of integrated area (%Ti) between 150 and 10,000 bp from the 2200 TapeStation electropherogram was calculated to measure gDNA degradation. Refrigerated EDTA samples yielded gDNA with low %Ti (high quality). Heparinized samples stored at room temperature yielded gDNA of worst quality. Downstream analysis demonstrated that the quality of the gDNA correlated with the quality of the data; samples with high %Ti generated significantly lower levels of high molecular weight amplicons. Recommendations from these analyses include storing blood samples intended for nucleic acid isolation in EDTA tubes at 4 °C for long term storage (>10 days). gDNA should be extracted within 3 days when blood is stored at room temperature regardless of the container. Finally, refrigerated heparinized samples should not be stored longer than 9 days if expecting high quality gDNA isolates. Laboratories should consider many factors, in addition to the results obtained herein, to update their policies for sample acceptance for gDNA extraction intended for molecular genetic testing.

  19. Cell wall degrading isoenzyme profiles of Trichoderma biocontrol strains show correlation with rDNA species

    Institute of Scientific and Technical Information of China (English)

    Sanz L; Hermosa M R; González F J; Monte E

    2004-01-01

    @@ Species of the fungus Trichoderma, a genus of Hyphomycetes, are ubiquitous in the environment, but especially in soil. They have been used in a wide range of commercial applications including the production of hydrolases and in the biological control of plant diseases. A fundamental part of the Trichoderma antifungal system consists of a series of genes coding for a surprising variety of extracellular cell wall degrading enzymes (CWDE).Characterisation and identification of strains at the species level is the first step in utilizing the full potential of fungi in specific applications. One aim when isolating Trichoderma strains is to identify those which can be used in new agricultural and industrial applications. In the past it was not uncommon that biocontrol strains were defined as T. harzianum Rifai, due to the limited classification system of the genus Trichoderma. In recent years, several PCR-based molecular techniques have been used to detect and discriminate among microorganisms. Sequence analysis of the ITS regions of the ribosomal DNA and gene fragments as those corresponding to tef1 gene have been helpful in the neotypification, description and characterization of species in the genus Trichoderna.Another useful method for the identification of Trichoderma strains is the randomly amplified polymorphic DNA (RAPD) technique.Isozyme polymorphisms evaluation of five putative extracellular lytic enzymes loci (β-1,3-glucanase, β-1,6-glucanase, cellulase, chitinase and protease antivities) were carried out using representative strains of defined molecular groups. CWDE groupings obtained from biocontrol strains are discussed in relation to their phylogenetic location and antifungal activities.Compiling morphological, biochemical and sequence information data into a common database would provide a useful resource that could be used to accurately name new haplotypes identified in the future and correctly place them within the genus Trichoderma.

  20. MiniSTR multiplex systems based on non-CODIS loci for analysis of degraded DNA samples.

    Science.gov (United States)

    Asamura, H; Fujimori, S; Ota, M; Fukushima, H

    2007-11-15

    We describe two short amplicon autosomal short tandem repeat (miniSTR) quadruplex systems for eight loci D1S1171, D2S1242, D3S1545, D4S2366, D12S391, D16S3253, D20S161, and D21S1437, unlinked from the combined DNA index system (non-CODIS) loci, using newly designed primer sets. The results of an assay of 411 Japanese individuals showed that polymerase chain reaction (PCR) products within the eight loci were less than 150bp in size, without the seven additional bases for adenylation. The frequency distributions in the loci showed no deviations from Hardy-Weinberg equilibrium expectations. The accumulated power of discrimination and power of exclusion for the eight loci were 0.9999999991 and 0.998, respectively. For assay of highly degraded DNA, including artificially degraded samples and the degraded forensic casework samples assessed with the present miniSTR quadruplex systems, the systems proved quite effective in analyzing degraded DNA.

  1. Comparison of three different DNA extraction methods from a highly degraded biological material.

    Science.gov (United States)

    Kuś, M; Ossowski, A; Zielińska, G

    2016-05-01

    The identification of unknown victims is one of the most challenging tasks faced by forensic medicine. This is due to the rapid decomposition of tissues, beginning at the moment of death and caused by released enzymes and microbial activity. Decay is directly associated with the decomposition of soft tissues and also the degradation of genetic material inside cells. Decomposition rates vary depending on a number of environmental factors, including temperature, humidity, season, and soil properties. Decomposition also differs between bodies left in the open air or buried. To date, forensic medicine has identified mainly people who were the victims of various types of criminal offences. However, with advances in identification methods, increasingly frequent attempts are made to identify the victims of armed conflicts, crimes of totalitarian regimes, or genocide. The aim of the study was to compare three different methods for the extraction of nuclear DNA from material considered in forensic medicine as difficult to handle, i.e. fragments of bones and teeth, and to determine the performance of these methods and their suitability for identification procedures.

  2. Biological evaluation of a cytotoxic 2-substituted benzimidazole copper(II) complex: DNA damage, antiproliferation and apoptotic induction activity in human cervical cancer cells.

    Science.gov (United States)

    Qiao, Xin; Ma, Zhong-Ying; Shao, Jia; Bao, Wei-Guo; Xu, Jing-Yuan; Qiang, Zhao-Yan; Lou, Jian-Shi

    2014-02-01

    Exploring novel chemotherapeutic agents is a great challenge in cancer medicine. To that end, 2-substituted benzimidazole copper(II) complex, [Cu(BMA)Cl2]·(CH3OH) (1) [BMA = N,N'-bis(benzimidazol-2-yl-methyl)amine], was synthesized and its cytotoxicity was characterized. The interaction between complex 1 and calf thymus DNA was detected by spectroscopy methods. The binding constant (K b = 1.24 × 10(4 )M(-1)) and the apparent binding constant (K app = 6.67 × 10(6 )M(-1)) of 1 indicated its moderate DNA affinity. Complex 1 induced single strand breaks of pUC19 plasmid DNA in the presence of H2O2 through an oxidative pathway. Cytotoxicity studies proved that complex 1 could inhibit the proliferation of human cervical carcinoma cell line HeLa in both time- and dose-dependent manners. The results of nuclei staining by Hoechst 33342 and alkaline single-cell gel electrophoresis proved that complex 1 caused cellular DNA damage in HeLa cells. Furthermore, treatment of HeLa cells with 1 resulted in S-phase arrest, loss of mitochondrial potential, and up-regulation of caspase-3 and -9 in HeLa cells, suggesting that complex 1 was capable of inducing apoptosis in cancer cells through the intrinsic mitochondrial pathway.

  3. Assessing PreCR™ repair enzymes for restoration of STR profiles from artificially degraded DNA for human identification.

    Science.gov (United States)

    Robertson, James M; Dineen, Shauna M; Scott, Kristina A; Lucyshyn, Jonathan; Saeed, Maria; Murphy, Devonie L; Schweighardt, Andrew J; Meiklejohn, Kelly A

    2014-09-01

    Forensic scientists have used several approaches to obtain short tandem repeat (STR) profiles from compromised DNA samples, including supplementing the polymerase chain reaction (PCR) with enhancers and using procedures yielding reduced-length amplicons. For degraded DNA, the peak intensities of the alleles separated by electrophoresis generally decrease as the length of the allele increases. When the intensities of the alleles decrease below an established threshold, they are described as drop-outs, thus contributing to a partial STR profile. This work assesses the use of repair enzymes to improve the STR profiles from artificially degraded DNA. The commercial PreCR™ repair kit of DNA repair enzymes was tested on both purified DNA and native DNA in body fluids exposed to oxidizing agents, hydrolytic conditions, ultraviolet (UV) and ionizing radiation, and desiccation. The strategy was to restrict the level of DNA damage to that which yields partial STR profiles in order to test for allele restoration as opposed to simple allele enhancement. Two protocols were investigated for allele restoration: a sequential protocol using the manufacturer's repair procedure and a modified protocol reportedly designed for optimal STR analysis of forensic samples. Allele restoration was obtained with both protocols, but the peak height appeared to be higher for the modified protocol (determined by Mann-Kendall Trend Test). The success of the approach using the PreCR™ repair enzymes was sporadic; it led to allele restoration as well as allele drop-out. Additionally, allele restoration with the PreCR™ enzymes was compared with restoration by alternative, but commonly implemented approaches using Restorase™, PCRBoost™, bovine serum albumin (BSA) and the Minifiler™ STR system. The alternative methods were also successful in improving the STR profile, but their success also depended on the quality of the template encountered. Our results indicate the PreCR™ repair kit may

  4. Construction of two fluorescence-labeled non-combined DNA index system miniSTR multiplex systems to analyze degraded DNA samples in the Chinese Han Population.

    Science.gov (United States)

    Bai, Xue; Li, Shujin; Cong, Bin; Li, Xia; Guo, Xia; He, Lujun; Ye, Jian; Pei, Li

    2010-09-01

    MiniSTR loci have been demonstrated to be an effective approach in recovering genetic information from degraded specimens, because of the reduced PCR amplicon sizes which improved the PCR efficiency. Eight non-combined DNA index system miniSTR loci suitable for the Chinese Han Population were analyzed in 300 unrelated Chinese Han individuals using two novel five fluorescence-labeled miniSTR multiplex systems(multiplex I: D10S1248, D2S441, D1S1677 and D9S2157; multiplex II: D9S1122, D10S1435, D12ATA63, D2S1776 and Amelogenin). The allele frequency distribution and forensic parameters in the Chinese Han Population were reported in this article. The Exact Test demonstrated that all loci surveyed here were found to be no deviation from Hardy-Weinberg equilibrium. The accumulated power of discrimination and power of exclusion for the eight loci were 0.999999992 and 0.98, respectively. The highly degraded DNA from artificially degraded samples and the degraded forensic case work samples was assessed with the two miniSTR multiplex systems, and the results showed that the systems were quite effective.

  5. Degradation and half-life of DNA present in biomass from a genetically-modified organism during land application.

    Science.gov (United States)

    Halter, Mathew C; Zahn, James A

    2017-02-01

    White biotechnology has made a positive impact on the chemical industry by providing safer, more efficient chemical manufacturing processes that have reduced the use of toxic chemicals, harsh reaction conditions, and expensive metal catalysts, which has improved alignment with the principles of Green Chemistry. The genetically-modified (GM) biocatalysts that are utilized in these processes are typically separated from high-value products and then recycled, or eliminated. Elimination routes include disposal in sanitary landfills, incineration, use as a fuel, animal feed, or reuse as an agricultural soil amendment or other value-added products. Elimination routes that have the potential to impact the food chain or environment have been more heavily scrutinized for the fate and persistence of biological products. In this study, we developed and optimized a method for monitoring the degradation of strain-specific DNA markers from a genetically-modified organism (GMO) used for the commercial production of 1,3-propanediol. Laboratory and field tests showed that a marker for heterologous DNA in the GM organism was no longer detectable by end-point polymerase chain reaction (PCR) after 14 days. The half-life of heterologous DNA was increased by 17% (from 42.4 to 49.7 h) after sterilization of the soil from a field plot, which indicated that abiotic factors were important in degradation of DNA under field conditions. There was no evidence for horizontal transfer of DNA target sequences from the GMO to viable organisms present in the soil.

  6. Lactoferrin Directly Scavenges Hydroxyl Radicals and Undergoes Oxidative Self-Degradation: A Possible Role in Protection against Oxidative DNA Damage

    Directory of Open Access Journals (Sweden)

    Yuki Ogasawara

    2014-01-01

    Full Text Available In this study, we examined the protective effect of lactoferrin against DNA damage induced by various hydroxyl radical generation systems. Lactoferrin (LF was examined with regard to its potential role as a scavenger against radical oxygen species using bovine milk LF. Native LF, iron-saturated LF (holo-LF, and apolactoferrin (apo-LF effectively suppressed strand breaks in plasmid DNA due to hydroxyl radicals produced by the Fenton reaction. In addition, both native LF and holo-LF clearly protected calf thymus DNA from fragmentation due to ultraviolet irradiation in the presence of H2O2. We also demonstrated a protective effect of all three LF molecules against 8-hydroxydeoxyguanosine (8-OHdG formation in calf thymus DNA following ultraviolet (UV irradiation with H2O2. Our results clearly indicate that native LF has reactive oxygen species-scavenging ability, independent of its nature as a masking component for transient metals. We also demonstrated that the protective effect of LF against oxidative DNA damage is due to degradation of LF itself, which is more susceptible to degradation than other bovine milk proteins.

  7. Combined treatment with vitamin B12b and ascorbic acid causes in vitro DNA degradation in tumor cells.

    Science.gov (United States)

    Medvedev, A I; Akatov, V S; Kreshchenko, N D; Solov'eva, M E; Leshchenko, V V; Lezhnev, E I; Yakubovskaya, R I

    2001-04-01

    Incubation of Ehrlich ascites carcinoma and HEp-2 human epidermoid laryngeal carcinoma cells with hydroxycobalamin (vitamin B12b) and ascorbic acid induced generation and accumulation of double-stranded DNA fragments (23,000 b.p. and longer) in cells. The same vitamins alone in the same concentrations produced no such effects. DNA degradation in HEp-2 cells caused by long-term (4 h) incubation with 5-25 microM hydroxycobalamin and ascorbic acid (1:10-1:40 molar ratio) at 37 degrees C was comparable with that induced by gamma-irradiation in a dose of 150 Gy at 4 degrees C.

  8. Proteasomal degradation of herpes simplex virus capsids in macrophages releases DNA to the cytosol for recognition by DNA sensors

    NARCIS (Netherlands)

    Horan, K.A.; Hansen, K.; Jakobsen, M.R.; Holm, C.K.; Soby, S.; Unterholzner, L.; Thompson, M.; West, J.A.; Iversen, M.B.; Rasmussen, S.B.; Ellermann-Eriksen, S.; Kurt-Jones, E.; Landolfo, S.; Damania, B.; Melchjorsen, J.; Bowie, A.G.; Fitzgerald, K.A.; Paludan, S.R.

    2013-01-01

    The innate immune system is important for control of infections, including herpesvirus infections. Intracellular DNA potently stimulates antiviral IFN responses. It is known that plasmacytoid dendritic cells sense herpesvirus DNA in endosomes via TLR9 and that nonimmune tissue cells can sense herpes

  9. Forensic genetic SNP typing of low-template DNA and highly degraded DNA from crime case samples

    DEFF Research Database (Denmark)

    Børsting, Claus; Mogensen, Helle Smidt; Morling, Niels

    2013-01-01

    Heterozygote imbalances leading to allele drop-outs and disproportionally large stutters leading to allele drop-ins are known stochastic phenomena related to STR typing of low-template DNA (LtDNA). The large stutters and the many drop-ins in typical STR stutter positions are artifacts from the PCR...

  10. Nuclear translocation of p21{sup WAF1/CIP1} protein prior to its cytosolic degradation by UV enhances DNA repair and survival

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Young; Kim, Hee Suk; Kim, Joo Young [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Sohn, Jeongwon, E-mail: biojs@korea.ac.kr [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of)

    2009-12-25

    We previously reported that UV induced rapid proteasomal degradation of p21 protein in an ubiquitination-independent manner. Here, UV-induced p21 proteolysis was found to occur in the cytosol. Before cytosolic degradation, however, p21 protein translocated to and transiently accumulated in the nucleus. Nuclear translocation of p21 was not required for its degradation, but rather promoted DNA repair and cell survival. Overexpression of the wild type p21, but not the one with defective nuclear localization signal (NLS), reduced UV-induced DNA damage and cell death. Some of p21 protein translocated to the nucleus were associated with chromatin-bound PCNA and saved from UV-induced proteolysis. These data together show that p21 translocates to the nucleus to participate in DNA repair, while the rest is rapidly degraded in the cytosol. We propose that our findings reflect a mechanism to facilitate removal of damaged cells, enhancing DNA repair at the same time.

  11. Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

    Science.gov (United States)

    Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

    2002-01-01

    A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

  12. An evidence on G2/M arrest, DNA damage and caspase mediated apoptotic effect of biosynthesized gold nanoparticles on human cervical carcinoma cells (HeLa)

    Energy Technology Data Exchange (ETDEWEB)

    Jeyaraj, M. [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India); Arun, R. [Department of Biomedical Sciences, Bharathidasan University, Tiruchirappalli 620024 (India); Sathishkumar, G. [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India); MubarakAli, D. [Central Inter-Disciplinary Research Facility, Mahatma Gandhi Medical College and Research Institute Campus, Pondicherry 607402 (India); Rajesh, M.; Sivanandhan, G.; Kapildev, G.; Manickavasagam, M. [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India); Thajuddin, N. [Department of Microbiology, Bharathidasan University, Tiruchirappalli 620024 (India); Ganapathi, A., E-mail: aganapathi2007@gmail.com [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India)

    2014-04-01

    Highlights: • Gold nanoparticles (AuNPs) have been synthesized using Podophyllum hexandrum L. • AuNPs induces the oxidative stress to cell death in human cervical carcinoma cells. • It activates the caspase-cascade to cellular death. • It is actively blocks G2/M phase of cell cycle. - Abstract: Current prospect of nanobiotechnology involves in the greener synthesis of nanostructured materials particularly noble metal nanoparticles for various biomedical applications. In this study, biologically (Podophyllum hexandrum L.) synthesized crystalline gold nanoparticles (AuNPs) with the size range between 5 and 35 nm were screened for its anticancereous potential against human cervical carcinoma cells (HeLa). Stoichiometric proportion of the reaction mixture and conditions were optimized to attain stable nanoparticles with narrow size range. Different high throughput techniques like transmission electron microscope (TEM), X-ray diffraction (XRD) and UV–vis spectroscopy were adopted for the physio-chemical characterization of AuNPs. Additionally, Fourier transform infrared spectroscopy (FTIR) study revealed that the water soluble fractions present in the plant extract solely influences the reduction of AuNPs. Sublimely, synthesized AuNPs exhibits an effective in vitro anticancer activity against HeLa cells via induction of cell cycle arrest and DNA damage. Furthermore, it was evidenced that AuNPs treated cells are undergone apoptosis through the activation of caspase cascade which subsequently leads to mitochondrial dysfunction. Thereby, this study proves that biogenic colloidal AuNPs can be developed as a promising drug candidature for human cervical cancer therapy.

  13. Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation.

    Science.gov (United States)

    Vallerga, María Belén; Mansilla, Sabrina F; Federico, María Belén; Bertolin, Agustina P; Gottifredi, Vanesa

    2015-12-01

    After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates.

  14. 16S ribosomal DNA clone libraries to reveal bacterial diversity in anaerobic reactor-degraded tetrabromobisphenol A.

    Science.gov (United States)

    Peng, Xingxing; Zhang, Zaili; Zhao, Ziling; Jia, Xiaoshan

    2012-05-01

    Microorganisms able to rapidly degrade tetrabromobisphenol A (TBBPA) were domesticated in an anaerobic reactor and added to gradually increased concentrations of TBBPA. After 240 days of domestication, the degradation rate reached 96.0% in cultivated batch experiments lasting 20 days. The optimum cultivating temperature and pH were 30°C and 7.0. The bacterial community's composition and diversity in the reactor was studied by comparative analysis with 16S ribosomal DNA clone libraries. Amplified rDNA restriction analysis of 200 clones from the library indicate that the rDNA richness was high (Coverage C 99.5%) and that evenness was not high (Shannon-Weaver index 2.42). Phylogenetic analysis of 63 bacterial sequences from the reactor libraries demonstrated the presence of Betaproteobacteria (33.1%), Gammaproteobacteria (18.7%), Bacteroidetes (13.9%), Firmicutes (11.4%), Chloroflexi (3.6%), Actinobacteria (0.6%), the candidate division TM7 (4.2%) and other unknown, uncultured bacterial groups (14.5%). Comamonas, Achromobacter, Pseudomonas and Flavobacterium were the dominant types.

  15. Sliding Window Analyses for Optimal Selection of Mini-Barcodes, and Application to 454-Pyrosequencing for Specimen Identification from Degraded DNA

    DEFF Research Database (Denmark)

    Boyer, Stephane; Brown, Samuel D J; Collins, Rupert A

    2012-01-01

    DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI) is over 600 base pairs (bp), amplifi......DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI) is over 600 base pairs (bp...... primers were designed and used to amplify degraded earthworm (prey) DNA from 46 landsnail (predator) faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative...

  16. Distortion of genetically modified organism quantification in processed foods: influence of particle size compositions and heat-induced DNA degradation.

    Science.gov (United States)

    Moreano, Francisco; Busch, Ulrich; Engel, Karl-Heinz

    2005-12-28

    Milling fractions from conventional and transgenic corn were prepared at laboratory scale and used to study the influence of sample composition and heat-induced DNA degradation on the relative quantification of genetically modified organisms (GMO) in food products. Particle size distributions of the obtained fractions (coarse grits, regular grits, meal, and flour) were characterized using a laser diffraction system. The application of two DNA isolation protocols revealed a strong correlation between the degree of comminution of the milling fractions and the DNA yield in the extracts. Mixtures of milling fractions from conventional and transgenic material (1%) were prepared and analyzed via real-time polymerase chain reaction. Accurate quantification of the adjusted GMO content was only possible in mixtures containing conventional and transgenic material in the form of analogous milling fractions, whereas mixtures of fractions exhibiting different particle size distributions delivered significantly over- and underestimated GMO contents depending on their compositions. The process of heat-induced nucleic acid degradation was followed by applying two established quantitative assays showing differences between the lengths of the recombinant and reference target sequences (A, deltal(A) = -25 bp; B, deltal(B) = +16 bp; values related to the amplicon length of the reference gene). Data obtained by the application of method A resulted in underestimated recoveries of GMO contents in the samples of heat-treated products, reflecting the favored degradation of the longer target sequence used for the detection of the transgene. In contrast, data yielded by the application of method B resulted in increasingly overestimated recoveries of GMO contents. The results show how commonly used food technological processes may lead to distortions in the results of quantitative GMO analyses.

  17. Correlative Analysis on the Relationship between PMI and DNA Degradation of Cell Nucleus in Human Different Tissues

    Institute of Scientific and Technical Information of China (English)

    SHU Xiji; LIU Yaling; REN Liang; HE Fanggang; ZHOU Hongyan; LIU Lijiang; LIU Liang

    2005-01-01

    To determining the postmortem interval (PMI) through quantitative analysis of the DNA degradation of cell nucleus in human brain and spleen by using image analysis technique (IAT). The brain and spleen tissues from 32 cadavers with known PMI were collected, subjected to cell smear every 1 h within the first 5-36 h after death, stained by Feulgen-Van's staining, Three indices reflecting DNA in brain cells (astrocytes) and splenic lymphocytes, including integral optical density (IOD), average optical density (AOD), average gray (AG) were measured by employing the mage analysis instrument. The results showed that IOD and AOD declined and AG increased with the prolongation of dead time within 5-36 h. A correlation between the PMI and gray parameters (IOD,AOD and AG) was identified and the corresponding regression equation was obtained. The parameters (IOD,AOD and AG) were proved to be effective quantitative indicators for accurate estimation of PMI within 5-36 h after death.

  18. Forensic genetic SNP typing of low-template DNA and highly degraded DNA from crime case samples

    DEFF Research Database (Denmark)

    Børsting, Claus; Mogensen, Helle Smidt; Morling, Niels

    2013-01-01

    were correctly typed in the 25 pg experiments and only one allele drop-out (error rate: 0.07%) was observed in the consensus profiles. A total of 28 crime case samples were selected for typing with the sensitized SNPforID protocol. The samples were previously typed with old STR kits during the crime...... the heterozygote balance. Allele drop-ins were only observed in experiments with 25 pg of DNA and not in experiments with 50 and 100 pg of DNA. The allele drop-in rate in the 25 pg experiments was 0.06% or 100 times lower than what was previously reported for STR typing of LtDNA. A composite model and two...... polymerase used in the original protocol was replaced with the AmpFℓSTR(®) SEfiler Plus™ Master Mix, which was developed specifically for challenging forensic samples. All the crime case samples were successfully typed with the SNPforID multiplex assay and the match probabilities ranged from 1.1×10(-15) to 7...

  19. Sliding window analyses for optimal selection of mini-barcodes, and application to 454-pyrosequencing for specimen identification from degraded DNA.

    Directory of Open Access Journals (Sweden)

    Stephane Boyer

    Full Text Available DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI is over 600 base pairs (bp, amplification fails when the DNA molecule is degraded into smaller fragments. However, relevant information for specimen identification may not be evenly distributed along the barcoding region, and a shorter target can be sufficient for identification purposes. This study proposes a new, widely applicable, method to compare the performance of all potential 'mini-barcodes' for a given molecular marker and to objectively select the shortest and most informative one. Our method is based on a sliding window analysis implemented in the new R package SPIDER (Species IDentity and Evolution in R. This method is applicable to any taxon and any molecular marker. Here, it was tested on earthworm DNA that had been degraded through digestion by carnivorous landsnails. A 100 bp region of 16 S rDNA was selected as the shortest informative fragment (mini-barcode required for accurate specimen identification. Corresponding primers were designed and used to amplify degraded earthworm (prey DNA from 46 landsnail (predator faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative region of the gene they aim to amplify from degraded DNA. The method and tools provided here, can be particularly useful (1 when dealing with degraded DNA for which only small fragments can be amplified, (2 for cases where no consensus has yet been reached on the appropriate barcode gene, or (3 to allow direct analysis of short reads derived from massively parallel sequencing without the need for bioinformatic consolidation.

  20. Purification of Single-Stranded cDNA Based on RNA Degradation Treatment and Adsorption Chromatography.

    Science.gov (United States)

    Trujillo-Esquivel, Elías; Franco, Bernardo; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Mora-Montes, Héctor M

    2016-08-01

    Analysis of gene expression is a common research tool to study networks controlling gene expression, the role of genes with unknown function, and environmentally induced responses of organisms. Most of the analytical tools used to analyze gene expression rely on accurate cDNA synthesis and quantification to obtain reproducible and quantifiable results. Thus far, most commercial kits for isolation and purification of cDNA target double-stranded molecules, which do not accurately represent the abundance of transcripts. In the present report, we provide a simple and fast method to purify single-stranded cDNA, exhibiting high purity and yield. This method is based on the treatment with RNase H and RNase A after cDNA synthesis, followed by separation in silica spin-columns and ethanol precipitation. In addition, our method avoids the use of DNase I to eliminate genomic DNA from RNA preparations, which improves cDNA yield. As a case report, our method proved to be useful in the purification of single-stranded cDNA from the pathogenic fungus Sporothrix schenckii.

  1. Phosphorylation of Hdmx mediates its Hdm2- and ATM-dependent degradation in response to DNA damage.

    Science.gov (United States)

    Pereg, Yaron; Shkedy, Dganit; de Graaf, Petra; Meulmeester, Erik; Edelson-Averbukh, Marina; Salek, Mogjiborahman; Biton, Sharon; Teunisse, Amina F A S; Lehmann, Wolf D; Jochemsen, Aart G; Shiloh, Yosef

    2005-04-05

    Maintenance of genomic stability depends on the DNA damage response, an extensive signaling network that is activated by DNA lesions such as double-strand breaks (DSBs). The primary activator of the mammalian DSB response is the nuclear protein kinase ataxia-telangiectasia, mutated (ATM), which phosphorylates key players in various arms of this network. The activation and stabilization of the p53 protein play a major role in the DNA damage response and are mediated by ATM-dependent posttranslational modifications of p53 and Mdm2, a ubiquitin ligase of p53. p53's response to DNA damage also depends on Mdm2-dependent proteolysis of Mdmx, a homologue of Mdm2 that represses p53's transactivation function. Here we show that efficient damage-induced degradation of human Hdmx depends on functional ATM and at least three sites on the Hdmx that are phosphorylated in response to DSBs. One of these sites, S403, is a direct ATM target. Accordingly, each of these sites is important for Hdm2-mediated ubiquitination of Hdmx after DSB induction. These results demonstrate a sophisticated mechanism whereby ATM fine-tunes the optimal activation of p53 by simultaneously modifying each player in the process.

  2. Degradation of mitochondrial DNA in cryoprotectant-treated hard coral (Echinopora spp.) oocytes.

    Science.gov (United States)

    Tsai, Sujune; Chen, Jiann-Chu; Spikings, Emma; Li, Jan-Jung; Lin, Chiahsin

    2015-06-01

    A critical step for successful cryopreservation is to determine the optimal cryoprotectant treatment that can provide protective effects against cryoinjury during freezing and with minimal toxicity. Most cryoprotectants have chemical and osmotic effects when used at high concentrations. Cryoprotectants can damage coral mitochondrial distributions and membrane potentials, which results in reduced ATP production. As mitochondrial DNA (mtDNA) encodes for components of the electron transport chain (ETC) and plays a critical role in ATP synthesis capacity, we determined the effects of cryoprotectants on mtDNA in hard coral (Echinopora spp.) oocytes using quantitative real-time PCR. Our results showed that an insult from a cryoprotectant may be compensated for by the genetic defense mechanisms of these cells. Methanol was found to have the least effect on coral oocytes with regard to their energy status. A single oocyte without cryoprotectant treatment produced an average of 4,220,645 ± 169,990 mtDNA copies, which was greater than that in mammals. However, relatively lower mtDNA copy numbers (<2,000,000) were observed when oocytes were treated with dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), or glycerol at a concentration of 3 M for 20 min. These results provide direct evidence that hard coral (Echinopora spp.) oocytes are extremely susceptible to cryoprotectants and support the concerns with regard to the adverse effects of cryoprotectants.

  3. SCF(Cyclin F)-dependent degradation of CDC6 suppresses DNA re-replication

    DEFF Research Database (Denmark)

    Walter, David; Hoffmann, Saskia; Komseli, Eirini-Stavroula;

    2016-01-01

    Maintenance of genome stability requires that DNA is replicated precisely once per cell cycle. This is believed to be achieved by limiting replication origin licensing and thereby restricting the firing of each replication origin to once per cell cycle. CDC6 is essential for eukaryotic replicatio...

  4. Early apoptotic vascular signaling is determined by Sirt1 through nuclear shuttling, forkhead trafficking, bad, and mitochondrial caspase activation.

    Science.gov (United States)

    Hou, Jinling; Chong, Zhao Zhong; Shang, Yan Chen; Maiese, Kenneth

    2010-05-01

    Complications of diabetes mellitus (DM) weigh heavily upon the endothelium that ultimately affect multiple organ systems. These concerns call for innovative treatment strategies that employ molecular pathways responsible for cell survival and longevity. Here we show in a clinically relevant model of DM with elevated D-glucose that endothelial cell (EC) SIRT1 is vital for the prevention of early membrane apoptotic phosphatidylserine externalization and subsequent DNA degradation supported by studies with modulation of SIRT1 activity and gene knockdown of SIRT1. Furthermore, during elevated D-glucose exposure, we show that SIRT1 is sequestered in the cytoplasm of ECs, but specific activation of SIRT1 shuttles the protein to the nucleus to allow for cytoprotection. The ability of SIRT1 to avert apoptosis employs the activation of protein kinase B (Akt1), the post-translational phosphorylation of the forkhead member FoxO3a, the blocked trafficking of FoxO3a to the nucleus, and the inhibition of FoxO3a to initiate a "pro-apoptotic" program as shown by complimentary gene knockdown studies of FoxO3a. Vascular apoptotic oversight by SIRT1 extends to the direct modulation of mitochondrial membrane permeability, cytochrome c release, Bad activation, and caspase 1 and 3 activation, since inhibition of SIRT1 activity and gene knockdown of SIRT1 significantly accentuate cascade progression while SIRT1 activation abrogates these apoptotic elements. Our work identifies vascular SIRT1 and its control over early apoptotic membrane signaling, Akt1 activation, post-translational modification and trafficking of FoxO3a, mitochondrial permeability, Bad activation, and rapid caspase induction as new avenues for the treatment of vascular complications during DM.

  5. Bacteria capable of degrading anthracene, phenanthrene, and fluoranthene as revealed by DNA based stable-isotope probing in a forest soil.

    Science.gov (United States)

    Song, Mengke; Jiang, Longfei; Zhang, Dayi; Luo, Chunling; Wang, Yan; Yu, Zhiqiang; Yin, Hua; Zhang, Gan

    2016-05-05

    Information on microorganisms possessing the ability to metabolize different polycyclic aromatic hydrocarbons (PAHs) in complex environments helps in understanding PAHs behavior in natural environment and developing bioremediation strategies. In the present study, stable-isotope probing (SIP) was applied to investigate degraders of PAHs in a forest soil with the addition of individually (13)C-labeled phenanthrene, anthracene, and fluoranthene. Three distinct phylotypes were identified as the active phenanthrene-, anthracene- and fluoranthene-degrading bacteria. The putative phenanthrene degraders were classified as belonging to the genus Sphingomona. For anthracene, bacteria of the genus Rhodanobacter were the putative degraders, and in the microcosm amended with fluoranthene, the putative degraders were identified as belonging to the phylum Acidobacteria. Our results from DNA-SIP are the first to directly link Rhodanobacter- and Acidobacteria-related bacteria with anthracene and fluoranthene degradation, respectively. The results also illustrate the specificity and diversity of three- and four-ring PAHs degraders in forest soil, contributes to our understanding on natural PAHs biodegradation processes, and also proves the feasibility and practicality of DNA-based SIP for linking functions with identity especially uncultured microorganisms in complex microbial biota.

  6. A paradoxical relationship between Resveratrol and copper (II with respect to degradation of DNA and RNA [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Siddharth Subramaniam

    2016-03-01

    Full Text Available Resveratrol (R, a plant polyphenol, is known to reduce Cu (II to Cu (I generating reactive oxygen species that can cleave plasmid DNA. Here we report a surprising observation of a paradoxical relationship between R and Cu whereby plasmid DNA cleaving / degrading activity of R-Cu increased progressively as the ratio of R to Cu was increased i.e., the concentration of Cu was successively reduced with respect to a fixed concentration R. Whereas cleavage of plasmid DNA occurred at low molar ratios of R to Cu, at higher ratios, complete degradation of DNA was achieved. By further increasing the ratio, whereby the concentration of Cu was reduced to very low levels, the DNA degrading activity of R-Cu was lost. This paradoxical relationship is also seen with respect to eukaryotic genomic DNA and RNA. Since R-Cu may have anti-cancer and anti-viral activities, our findings may not only help to improve the therapeutic efficacy of R-Cu but also reduce its toxic side effects with the use of low concentration of Cu.

  7. A mathematical model of DNA degradation: Possible role of magnetic nanoparticles

    CERN Document Server

    Binhi, V N

    2007-01-01

    A mathematical model of genome degradation is proposed that takes into account a variable rate of mutation and increasing number of cells in a developing human organism. The model explains known properties of cancer development, in particular, a synergism between different mutagens and an increased probability of cancer in the early years of life. An iteration equation is suggested that uses only a few model parameters and describes basic regularities observed in cancer onset. In the model context, relatively small chronic variations in the intracellular content of free radicals may markedly affect the probability of a cell to become a cancer cell. On the other hand, magnetic nanoparticles are shown to be an endogenous source of chronic magnetic exposure that increases the local concentration of free radicals. An enhanced level of leukaemia in early childhood is assumed to originate from magnetic nanoparticles located in hematopoietic stem cells.

  8. Targeted rapid amplification of cDNA ends (T-RACE)--an improved RACE reaction through degradation of non-target sequences.

    Science.gov (United States)

    Bower, Neil I; Johnston, Ian A

    2010-11-01

    Amplification of the 5' ends of cDNA, although simple in theory, can often be difficult to achieve. We describe a novel method for the specific amplification of cDNA ends. An oligo-dT adapter incorporating a dUTP-containing PCR primer primes first-strand cDNA synthesis incorporating dUTP. Using the Cap finder approach, another distinct dUTP containing adapter is added to the 3' end of the newly synthesized cDNA. Second-strand synthesis incorporating dUTP is achieved by PCR, using dUTP-containing primers complimentary to the adapter sequences incorporated in the cDNA ends. The double-stranded cDNA-containing dUTP serves as a universal template for the specific amplification of the 3' or 5' end of any gene. To amplify the ends of cDNA, asymmetric PCR is performed using a single gene-specific primer and standard dNTPs. The asymmetric PCR product is purified and non-target transcripts containing dUTP degraded by Uracil DNA glycosylase, leaving only those transcripts produced during the asymmetric PCR. Subsequent PCR using a nested gene-specific primer and the 3' or 5' T-RACE primer results in specific amplification of cDNA ends. This method can be used to specifically amplify the 3' and 5' ends of numerous cDNAs from a single cDNA synthesis reaction.

  9. FOXO3a governs early and late apoptotic endothelial programs during elevated glucose through mitochondrial and caspase signaling.

    Science.gov (United States)

    Hou, Jinling; Chong, Zhao Zhong; Shang, Yan Chen; Maiese, Kenneth

    2010-06-10

    Mechanisms that preserve endothelial cell (EC) integrity remain elusive, but are critical for new strategies directed against endocrine disorders such as diabetes mellitus (DM). Here we demonstrate in primary cerebral ECs with a clinically relevant model of elevated d-glucose that Akt1 and the post-translational modification and subcellular trafficking of the forkhead transcription factor FoxO3a are critical for early apoptotic membrane signaling and subsequent degradation of nuclear DNA. FoxO3a also directly governs apoptotic mitochondrial signal transduction pathways, since gene knockdown of FoxO3a prevents mitochondrial membrane depolarization as well as the release of cytochrome c. Control of this apoptotic cascade extends to the rapid and progressive activation of caspases. The presence of FoxO3a is necessary for cleaved (active) caspase 1 and 3 expression, since loss of FoxO3a abrogates the induction of caspase activity. Our work identifies Akt1, FoxO3a and closely aligned pathways as key therapeutic targets during impaired glucose tolerance and DM.

  10. Degradation and detection of transgenic Bacillus thuringiensis DNA and proteins in flour of three genetically modified rice events submitted to a set of thermal processes.

    Science.gov (United States)

    Wang, Xiaofu; Chen, Xiaoyun; Xu, Junfeng; Dai, Chen; Shen, Wenbiao

    2015-10-01

    This study aimed to investigate the degradation of three transgenic Bacillus thuringiensis (Bt) genes (Cry1Ab, Cry1Ac, and Cry1Ab/Ac) and the corresponding encoded Bt proteins in KMD1, KF6, and TT51-1 rice powder, respectively, following autoclaving, cooking, baking, or microwaving. Exogenous Bt genes were more stable than the endogenous sucrose phosphate synthase (SPS) gene, and short DNA fragments were detected more frequently than long DNA fragments in both the Bt and SPS genes. Autoclaving, cooking (boiling in water, 30 min), and baking (200 °C, 30 min) induced the most severe Bt protein degradation effects, and Cry1Ab protein was more stable than Cry1Ac and Cry1Ab/Ac protein, which was further confirmed by baking samples at 180 °C for different periods of time. Microwaving induced mild degradation of the Bt and SPS genes, and Bt proteins, whereas baking (180 °C, 15 min), cooking and autoclaving led to further degradation, and baking (200 °C, 30 min) induced the most severe degradation. The findings of the study indicated that degradation of the Bt genes and proteins somewhat correlated with the treatment intensity. Polymerase chain reaction, enzyme-linked immunosorbent assay, and lateral flow tests were used to detect the corresponding transgenic components. Strategies for detecting transgenic ingredients in highly processed foods are discussed.

  11. The α2 helix in the DNA ligase IV BRCT-1 domain is required for targeted degradation of ligase IV during adenovirus infection.

    Science.gov (United States)

    Gilson, Timra; Greer, Amy E; Vindigni, Alessandro; Ketner, Gary; Hanakahi, Leslyn A

    2012-07-05

    In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood. Through genetic analysis, we identified the α2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 α2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 α2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV.

  12. The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM.

    Science.gov (United States)

    Daugaard, Iben; Kjeldsen, Tina E; Hager, Henrik; Hansen, Lise Lotte; Wojdacz, Tomasz K

    2015-12-01

    Readily accessible formalin-fixed paraffin embedded (FFPE) tissues are a highly valuable source of genetic material for molecular analyses in both research and in vitro diagnostics but frequently genetic material in those samples is highly degraded. With locus-specific methylation changes being widely investigated for use as biomarkers in various aspects of clinical disease management, we aimed to evaluate to what extent standard laboratory procedures can approximate the quality of the DNA extracted from FFPE samples prior to methylation analyses. DNA quality in 107 FFPE non-small cell lung cancer (NSCLC) samples was evaluated using spectrophotometry and gel electrophoresis. Subsequently, the quality assessment results were correlated with the results of locus specific methylation assessment with methylation sensitive high resolution melting (MS-HRM). The correlation of template quality with PCR amplification performance and HRM based methylation detection indicated a significant influence of DNA quality on PCR amplification but not on methylation assessment. In conclusion, standard laboratory procedures fairly well approximate DNA degradation of FFPE samples and DNA degradation does not seem to considerably affect locus-specific methylation assessment by MS-HRM.

  13. Upregulation of Phagocytic Clearance of Apoptotic Cells by Autoimmune Regulator

    Institute of Scientific and Technical Information of China (English)

    石亮; 胡丽华; 李一荣

    2010-01-01

    To investigate the effect of autoimmune regulator(AIRE) on phagocytic clearance of apoptotic cells,a recombinant expression vector containing full-length human AIRE cDNA was transfected into 16HBE cells.After incubation with transfected 16HBE cells,engulfment of apoptotic HL-60 cells induced by camptothecin was detected by myeloperoxidase(MPO) staining.The change in the expression of Rac 1 in transfected 16HBE cells was determined by RT-PCR and Western blotting.The results showed that the phagocytosis perce...

  14. Immunosuppressive effects of apoptotic cells

    Science.gov (United States)

    Voll, Reinhard E.; Herrmann, Martin; Roth, Edith A.; Stach, Christian; Kalden, Joachim R.; Girkontaite, Irute

    1997-11-01

    Apoptotic cell death is important in the development and homeostasis of multicellular organisms and is a highly controlled means of eliminating dangerous, damaged or unnecessary cells without causing an inflammatory response or tissue damage,. We now show that the presence of apoptotic cells during monocyte activation increases their secretion of the anti-inflammatory and immunoregulatory cytokine interleukin 10 (IL-10) and decreases secretion of the proinflammatory cytokines tumour necrosis factor-α (TNF-α), IL-1 and IL-12. This may inhibit inflammation and contribute to impaired cell-mediated immunity in conditions associated with increased apoptosis, such as viral infections, pregnancy, cancer and exposure to radiation.

  15. Oral administration of copper to rats leads to increased lymphocyte cellular DNA degradation by dietary polyphenols: implications for a cancer preventive mechanism.

    Science.gov (United States)

    Khan, Husain Y; Zubair, Haseeb; Ullah, Mohd F; Ahmad, Aamir; Hadi, Sheikh M

    2011-12-01

    To account for the observed anticancer properties of plant polyphenols, we have earlier proposed a mechanism which involves the mobilization of endogenous copper ions by polyphenols leading to the generation of reactive oxygen species (ROS) that serve as proximal DNA cleaving agents and lead to cell death. Over the last decade we have proceeded to validate our hypothesis with considerable success. As a further confirmation of our hypothesis, in this paper we first show that oral administration of copper to rats leads to elevated copper levels in lymphocytes. When such lymphocytes with a copper overload were isolated and treated with polyphenols EGCG, genistein and resveratrol, an increased level of DNA breakage was observed. Further, preincubation of lymphocytes having elevated copper levels with the membrane permeable copper chelator neocuproine, resulted in inhibition of polyphenol induced DNA degradation. However, membrane impermeable chelator of copper bathocuproine, as well as iron and zinc chelators were ineffective in causing such inhibition in DNA breakage, confirming the involvement of endogenous copper in polyphenol induced cellular DNA degradation. It is well established that serum and tissue concentrations of copper are greatly increased in various malignancies. In view of this fact, the present results further confirm our earlier findings and strengthen our hypothesis that an important anticancer mechanism of plant polyphenols could be the mobilization of intracellular copper leading to ROS-mediated cellular DNA breakage. In this context, it may be noted that cancer cells are under considerable oxidative stress and increasing such stress to cytotoxic levels could be a successful anticancer approach.

  16. Human DNA Ligase I Interacts with and Is Targeted for Degradation by the DCAF7 Specificity Factor of the Cul4-DDB1 Ubiquitin Ligase Complex.

    Science.gov (United States)

    Peng, Zhimin; Liao, Zhongping; Matsumoto, Yoshihiro; Yang, Austin; Tomkinson, Alan E

    2016-10-14

    The synthesis, processing, and joining of Okazaki fragments during DNA replication is complex, requiring the sequential action of a large number of proteins. Proliferating cell nuclear antigen, a DNA sliding clamp, interacts with and coordinates the activity of several DNA replication proteins, including the enzymes flap endonuclease 1 (FEN-1) and DNA ligase I that complete the processing and joining of Okazaki fragments, respectively. Although it is evident that maintaining the appropriate relative stoichiometry of FEN-1 and DNA ligase I, which compete for binding to proliferating cell nuclear antigen, is critical to prevent genomic instability, little is known about how the steady state levels of DNA replication proteins are regulated, in particular the proteolytic mechanisms involved in their turnover. Because DNA ligase I has been reported to be ubiquitylated, we used a proteomic approach to map ubiquitylation sites and screen for DNA ligase I-associated E3 ubiquitin ligases. We identified three ubiquitylated lysine residues and showed that DNA ligase I interacts with and is targeted for ubiquitylation by DCAF7, a specificity factor for the Cul4-DDB1 complex. Notably, knockdown of DCAF7 reduced the degradation of DNA ligase I in response to inhibition of proliferation and replacement of ubiquitylated lysine residues reduced the in vitro ubiquitylation of DNA ligase I by Cul4-DDB1 and DCAF7. In contrast, a different E3 ubiquitin ligase regulates FEN-1 turnover. Thus, although the expression of many of the genes encoding DNA replication proteins is coordinately regulated, our studies reveal that different mechanisms are involved in the turnover of these proteins.

  17. Detection of methylglyoxal as a degradation product of DNA and nucleic acid components treated with strong acid.

    Science.gov (United States)

    Chaplen, F W; Fahl, W E; Cameron, D C

    1996-05-01

    The 1,2-diaminobenzene derivation assay for methylglyoxal in biological systems involves the use of perchloric acid, both as a deproteinizing agent and to prevent the spontaneous formation of methylglyoxal from glycolytic pathway intermediates. However, while using a modification of the standard literature assay to measure methylglyoxal in Chinese hamster ovary cells, we found that oxidation of nucleic acids and related compounds by perchloric or trichloroacetic acid results in the formation of methylglyoxal. Compounds containing 2-deoxyribose gave higher levels of methylglyoxal than those containing ribose; purine nucleotides and deoxynucleotides gave more methylglyoxal than did the pyrimidines. Nucleic acids were the most susceptible to degradation, with 12-fold more methylglyoxal being formed from DNA than RNA. Oxidation of nucleic acids increased with higher temperatures and with decreasing nucleic acid fragment size. Another product of nucleic acid oxidation was 2,3-butanedione, the 1,2-diaminobenzene derivative of which is sometimes used as an internal standard during methylglyoxal measurement. Unless accounted for during the assay procedure, the generation of methylglyoxal and 2,3-butanedione due to the oxidation of nucleic acids may lead to substantial errors in the determination of methylglyoxal concentrations in biological systems.

  18. Antioxidant properties of neohesperidin dihydrochalcone: inhibition of hypochlorous acid-induced DNA strand breakage, protein degradation, and cell death.

    Science.gov (United States)

    Choi, Je-Min; Yoon, Byoung-Seok; Lee, Sang-Kyou; Hwang, Jae-Kwan; Ryang, Ryung

    2007-02-01

    Neohesperidin dihydrochalcone (NHDC), a non-nutritive sweetening agent, is simply produced by hydrogenation of neohesperidin. The aim of this study is to evaluate the antioxidant and radical scavenging properties of neohesperidin dihydrochalcone and other structurally related compounds (phloridzin, neohesperidin) toward different reactive radical and oxygen species including .ABTS+, .O2-, .OH, H2O2, and HOCl in vitro. NHDC showed remarkable radical scavenging activity against stable radical and reactive oxygen species (ROS) in concentration dependent manner. Especially, NHDC was the most potent inhibitor of H2O2 and HOCl. NHDC showed HOCl scavenging activity of 93.5% and H2O2 scavenging property of 73.5% which was more than those of all the tested compounds including ascorbic acid and BHT. Moreover, NHDC could inhibit protein degradation, plasmid DNA strand cleavage and HIT-T15, HUVEC cell death from HOCl attack while mannitol, BHT, and ascorbic acid could not protect them effectively. These results suggest that NHDC is a potent antioxidant, especially it is evaluated as a novel HOCl scavenger. This study implies the possibility of therapeutic effect of NHDC on ROS-related inflammatory diseases.

  19. Resveratrol engages selective apoptotic signals in gastric adenocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    William L Riles; Jason Erickson; Sanjay Nayyar; Mary Jo Atten; Bashar M Attar; Oksana Holian

    2006-01-01

    AIM: To investigate the intracellular apoptotic signals engaged by resveratrol in three gastric adenocarcinoma cancer cell lines, two of which (AGS and SNU-1) express p53 and one (KATO-Ⅲ) with deleted p53.METHODS: Nuclear fragmentation was used to quantitate apoptotic cells; caspase activity was determined by photometric detection of cleaved substrates; formation of oxidized cytochrome C was used to measure cytochrome C activity, and Western blot analysis was used to determine protein expression.RESULTS: Gastric cancer cells, irrespective of their p53 status, responded to resveratrol with fragmentation of DNA and cleavage of nuclear lamins A and B and PARP, Resveratrol, however, has no effect on mitochondria-associated apoptotic proteins Bcl-2, Bclxl, Bax, Bid or Smac/Diablo, and did not promote subcellular redistribution of cytochrome C, indicating that resveratrol-induced apoptosis of gastric carcinoma cells does not require breakdown of mitochondrial membrane integrity. Resveratrol up-regulated p53 protein in SNU-1 and AGS cells but there was a difference in response of intracellular apoptotic signals between these cell lines.SNU-1 cells responded to resveratrol treatment with down-regulation of survivin, whereas in AGS and KATO-Ⅲ cells resveratrol stimulated caspase 3 and cytochrome C oxidase activities.CONCLUSION: These findings indicate that even within a specific cancer the intracellular apoptotic signals engaged by resveratrol are cell type dependent and suggest that such differences may be related to differentiation or lack of differentiation of these cells.

  20. Mouse zygotes respond to severe sperm DNA damage by delaying paternal DNA replication and embryonic development.

    Directory of Open Access Journals (Sweden)

    Joanna E Gawecka

    Full Text Available Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF. SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B followed by irreversible DNA degradation by a nuclease(s. Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development.

  1. Spectral, thermal, kinetic, molecular modeling and eukaryotic DNA degradation studies for a new series of albendazole (HABZ) complexes

    Science.gov (United States)

    El-Metwaly, Nashwa M.; Refat, Moamen S.

    2011-01-01

    This work represents the elaborated investigation for the ligational behavior of the albendazole ligand through its coordination with, Cu(II), Mn(II), Ni(II), Co(II) and Cr(III) ions. Elemental analysis, molar conductance, magnetic moment, spectral studies (IR, UV-Vis and ESR) and thermogravimetric analysis (TG and DTG) have been used to characterize the isolated complexes. A deliberate comparison for the IR spectra reveals that the ligand coordinated with all mentioned metal ions by the same manner as a neutral bidentate through carbonyl of ester moiety and NH groups. The proposed chelation form for such complexes is expected through out the preparation conditions in a relatively acidic medium. The powder XRD study reflects the amorphous nature for the investigated complexes except Mn(II). The conductivity measurements reflect the non-electrolytic feature for all complexes. In comparing with the constants for the magnetic measurements as well as the electronic spectral data, the octahedral structure was proposed strongly for Cr(III) and Ni(II), the tetrahedral for Co(II) and Mn(II) complexes but the square-pyramidal for the Cu(II) one. The thermogravimetric analysis confirms the presence or absence of water molecules by any type of attachments. Also, the kinetic parameters are estimated from DTG and TG curves. ESR spectrum data for Cu(II) solid complex confirms the square-pyramidal state is the most fitted one for the coordinated structure. The albendazole ligand and its complexes are biologically investigated against two bacteria as well as their effective effect on degradation of calf thymus DNA.

  2. Evaluation ofin vitro antioxidant and apoptotic activities ofCyperus rotundus

    Institute of Scientific and Technical Information of China (English)

    Kilani-Jaziri Soumaya; Ghedira Zied; Nasr Nouha; Krifa Mounira; Ghedira Kamel; Franca Dijoux Marie Genvive; Ghedira Chekir Leila

    2014-01-01

    Objective:To evaluate in vitro antioxidant and apoptotic activities ofCyperus rotundus(C. rotundus).Methods:The phytochemical study and the antioxidant activities of both methanol and aqueous extracts fromC. rotundus aerial part were determined.In addition, these extracts were also investigated for their cytotoxic and apoptotic activities.The major compound of the methanol extract was isolated.Both methanol and aqueous extracts(300,150, and50 μg/mL) were evaluated for their antioxidant activity by the xanthine/xanthine oxidase assay system.However, 16,8, and4 mg/mL of each extract were tested to investigate theirOH. formation scavenging potential.Aqueous extract(800,400, and200 μg/mL) and methanol extract(350,175, and88 μg/mL) were tested against lipid peroxidation, induced by75 μMH2O2.The cytotoxicity(byMTT assay) andcellDNA fragmentation of both extracts were evaluated towardsK562 andL1210 cell lines. The major compound was obtained from the butanol fraction of methanol extract and its structure was determined byRMN spectroscopic analysis.Results:The methanol and aqueous extracts showed respectively,88% and19% inhibition of xanthine oxidase activity.Yet, the same extracts inhibited lipid peroxidation by61.5% and42.0%, respectively.Both extracts inhibitedOH. formation by27.1% and25.3%, respectively.Only methanol extract inducedDNA degradation. Orientin was determined as the major compound isolated from the butanol fraction of methanol extract.Conclusions:It appears thatC. rotundus extracts exhibit a potential use as a natural antioxidant and an apoptosis inducer.

  3. Apoptotic markers in protozoan parasites

    Directory of Open Access Journals (Sweden)

    Fasel Nicolas

    2010-11-01

    Full Text Available Abstract The execution of the apoptotic death program in metazoans is characterized by a sequence of morphological and biochemical changes that include cell shrinkage, presentation of phosphatidylserine at the cell surface, mitochondrial alterations, chromatin condensation, nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies. Methodologies for measuring apoptosis are based on these markers. Except for membrane blebbing and formation of apoptotic bodies, all other events have been observed in most protozoan parasites undergoing cell death. However, while techniques exist to detect these markers, they are often optimised for metazoan cells and therefore may not pick up subtle differences between the events occurring in unicellular organisms and multi-cellular organisms. In this review we discuss the markers most frequently used to analyze cell death in protozoan parasites, paying special attention to changes in cell morphology, mitochondrial activity, chromatin structure and plasma membrane structure/permeability. Regarding classical regulators/executors of apoptosis, we have reviewed the present knowledge of caspase-like and nuclease activities.

  4. Apoptotic markers in protozoan parasites.

    Science.gov (United States)

    Jiménez-Ruiz, Antonio; Alzate, Juan Fernando; Macleod, Ewan Thomas; Lüder, Carsten Günter Kurt; Fasel, Nicolas; Hurd, Hilary

    2010-11-09

    The execution of the apoptotic death program in metazoans is characterized by a sequence of morphological and biochemical changes that include cell shrinkage, presentation of phosphatidylserine at the cell surface, mitochondrial alterations, chromatin condensation, nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies. Methodologies for measuring apoptosis are based on these markers. Except for membrane blebbing and formation of apoptotic bodies, all other events have been observed in most protozoan parasites undergoing cell death. However, while techniques exist to detect these markers, they are often optimised for metazoan cells and therefore may not pick up subtle differences between the events occurring in unicellular organisms and multi-cellular organisms.In this review we discuss the markers most frequently used to analyze cell death in protozoan parasites, paying special attention to changes in cell morphology, mitochondrial activity, chromatin structure and plasma membrane structure/permeability. Regarding classical regulators/executors of apoptosis, we have reviewed the present knowledge of caspase-like and nuclease activities.

  5. Temporal activation of anti- and pro-apoptotic factors in human gingival fibroblasts infected with the periodontal pathogen, Porphyromonas gingivalis: potential role of bacterial proteases in host signalling

    Directory of Open Access Journals (Sweden)

    Takehara Tadamichi

    2006-03-01

    Full Text Available Abstract Background Porphyromonas gingivalis is the foremost oral pathogen of adult periodontitis in humans. However, the mechanisms of bacterial invasion and the resultant destruction of the gingival tissue remain largely undefined. Results We report host-P. gingivalis interactions in primary human gingival fibroblast (HGF cells. Quantitative immunostaining revealed the need for a high multiplicity of infection for optimal infection. Early in infection (2–12 h, P. gingivalis activated the proinflammatory transcription factor NF-kappa B, partly via the PI3 kinase/AKT pathway. This was accompanied by the induction of cellular anti-apoptotic genes, including Bfl-1, Boo, Bcl-XL, Bcl2, Mcl-1, Bcl-w and Survivin. Late in infection (24–36 h the anti-apoptotic genes largely shut down and the pro-apoptotic genes, including Nip3, Hrk, Bak, Bik, Bok, Bax, Bad, Bim and Moap-1, were activated. Apoptosis was characterized by nuclear DNA degradation and activation of caspases-3, -6, -7 and -9 via the intrinsic mitochondrial pathway. Use of inhibitors revealed an anti-apoptotic function of NF-kappa B and PI3 kinase in P. gingivalis-infected HGF cells. Use of a triple protease mutant P. gingivalis lacking three major gingipains (rgpA rgpB kgp suggested a role of some or all these proteases in myriad aspects of bacteria-gingival interaction. Conclusion The pathology of the gingival fibroblast in P. gingivalis infection is affected by a temporal shift from cellular survival response to apoptosis, regulated by a number of anti- and pro-apoptotic molecules. The gingipain group of proteases affects bacteria-host interactions and may directly promote apoptosis by intracellular proteolytic activation of caspase-3.

  6. Apoptotic HPV positive cancer cells exhibit transforming properties.

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    Emilie Gaiffe

    Full Text Available Previous studies have shown that DNA can be transferred from dying engineered cells to neighboring cells through the phagocytosis of apoptotic bodies, which leads to cellular transformation. Here, we provide evidence of an uptake of apoptotic-derived cervical cancer cells by human mesenchymal cells. Interestingly, HeLa (HPV 18+ or Ca Ski (HPV16+ cells, harboring integrated high-risk HPV DNA but not C-33 A cells (HPV-, were able to transform the recipient cells. Human primary fibroblasts engulfed the apoptotic bodies effectively within 30 minutes after co-cultivation. This mechanism is active and involves the actin cytoskeleton. In situ hybridization of transformed fibroblasts revealed the presence of HPV DNA in the nucleus of a subset of phagocytosing cells. These cells expressed the HPV16/18 E6 gene, which contributes to the disruption of the p53/p21 pathway, and the cells exhibited a tumorigenic phenotype, including an increased proliferation rate, polyploidy and anchorage independence growth. Such horizontal transfer of viral oncogenes to surrounding cells that lack receptors for HPV could facilitate the persistence of the virus, the main risk factor for cervical cancer development. This process might contribute to HPV-associated disease progression in vivo.

  7. A Column Experiment To Determine Black Shale Degradation And Colonization By Means of δ13C and 14C Analysis Of Phospholipid Fatty Acids And DNA Extraction

    Science.gov (United States)

    Seifert, A.; Gleixner, G.

    2008-12-01

    We investigated the degradation of black shale organic matter by microbial communities. We inoculated two columns respectively, with the fungi Schizophyllum commune, the gram-positive bacterium Pseudomonas putida and the gram-negative bacteria Streptomyces griseus and Streptomyces chartreusis. These microorganisms are known to degrade a wide variety of organic macromolecules. Additionally, we had two sets of control columns. To one set the same nutrient solution was added as to the inoculated columns and to the other set only sterile deionised water was supplied. All columns contained 1.5 kg of freshly crushed not autoclaved black shale material with a particle size of 0.63-2 mm. The columns were incubated at 28° C and 60% humidity in the dark. The aim was to investigate, which microorganisms live on black shales and if these microorganisms are able to degrade ancient organic matter. We used compound specific stable isotope measurement techniques and compound specific 14C-dating methods. After 183 days PLFAs were extracted from the columns to investigate the microbial community, furthermore we extracted on one hand total-DNA of column material and on the other hand DNA from pure cultures isolates which grew on Kinks-agar B, Starch-casein-nitrate-agar (SCN) and on complete-yeast-medium-agar (CYM). According to the PLFA analysis bacteria dominated in the columns, whereas in pure cultures more fungi were isolated. A principal component analysis revealed differences between the columns in accordance with the inoculation, but it seems that the inoculated microorganisms were replaced by the natural population. For AMS measurements palmitic acid (C 16:0) was re-isolated from total-PLFA-extract with a preparative fraction collector (PFC). Preliminary results of the study revealed that microorganisms are able to degrade black shale material and that PLFA analysis are useful methods to be combined with analysis of stable isotope and 14C measurements to study microbial

  8. Hepatitis C virus core protein inhibits E6AP expression via DNA methylation to escape from ubiquitin-dependent proteasomal degradation.

    Science.gov (United States)

    Kwak, Juri; Shim, Joo Hee; Tiwari, Indira; Jang, Kyung Lib

    2016-09-28

    The E6-associated protein (E6AP) is a ubiquitin ligase that mediates ubiquitination and proteasomal degradation of hepatitis C virus (HCV) core protein. Given the role of HCV core protein as a major component of the viral nucleocapsid, as well as a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis, HCV has likely evolved a strategy to counteract the host anti-viral defense mechanism of E6AP and maximize its potential to produce infectious virus particles. In the present study, we found that HCV core protein derived from either ectopic expression or HCV infection inhibits E6AP expression via promoter hypermethylation in human hepatocytes. As a result, the potential of E6AP to ubiquitinate and degrade HCV core protein through the ubiquitin-proteasome system was severely impaired, which in turn led to stimulation of virus propagation. The effects of HCV core protein were almost completely abolished when the E6AP level was restored by ectopic expression of E6AP, treatment with a universal DNA methyltransferase (DNMT) inhibitor, 5-Aza-2'dC, or knock-down of DNMT1. In conclusion, HCV core protein inhibits E6AP expression via DNA methylation to protect itself from ubiquitin-dependent proteasomal degradation and stimulate virus propagation, providing a potential target for the development of anti-viral drugs against HCV.

  9. Phosphorylation of Hdmx mediates its Hdm2- and ATM-dependent degradation in response to DNA damage

    OpenAIRE

    Pereg, Yaron; Shkedy, Dganit; de Graaf, Petra; Meulmeester, Erik; Edelson-Averbukh, Marina; Salek, Mogjiborahman; Biton, Sharon; Teunisse, Amina F.A.S.; Lehmann, Wolf D.; Jochemsen, Aart G.; Shiloh, Yosef

    2005-01-01

    Maintenance of genomic stability depends on the DNA damage response, an extensive signaling network that is activated by DNA lesions such as double-strand breaks (DSBs). The primary activator of the mammalian DSB response is the nuclear protein kinase ataxia–telangiectasia, mutated (ATM), which phosphorylates key players in various arms of this network. The activation and stabilization of the p53 protein play a major role in the DNA damage response and are mediated by ATM-dependent posttransl...

  10. Wnt1 Neuroprotection Translates into Improved Neurological Function during Oxidant Stress and Cerebral Ischemia Through AKT1 and Mitochondrial Apoptotic Pathways

    Directory of Open Access Journals (Sweden)

    Zhao Zhong Chong

    2010-01-01

    Full Text Available Although essential for the development of the nervous system, Wnt1 also has been associated with neurodegenerative disease and cognitive loss during periods of oxidative stress. Here we show that endogenous expression of Wnt1 is suppressed during oxidative stress in both in vitro and in vivo experimental models. Loss of endogenous Wnt1 signaling directly correlates with neuronal demise and increased functional deficit, illustrating that endogenous neuronal Wnt1 offers a vital level of intrinsic cellular protection against oxidative stress. Furthermore, transient overexpression of Wnt1 or application of exogenous Wnt1 recombinant protein is necessary to preserve neurological function and rescue neurons from apoptotic membrane phosphatidylserine externalization and genomic DNA degradation, since blockade of Wnt1 signaling with a Wnt1 antibody or dickkopf related protein 1 abrogates neuronal protection by Wnt1. Wnt1 ultimately relies upon the activation of Akt1, the modulation of mitochondrial membrane permeability, and the release of cytochrome c to control the apoptotic cascade, since inhibition of Wnt1 signaling, the phosphatidylinositol 3-kinase pathway, or Akt1 activity abrogates the ability of Wnt1 to block these apoptotic components. Our work identifies Wnt1 and its downstream signaling as cellular targets with high clinical potential for novel treatment strategies for multiple disorders precipitated by oxidative stress.

  11. Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways.

    Science.gov (United States)

    Chong, Zhao Zhong; Shang, Yan Chen; Hou, Jinling; Maiese, Kenneth

    2010-01-01

    Although essential for the development of the nervous system, Wnt1 also has been associated with neurodegenerative disease and cognitive loss during periods of oxidative stress. Here we show that endogenous expression of Wnt1 is suppressed during oxidative stress in both in vitro and in vivo experimental models. Loss of endogenous Wnt1 signaling directly correlates with neuronal demise and increased functional deficit, illustrating that endogenous neuronal Wnt1 offers a vital level of intrinsic cellular protection against oxidative stress. Furthermore, transient overexpression of Wnt1 or application of exogenous Wnt1 recombinant protein is necessary to preserve neurological function and rescue neurons from apoptotic membrane phosphatidylserine externalization and genomic DNA degradation, since blockade of Wnt1 signaling with a Wnt1 antibody or dickkopf related protein 1 abrogates neuronal protection by Wnt1. Wnt1 ultimately relies upon the activation of Akt1, the modulation of mitochondrial membrane permeability, and the release of cytochrome c to control the apoptotic cascade, since inhibition of Wnt1 signaling, the phosphatidylinositol 3-kinase pathway, or Akt1 activity abrogates the ability of Wnt1 to block these apoptotic components. Our work identifies Wnt1 and its downstream signaling as cellular targets with high clinical potential for novel treatment strategies for multiple disorders precipitated by oxidative stress.

  12. Involvement of advanced lipooxidation end products (ALEs) and protein oxidation in the apoptotic actions of nitric oxide in insulin secreting RINm5F cells.

    Science.gov (United States)

    Cahuana, Gladys M; Tejedo, Juan R; Jiménez, Juan; Ramírez, Remedios; Sobrino, Francisco; Bedoya, Francisco J

    2003-11-15

    We have explored the impact of nitric oxide (NO) exposure on oxidation damage of lipids, and proteins, and the contribution of this type of damage to the activation of the apoptotic program in insulin secreting RINm5F cells. Exposure of cells to NO donors and to interleukin-1 beta (IL-1beta) led to generation of lipooxidation products such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). Addition of superoxide dismutase (SOD) and catalase (Cat) to cells decreased by 50% MDA and 4-HNE production induced by IL-1beta. Over-expression of Mn-SOD in cells conferred a remarkable decrease (75%) in IL-1beta-induced lipid peroxidation. These data suggest that peroxynitrite (ONOO(-)) mediates peroxidative damage to lipids in this cell system. Inhibitors of advanced lipooxidation end products (ALEs) formation such as aminoguanidine (AG) and pyridoxamine (PM) prevented partially apoptotic events triggered by NO such as DNA fragmentation, caspase-3 activation and cytochrome c release from mitochondria. These findings indicate that ALEs are involved in NO-induced apoptosis. In fact, NO-induced carbonylation of PARP protein preceded its apoptotic degradation and inhibitors of ALEs formation prevented both events. We thus propose that carbonylation of proteins is instrumental in linking NO-dependent lipid oxidation and apoptosis in this cell system.

  13. DNA degradation in genetically modified rice with Cry1Ab by food processing methods: implications for the quantification of genetically modified organisms.

    Science.gov (United States)

    Xing, Fuguo; Zhang, Wei; Selvaraj, Jonathan Nimal; Liu, Yang

    2015-05-01

    Food processing methods contribute to DNA degradation, thereby affecting genetically modified organism detection and quantification. This study evaluated the effect of food processing methods on the relative transgenic content of genetically modified rice with Cry1Ab. In steamed rice and rice noodles, the levels of Cry1Ab were ⩾ 100% and processing methods of sweet rice wine had the most severe degradation effects on Pubi and Cry1Ab. In steamed rice and rice noodles, Cry1Ab was the most stable, followed by SPS and Pubi. However, in rice crackers and sweet rice wine, SPS was the most stable, followed by Cry1Ab and Pubi. Therefore, Cry1Ab is a better representative of transgenic components than is Pubi because the levels of Cry1Ab were less affected compared to Pubi.

  14. Apoptotic Capacity and Risk of Squamous Cell Carcinoma of the Head and Neck

    Science.gov (United States)

    Liu, Zhensheng; Liu, Hongliang; Han, Peng; Gao, Fengqin; Dahlstrom, Kristina R.; Li, Guojun; Owzar, Kouros; Zevallos, Jose P.; Sturgis, Erich M.; Wei, Qingyi

    2017-01-01

    Background Tobacco smoke and alcohol drinking are the major risk factors for squamous cell carcinoma of the head and neck (SCCHN). Smoking and drinking cause DNA damage leading to apoptosis, and insufficient apoptotic capacity may favor development of cancer because of the dysfunction of removing damaged cells. In the present study, we investigated the association between camptothecin (CPT)-induced apoptotic capacity and risk of SCCHN in a North American population. Methods In a case-control study of 708 SCCHN patients and 685 matched cancer-free controls, we measured apoptotic capacity in cultured peripheral blood lymphocytes (PBLs) in response to in vitro exposure to CPT by using the flow cytometry-based method. Results We found that the mean level of apoptotic capacity in the cases (45.9±23.3%) was significantly lower than that in the controls (49.0±23.1%) (P = 0.002). When we used the median level of apoptotic capacity in the controls as the cutoff value for calculating adjusted odds ratios (ORs), subjects with a reduced apoptotic capacity had an increased risk (adjusted OR = 1.42, 95% confidence interval [CI] = 1.13–1.78, P = 0.002), especially for those who were age ≥57 (1.73, 1.25–2.38, 0.0009), men (1.76, 1.36–2.27, < 0.0001) and ever drinkers (1.67, 1.27–2.21, 0.0003), and these variables significantly interacted with apoptotic capacity (Pinteraction = 0.015, 0.005 and 0.009, respectively). A further fitted prediction model suggested that the inclusion of apoptotic capacity significantly improved in the prediction of SCCHN risk. Conclusion Individuals with a reduced CPT-induced apoptotic capacity may be at an increased risk of developing SCCHN, and apoptotic capacity may be a biomarker for susceptibility to SCCHN. PMID:28033527

  15. Apoptotic engulfment pathway and schizophrenia.

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    Xiangning Chen

    Full Text Available BACKGROUND: Apoptosis has been speculated to be involved in schizophrenia. In a previously study, we reported the association of the MEGF10 gene with the disease. In this study, we followed the apoptotic engulfment pathway involving the MEGF10, GULP1, ABCA1 and ABCA7 genes and tested their association with the disease. METHODOLOGY/PRINCIPAL FINDINGS: Ten, eleven and five SNPs were genotyped in the GULP1, ABCA1 and ABCA7 genes respectively for the ISHDSF and ICCSS samples. In all 3 genes, we observed nominally significant associations. Rs2004888 at GULP1 was significant in both ISHDSF and ICCSS samples (p = 0.0083 and 0.0437 respectively. We sought replication in independent samples for this marker and found highly significant association (p = 0.0003 in 3 Caucasian replication samples. But it was not significant in the 2 Chinese replication samples. In addition, we found a significant 2-marker (rs2242436 * rs3858075 interaction between the ABCA1 and ABCA7 genes in the ISHDSF sample (p = 0.0022 and a 3-marker interaction (rs246896 * rs4522565 * rs3858075 amongst the MEGF10, GULP1 and ABCA1 genes in the ICCSS sample (p = 0.0120. Rs3858075 in the ABCA1 gene was involved in both 2- and 3-marker interactions in the two samples. CONCLUSIONS/SIGNIFICANCE: From these data, we concluded that the GULP1 gene and the apoptotic engulfment pathway are involved in schizophrenia in subjects of European ancestry and multiple genes in the pathway may interactively increase the risks to the disease.

  16. Apoptotic engulfment pathway and schizophrenia.

    LENUS (Irish Health Repository)

    Chen, Xiangning

    2009-01-01

    BACKGROUND: Apoptosis has been speculated to be involved in schizophrenia. In a previously study, we reported the association of the MEGF10 gene with the disease. In this study, we followed the apoptotic engulfment pathway involving the MEGF10, GULP1, ABCA1 and ABCA7 genes and tested their association with the disease. METHODOLOGY\\/PRINCIPAL FINDINGS: Ten, eleven and five SNPs were genotyped in the GULP1, ABCA1 and ABCA7 genes respectively for the ISHDSF and ICCSS samples. In all 3 genes, we observed nominally significant associations. Rs2004888 at GULP1 was significant in both ISHDSF and ICCSS samples (p = 0.0083 and 0.0437 respectively). We sought replication in independent samples for this marker and found highly significant association (p = 0.0003) in 3 Caucasian replication samples. But it was not significant in the 2 Chinese replication samples. In addition, we found a significant 2-marker (rs2242436 * rs3858075) interaction between the ABCA1 and ABCA7 genes in the ISHDSF sample (p = 0.0022) and a 3-marker interaction (rs246896 * rs4522565 * rs3858075) amongst the MEGF10, GULP1 and ABCA1 genes in the ICCSS sample (p = 0.0120). Rs3858075 in the ABCA1 gene was involved in both 2- and 3-marker interactions in the two samples. CONCLUSIONS\\/SIGNIFICANCE: From these data, we concluded that the GULP1 gene and the apoptotic engulfment pathway are involved in schizophrenia in subjects of European ancestry and multiple genes in the pathway may interactively increase the risks to the disease.

  17. To the nucleolar density and size in apoptotic human leukemic myeloblasts produced in vitro by Trichostatin A

    Directory of Open Access Journals (Sweden)

    K Smetana

    2009-08-01

    Full Text Available The present study was designed to provide more information on nucleoli in apoptotic cells, which were represented in the present study by cultured leukemic myeloblasts (Kasumi-1 cells. The apoptotic process in these cells was produced by trichostatin A (TSA that is a histone deacetylase inhibitor with strong cytostatic effects. The selected TSA concentration added to cultures facilitated to study apoptotic and notapoptotic cells in one and the same specimen. The nucleolar diameter and density were determined using computer assisted measurement and densitometry in specimens stained for RNA. In comparison with not-apoptotic cells, in apoptotic cells, nucleolar mean diameter did not change significantly and nucleolar RNA density was also not apparently different. On the other hand, the cytoplasmic RNA density in apoptotic cells was markedly reduced. Thus it seemed to be possible that the transcribed RNA remained “frozen” within the nucleolus but its transport to the cytoplasm decreased or stopped. However, the possibility of the RNA degradation in the cytoplasm of apoptotic cells based on the present study cannot be eliminated. At this occasion it should be added that AgNORs reflecting nucleolar biosynthetic and cell proliferation activity in apoptotic cells decreased in number or disappeared. The presented results also indicated that large nucleoli intensely stained for RNA need not be necessarily related to the high nucleolar biosynthetic or cell proliferation activity and may be also present in apoptotic cells responding to the cytostatic treatment.

  18. Autosomal and Y-STR analysis of degraded DNA from the 120-year-old skeletal remains of Ezekiel Harper.

    Science.gov (United States)

    Ambers, Angie; Gill-King, Harrell; Dirkmaat, Dennis; Benjamin, Robert; King, Jonathan; Budowle, Bruce

    2014-03-01

    The 120-year-old skeletal remains of Confederate Civil War soldier Captain Ezekiel "Zeke" Harper were exhumed by court order in January 2011 for DNA analysis. The goal of the DNA testing was to support or refute whether Captain Harper had fathered a son (Earl J. Maxwell) with his Native American maid prior to his murder in 1892. Bones with adequate structural integrity (left tibia, right tibia, right femur, mandible, four teeth) were retrieved from the burial site and sent to the Institute of Applied Genetics in Fort Worth, Texas for analysis. Given the age and condition of the remains, three different extraction methods were used to maximize the probability of DNA recovery. The majority of the DNA isolates from over fifty separate bone sections yielded partial autosomal STR genotypes and partial Y-STR haplotypes. After comparing the partial results for concordance, consensus profiles were generated for comparison to reference samples from alleged family members. Considering the genetic recombination that occurs in autosomal DNA over the generations within a family, Y-STR analysis was determined to be the most appropriate and informative approach for determining potential kinship. Two of Earl J. Maxwell's grandsons submitted buccal samples for comparison. The Y-STR haplotypes obtained from both of these reference samples were identical to each other and to the alleles in Ezekiel Harper's consensus profile at all 17 loci examined. This Y-STR haplotype was not found in either of two major Y-STR population databases (U.S. Y-STR database and YHRD). The fact that the Y-STR haplotype obtained from Ezekiel's skeletal remains and Earl's grandsons is not found in either population database demonstrates its rarity and further supports a paternal lineage relationship among them. Results of the genetic analyses are consistent with the hypothesis that Earl J. Maxwell is the son of Ezekiel Harper.

  19. DNA rearrangement has occurred in the carbazole-degradative plasmid pCAR1 and the chromosome of its unsuitable host, Pseudomonas fluorescens Pf0-1.

    Science.gov (United States)

    Shintani, Masaki; Matsumoto, Takashi; Yoshikawa, Hirofumi; Yamane, Hisakazu; Ohkuma, Moriya; Nojiri, Hideaki

    2011-12-01

    The carbazole-degradative plasmid pCAR1 carries the class II transposon Tn4676, which contains the car and ant genes, essential for conversion of carbazole into anthranilate, and anthranilate into catechol, respectively. In our previous study, DNA rearrangements in pCAR1 were frequently detected in the host Pseudomonas fluorescens Pf0-1 in the presence of carbazole, resulting in the improvement of host survivability. Several Pf0-1 mutants harbouring pCAR1 were isolated, and deletion of DNA in the plasmid ant gene was found. Here, we compared genome sequences of the parent strain Pf0-1L(pCAR1::rfp) and one of its mutants, 5EP83, to assess whether other DNA rearrangements occurred in either the plasmid or the host chromosome. We found transposition of Tn4676 into the 5EP83 chromosome. In addition, ISPre1 had transposed into the car gene intergenic region on the pCAR1-derivative plasmid of 5EP83, which inhibited car transcription. As a result of these transpositions, 5EP83 was able to metabolize carbazole due to the Tn4676 on its chromosome, although the car genes on its plasmid were non-functional. We also found that one copy of duplicate carAa genes had been deleted, and that ISPre4 had transposed into both the host chromosome and the plasmid. Our findings suggest that Pf0-1 harbouring pCAR1 is subjected to DNA rearrangements not only on the plasmid but also on its chromosome in the presence of carbazole.

  20. Intercellular transfer of apoptotic signals via electrofusion

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jin Suk; Lee, Wilson; McCulloch, Christopher A., E-mail: christopher.mcculloch@utoronto.ca

    2012-05-01

    We determined whether cells that are induced to undergo anoikis by matrix detachment can initiate apoptosis in healthy cells following electroporation-induced fusion. Separate populations of MDCK cells undergoing anoikis and stained with FITC-annexin or viable MDCK cells that were labeled with spectrally discrete fluorescent beads were electroporated. Cells were analyzed by flow cytometry for enumeration of viable cells with beads, apoptotic cells or fused cells. Electroporation promoted a 49-fold increase of the percentage of viable cells that had fused with apoptotic cells. Apoptotic cell-viable cell fusions were 8-fold more likely to not attach to cell culture plastic and 2.3-fold less likely to proliferate after 24 hr incubation than viable cell fusion controls. These data demonstrate that apoptotic signals can be transferred between cells by electrofusion, possibly suggesting a novel investigative approach for optimizing targeted cell deletion in cancer treatment.

  1. 一种快速分离细胞凋亡片段化DNA的简单方法--NP-40法%A rapid and simple method for the isolation of apoptotic DNA fragments

    Institute of Scientific and Technical Information of China (English)

    熊世勤; 朱锡华; 黄云辉

    2000-01-01

    目的鉴定细胞凋亡的一个重要生化指标是凋亡细胞的核DNA通过琼脂糖凝胶电泳呈现典现的DNA"梯状"图谱.传统分离凋亡细胞片段化DNA的方法多采用酚/氯仿抽提.因此,有必要探寻一种更为灵敏、简单的分离凋亡片段化DNA的新方法.方法通过比较传统分离凋亡片段化DNA的方法,并对HERRMANN等[1]报道的方法加以改进.结果本文介绍一种快速分离凋亡DNA片段化的新方法(NP-40法).结论这种简单、快速的分离方法无需酚/氯仿反复抽提,无需大量的细胞样品,并且凋亡DNA片段化梯状图谱清晰,适用于大多数凋亡细胞片段化DNA的分离.

  2. Staphylococcus aureus alpha-toxin-induced cell death : predominant necrosis despite apoptotic caspase activation

    NARCIS (Netherlands)

    Essmann, F; Bantel, H; Totzke, G; Engels, I H; Sinha, B; Schulze-Osthoff, K; Jänicke, R U

    2003-01-01

    Recent data suggest that alpha-toxin, the major hemolysin of Staphylococcus aureus, induces cell death via the classical apoptotic pathway. Here we demonstrate, however, that although zVAD-fmk or overexpression of Bcl-2 completely abrogated caspase activation and internucleosomal DNA fragmentation,

  3. NKLP27: a teleost NK-lysin peptide that modulates immune response, induces degradation of bacterial DNA, and inhibits bacterial and viral infection.

    Science.gov (United States)

    Zhang, Min; Li, Mo-fei; Sun, Li

    2014-01-01

    NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis) NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides.

  4. NKLP27: a teleost NK-lysin peptide that modulates immune response, induces degradation of bacterial DNA, and inhibits bacterial and viral infection.

    Directory of Open Access Journals (Sweden)

    Min Zhang

    Full Text Available NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides.

  5. In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer.

    LENUS (Irish Health Repository)

    Murphy, Therese M

    2011-01-01

    Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC).

  6. Investigating migration inhibition and apoptotic effects of Fomitopsis pinicola chloroform extract on human colorectal cancer SW-480 cells.

    Directory of Open Access Journals (Sweden)

    Yaqin Wang

    Full Text Available BACKGROUND: Fomitopsis pinicola (Sw. Ex Fr.m Karst (FPK which belongs to the Basidiomycota fungal class is one of the most popular medical fungi in China. It has been used for many diseases: cancer, heart diseases, diabetes and so on. However, little study on the pro-apoptotic effect and migration inhibition of FPK chloroform extract (FPKc has been reported and the possible involved mechanism has not been illuminated. METHODOLOGY/PRINCIPAL FINDINGS: Chemical analysis was performed by HPLC which showed ergosterol (ES concentration was 105 µg/mg. MTT assay revealed that FPKc could selectively inhibit SW-480 cells viability with the IC50 of 190.28 µg/ml. Wound healing and transwell assay indicated that FPKc could inhibit the migration of SW-480 cells obviously, FPKc could also dramatically decreased the matrix metalloproteinases-2, 9 (MMP-2 and MMP-9 expression. Annexin V-FITC/PI staining, nuclear Hoechst 33342 staining and DNA fragmentation analysis revealed that FPKc and ES could induce SW-480 cells apoptosis. The apoptosis process closely involved in ROS accumulation and depletion of GSH, activation of caspase 3, poly (ADP-ribose polymerase (PARP degradation. FPKc could also up-regulate P53 expression and thus lead to G1 phase arrest. When SW-480 cells were pretreated with N-acetylcysteine (NAC, the ROS generation, cell viability and apoptotic ratio were partially declined, which indicated that ROS was vertical in the pro-apoptosis process induced by FPKc. Moreover, in the whole process, ES which has been previously found in FPKc had the similar effect to FPKc. Thus we could conclude that ES, as one of the highest abundant components in FPKc, might also be one of the active constituents. CONCLUSION/SIGNIFICANCE: FPKc could inhibit the migration of SW-480 cells, induce SW-480 cells G1 phase arrest and cause ROS-mediated apoptosis effect. And ES might be one of the effective constituents in the whole process.

  7. FSAP-mediated nucleosome release from late apoptotic cells is inhibited by autoantibodies present in SLE.

    Science.gov (United States)

    Marsman, Gerben; Stephan, Femke; de Leeuw, Karina; Bulder, Ingrid; Ruinard, Jessica T; de Jong, Jan; Westra, Johanna; Bultink, Irene E M; Voskuyl, Alexandre E; Aarden, Lucien A; Luken, Brenda M; Kallenberg, Cees G M; Zeerleder, Sacha

    2016-03-01

    Inefficient clearance of apoptotic cells and the subsequent exposure of the immune system to nuclear contents are crucially involved in the pathogenesis of systemic lupus erythematosus (SLE). Factor VII-activating protease (FSAP) is activated in serum upon contact with dead cells, and releases nucleosomes from late apoptotic cells into the extracellular environment. We investigated whether FSAP-mediated nucleosome release from late apoptotic cells is affected in SLE patients. Nucleosome release in sera of 27 SLE patients and 30 healthy controls was investigated by incubating late apoptotic Jurkat cells with serum and analyzing the remaining DNA content by flow cytometry. We found that nucleosome release in sera of SLE patients with high disease activity was significantly decreased when compared with that in SLE sera obtained during low disease activity or from healthy individuals. Upon removal of IgG/IgM antibodies from SLE sera, nucleosome release was restored. Similarly, monoclonal antinuclear antibodies inhibited nucleosome release in healthy donor serum or by plasma-purified FSAP. This inhibition was lost when Fab fragments were used, suggesting that antigen cross-linking is involved. In conclusion, FSAP-mediated nucleosome release from late apoptotic cells is greatly impaired in SLE patient sera, possibly hampering the clearance of these cells and thereby propagating inflammation.

  8. Apoptotic activity in Libyan breast cancer

    Directory of Open Access Journals (Sweden)

    Boder Jamela

    2012-06-01

    Full Text Available Abstract Background We evaluated the relationship of the apoptotic activity index (AI and the standardized mitotic-apoptotic ratio (SMI/AI with clinicopathological features and prognosis in Libyan female breast cancer (BC patients. We then compared our results with corresponding results in Finnish and Nigerian female BC patients. Methods Histological samples of breast carcinoma from 130 patients were retrospectively studied: an estimation of the apoptotic activity per square millimeter (expressed as apoptotic activity index (AI, and standardized mitotic-apoptotic ratio (SMI/AI was made, and the results compared with the clinicopathological features and the patient’s survival. Results There was a statistically significant correlation between the AI and most of the clinicopathological features; the strongest association was observed for clinical stage lymph node (LN status (P = 0.005. There were also correlations between AI and histological grade (P = 0.035, large tumor size (P = 0.011 and the clinical stage (P = 0.009. There were, however, prominent AI differences between Libyan, Nigerian and Finnish populations. The mean values of AI and SMI/AI in Libyan BC patients were 12.8 apoptotic figures per square millimeter and 2.8, respectively. The Libyan AI is slightly higher than in Nigeria, but much higher than in Finland. The differences between countries are seen throughout the samples as well as being present in certain subgroups. The survival analysis indicated that short survival time was associated with high apoptotic indices values and so can identify aggressive tumors and provide significant prognostic support. The cutoff (4 and 18 apoptosis/mm2 of AI might be applied as a quantitative criterion for Libyan BC to separate the patients into good, moderate and bad prognosis groups. Conclusions The results indicated that the differences in AI among the three countries may be due to the known variation in the distribution of

  9. Reversal of Apoptotic Resistance by Lycium barbarum Glycopeptide 3 in Aged T Cells

    Institute of Scientific and Technical Information of China (English)

    LONG-GUO YUAN; HONG-BIN DENG; LI-HUI CHEN; DIAN-DONG LI; QI-YANG HE

    2008-01-01

    Objective To study whether Lycium barbarian glycopeptide 3 (LBGP3) affects T cell apeptosis in aged mice. Methods LBGP3 was purified with DEAE cellulose and Sephadex columns. Apoptotic "sub-Gl peak" was detected by flow cytometry and DNA ladder was resolved by agarose gel electrophoresis. Levels of IFN-γ, and IL-10 were measured with specific kits and mRNA expression was detected by RT-PCR. Apoptosis-related proteins of FLIP, FasL, and Bcl-2 were determined by Western blotting. Resdts LBGP3 was purified from Fructus Lycii water extracts and identified as a 41 kD glycopeptide.Treatment with 200 μg/mL LBGP3 increased the apoptotic rate of T cells from aged mice and showed a similar DNA ladder pattern to that in young T ceils. The reversal of apoptotic resistance was involved in down-regulating the expression of Bcl-2 and FLIP, and up-regulating the expression of FasL. Conclusion Lycium barbarum glycopeptide 3 reverses apoptotic resistance of aged T cells by modulating the expression of apoptosis-related molecules.

  10. Apoptotic-like phenotype triggered by hydrogen peroxide and amphotericin B in the fungus Rhizopus arrhizus.

    Science.gov (United States)

    Wang, Sibu; Li, Ruoyu; Yu, Jin

    2014-12-01

    Rhizopus is the most common genus of invasive mucormycosis, whose prognosis and outcome was not improved over the past decades. We studied the apoptotic-like phenotype in Rhizopus arrhizus exposed to hydrogen peroxide (H2 O2 ) and amphotericin B (AMB). The strain provided by Fungal Genetic Stock centre was studied about the apoptotic-like phenotype treated with different concentrations of H2 O2 and AMB, and then analyzed by fluorescent microscopy (observed by Annexin-V/FITC and TUNEL staining), flow cytometry (stained with DHR123/PI), and DNA agarose gel electrophores. When R. arrhizus was treated with H2 O2 and AMB, there was a loss of viability associated with different phenotype of apoptosis makers. Membrane externalization of phosphatidylserine (PS) on the cell surface, DNA fragmentation, chromatin condensation can be induced and observed obviously by Annexin-V/FITC, DAPI and TUNEL staining. DNA smear not DNA ladder was also visible in R. arrhizus. Flowcytometry of R. arrhizus cells revealed not only the increase of apoptosis cell stained with DHR123 under the nonfungicida doses but dead cells stained with PI under the fungicida concentrations.This study indicated that both H2 O2 and AMB could induce the apoptotic-like phenotype in R. arrhizus.

  11. Modeling and time-dependent dynamics of processes of stimulated depolymerization, auto-repairing, degradation and radiation curing of DNA macromolecules and biopolymers at separated and combined actions of ionizing irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Vysotskii, Vladimir I. E-mail: viv@vhome.kiev.ua; Pinchuk, Anatoliy O.; Kornilova, Alla A.; Samoylenko, Igor I

    2001-12-01

    The time-dependent dynamics of the formation, relaxation and auto-repairing of double breaks of DNA macromolecules at the combined radiation action and non-radiation processes of degradation (e.g. by free radicals) were considered. The auto-repairing of DNA double breaks is connected with the peculiarities of long-range interaction of nucleotide charges, atoms and molecules in the intracellular milieu. The properties of intracellular liquid and the characteristics of force interaction between the end-pairs of nucleotides in the area of DNA break in response to radiation are changed. Each kind of radiation is characterized by a certain effectiveness of the double DNA break formation but simultaneously one creates the conditions for their liquidation. On the basis of the analysis and correlation of these processes the time-dependent theory for DNA degradation was created, including hormesis phenomenon, radiation antagonism, the validity of anomaly influence of low and large doses at sharp and chronic radiation and other effects. The qualitative and quantitative correspondences of the theory and experimental results of radiation biology were obtained.

  12. The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Eun-Ah [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From

  13. Stabilization Of Apoptotic Cells: Generation Of Zombie Cells

    Directory of Open Access Journals (Sweden)

    José A. Sánchez Alcázar

    2015-08-01

    Stabilization of apoptotic cells can be used for reliable detection and quantification of apoptosis in cultured cells and may allow a safer administration of apoptotic cells in clinical applications. Furthermore, it opens new avenues in the functional reconstruction of apoptotic cells for longer preservation.

  14. Investigation of the apoptotic way induced by digallic acid in human lymphoblastoid TK6 cells

    Directory of Open Access Journals (Sweden)

    Bhouri Wissem

    2012-06-01

    Full Text Available Abstract Background The digallic acid (DGA purified from Pistacia lentiscus. L fruits was investigated for its antiproliferative and apoptotic activities on human lymphoblastoid TK6 cells. Methods We attempt to characterize the apoptotic pathway activated by DGA. Apoptosis was detected by DNA fragmentation, PARP cleavage and by evaluating caspase activities. Results The inhibition of lymphoblastoid cell proliferation was noted from 8.5 μg/ml of DGA. The induction of apoptosis was confirmed by DNA fragmentation and PARP cleavage. We have demonstrated that DGA induces apoptosis by activating the caspase-8 extrinsic pathway. Caspase-3 was also activated in a dose dependent manner. Conclusion In summary, DGA exhibited an apoptosis inductor effect in TK6 cells revealing thus its potential as a cancer-preventive agent.

  15. Apoptotic death sensor: an organelle's alter ego?

    Science.gov (United States)

    Bratton, S B; Cohen, G M

    2001-06-01

    Caspases are intracellular cysteine proteases that are primarily responsible for the stereotypic morphological and biochemical changes that are associated with apoptosis. Caspases are often activated by the apoptotic protease-activating factor 1 (APAF-1) apoptosome, a complex that is formed following mitochondrial release of cytochrome c in response to many death-inducing stimuli. Both pro- and anti-apoptotic BCL-2 family members regulate apoptosis, primarily by their effects on mitochondria, whereas many inhibitor of apoptosis proteins (IAPs) regulate apoptosis by directly inhibiting distinct caspases. Exposure of cells to chemicals and radiation, as well as loss of trophic stimuli, perturb cellular homeostasis and, depending on the type of cellular stress, particular or multiple organelles appear to 'sense' the damage and signal the cell to undergo apoptosis by stimulating the formation of unique and/or common caspase-activating complexes.

  16. BAD-mediated apoptotic pathway is associated with human cancer development.

    Science.gov (United States)

    Stickles, Xiaomang B; Marchion, Douglas C; Bicaku, Elona; Al Sawah, Entidhar; Abbasi, Forough; Xiong, Yin; Bou Zgheib, Nadim; Boac, Bernadette M; Orr, Brian C; Judson, Patricia L; Berry, Amy; Hakam, Ardeshir; Wenham, Robert M; Apte, Sachin M; Berglund, Anders E; Lancaster, Johnathan M

    2015-04-01

    The malignant transformation of normal cells is caused in part by aberrant gene expression disrupting the regulation of cell proliferation, apoptosis, senescence and DNA repair. Evidence suggests that the Bcl-2 antagonist of cell death (BAD)-mediated apoptotic pathway influences cancer chemoresistance. In the present study, we explored the role of the BAD-mediated apoptotic pathway in the development and progression of cancer. Using principal component analysis to derive a numeric score representing pathway expression, we evaluated clinico-genomic datasets (n=427) from corresponding normal, pre-invasive and invasive cancers of different types, such as ovarian, endometrial, breast and colon cancers in order to determine the associations between the BAD-mediated apoptotic pathway and cancer development. Immunofluorescence was used to compare the expression levels of phosphorylated BAD [pBAD (serine-112, -136 and -155)] in immortalized normal and invasive ovarian, colon and breast cancer cells. The expression of the BAD-mediated apoptotic pathway phosphatase, PP2C, was evaluated by RT-qPCR in the normal and ovarian cancer tissue samples. The growth-promoting effects of pBAD protein levels in the immortalized normal and cancer cells were assessed using siRNA depletion experiments with MTS assays. The expression of the BAD-mediated apoptotic pathway was associated with the development and/or progression of ovarian (n=106, pcancers, as well as with ovarian endometriosis (n=20, pcancer cells compared to the immortalized normal cells, whereas PP2C gene expression was lower in the cancer compared to the ovarian tumor tissue samples (n=76, pcancer cells. The BAD-mediated apoptotic pathway is thus associated with the development of human cancers likely influenced by the protein levels of pBAD.

  17. Non-apoptotic function of apoptotic proteins in the development of Malpighian tubules of Drosophila melanogaster

    Indian Academy of Sciences (India)

    Madhu G Tapadia; Naveen K Gautam

    2011-08-01

    Drosophila metamorphosis is characterized by the histolysis of larval structures by programmed cell death, which paves the way for the establishment of adult-specific structures under the influence of the steroid hormone ecdysone. Malpighian tubules function as an excretory system and are one of the larval structures that are not destroyed during metamorphosis and are carried over to adulthood. The pupal Malpighian tubules evade destruction in spite of expressing apoptotic proteins, Reaper, Hid, Grim, Dronc and Drice. Here we show that in the Malpighian tubules expression of apoptotic proteins commences right from embryonic development and continues throughout the larval stages. Overexpression of these proteins in the Malpighian tubules causes larval lethality resulting in malformed tubules. The number and regular organization of principal and stellate cells of Malpighian tubules is disturbed, in turn disrupting the physiological functioning of the tubules as well. Strikingly, the localization of -tubulin, F-actin and Disclarge (Dlg) is also disrupted. These results suggest that the apoptotic proteins could be having non-apoptotic function in the development of Malpighian tubules.

  18. Targeted rapid amplification of cDNA ends (T-RACE)—an improved RACE reaction through degradation of non-target sequences

    OpenAIRE

    Neil I Bower; Johnston, Ian A

    2010-01-01

    Amplification of the 5' ends of cDNA, although simple in theory, can often be difficult to achieve. We describe a novel method for the specific amplification of cDNA ends. An oligo-dT adapter incorporating a dUTP-containing PCR primer primes first-strand cDNA synthesis incorporating dUTP. Using the Cap finder approach, another distinct dUTP containing adapter is added to the 3' end of the newly synthesized cDNA. Second-strand synthesis incorporating dUTP is achieved by PCR, using dUTP-contain...

  19. Effect of ALD-DNA derived from cells with different apoptotic levels on the induction of anti-double-stranded DNA antibody%不同凋亡水平T细胞来源的ALD-DNA对抗双链DNA抗体产生的影响

    Institute of Scientific and Technical Information of China (English)

    温振科; 曹清华; 徐林; 徐薇; 储以微; 熊思东

    2007-01-01

    研究不同凋亡水平T细胞来源的ALD-DNA(activated lymphocyte-derived DNA)对抗双链DNA抗体产生的影响.用尼龙毛柱法分离小鼠脾脏T细胞,分别提取不同凋亡水平T细胞的DNA定义为未凋亡ALD-DNA、低凋亡水平ALD-DNA、中凋亡水平ALD-DNA和高凋亡水平ALD-DNA,并免疫同系BALB/c小鼠;用ELISA方法检测血清中IgG类抗双链DNA抗体的水平及其亚型;用考马斯亮蓝法检测免疫小鼠尿蛋白的含量.结果显示,ALD-DNA的凋亡水平与其诱导产生的抗双链DNA抗体水平成正相关,二者的相关系数r=0.852;不同凋亡水平的ALD-DNA诱导的抗双链DNA抗体均以IgG1为主,但IgG2a和IgG2b的水平在高凋亡ALD-DNA免疫组有明显增高;低凋亡ALD-DNA、中凋亡ALD-DNA和高凋亡ALD-DNA免疫小鼠均有显著蛋白尿形成,并且高凋亡ALD-DNA免疫组的尿蛋白含量有明显增高.该结果表明,高凋亡水平的ALD-DNA免疫同系小鼠更易诱导高水平的致病性IgG类抗双链DNA抗体产生,凋亡DNA可能在系统性红斑狼疮(SLE)发生发展过程中发挥了重要作用,这为我们能更加深入的理解SLE的发生机制提供了有力的实验依据.

  20. Apoptotic lymphocytes of H. sapiens lose nucleosomes in GC-rich promoters.

    Science.gov (United States)

    Hosid, Sergey; Ioshikhes, Ilya

    2014-07-01

    We analyzed two sets of human CD4+ nucleosomal DNA directly sequenced by Illumina (Solexa) high throughput sequencing method. The first set has ∼40 M sequences and was produced from the normal CD4+ T lymphocytes by micrococcal nuclease. The second set has ∼44 M sequences and was obtained from peripheral blood lymphocytes by apoptotic nucleases. The different nucleosome sets showed similar dinucleotide positioning AA/TT, GG/CC, and RR/YY (R is purine, Y--pyrimidine) patterns with periods of 10-10.4 bp. Peaks of GG/CC and AA/TT patterns were shifted by 5 bp from each other. Two types of promoters in H. sapiens: AT and GC-rich were identified. AT-rich promoters in apoptotic cell had +1 nucleosome shifts 50-60 bp downstream from those in normal lymphocytes. GC-rich promoters in apoptotic cells lost 80% of nucleosomes around transcription start sites as well as in total DNA. Nucleosome positioning was predicted by combination of {AA, TT}, {GG, CC}, {WW, SS} and {RR, YY} patterns. In our study we found that the combinations of {AA, TT} and {GG, CC} provide the best results and successfully mapped 33% of nucleosomes 147 bp long with precision ±15 bp (only 31/147 or 21% is expected).

  1. Apoptotic cell clearance: basic biology and therapeutic potential.

    Science.gov (United States)

    Poon, Ivan K H; Lucas, Christopher D; Rossi, Adriano G; Ravichandran, Kodi S

    2014-03-01

    The prompt removal of apoptotic cells by phagocytes is important for maintaining tissue homeostasis. The molecular and cellular events that underpin apoptotic cell recognition and uptake, and the subsequent biological responses, are increasingly better defined. The detection and disposal of apoptotic cells generally promote an anti-inflammatory response at the tissue level, as well as immunological tolerance. Consequently, defects in apoptotic cell clearance have been linked with various inflammatory diseases and autoimmunity. Conversely, under certain conditions, such as the killing of tumour cells by specific cell-death inducers, the recognition of apoptotic tumour cells can promote an immunogenic response and antitumour immunity. Here, we review the current understanding of the complex process of apoptotic cell clearance in physiology and pathology, and discuss how this knowledge could be harnessed for new therapeutic strategies.

  2. Relationship between apoptotic markers in semen from fertile men and demographic, hormonal and seminal characteristics

    Institute of Scientific and Technical Information of China (English)

    Ina O Specht; Marcello Spanò; Karin S Hougaard; Gian C Manicardi; Davide Bizzaro; Gunnar Toft; Aleksander Giwercman; Jens-Peter E Bonde

    2012-01-01

    Apoptosis in the testis has two putative roles during normal spermatogenesis; limitation of the germ ceil population to numbers that can be supported by the Sertoli cells,and,possibly,selective depletion of meiotic and postmeiotic abnormal germ cells.We investigated the demographic and biological correlates of the pro-apoptotic marker Fas and the anti-apoptotic marker Bcl-xL in sperm cells of fertile men.Six hundred and four men from Greenland,Poland and Ukraine were consecutively enrolled during their pregnant wife's antenatal visits.Semen analysis was performed as recommended by the World Health Organization.Immunofluorescence coupled to flow cytometry was utilized for detection of apoptotic markers in the sperm cell.DNA damage was assessed by flow cytometry using both the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay.The percentage of Fas-positive sperm cells was higher in men with high total sperm count (P<O.01),more motile sperms (P=O.04) and fewer sperm head defects (P=O.05).These associations were consistent within and across study regions.Furthermore,testosterone,follicle-stimulating hormone (FSH) and sexual hormone-binding globulin (SHBG) were significantly negatively correlated with Fas within and across regions as well.The data indicated no association between the anti-apoptotic Bcl-xL marker and semen or personal characteristics.The finding of Fas-positive sperm cells associated with better semen quality in a cohort of spouses of pregnant women seems different from previous data obtained in infertile men and warrants further investigation to clarifv the biological significance of sperm apoptotic markers.

  3. Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures.

    Science.gov (United States)

    Lepsch, Lucilia B; Planeta, Cleopatra S; Scavone, Critoforo

    2015-01-01

    To study cocaine's toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2) and/or neuronal nucleus protein (NeuN) staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mechanism, confirmed by TUNEL assay. Therefore, the present paper shows that cocaine causes apoptotic cell death and inhibition of the neurite prolongation in striatal and mesencephalic cell culture. These data suggest that if similar neuronal damage could be produced in the developing human brain, it could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following prenatal exposure to cocaine.

  4. Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures

    Directory of Open Access Journals (Sweden)

    Lucilia B. Lepsch

    2015-01-01

    Full Text Available To study cocaine’s toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2 and/or neuronal nucleus protein (NeuN staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mechanism, confirmed by TUNEL assay. Therefore, the present paper shows that cocaine causes apoptotic cell death and inhibition of the neurite prolongation in striatal and mesencephalic cell culture. These data suggest that if similar neuronal damage could be produced in the developing human brain, it could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following prenatal exposure to cocaine.

  5. Proinflammatory cytokines activate the intrinsic apoptotic pathway in beta-cells

    DEFF Research Database (Denmark)

    Grunnet, Lars G; Aikin, Reid; Tonnesen, Morten F;

    2009-01-01

    of the intrinsic apoptotic pathway and the role of the two proapoptotic Bcl-2 proteins, Bad and Bax, were examined in beta-cells. RESEARCH DESIGN AND METHODS: Human and rat islets and INS-1 cells were exposed to a combination of proinflammatory cytokines (interleukin-1beta, interferon-gamma, and/or tumor necrosis......OBJECTIVE: Proinflammatory cytokines are cytotoxic to beta-cells and have been implicated in the pathogenesis of type 1 diabetes and islet graft failure. The importance of the intrinsic mitochondrial apoptotic pathway in cytokine-induced beta-cell death is unclear. Here, cytokine activation...... to investigate the role of Bad and Bax activation, respectively. RESULTS: We found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser136, mitochondrial stress, cytochrome c release, activation of caspase-9 and -3, and DNA fragmentation. Inhibition of Bad Ser136...

  6. Effects of combined prenatal stress and toluene exposure on apoptotic neurodegeneration in cerebellum and hippocampus of rats

    DEFF Research Database (Denmark)

    Ladefoged, Ole; Hougaard, Karin Sørig; Hass, Ulla

    2004-01-01

    the offspring for developmental neurotoxicity and level of apoptosis in the brain. The number of apoptotic cells in cerebellum postnatal day 22, 24, and 27 and in hippocampus (postnatal day 22, 24, and 27) were counted after visualization by the TUNEL staining or measured by DNA-laddering technique. Caspase-3...... activity was determined in cerebellum (postnatal day 6, 22, 24, and 27) and in hippocampus (postnatal day 6 and 22). TUNEL staining and DNA-laddering technique showed a marked decrease in number of apoptotic cells from postnatal day 22 to 27 in both cerebellum and hippocampus. Apparently, a peak...... in the number of TUNEL positive cells was identified in cerebellum at postnatal day 22. There was no statistically significant influence of exposure except that DNA-laddering in cerebellum at postnatal day 27 was increased by toluene exposure. Caspase-3 activity decreased in cerebellum and hippocampus with age...

  7. NEDD4-mediated HSF1 degradation underlies α-synucleinopathy.

    Science.gov (United States)

    Kim, Eunhee; Wang, Bin; Sastry, Namratha; Masliah, Eliezer; Nelson, Peter T; Cai, Huaibin; Liao, Francesca-Fang

    2016-01-15

    Cellular protein homeostasis is achieved by a delicate network of molecular chaperones and various proteolytic processes such as ubiquitin-proteasome system (UPS) to avoid a build-up of misfolded protein aggregates. The latter is a common denominator of neurodegeneration. Neurons are found to be particularly vulnerable to toxic stress from aggregation-prone proteins such as α-synuclein. Induction of heat-shock proteins (HSPs), such as through activated heat shock transcription factor 1 (HSF1) via Hsp90 inhibition, is being investigated as a therapeutic option for proteinopathic diseases. HSF1 is a master stress-protective transcription factor which activates genes encoding protein chaperones (e.g. iHsp70) and anti-apoptotic proteins. However, whether and how HSF1 is dysregulated during neurodegeneration has not been studied. Here, we discover aberrant HSF1 degradation by aggregated α-synuclein (or α-synuclein-induced proteotoxic stress) in transfected neuroblastoma cells. HSF1 dysregulation via α-synuclein was confirmed by in vivo assessment of mouse and in situ studies of human specimens with α-synucleinopathy. We demonstrate that elevated NEDD4 is implicated as the responsible ubiquitin E3 ligase for HSF1 degradation through UPS. Furthermore, pharmacologically induced SIRT1-mediated deacetylation can attenuate aberrant NEDD4-mediated HSF1 degradation. Indeed, we define the acetylation status of the Lys 80 residue located in the DNA-binding domain of HSF1 as a critical factor in modulating HSF1 protein stability in addition to its previously identified role in the transcriptional activity. Together with the finding that preserving HSF1 can alleviate α-synuclein toxicity, this study strongly suggests that aberrant HSF1 degradation is a key neurodegenerative mechanism underlying α-synucleinopathy.

  8. Evaluation of anti-apoptotic activity of different dietary antioxidants in renal cell carcinoma against hydrogen peroxide

    Institute of Scientific and Technical Information of China (English)

    Garg Neeraj K; Mangal Sharad; Sahu Tejram; Mehta Abhinav; Vyas Suresh P; Tyagi Rajeev K

    2011-01-01

    Objective: To evaluate the anti-apoptotic and radical scavenging activities of dietary phenolics, namely ascorbic acid, -tocopherol acetate, citric acid, salicylic acid, and estimate H2O2-induced apoptosis in renal cell carcinoma cells. Methods: The intracellular antioxidant potency of antioxidants was investigated. H2O2-induced apoptosis in RCC-26 was assayed with the following parameters: cell viability (% apoptosis), nucleosomal damage and DNA fragmentation, bcl-2 levels and flow cytometery analysis (ROS production evaluation). Results: The anticancer properties of antioxidants such as ascorbic acid, - tocopherol acetate, citric acid, salicylic acid with perdurable responses were investigated. It was observed that these antioxidants had protective effect (anti-apoptotic activity) against hydrogen peroxide (H2O2) in renal cell carcinoma (RCC-26) cell line. Conclusions: This study reveals and proves the anticancer properties. However, in cancer cell lines anti-apoptotic activity can indirectly reflect the cancer promoter activity through radicals scavenging, and significantly protect nucleus and bcl-2.

  9. The regulation of apoptotic cell death

    Directory of Open Access Journals (Sweden)

    G.P. Amarante-Mendes

    1999-09-01

    Full Text Available Apoptosis is a fundamental biological phenomenon in which the death of a cell is genetically and biochemically regulated. Different molecules are involved in the regulation of the apoptotic process. Death receptors, coupled to distinct members of the caspases as well as other adapter molecules, are involved in the initiation of the stress signals (The Indictment. Members of the Bcl-2 family control at the mitochondrial level the decision between life and death (The Judgement. The effector caspases are responsible for all morphological and biochemical changes related to apoptosis including the "eat-me" signals perceived by phagocytes and neighboring cells (The Execution. Finally, apoptosis would have little biological significance without the recognition and removal of the dying cells (The Burial.

  10. The regulation of apoptotic cell death

    Directory of Open Access Journals (Sweden)

    Amarante-Mendes G.P.

    1999-01-01

    Full Text Available Apoptosis is a fundamental biological phenomenon in which the death of a cell is genetically and biochemically regulated. Different molecules are involved in the regulation of the apoptotic process. Death receptors, coupled to distinct members of the caspases as well as other adapter molecules, are involved in the initiation of the stress signals (The Indictment. Members of the Bcl-2 family control at the mitochondrial level the decision between life and death (The Judgement. The effector caspases are responsible for all morphological and biochemical changes related to apoptosis including the "eat-me" signals perceived by phagocytes and neighboring cells (The Execution. Finally, apoptosis would have little biological significance without the recognition and removal of the dying cells (The Burial.

  11. Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strains.

    Science.gov (United States)

    Sukhanova, Maria V; D'Herin, Claudine; Boiteux, Serge; Lavrik, Olga I

    2014-10-01

    To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [(32)P]-labeled photoreactive partial DNA duplexes containing a 3'-ss/ds-junction (3'-junction) or a 5'-ss/ds-junction (5'-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3'-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5'-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5'-junction. The results show that RPAp70 crosslinked to DNA with a 5'-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome.

  12. The flexibility of UV-inducible mutation in Deinococcus ficus as evidenced by the existence of the imuB-dnaE2 gene cassette and generation of superior feather degrading bacteria.

    Science.gov (United States)

    Zeng, You-Hong; Shen, Fo-Ting; Tan, Chen-Chung; Huang, Chieh-Chen; Young, Chiu-Chung

    2011-12-20

    The lexA-imuB-dnaE2 gene cassette contributing to the TLS (translesion synthesis) polymerase activity and can easily cause mutation after DNA damage in many bacteria. But it was previously thought that TLS polymerase activity was unlikely to exist in the radio-resistant genus Deinococcus. In our preliminary studies, the lexA-imuB-dnaE2 gene cassette was found in a newly isolated feather-degrading Deinococcus ficus. Here we have attempted to determine the imuB gene sequence from another Deinococcus species namely D. grandis, by using the newly designed primers. The destroying of either imuB or dnaE2 gene in D. ficus leads to the increase in UV sensitivity and decrease in UV-induced mutations, which demonstrated the existence of TLS polymerase activity in D. ficus. In the presence of lexA-imuB-dnaE2, it is possible to obtain mutants with various keratinolytic activities after UV exposure. The keratinolytic activity of mutant strain CC-ZG207 increased by approximately twofold during growth in liquid feather medium. In contrast, the mutant strain CC-ZG227 showed only half of the keratinolytic activity compared with the wild type strain. By utilizing SDS-PAGE and zymogram profile analysis, the change in the protease activity was observed. We have proposed that the superior mutants of D. ficus can be created under UV stress, which is mediated by the lexA-imuB-dnaE2 gene cassette.

  13. Innate recognition of apoptotic cells: novel apoptotic cell-associated molecular patterns revealed by crossreactivity of anti-LPS antibodies.

    Science.gov (United States)

    Tennant, I; Pound, J D; Marr, L A; Willems, J J L P; Petrova, S; Ford, C A; Paterson, M; Devitt, A; Gregory, C D

    2013-05-01

    Cells dying by apoptosis are normally cleared by phagocytes through mechanisms that can suppress inflammation and immunity. Molecules of the innate immune system, the pattern recognition receptors (PRRs), are able to interact not only with conserved structures on microbes (pathogen-associated molecular patterns, PAMPs) but also with ligands displayed by apoptotic cells. We reasoned that PRRs might therefore interact with structures on apoptotic cells - apoptotic cell-associated molecular patterns (ACAMPs) - that are analogous to PAMPs. Here we show that certain monoclonal antibodies raised against the prototypic PAMP, lipopolysaccharide (LPS), can crossreact with apoptotic cells. We demonstrate that one such antibody interacts with a constitutively expressed intracellular protein, laminin-binding protein, which translocates to the cell surface during apoptosis and can interact with cells expressing the prototypic PRR, mCD14 as well as with CD14-negative cells. Anti-LPS cross reactive epitopes on apoptotic cells colocalised with annexin V- and C1q-binding sites on vesicular regions of apoptotic cell surfaces and were released associated with apoptotic cell-derived microvesicles (MVs). These results confirm that apoptotic cells and microbes can interact with the immune system through common elements and suggest that anti-PAMP antibodies could be used strategically to characterise novel ACAMPs associated not only with apoptotic cells but also with derived MVs.

  14. Genotoxic and apoptotic effects of Goeckerman therapy for psoriasis

    Energy Technology Data Exchange (ETDEWEB)

    Borska, L.; Andrys, C.; Krejsek, J.; Hamakova, K.; Kremlacek, J.; Palicka, V.; Ranna, D.; Fiala, Z. [Charles University Prague, Prague (Czech Republic). Faculty of Medicine

    2010-03-15

    Goeckerman therapy (GT) for psoriasis is based on cutaneous application of crude coal tar (polycyclic aromatic hydrocarbons (PAH)) and exposure to ultraviolet radiation (UVR). PAH and UVR are mutagenic, carcinogenic and immunotoxic agents that promote apoptosis. We evaluated dermal absorption of PAH as well as the genotoxic and apoptotic effects of GT in 20 patients with psoriasis, by determining numbers of chromosomal abnormalities in peripheral lymphocytes, and levels of 1-hydroxypyrene (1-OHP), p53 protein and soluble FasL (sFasL) in urine and/or blood, before and after GT. Psoriasis Area and Severity Index (PASI) score was used to evaluate clinical efficacy of GT. Compared with pre-treatment levels, there was a significant increase in urine 1-OHP, indicating a high degree of dermal absorption of PAH (P <0.01). We also found a significant increase in the number of chromosomal abnormalities in peripheral blood lymphocytes (P <0.001), suggesting that GT is genotoxic; significantly increased p53 protein in plasma (P <0.05), an indicator of cell response to DNA damage; and significantly increased sFasL in serum (P <0.01), an indicator of apoptosis. The PASI score was significantly decreased after GT (P <0.001), confirming clinical benefit of this treatment. Our results demonstrate high dermal absorption of PAH during GT and provide evidence that GT promotes genotoxicity and apoptosis.

  15. Genes of the mitochondrial apoptotic pathway in Mytilus galloprovincialis.

    Directory of Open Access Journals (Sweden)

    Noelia Estévez-Calvar

    Full Text Available Bivalves play vital roles in marine, brackish, freshwater and terrestrial habitats. In recent years, these ecosystems have become affected through anthropogenic activities. The ecological success of marine bivalves is based on the ability to modify their physiological functions in response to environmental changes. One of the most important mechanisms involved in adaptive responses to environmental and biological stresses is apoptosis, which has been scarcely studied in mollusks, although the final consequence of this process, DNA fragmentation, has been frequently used for pollution monitoring. Environmental stressors induce apoptosis in molluscan cells via an intrinsic pathway. Many of the proteins involved in vertebrate apoptosis have been recognized in model invertebrates; however, this process might not be universally conserved. Mytilus galloprovincialis is presented here as a new model to study the linkage between molecular mechanisms that mediate apoptosis and marine bivalve ecological adaptations. Therefore, it is strictly necessary to identify the key elements involved in bivalve apoptosis. In the present study, six mitochondrial apoptotic-related genes were characterized, and their gene expression profiles following UV irradiation were evaluated. This is the first step for the development of potential biomarkers to assess the biological responses of marine organisms to stress. The results confirmed that apoptosis and, more specifically, the expression of the genes involved in this process can be used to assess the biological responses of marine organisms to stress.

  16. Apoptotic pathway induced by noscapine in human myelogenous leukemic cells.

    Science.gov (United States)

    Heidari, Nastaran; Goliaei, Bahram; Moghaddam, Parvaneh Rahimi; Rahbar-Roshandel, Nahid; Mahmoudian, Massoud

    2007-11-01

    It has been shown that noscapine, an opium-derived phthalideisoquinoline alkaloid that is currently being used as an oral antitussive drug, induces apoptosis in myeloid leukemia cells. The molecular mechanism responsible for the anticancer effects of noscapine is poorly understood. In the current study, the apoptotic effects of noscapine on two myeloid cell lines, apoptosis-proficient HL60 cells and apoptosis-resistant K562 cells, were analyzed. An increase in the activity of caspase-2, -3, -6, -8 and -9, poly(ADP ribose) polymerase cleavage, detection of phosphatidylserine on the outer layer of the cell membrane, nucleation of chromatin, and DNA fragmentation suggested the induction of apoptosis. Noscapine increased the Bax/Bcl-2 ratio with a significant decrease of Bcl-2 expression accompanied with Bcl-2 phosphorylation. Using an inhibitory approach, the activation of the caspase cascade involved in the noscapine-induced apoptosis was analyzed. We observed no inhibitory effect of the caspase-8 inhibitor on caspase-9 activity. In view of these results and taking into consideration that K562 cells are Fas-null, we suggested that caspase-8 is activated in a Fas-independent manner downstream of caspase-9. In conclusion, noscapine can induce apoptosis in both apoptosis-proficient and apoptosis-resistant leukemic cells, and it can be a novel candidate in the treatment of hematological malignancies.

  17. Thyroid hormone regulation of apoptotic tissue remodeling during anuran metamorphosis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Anuran metamorphosis involves systematic transformations of individual organs in a thyroid hormone (TH)-dependent manner. Morphological and cellular studies have shown that the removal of larval or gans/tissues such the tail and the tadpole intestinal epithelium is through programmed cell death or apop tosis. Recent molecular investigations suggest that TH regulates metamorphosis by regulating target gene expression through thyroid hormone receptors (TRs), which are DNA-binding transcription factors. Cloning and characterization of TH response genes show that diverse groups of early response genes are induced by TH. The products of these TH response genes are believed to directly or indirectly affect the expression and/or functions of cell death genes, which are conserved at both sequence and function levels in different animal species. A major challenge for future research lies at determining the signaling pathways leading to the activation of apoptotic processes and whether different death genes are involved in the regulation of apoptosis in different tissues/organs to effect tissue-specific transformations.

  18. Modulating effect of SIRT1 activation induced by resveratrol on Foxo1-associated apoptotic signalling in senescent heart.

    Science.gov (United States)

    Sin, Thomas K; Yu, Angus P; Yung, Benjamin Y; Yip, Shea Ping; Chan, Lawrence W; Wong, Cesar S; Ying, Michael; Rudd, John A; Siu, Parco M

    2014-06-15

    Elevations of cardiomyocyte apoptosis and fibrotic deposition are major characteristics of the ageing heart. Resveratrol, a polyphenol in grapes and red wine, is known to improve insulin resistance and increase mitochondrial biogenesis through the SIRT1-PGC-1α signalling axis. Recent studies attempted to relate SIRT1 activation by resveratrol to the regulation of apoptosis in various disease models of cardiac muscle. In the present study, we tested the hypothesis that long-term (8-month) treatment of resveratrol would activate SIRT1 and improve the cardiac function of senescent mice through suppression of Foxo1-associated pro-apoptotic signalling. Our echocardiographic measurements indicated that the cardiac systolic function measured as fractional shortening and ejection fraction was significantly reduced in aged mice when compared with the young mice. These reductions, however, were not observed in resveratrol-treated hearts. Ageing significantly reduced the deacetylase activity, but not the protein abundance of SIRT1 in the heart. This reduction was accompanied by increased acetylation of the Foxo1 transcription factor and transactivation of its target, pro-apoptotic Bim. Subsequent analyses indicated that pro-apoptotic signalling measured as p53, Bax and apoptotic DNA fragmentation was up-regulated in the heart of aged mice. In contrast, resveratrol restored SIRT1 activity and suppressed elevations of Foxo1 acetylation, Bim and pro-apoptotic signalling in the aged heart. In parallel, resveratrol also attenuated the ageing-induced elevations of fibrotic collagen deposition and markers of oxidative damage including 4HNE and nitrotyrosine. In conclusion, these novel data demonstrate that resveratrol mitigates pro-apoptotic signalling in senescent heart through a deacetylation mechanism of SIRT1 that represses the Foxo1-Bim-associated pro-apoptotic signalling axis.

  19. Poncirin Induces Apoptosis in AGS Human Gastric Cancer Cells through Extrinsic Apoptotic Pathway by up-Regulation of Fas Ligand.

    Science.gov (United States)

    Saralamma, Venu Venkatarame Gowda; Nagappan, Arulkumar; Hong, Gyeong Eun; Lee, Ho Jeong; Yumnam, Silvia; Raha, Suchismita; Heo, Jeong Doo; Lee, Sang Joon; Lee, Won Sup; Kim, Eun Hee; Kim, Gon Sup

    2015-09-18

    Poncirin, a natural bitter flavanone glycoside abundantly present in many species of citrus fruits, has various biological benefits such as anti-oxidant, anti-microbial, anti-inflammatory and anti-cancer activities. The anti-cancer mechanism of Poncirin remains elusive to date. In this study, we investigated the anti-cancer effects of Poncirin in AGS human gastric cancer cells (gastric adenocarcinoma). The results revealed that Poncirin could inhibit the proliferation of AGS cells in a dose-dependent manner. It was observed Poncirin induced accumulation of sub-G1 DNA content, apoptotic cell population, apoptotic bodies, chromatin condensation, and DNA fragmentation in a dose-dependent manner in AGS cells. The expression of Fas Ligand (FasL) protein was up-regulated dose dependently in Poncirin-treated AGS cells Moreover, Poncirin in AGS cells induced activation of Caspase-8 and -3, and subsequent cleavage of poly(ADP-ribose) polymerase (PARP). Inhibitor studies' results confirm that the induction of caspase-dependent apoptotic cell death in Poncirin-treated AGS cells was led by the Fas death receptor. Interestingly, Poncirin did not show any effect on mitochondrial membrane potential (ΔΨm), pro-apoptotic proteins (Bax and Bak) and anti-apoptotic protein (Bcl-xL) in AGS-treated cells followed by no activation in the mitochondrial apoptotic protein caspase-9. This result suggests that the mitochondrial-mediated pathway is not involved in Poncirin-induced cell death in gastric cancer. These findings suggest that Poncirin has a potential anti-cancer effect via extrinsic pathway-mediated apoptosis, possibly making it a strong therapeutic agent for human gastric cancer.

  20. Poncirin Induces Apoptosis in AGS Human Gastric Cancer Cells through Extrinsic Apoptotic Pathway by up-Regulation of Fas Ligand

    Directory of Open Access Journals (Sweden)

    Venu Venkatarame Gowda Saralamma

    2015-09-01

    Full Text Available Poncirin, a natural bitter flavanone glycoside abundantly present in many species of citrus fruits, has various biological benefits such as anti-oxidant, anti-microbial, anti-inflammatory and anti-cancer activities. The anti-cancer mechanism of Poncirin remains elusive to date. In this study, we investigated the anti-cancer effects of Poncirin in AGS human gastric cancer cells (gastric adenocarcinoma. The results revealed that Poncirin could inhibit the proliferation of AGS cells in a dose-dependent manner. It was observed Poncirin induced accumulation of sub-G1 DNA content, apoptotic cell population, apoptotic bodies, chromatin condensation, and DNA fragmentation in a dose-dependent manner in AGS cells. The expression of Fas Ligand (FasL protein was up-regulated dose dependently in Poncirin-treated AGS cells Moreover, Poncirin in AGS cells induced activation of Caspase-8 and -3, and subsequent cleavage of poly(ADP-ribose polymerase (PARP. Inhibitor studies’ results confirm that the induction of caspase-dependent apoptotic cell death in Poncirin-treated AGS cells was led by the Fas death receptor. Interestingly, Poncirin did not show any effect on mitochondrial membrane potential (ΔΨm, pro-apoptotic proteins (Bax and Bak and anti-apoptotic protein (Bcl-xL in AGS-treated cells followed by no activation in the mitochondrial apoptotic protein caspase-9. This result suggests that the mitochondrial-mediated pathway is not involved in Poncirin-induced cell death in gastric cancer. These findings suggest that Poncirin has a potential anti-cancer effect via extrinsic pathway-mediated apoptosis, possibly making it a strong therapeutic agent for human gastric cancer.

  1. An isothermal primer extension method for whole genome amplification of fresh and degraded DNA: applications in comparative genomic hybridization, genotyping and mutation screening.

    Science.gov (United States)

    Lee, Cheryl I P; Leong, Siew Hong; Png, Adrian E H; Choo, Keng Wah; Syn, Christopher; Lim, Dennis T H; Law, Hai Yang; Kon, Oi Lian

    2006-01-01

    We describe a protocol that uses a bioinformatically optimized primer in an isothermal whole genome amplification (WGA) reaction. Overnight incubation at 37 degrees C efficiently generates several hundred- to several thousand-fold increases in input DNA. The amplified product retains reasonably faithful quantitative representation of unamplified whole genomic DNA (gDNA). We provide protocols for applying this isothermal primer extension WGA protocol in three different techniques of genomic analysis: comparative genomic hybridization (CGH), genotyping at simple tandem repeat (STR) loci and screening for single base mutations in a common monogenic disorder, beta-thalassemia. gDNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues can also be amplified with this protocol.

  2. Apoptosis and DNA Methylation

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Huan X.; Hackett, James A. [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Nestor, Colm [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Dunican, Donncha S.; Madej, Monika; Reddington, James P. [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Pennings, Sari [Queen' s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ (United Kingdom); Harrison, David J. [Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Meehan, Richard R., E-mail: Richard.Meehan@hgu.mrc.ac.uk [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom)

    2011-04-01

    Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG.

  3. Apoptosis and DNA Methylation

    Directory of Open Access Journals (Sweden)

    Richard R. Meehan

    2011-04-01

    Full Text Available Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG.

  4. Cytotoxic, genotoxic and apoptotic effects of naringenin-oxime relative to naringenin on normal and cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Abdurrahim Kocyigit; Ismail Koyuncu; Murat Dikilitas; Fatemeh Bahadori; Baki Turkkan

    2016-01-01

    Objective: To assess and compare the cytotoxic, genotoxic, apoptotic and reactive oxygen species (ROS) generating effects of naringenin (NG) and its new derived compound naringenin-oxime (NG-Ox) on MCF-7, HT-29, PC-12 cancer and L-929 normal cell lines. Methods: The cells were incubated with different doses of NG-Ox and NG (50–1 000 mmol/L) for 24 h. The cell viability was assessed based on ATP cell viability assay. Intracellular accumulation of ROS was determined using the fluorescent probes 2070-dichlorodihydrofluorescin diacetate. Genotoxic effects were evaluated by alkaline single cell gel electrophoresis assay (comet assay) and, the apoptotic effect was evaluated by acridine orange staining at below the IC50 levels. Results: Both NG-Ox and NG exhibited cytotoxic, genotoxic and apoptotic effects and resulted in increased ROS values in a dose-dependent manner. The effects were more pronounced on cancer cell lines. The cytotoxic, genotoxic and apoptotic effects of NG-Ox were higher than that of NG in all cell lines. Significant correlations were observed be-tween cell viability, DNA damage, apoptosis and ROS, in all cell lines exposed to either NG-Ox or NG. Conclusions: This study showed that both NG-Ox and NG possess cytotoxic, genotoxic and apoptotic activities through the production of ROS on cells, NG-Ox being the more effective one. Therefore, derived compound of NG might be used as antiproliferative agents for the treatment of cancer.

  5. BAG1: the guardian of anti-apoptotic proteins in acute myeloid leukemia.

    Science.gov (United States)

    Aveic, Sanja; Pigazzi, Martina; Basso, Giuseppe

    2011-01-01

    BCL2 associated Athano-Gene 1 (BAG1) is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance.

  6. BAG1: the guardian of anti-apoptotic proteins in acute myeloid leukemia.

    Directory of Open Access Journals (Sweden)

    Sanja Aveic

    Full Text Available BCL2 associated Athano-Gene 1 (BAG1 is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance.

  7. Vascular endothelial growth factor enhances macrophage clearance of apoptotic cells

    Science.gov (United States)

    Dalal, Samay; Horstmann, Sarah A.; Richens, Tiffany R.; Tanaka, Takeshi; Doe, Jenna M.; Boe, Darren M.; Voelkel, Norbert F.; Taraseviciene-Stewart, Laimute; Janssen, William J.; Lee, Chun G.; Elias, Jack A.; Bratton, Donna; Tuder, Rubin M.; Henson, Peter M.; Vandivier, R. William

    2012-01-01

    Efficient clearance of apoptotic cells from the lung by alveolar macrophages is important for the maintenance of tissue structure and function. Lung tissue from humans with emphysema contains increased numbers of apoptotic cells and decreased levels of vascular endothelial growth factor (VEGF). Mice treated with VEGF receptor inhibitors have increased numbers of apoptotic cells and develop emphysema. We hypothesized that VEGF regulates apoptotic cell clearance by alveolar macrophages (AM) via its interaction with VEGF receptor 1 (VEGF R1). Our data show that the uptake of apoptotic cells by murine AMs and human monocyte-derived macrophages is inhibited by depletion of VEGF and that VEGF activates Rac1. Antibody blockade or pharmacological inhibition of VEGF R1 activity also decreased apoptotic cell uptake ex vivo. Conversely, overexpression of VEGF significantly enhanced apoptotic cell uptake by AMs in vivo. These results indicate that VEGF serves a positive regulatory role via its interaction with VEGF R1 to activate Rac1 and enhance AM apoptotic cell clearance. PMID:22307908

  8. The role of macrophages in the removal of apoptotic B-cells in the sheep ileal Peyer's patch.

    Science.gov (United States)

    Bhogal, Hardeep S; Kennedy, Laurie J; Babic, Kelly; Reynolds, John D

    2004-06-01

    In the process of generating the cells that populate the sheep's B-cell pool, the ileal Peyer's patch (PP) produces an immense number of B-cells and then destroys most of them by apoptosis. Rapid clearance of these apoptotic cells is essential for tissue homeostasis and for preventing pathology. Macrophages comprise a small percentage of cells in the follicles. They resemble macrophages found in other tissues and can be identified by the expression of MHC Class II and CD14. In this study, enriched macrophages co-cultured with apoptotic ileal PP cells showed increased DNA content as they ingested apoptotic cells. The higher the proportion of apoptotic cells in culture the greater the increase in DNA content of the macrophages. This occurred when B-cell apoptosis was initiated by a period in culture or in response to treating the animals with steroids. Thus, macrophages resident in the ileal PP follicle mediate the phagocytosis and removal of discarded B-cells.

  9. Transcellular degradation of axonal mitochondria.

    Science.gov (United States)

    Davis, Chung-ha O; Kim, Keun-Young; Bushong, Eric A; Mills, Elizabeth A; Boassa, Daniela; Shih, Tiffany; Kinebuchi, Mira; Phan, Sebastien; Zhou, Yi; Bihlmeyer, Nathan A; Nguyen, Judy V; Jin, Yunju; Ellisman, Mark H; Marsh-Armstrong, Nicholas

    2014-07-01

    It is generally accepted that healthy cells degrade their own mitochondria. Here, we report that retinal ganglion cell axons of WT mice shed mitochondria at the optic nerve head (ONH), and that these mitochondria are internalized and degraded by adjacent astrocytes. EM demonstrates that mitochondria are shed through formation of large protrusions that originate from otherwise healthy axons. A virally introduced tandem fluorophore protein reporter of acidified mitochondria reveals that acidified axonal mitochondria originating from the retinal ganglion cell are associated with lysosomes within columns of astrocytes in the ONH. According to this reporter, a greater proportion of retinal ganglion cell mitochondria are degraded at the ONH than in the ganglion cell soma. Consistently, analyses of degrading DNA reveal extensive mtDNA degradation within the optic nerve astrocytes, some of which comes from retinal ganglion cell axons. Together, these results demonstrate that surprisingly large proportions of retinal ganglion cell axonal mitochondria are normally degraded by the astrocytes of the ONH. This transcellular degradation of mitochondria, or transmitophagy, likely occurs elsewhere in the CNS, because structurally similar accumulations of degrading mitochondria are also found along neurites in superficial layers of the cerebral cortex. Thus, the general assumption that neurons or other cells necessarily degrade their own mitochondria should be reconsidered.

  10. Electroanalytical studies of sulfentrazone in protic medium, its degradation by the electro-Fenton process, and toxicity assessment using ss-DNA.

    Science.gov (United States)

    de A Lima, Augusto C; dos S Melo, Acácia M; Pires, Edjane Vieira; dos Santos Ferreira, Roberta Costa; Sant'Ana, Antônio E G; Goulart, Marília O F; de Abreu, Fabiane C

    2010-11-01

    Sulfentrazone is an herbicide used as a pre-plant incorporated or pre-emergence treatment. The electrochemical oxidation of sulfentrazone was studied, by cyclic, differential and square-wave voltammetry on unmodified and on glassy carbon nanotube-modified electrodes, and by controlled-potential coulometry and electrolysis. The voltammograms of sulfentrazone showed a main irreversible diffusion-controlled pH-dependent oxidation peak. The in situ DNA-damaging capacity of sulfentrazone was also investigated, employing double stranded ds-DNA-modified glassy carbon electrode, without evidence of interaction. On the other hand, in a solution of sulfentrazone and single stranded ss-DNA, the oxidation signals of the respective bases decreased concentration-dependently, indicating binding of sulfentrazone to guanine and adenine. The electro-Fenton method was employed to promote decontamination by eliminating the herbicide, resulting in almost 60% of mineralization.

  11. Inhibition of mitochondria responsible for the anti-apoptotic effects of melatonin during ischemia-reperfusion

    Institute of Scientific and Technical Information of China (English)

    HAN Yi-xiang; ZHANG Sheng-hui; WANG Xi-ming; WU Jian-bo

    2006-01-01

    Objective: To investigate a possible mechanism responsible for anti-apoptotic effects of melatonin and provide theoretical evidences for clinical therapy. Methods: Ischemia-reperfusion mediated neuronal cell injury model was constructed in cerebellar granule neurons (CGNs) by deprivation of glucose, serum and oxygen in media. After ischemia, melatonin was added to the test groups to reach differential concentration during reperfusion. DNA fragmentation, mitochondrial transmembrane potential,mitochondrial cytochrome c release and caspase-3 activity were observed after subjecting cerebellar granule neurons to oxygen-glucose deprivation (OGD). Results: The results showed that OGD induced typical cell apoptosis change, DNA ladder and apoptosis-related alterations in mitochondrial functions including depression of mitochondrial transmembrane potential (its maximal protection ratio was 73.26%) and release of cytochrome c (its maximal inhibition ratio was 42.52%) and the subsequent activation of caspase-3 (its maximal protection ratio was 59.32%) in cytoplasm. Melatonin reduced DNA damage and inhibited release of mitochondrial cytochrome c and activation of caspase-3. Melatonin can strongly prevent the OGD-induced loss of the mitochondria membrane potential. Conclusion: Our findings suggested that the direct inhibition of mitochondrial pathway might essentially contribute to its anti-apoptotic effects in neuronal ischemia-reperfusion.

  12. Etoposide Induces Mitochondria-Associated Apoptotic Cell Death in Human Gastric Carcinoma Cells

    Institute of Scientific and Technical Information of China (English)

    LI Jing-hua; CHEN Yue; WANG Jia-si; KONG Wei; JIN Ying-hua

    2008-01-01

    Recent observations indicate that the resistance of apoptosis is an important process of tumor metastasis and metastases are the cause of 90% of human cancer death.Etoposide,a semisynthetic derivative of the podophyllotoxins,is a clinically used anti-cancer reagent,but the effects of it on metastatic gastric carcinoma cells are totally unknown.In this study,etoposide induced apoptotic cell death in human gastric adenocareinoma cell line SGC-7901,derived from metastatic lymph nodes,as evidenced by the analysis of DNA fragmentation,apoptotic body formation,caspase activation,and apoptosis specific changes in cell morphology is demonstrated.The depolarization of mitochondrial membrane and the release of cytochrome c were most early events in etoposide treated SGC-7901 cells,and were followed by caspase-3 activation and PARP cleavage.Caspase-8 activation was not detected under the same condition.Thus,it was proposed that etoposide induces caspase-associated apoptotic cell death in human metastatic gastric carcinoma,which is initiated by mitochondrial cytochrome c release.

  13. p53 dependent apoptotic cell death induces embryonic malformation in Carassius auratus under chronic hypoxia.

    Directory of Open Access Journals (Sweden)

    Paramita Banerjee Sawant

    Full Text Available Hypoxia is a global phenomenon affecting recruitment as well as the embryonic development of aquatic fauna. The present study depicts hypoxia induced disruption of the intrinsic pathway of programmed cell death (PCD, leading to embryonic malformation in the goldfish, Carrasius auratus. Constant hypoxia induced the early expression of pro-apoptotic/tumor suppressor p53 and concomitant expression of the cell death molecule, caspase-3, leading to high level of DNA damage and cell death in hypoxic embryos, as compared to normoxic ones. As a result, the former showed delayed 4 and 64 celled stages and a delay in appearance of epiboly stage. Expression of p53 efficiently switched off expression of the anti-apoptotic Bcl-2 during the initial 12 hours post fertilization (hpf and caused embryonic cell death. However, after 12 hours, simultaneous downregulation of p53 and Caspase-3 and exponential increase of Bcl-2, caused uncontrolled cell proliferation and prevented essential programmed cell death (PCD, ultimately resulting in significant (p<0.05 embryonic malformation up to 144 hpf. Evidences suggest that uncontrolled cell proliferation after 12 hpf may have been due to downregulation of p53 abundance, which in turn has an influence on upregulation of anti-apoptotic Bcl-2. Therefore, we have been able to show for the first time and propose that hypoxia induced downregulation of p53 beyond 12 hpf, disrupts PCD and leads to failure in normal differentiation, causing malformation in gold fish embryos.

  14. p53 Dependent Apoptotic Cell Death Induces Embryonic Malformation in Carassius auratus under Chronic Hypoxia

    Science.gov (United States)

    Dasgupta, Subrata; Sawant, Bhawesh T.; Chadha, Narinder K.; Pal, Asim K.

    2014-01-01

    Hypoxia is a global phenomenon affecting recruitment as well as the embryonic development of aquatic fauna. The present study depicts hypoxia induced disruption of the intrinsic pathway of programmed cell death (PCD), leading to embryonic malformation in the goldfish, Carrasius auratus. Constant hypoxia induced the early expression of pro-apoptotic/tumor suppressor p53 and concomitant expression of the cell death molecule, caspase-3, leading to high level of DNA damage and cell death in hypoxic embryos, as compared to normoxic ones. As a result, the former showed delayed 4 and 64 celled stages and a delay in appearance of epiboly stage. Expression of p53 efficiently switched off expression of the anti-apoptotic Bcl-2 during the initial 12 hours post fertilization (hpf) and caused embryonic cell death. However, after 12 hours, simultaneous downregulation of p53 and Caspase-3 and exponential increase of Bcl-2, caused uncontrolled cell proliferation and prevented essential programmed cell death (PCD), ultimately resulting in significant (p<0.05) embryonic malformation up to 144 hpf. Evidences suggest that uncontrolled cell proliferation after 12 hpf may have been due to downregulation of p53 abundance, which in turn has an influence on upregulation of anti-apoptotic Bcl-2. Therefore, we have been able to show for the first time and propose that hypoxia induced downregulation of p53 beyond 12 hpf, disrupts PCD and leads to failure in normal differentiation, causing malformation in gold fish embryos. PMID:25068954

  15. Tomato lycopene attenuates myocardial infarction induced by isoproterenol:Electrocardiographic, biochemical and anti-apoptotic study

    Institute of Scientific and Technical Information of China (English)

    Aman Upaganlawar; Vaibhav Patel; Balaraman R

    2012-01-01

    Objective: To assess the protective effects of lycopene on electrocardiographic, hemodynamic, biochemical and apoptotic changes in isoproterenol induced myocardial infarction. Methods:Myocardial infarction was induced in rats by subcutaneous injection of isoproterenol (200 mg/kg) for two consecutive days at an interval of 24 h. Rats were treated with lycopene (10 mg/kg/day,p.o. ) for a period of 30 days and isoproterenol (ISO) was injected on the 29th and 30th day. At the end of experiment i.e. on the 31st day electrocardiographic, hemodynamic, biochemical and apoptotic changes were monitored from control and experimental groups. Results: ISO injected rats showed a significant alteration in electrocardiograph pattern and hemodynamic changes (i.e. systolic, diastolic and mean arterial pressure). It also showed significant increase in C-reactive protein, myeloperoxidase, nitrite levels and Caspase-3 protease activity. In addition, it also exhibited alteration in the levels of electrolytes (Na+, K+ and Ca2+), vitamin E, uric acid and serum protein. Gel electrophoresis of ISO injected rats showed increase in DNA fragmentation. Triphenyl tetrazolium chloride staining of the heart section shows increase area of infarction in ISO injected rats. Pre-co-treatment with lycopene significantly prevented the ISO induced alteration in ECG, haemodynamic, biochemical and apoptotic changes. Conclusions: The present result shows that treatment of lycopene in ISO injected rats significantly attenuates induced myocardial infarction.

  16. SATB1 Mediates Long-Range Chromatin Interactions: A Dual Regulator of Anti-Apoptotic BCL2 and Pro-Apoptotic NOXA Genes.

    Science.gov (United States)

    Yang, Yin; Wang, Zongdan; Sun, Luan; Shao, Lipei; Yang, Nan; Yu, Dawei; Zhang, Xin; Han, Xiao; Sun, Yujie

    2015-01-01

    Aberrant expression of special AT-rich binding protein 1 (SATB1), a global genomic organizer, has been associated with various cancers, which raises the question of how higher-order chromatin structure contributes to carcinogenesis. Disruption of apoptosis is one of the hallmarks of cancer. We previously demonstrated that SATB1 mediated specific long-range chromosomal interactions between the mbr enhancer located within 3'-UTR of the BCL2 gene and the promoter to regulate BCL2 expression during early apoptosis. In the present study, we used chromosome conformation capture (3C) assays and molecular analyses to further investigate the function of the SATB1-mediated higher-order chromatin structure in co-regulation of the anti-apoptotic BCL2 gene and the pro-apoptotic NOXA gene located 3.4Mb downstream on Chromosome 18. We demonstrated that the mbr enhancer spatially juxtaposed the promoters of BCL2 and NOXA genes through SATB1-mediated chromatin-loop in Jurkat cells. Decreased SATB1 levels switched the mbr-BCL2 loop to mbr-NOXA loop, and thus changed expression of these two genes. The SATB1-mediated dynamic switch of the chromatin loop structures was essential for the cooperative expression of the BCL2 and NOXA genes in apoptosis. Notably, the role of SATB1 was specific, since inhibition of SATB1 degradation by caspase-6 inhibitor or caspase-6-resistant SATB1 mutant reversed expression of BCL-2 and NOXA in response to apoptotic stimulation. This study reveals the critical role of SATB1-organized higher-order chromatin structure in regulating the dynamic equilibrium of apoptosis-controlling genes with antagonistic functions and suggests that aberrant SATB1 expression might contribute to cancer development by disrupting the co-regulated genes in apoptosis pathways.

  17. Endoplasmic Reticulum Stress Induces the Early Appearance of Pro-apoptotic and Anti-apoptotic Proteins in Neurons of Five Familial Alzheimer's Disease Mice

    Institute of Scientific and Technical Information of China (English)

    Hui Shen; Xiao-Dong Pan; Jing Zhang; Yu-Qi Zeng; Meng Zhou; Lu-Meng Yang; Bing Ye

    2016-01-01

    Background:Amyloid β (Aβ) deposits and the endoplasmic reticulum stress (ERS) are both well established in the development and progression ofAlzheimer's disease (AD).However,the mechanism and role of Aβ-induced ERS in AD-associated pathological progression remain to be elucidated.Methods:The five familial AD (5 ×FAD) mice and wild-type (WT) mice aged 2,7,and 12 months were used in the present study.Morris water maze test was used to evaluate their cognitive performance.Immunofluorescence and Western blot analyses were used to examine the dynamic changes of pro-apoptotic (CCAAT/enhancer-binding protein homologous protein [CHOP] and cleaved caspase-12) and anti-apoptotic factors (chaperone glucose-regulated protein [GRP] 78 and endoplasmic reticulum-associated protein degradation-associated ubiquitin ligase synovial apoptosis inhibitor 1 [SYVN1]) in the ERS-associated unfolded protein response (UPR) pathway.Results:Compared with age-matched WT mice,5 ×FAD mice showed higher cleaved caspase-3,lower neuron-positive staining at the age of 12 months,but earlier cognitive deficit at the age of 7 months (all P < 0.05).Interestingly,for 2-month-old 5×FAD mice,the related proteins involved in the ERS-associated UPR pathway,including CHOP,cleaved caspase-12,GRP 78,and SYVN1,were significantly increased when compared with those in age-matched WT mice (all P < 0.05).Moreover,ERS occurred mainly in neurons,not in astrocytes.Conclusions:These findings suggest that compared with those of age-matched WT mice,ERS-associated pro-apoptotic and anti-apoptoticproteins are upregulated in 2-month-old 5×FAD mice,consistent with intracellular Aβ aggregation in neurons.

  18. Nitric oxide induces cell death by regulating anti-apoptotic BCL-2 family members.

    Directory of Open Access Journals (Sweden)

    Colleen M Snyder

    Full Text Available Nitric oxide (NO activates the intrinsic apoptotic pathway to induce cell death. However, the mechanism by which this pathway is activated in cells exposed to NO is not known. Here we report that BAX and BAK are activated by NO and that cytochrome c is released from the mitochondria. Cells deficient in Bax and Bak or Caspase-9 are completely protected from NO-induced cell death. The individual loss of the BH3-only proteins, Bim, Bid, Puma, Bad or Noxa, or Bid knockdown in Bim(-/-/Puma(-/- MEFs, does not prevent NO-induced cell death. Our data show that the anti-apoptotic protein MCL-1 undergoes ASK1-JNK1 mediated degradation upon exposure to NO, and that cells deficient in either Ask1 or Jnk1 are protected against NO-induced cell death. NO can inhibit the mitochondrial electron transport chain resulting in an increase in superoxide generation and peroxynitrite formation. However, scavengers of ROS or peroxynitrite do not prevent NO-induced cell death. Collectively, these data indicate that NO degrades MCL-1 through the ASK1-JNK1 axis to induce BAX/BAK-dependent cell death.

  19. Degradation of fungal DNA in formalin-fixed paraffin-embedded sinus fungal balls hampers reliable sequence-based identification of fungi.

    Science.gov (United States)

    Cabaret, Odile; Toussain, Guillaume; Abermil, Nassera; Alsamad, Issam Abd; Botterel, Françoise; Costa, Jean-Marc; Papon, Jean-François; Bretagne, Stéphane

    2011-04-01

    Identification of the etiologic agent responsible for sinus fungal ball (SFB) is rarely obtained due to either the culture of patient specimens not being ordered or if cultures were inoculated they proved to be negative. Obviously, this has a significant impact on the design of appropriate therapeutic strategies. We investigated whether paraffin-embedded (PE) tissues, the only materials often available, were suitable for the correct identification of the responsible fungi. We obtained PE tissues of SFB from 16 different patients who had risk factors for invasive fungal infections. DNA was extracted using an automated extractor and the internal transcribed spacer (ITS) sequenced following amplification with two sets of primers designed to amplify >300 bp fragments. This was attempted in parallel with a real-time quantitative PCR assay targeting Aspergillus spp. mitochondrial DNA designed to amplify <150 bp fragments. ITS sequencing succeeded in appropriately identifying the etiologic agents in 10 of the 16 samples (nine Aspergillus fumigatus, one Lewia spp.). In contrast, the <150 bp PCR assay amplified all specimens correctly except the one involving Lewia spp. If fungal identification is warranted to understand the pathophysiology of SFB and guide clinicians, we cannot rely only on ITS sequencing of the DNA obtained from PE tissues. The main reason is probably due to the fact that formalin prevents amplification of long DNA fragments and consequently, frozen or fresh tissues should be employed.

  20. Macrophage recognition of ICAM-3 on apoptotic leukocytes.

    Science.gov (United States)

    Moffatt, O D; Devitt, A; Bell, E D; Simmons, D L; Gregory, C D

    1999-06-01

    Cells undergoing apoptosis are cleared rapidly by phagocytes, thus preventing tissue damage caused by loss of plasma membrane integrity. In this study, we show that the surface of leukocytes is altered during apoptosis such that the first Ig-like domain of ICAM-3 (CD50) can participate in the recognition and phagocytosis of the apoptotic cells by macrophages. Macrophage recognition of apoptotic cell-associated ICAM-3 was demonstrated both on leukocytes and, following transfection of exogenous ICAM-3, on nonleukocytes. The change in ICAM-3 was a consistent consequence of apoptosis triggered by various stimuli, suggesting that it occurs as part of a final common pathway of apoptosis. Alteration of ICAM-3 on apoptotic cells permitting recognition by macrophages resulted in a switch in ICAM-3-binding preference from the prototypic ICAM-3 counterreceptor, LFA-1, to an alternative macrophage receptor. Using mAbs to block macrophage/apoptotic cell interactions, we were unable to obtain evidence that either the alternative ICAM-3 counterreceptor alpha d beta 2 or the apoptotic cell receptor alpha v beta 3 was involved in the recognition of ICAM-3. By contrast, mAb blockade of macrophage CD14 inhibited ICAM-3-dependent recognition of apoptotic cells. These results show that ICAM-3 can function as a phagocytic marker of apoptotic leukocytes on which it acquires altered macrophage receptor-binding activity.

  1. New insights into estrogenic regulation of O6-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells: Co-degradation of ER-α and MGMT proteins by fulvestrant or O6-benzylguanine indicates fresh avenues for therapy

    Science.gov (United States)

    Paranjpe, Ameya; Bailey, Nathan I.; Konduri, Santhi; Bobustuc, George C.; Ali-Osman, Francis; Yusuf, Mohd. A.; Punganuru, Surendra R.; Madala, Hanumantha Rao; Basak, Debasish; Mostofa, AGM; Srivenugopal, Kalkunte S.

    2016-01-01

    Abstract Endocrine therapy using estrogen receptor-α (ER-α) antagonists for attenuating horm2one-driven cell proliferation is a major treatment modality for breast cancers. To exploit any DNA repair deficiencies associated with endocrine therapy, we investigated the functional and physical interactions of ER-α with O6-methylguanine DNA methyltransferase (MGMT), a unique DNA repair protein that confers tumor resistance to various anticancer alkylating agents. The ER-α -positive breast cancer cell lines (MCF-7, T47D) and ER- negative cell lines (MDAMB-468, MDAMB-231), and established inhibitors of ER-α and MGMT, namely, ICI-182,780 (Faslodex) and O6-benzylguanine, respectively, were used to study MGMT- ER interactions. The MGMT gene promoter was found to harbor one full and two half estrogen-responsive elements (EREs) and two antioxidant-responsive elements (AREs). MGMT expression was upregulated by estrogen, downregulated by tamoxifen in Western blot and promoter-linked reporter assays. Similarly, both transient and stable transfections of Nrf-2 (nuclear factor-erythroid 2-related factor-2) increased the levels of MGMT protein and activity 3 to 4-fold reflecting novel regulatory nodes for this drug-resistance determinant. Of the different ER-α antagonists tested, the pure anti-estrogen fulvestrant was most potent in inhibiting the MGMT activity in a dose, time and ER-α dependent manner, similar to O6-benzylguanine. Interestingly, fulvestrant exposure led to a degradation of both ER-α and MGMT proteins and O6-benzylguanine also induced a specific loss of ER-α and MGMT proteins in MCF-7 and T47D breast cancer cells with similar kinetics. Immunoprecipitation revealed a specific association of ER-α and MGMT proteins in breast cancer cells. Furthermore, silencing of MGMT gene expression triggered a decrease in the levels of both MGMT and ER-α proteins. The involvement of proteasome in the drug-induced degradation of both proteins was also demonstrated

  2. New insights into estrogenic regulation of O(6)-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells: Co-degradation of ER-α and MGMT proteins by fulvestrant or O(6)-benzylguanine indicates fresh avenues for therapy.

    Science.gov (United States)

    Paranjpe, Ameya; Bailey, Nathan I; Konduri, Santhi; Bobustuc, George C; Ali-Osman, Francis; Yusuf, Mohd A; Punganuru, Surendra R; Madala, Hanumantha Rao; Basak, Debasish; Mostofa, Agm; Srivenugopal, Kalkunte S

    2016-09-01

    Endocrine therapy using estrogen receptor-α (ER-α) antagonists for attenuating horm2one-driven cell proliferation is a major treatment modality for breast cancers. To exploit any DNA repair deficiencies associated with endocrine therapy, we investigated the functional and physical interactions of ER-α with O(6)-methylguanine DNA methyltransferase (MGMT), a unique DNA repair protein that confers tumor resistance to various anticancer alkylating agents. The ER-α -positive breast cancer cell lines (MCF-7, T47D) and ER- negative cell lines (MDAMB-468, MDAMB-231), and established inhibitors of ER-α and MGMT, namely, ICI-182,780 (Faslodex) and O(6)-benzylguanine, respectively, were used to study MGMT- ER interactions. The MGMT gene promoter was found to harbor one full and two half estrogen-responsive elements (EREs) and two antioxidant-responsive elements (AREs). MGMT expression was upregulated by estrogen, downregulated by tamoxifen in Western blot and promoter-linked reporter assays. Similarly, both transient and stable transfections of Nrf-2 (nuclear factor-erythroid 2-related factor-2) increased the levels of MGMT protein and activity 3 to 4-fold reflecting novel regulatory nodes for this drug-resistance determinant. Of the different ER-α antagonists tested, the pure anti-estrogen fulvestrant was most potent in inhibiting the MGMT activity in a dose, time and ER-α dependent manner, similar to O(6)-benzylguanine. Interestingly, fulvestrant exposure led to a degradation of both ER-α and MGMT proteins and O(6)-benzylguanine also induced a specific loss of ER-α and MGMT proteins in MCF-7 and T47D breast cancer cells with similar kinetics. Immunoprecipitation revealed a specific association of ER-α and MGMT proteins in breast cancer cells. Furthermore, silencing of MGMT gene expression triggered a decrease in the levels of both MGMT and ER-α proteins. The involvement of proteasome in the drug-induced degradation of both proteins was also demonstrated

  3. The cDNA 385C to A missense polymorphism of the endocannabinoid degrading enzyme fatty acid amide hydrolase (FAAH) is associated with overweight/obesity but not with binge eating disorder in overweight/obese women.

    Science.gov (United States)

    Monteleone, Palmiero; Tortorella, Alfonso; Martiadis, Vassilis; Di Filippo, Carmela; Canestrelli, Benedetta; Maj, Mario

    2008-05-01

    Endocannabinoids are involved in the modulation of eating behavior; hence, alterations of this system may play a role in obesity. Recently, a single nucleotide polymorphism (cDNA 385C to A) of the gene coding for fatty acid amide hydrolase (FAAH), the major degrading enzyme of endocannabinoids, has been found to be associated with obesity. However, the possibility that the FAAH gene cDNA 385C to A single nucleotide polymorphism (SNP) is associated to binge eating disorder (BED), a condition that frequently occurs in obese individuals, has not been investigated. In order to address this issue, we assessed the distribution of the cDNA 385C to A SNP in 115 overweight/obese subjects with BED, 74 non-BED patients with obesity and 110 normal weight healthy controls. As compared to healthy controls, the whole group of overweight/obese BED and non-BED patients had a significantly higher frequency of the CA genotype and the A allele of the FAAH gene cDNA 385C to A SNP. Moreover, the SNP resulted significantly correlated to the presence of overweight/obesity (F(2, 296)=3.58, P=0.02), but not to the occurrence of BED (F(2, 296)=0.98; P=0.3). The present study confirms previously published significant over-representations of the FAAH 385 A allele in overweight/obese subjects and presents new data in BED patients that the 385 mutation is not significantly associated with BED-related obesity.

  4. Apoptotic-like Leishmania exploit the host´s autophagy machinery to reduce T-cell-mediated parasite elimination

    Science.gov (United States)

    Crauwels, Peter; Bohn, Rebecca; Thomas, Meike; Gottwalt, Stefan; Jäckel, Florian; Krämer, Susi; Bank, Elena; Tenzer, Stefan; Walther, Paul; Bastian, Max; van Zandbergen, Ger

    2015-01-01

    Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmania parasites in human host macrophages. Our data showed—in contrast to viable parasites—that apoptotic-like parasites enter an LC3+, autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmania induced proliferation of CD4+ T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferation-reducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmania hijack the host cells´ autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis. PMID:25801301

  5. Cobra venom cytotoxins; apoptotic or necrotic agents?

    Science.gov (United States)

    Ebrahim, Karim; Shirazi, Farshad H; Mirakabadi, Abbas Zare; Vatanpour, Hossein

    2015-12-15

    Organs homeostasis is controlled by a dynamic balance between cell proliferation and apoptosis. Failure to induction of apoptosis has been implicated in tumor development. Cytotoxin-I (CTX-I) and cytotoxin-II (CTX-II) are two physiologically active polypeptides found in Caspian cobra venom. Anticancer activity and mechanism of cell death induced by these toxins have been studied. The toxins were purified by different chromatographic steps and their cytotoxicity and pattern of cell death were determined by MTT, LDH release, acridine orange/ethidium bromide (AO/EtBr) double staining, flow cytometric analysis, caspase-3 activity and neutral red assays. The IC50 of CTX-II in MCF-7, HepG2, DU-145 and HL-60 was 4.1 ± 1.3, 21.2 ± 4.4, 9.4 ± 1.8 μg/mL and 16.3 ± 1.9 respectively while the IC50 of this toxin in normal MDCK cell line was 54.5 ± 3.9 μg/mL. LDH release suddenly increase after a specific toxins concentrations in all cell lines. AO/EtBr double staining, flow cytometric analysis and caspase-3 activity assay confirm dose and time-dependent induction of apoptosis by both toxins. CTX-I and CTX-II treated cells lost their lysosomal membrane integrity and couldn't uptake neutral red day. CTX-I and CTX-II showed significant anticancer activity with minimum effects on normal cells and better IC50 compared to current anticancer drug; cisplatin. They induce their apoptotic effect via lysosomal pathways and release of cathepsins to cytosol. These effects were seen in limited rage of toxins concentrations and pattern of cell death rapidly changes to necrosis by increase in toxin's concentration. In conclusion, significant apoptogenic effects of these toxins candidate them as a possible anticancer agent.

  6. Fluorescent Probes Detecting the Phagocytic Phase of Apoptosis: Enzyme-Substrate Complexes of Topoisomerase and DNA

    Directory of Open Access Journals (Sweden)

    Candace L. Minchew

    2011-06-01

    Full Text Available In apoptosis, the initial self-driven suicide phase generates cellular corpses which are digested in the phagolysosomes of professional and amateur phagocytes during the subsequent waste-management phase. This ensures the complete elimination of the genetic material which often contains pathological, viral or cancerous DNA sequences. Although the phagocytic phase is critical for the efficient execution of apoptosis, there are currently few methods specifically adapted for its detailed visualization in the fixed tissue section format. To resolve this we developed new fluorescent probes for in situ research. The probes selectively visualize active phagocytic cells of any lineage (professional, amateur phagocytes or surrounding tissue cells which engulf and digest apoptotic cell DNA. These fluorescent probes are the covalently-bound enzyme-DNA intermediates produced in a topoisomerase reaction with specific “starting” oligonucleotides. They detect a specific marker of DNase II cleavage activity, which occurs exclusively in phagolysosomes of the cells that engulfed apoptotic nuclei. The probes provide snap-shot images of the digestion process occurring in cellular organelles responsible for the actual execution of phagocytic degradation of apoptotic cell corpses. We applied the probes for visualization of the phagocytic reaction in tissue sections of normal thymus and in several human lymphomas. We also discuss the nature, stability and properties of DNase II-type breaks as a marker of phagocytic activity. This development provides a useful fluorescent tool for studies of pathologies where clearance of dying cells is essential, such as cancers, inflammation, infection and auto-immune disorders.

  7. In vitro study of immunosuppressive effect of apoptotic cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Wen-jin; ZHENG Shu-sen

    2005-01-01

    Recent studies revealed that apoptotic cells are actively involved in immunosuppression and anti-inflammation. After being phagocytosed by macrophages, apoptotic cells can actively regulate cytokines secretion from lipopolysaccharide (LPS)-stimulated macrophages, in which the secretion of immunosuppressive cytokines such as interleukin-10 (IL-10) is increased while the pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFα), interleukin-1beta (IL-1β) and leukin-8 (IL-8) are suppressed. In this paper, we first present evidence that phagocytosed apoptotic cells regulate cytokine secretion of LPS-stimulated macrophages, but also inhibit the activation of T lymphocytes stimulated by ConA. These data suggest that apoptotic cells can alter the biological behavior of macrophages which gain immunosuppressive property.

  8. Apoptotic-like changes of boar spermatozoa in freezing media supplemented with different antioxidants.

    Science.gov (United States)

    Trzcińska, M; Bryła, M

    2015-01-01

    This study evaluated the effect of supplementing the freezing extender with exogenous anti-oxidants on apoptotic-like changes in post-thaw boar spermatozoa. A total of 36 ejaculates were resuspended in standard lactose-egg yolk-glycerol extender supplemented with antioxidant to final concentrations of 0 (as control), 2.5mM GSH (group I), 5.0 mM GSH (group II), 150 IU/mL SOD (group III), 300 IU/mL SOD (group IV), 200 IU/mL CAT (group V), 400 IU/mL CAT (group VI), 150 IU/mL SOD+200 IU/mL CAT (group VII), 300 IU/mL SOD+400 IU/mL CAT (group VIII). Sperm motility and apoptotic-like changes were determined before and after freeze-thawing. The various markers of apoptotic-like changes were measured: plasma membrane permeability by YO-PRO-1/PI assay, phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled Annexin-V, mitochondrial transmembrane potential detected by JC-1, and DNA fragmentation evaluated by TUNEL assay. The highest percentage of progressive motile sperm was noticed in group II (PM% 64.2±15.4) compared with control (PM% 36.8±5.5). The supplementation of 400 IU/mL CAT (group VI) revealed significant (Psperm survival compared with the control. Evaluation by TUNEL assay revealed that cryopreservation and thawing did not induce DNA fragmentation in boar spermatozoa.

  9. Histone demethylase Jmjd3 regulates osteoblast apoptosis through targeting anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bim.

    Science.gov (United States)

    Yang, Di; Okamura, Hirohiko; Teramachi, Jumpei; Haneji, Tatsuji

    2016-04-01

    Posttranslational modifications including histone methylation regulate gene transcription through directly affecting the structure of chromatin. Trimethylation of histone H3K27 (H3K27me3) contributes to gene silencing and the histone demethylase Jumonji domain-containing 3 (Jmjd3) specifically removes the methylation of H3K27me3, followed by the activation of gene expression. In the present study, we explored the roles of Jmjd3 in regulating osteoblast apoptosis. Knockdown of Jmjd3 promoted osteoblast apoptosis induced by serum deprivation with decreased mitochondrial membrane potential and increased levels of caspase-3 activation, PARP cleavage, and DNA fragmentation. B cell lymphoma-2 (Bcl-2), an anti-apoptotic protein, was down-regulated by knockdown of Jmjd3 through retaining H3K27me3 on its promoter region. Knockdown of Jmjd3 increased the pro-apoptotic activity of Bim through inhibiting ERK-dependent phosphorylation of Bim. Protein kinase D1 (PKD1), which stimulates ERK phosphorylation, decreased in the Jmjd3-knockdown cells and introduction of PKD1 relieved osteoblast apoptosis in the Jmjd3-knockdown cells through increasing ERK-regulated Bim phosphorylation. These results suggest that Jmjd3 regulates osteoblast apoptosis through targeting Bcl-2 expression and Bim phosphorylation.

  10. Apoptotic pathways as a therapeutic target for colorectal cancer treatment

    Institute of Scientific and Technical Information of China (English)

    Aman M Abraha; Ezra B Ketema

    2016-01-01

    Colorectal cancer is the second leading cause of death from cancer among adults. The disease begins as a benign adenomatous polyp, which develops into an advanced adenoma with high-grade dysplasia and then progresses to an invasive cancer. Appropriate apoptotic signaling is fundamentally important to preserve a healthy balance between cell death and cell survival and in maintaining genome integrity. Evasion of apoptotic pathway has been established as a prominent hallmark of several cancers. During colorectal cancer development, the balance between the rates of cell growth and apoptosis that maintains intestinal epithelial cell homeostasis gets progressively disturbed. Evidences are increasingly available to support the hypothesis that failure of apoptosis may be an important factor in the evolution of colorectal cancer and its poor response to chemotherapy and radiation. The other reason for targeting apoptotic pathway in the treatment of cancer is based on the observation that this process is deregulated in cancer cells but not in normal cells. As a result, colorectal cancer therapies designed to stimulate apoptosis in target cells would play a critical role in controlling its development and progression. A better understanding of the apoptotic signaling pathways, and the mechanisms by which cancer cells evade apoptotic death might lead to effective therapeutic strategies to inhibit cancer cell proliferation with minimal toxicity and high responses to chemotherapy. In this review, we analyzed the current understanding and future promises of apoptotic pathways as a therapeutic target in colorectal cancer treatment.

  11. Human CD14 mediates recognition and phagocytosis of apoptotic cells.

    Science.gov (United States)

    Devitt, A; Moffatt, O D; Raykundalia, C; Capra, J D; Simmons, D L; Gregory, C D

    1998-04-02

    Cells undergoing programmed cell death (apoptosis) are cleared rapidly in vivo by phagocytes without inducing inflammation. Here we show that the glycosylphosphatidylinositol-linked plasma-membrane glycoprotein CD14 on the surface of human macrophages is important for the recognition and clearance of apoptotic cells. CD14 can also act as a receptor that binds bacterial lipopolysaccharide (LPS), triggering inflammatory responses. Overstimulation of CD14 by LPS can cause the often fatal toxic-shock syndrome. Here we show that apoptotic cells interact with CD14, triggering phagocytosis of the apoptotic cells. This interaction depends on a region of CD14 that is identical to, or at least closely associated with, a region known to bind LPS. However, apoptotic cells, unlike LPS, do not provoke the release of pro-inflammatory cytokines from macrophages. These results indicate that clearance of apoptotic cells is mediated by a receptor whose interactions with 'non-self' components (LPS) and 'self' components (apoptotic cells) produce distinct macrophage responses.

  12. Cytosolic pro-apoptotic SPIKE induces mitochondrial apoptosis in cancer.

    Science.gov (United States)

    Nikolic, Ivana; Kastratovic, Tatjana; Zelen, Ivanka; Zivanovic, Aleksandar; Arsenijevic, Slobodan; Mitrovic, Marina

    2010-04-30

    Proteins of the BCL-2 family are important regulators of apoptosis. The BCL-2 family includes three main subgroups: the anti-apoptotic group, such as BCL-2, BCL-XL, BCL-W, and MCL-1; multi-domain pro-apoptotic BAX, BAK; and pro-apoptotic "BH3-only" BIK, PUMA, NOXA, BID, BAD, and SPIKE. SPIKE, a rare pro-apoptotic protein, is highly conserved throughout the evolution, including Caenorhabditis elegans, whose expression is downregulated in certain tumors, including kidney, lung, and breast. In the literature, SPIKE was proposed to interact with BAP31 and prevent BCL-XL from binding to BAP31. Here, we utilized the Position Weight Matrix method to identify SPIKE to be a BH3-only pro-apoptotic protein mainly localized in the cytosol of all cancer cell lines tested. Overexpression of SPIKE weakly induced apoptosis in comparison to the known BH3-only pro-apoptotic protein BIK. SPIKE promoted mitochondrial cytochrome c release, the activation of caspase 3, and the caspase cleavage of caspase's downstream substrates BAP31 and p130CAS. Although the informatics analysis of SPIKE implicates this protein as a member of the BH3-only BCL-2 subfamily, its role in apoptosis remains to be elucidated.

  13. Teratogen-induced apoptotic cell death: does the apoptotic machinery act as a protector of embryos exposed to teratogens?

    Science.gov (United States)

    Torchinsky, Arkady; Fein, Amos; Toder, Vladimir

    2005-12-01

    Considerable evidence has been collected demonstrating that many teratogens induce apoptotic cell death in embryonic structures that turn out to be malformed in fetuses and newborns. Apoptosis is a genetically regulated process that is realized by the activation of death and pro-survival signaling cascades, and the interplay between these cascades determines whether the cell exposed to apoptotic stimuli dies or survives. Therefore, there is intense interest in understanding how the apoptotic machinery functions in embryos exposed to teratogens. However, the interpretation of the results obtained remains problematic. The main problem is that excessive embryonic cell death, regardless of its nature, if uncompensated for, ultimately leads to maldevelopment or embryonic death. Therefore, we can easily interpret results when the intensity of teratogen-induced cell death and the severity or incidence of teratogen-induced anomalies directly correlate with each other. However, when teratogen-induced cell death is not followed by the formation of anomalies, a usual explanation is that teratogen-induced apoptotic cell death contributes to the renewal of teratogen-targeted cell populations by promoting the removal of injured cells. It is clear that such an explanation leaves vague the role of the anti-apoptotic signaling mechanism (and, hence, the apoptotic machinery as a whole) with respect to protecting the embryo against teratogenic stress. In this review, we summarize the data from studies addressing the function of the apoptotic machinery in embryos exposed to teratogens, and then we discuss approaches to interpreting the results of these studies. We hypothesize that activation of a proapoptotic signaling in teratogen-targeted cell populations is a necessary condition for an anti-apoptotic signaling that counteracts the process of maldevelopment to be activated. If such a scenario is true, we need to modify our approaches to choosing molecular targets for studies

  14. The Acetone Extract of Sclerocarya birrea (Anacardiaceae Possesses Antiproliferative and Apoptotic Potential against Human Breast Cancer Cell Lines (MCF-7

    Directory of Open Access Journals (Sweden)

    Nicoline Fri Tanih

    2013-01-01

    Full Text Available Interesting antimicrobial data from the stem bark of Sclerocarya birrea, which support its use in traditional medicine for the treatment of many diseases, have been delineated. The current study was aimed to further study some pharmacological and toxicological properties of the plant to scientifically justify its use. Anticancer activity of water and acetone extracts of S. birrea was evaluated on three different cell lines, HT-29, HeLa, and MCF-7 using the cell titre blue viability assay in 96-well plates. Apoptosis was evaluated using the acridine orange and propidium iodide staining method, while morphological structure of treated cells was examined using SEM. The acetone extract exhibited remarkable antiproliferative activities on MCF-7 cell lines at dose- and time-dependent manners (24 h and 48 h of incubation. The extract also exerted apoptotic programmed cell death in MCF-7 cells with significant effect on the DNA. Morphological examination also displayed apoptotic characteristics in the treated cells, including clumping, condensation, and culminating to budding of the cells to produce membrane-bound fragmentation, as well as formation of apoptotic bodies. The acetone extract of S. birrea possesses antiproliferative and apoptotic potential against MCF-7-treated cells and could be further exploited as a potential lead in anticancer therapy.

  15. Low expression of pro-apoptotic Bcl-2 family proteins sets the apoptotic threshold in Waldenström macroglobulinemia.

    Science.gov (United States)

    Gaudette, B T; Dwivedi, B; Chitta, K S; Poulain, S; Powell, D; Vertino, P; Leleu, X; Lonial, S; Chanan-Khan, A A; Kowalski, J; Boise, L H

    2016-01-28

    Waldenström macroglobulinemia (WM) is a proliferative disorder of IgM-secreting, lymphoplasmacytoid cells that inhabit the lymph nodes and bone marrow. The disease carries a high prevalence of activating mutations in MyD88 (91%) and CXCR4 (28%). Because signaling through these pathways leads to Bcl-xL induction, we examined Bcl-2 family expression in WM patients and cell lines. Unlike other B-lymphocyte-derived malignancies, which become dependent on expression of anti-apoptotic proteins to counter expression of pro-apoptotic proteins, WM samples expressed both pro- and anti-apoptotic Bcl-2 proteins at low levels similar to their normal B-cell and plasma cell counterparts. Three WM cell lines expressed pro-apoptotic Bcl-2 family members Bim or Bax and Bak at low levels, which determined their sensitivity to inducers of intrinsic apoptosis. In two cell lines, miR-155 upregulation, which is common in WM, was responsible for the inhibition of FOXO3a and Bim expression. Both antagonizing miR-155 to induce Bim and proteasome inhibition increased the sensitivity to ABT-737 in these lines indicating a lowering of the apoptotic threshold. In this manner, treatments that increase pro-apoptotic protein expression increase the efficacy of agents treated in combination in addition to direct killing.

  16. Melittin induces apoptotic features in Candida albicans

    Energy Technology Data Exchange (ETDEWEB)

    Park, Cana [School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, 1370 Sankyuk-dong, Puk-ku, Daegu 702-701 (Korea, Republic of); Lee, Dong Gun, E-mail: dglee222@knu.ac.kr [School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, 1370 Sankyuk-dong, Puk-ku, Daegu 702-701 (Korea, Republic of)

    2010-03-26

    Melittin is a well-known antimicrobial peptide with membrane-active mechanisms. In this study, it was found that Melittin exerted its antifungal effect via apoptosis. Candida albicans exposed to Melittin showed the increased reactive oxygen species (ROS) production, measured by DHR-123 staining. Fluorescence microscopy staining with FITC-annexin V, TUNEL and DAPI further confirmed diagnostic markers of yeast apoptosis including phosphatidylserine externalization, and DNA and nuclear fragmentation. The current study suggests that Melittin possesses an antifungal effect with another mechanism promoting apoptosis.

  17. Expression of leptin and its receptor in female breast cancer in relation with selected apoptotic markers.

    Directory of Open Access Journals (Sweden)

    Stanislaw Sulkowski

    2008-04-01

    Full Text Available Leptin and its receptor may be engaged in pathogenesis of breast cancer among various human tumors. In vitro investigations showed leptin-mediated escalation of estrogen synthesis and boosted activity of estrogen receptor ERalpha. Furthermore, leptin induced growth of malignant cells, counteracted apoptosis and stimulated cell migration as well as overexpression of angiogenic factors and degrading enzymes that split network of intercellular matrix. On the other side, leptin has been reported to favor apoptosis, lately. Proapoptotic effect of leptin action was revealed in interstitial cells of bone marrow and adipocytes. Our past reports provide evidences for overexpression of leptin and its receptor in breast cancer in comparison with benign mammary lesions. In current study we aimed at assessment of eventual relationships between leptin, leptin receptor and selected protein regulators of apoptosis in breast cancer. We applied immunohistochemistry for leptin, leptin receptor, anti-apoptotic Bcl-2 and Bcl-xL as well as pro-apoptotic Bak and Bax expression assessment in 106 cases of human breast cancers. The immunoreaction was graded and statistically evaluated. Expression of leptin was positively correlated with Bcl-xL, Bak and Bax (p<0.001, r=0.614; p<0.001, r=0.518; p<0.001, r=0.511, respectively. Statistical significances were noted between expression of leptin receptor and Bcl-xL or Bax (p=0.011, r=0.210; p<0.001, r=0.313, respectively. No correlation was encountered between leptin and Bcl-2, either leptin receptor and Bcl-2 or leptin receptor and Bak. On the basis of obtained results, leptin system could interfere in balance among expressions of pro- and anti-apoptotic proteins and regulate cell turnover and--by means of it--facilitate breast cancer progression.

  18. Induction of apoptotic cell death by putrescine

    DEFF Research Database (Denmark)

    Takao, Koichi; Rickhag, Karl Mattias; Hegardt, Cecilia

    2006-01-01

    The polyamines are essential for cellular growth and differentiation. Ornithine decarboxylase (ODC), which catalyses the first step in the biosynthesis of the polyamines, has a very fast turnover and is subject to a strong feedback control by the polyamines. In the present study, we show that ove......The polyamines are essential for cellular growth and differentiation. Ornithine decarboxylase (ODC), which catalyses the first step in the biosynthesis of the polyamines, has a very fast turnover and is subject to a strong feedback control by the polyamines. In the present study, we show...... for their growth. The induction of cell death was correlated with a dramatic increase in cellular putrescine levels. Analysis using flow cytometry revealed perturbed cell cycle kinetics, with a large accumulation of cells with sub-G1 amounts of DNA, which is a typical sign of apoptosis. Another strong indication...

  19. Leber's hereditary optic neuropathy (LHON) pathogenic mutations induce mitochondrial-dependent apoptotic death in transmitochondrial cells incubated with galactose medium.

    Science.gov (United States)

    Ghelli, Anna; Zanna, Claudia; Porcelli, Anna Maria; Schapira, Anthony H V; Martinuzzi, Andrea; Carelli, Valerio; Rugolo, Michela

    2003-02-01

    Leber's hereditary optic neuropathy (LHON), a maternally inherited form of central vision loss, is associated with mitochondrial DNA pathogenic point mutations affecting different subunits of complex I. We here report that osteosarcoma-derived cytoplasmic hybrids (cybrid) cell lines harboring one of the three most frequent LHON pathogenic mutations, at positions 11778/ND4, 3460/ND1, and 14484/ND6, undergo cell death when galactose replaces glucose in the medium, contrary to control cybrids that maintain some growth capabilities. This is a well known way to produce a metabolic stress, forcing the cells to rely on the mitochondrial respiratory chain to produce ATP. We demonstrate that LHON cybrid cell death is apoptotic, showing chromatin condensation and nuclear DNA laddering. Moreover, we also document the mitochondrial involvement in the activation of the apoptotic cascade, as shown by the increased release of cytochrome c into the cytosol in LHON cybrid cells as compared with controls. Cybrids bearing the 3460/ND1 and 14484/ND6 mutations seemed more readily prone to undergo apoptosis as compared with the 11778/ND4 mutation. In conclusion, LHON cybrid cells forced by the reduced rate of glycolytic flux to utilize oxidative metabolism are sensitized to an apoptotic death through a mechanism involving mitochondria.

  20. Apoptotic resistance to ionizing radiation in pediatric B-precursor acute lymphoblastic leukemia frequently involves increased NF-kappaB survival pathway signaling.

    Science.gov (United States)

    Weston, Victoria J; Austen, Belinda; Wei, Wenbin; Marston, Eliot; Alvi, Azra; Lawson, Sarah; Darbyshire, Philip J; Griffiths, Mike; Hill, Frank; Mann, Jill R; Moss, Paul A H; Taylor, A Malcolm R; Stankovic, Tatjana

    2004-09-01

    To investigate possible causes of the variable response to treatment in pediatric B-precursor acute lymphoblastic leukemia (ALL) and to establish potential novel therapeutic targets, we used ionizing radiation (IR) exposure as a model of DNA damage formation to identify tumors with resistance to p53-dependent apoptosis. Twenty-one of 40 ALL tumors responded normally to IR, exhibiting accumulation of p53 and p21 proteins and cleavage of caspases 3, 7, and 9 and of PARP1. Nineteen tumors exhibited apoptotic resistance and lacked PARP1 and caspase cleavage; although 15 of these tumors had normal accumulation of p53 and p21 proteins, examples exhibited abnormal expression of TRAF5, TRAF6, and cIAP1 after IR, suggesting increased NF-kappaB prosurvival signaling as the mechanism of apoptotic resistance. The presence of a hyperactive PARP1 mutation in one tumor was consistent with such increased NF-kappaB activity. PARP1 inhibition restored p53-dependent apoptosis after IR in these leukemias by reducing NF-kappaB DNA binding and transcriptional activity. In the remaining 4 ALL tumors, apoptotic resistance was associated with a TP53 mutation or with defective activation of p53. We conclude that increased NF-kappaB prosurvival signaling is a frequent mechanism by which B-precursor ALL tumors develop apoptotic resistance to IR and that PARP1 inhibition may improve the DNA damage response of these leukemias.

  1. EXTRACELLULAR DNA AND THE LEVEL OF ITS METHYLATION IN DIFFERENT RHEUMATIC DISEASES

    Directory of Open Access Journals (Sweden)

    N O Shubayeva

    2012-01-01

    Conclusion. RDs are characterized by the higher concentration of apoptotic and necrotic DNA, impaired exDNA methylation, varying complexification of exDNA with monometinic proteins, which is associated with the hyperproduction of autoantibodies (including anti-exDNA antibodies and inflammatory markers.

  2. Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells.

    Science.gov (United States)

    Rodríguez-Hernández, A; Navarro-Villarán, E; González, R; Pereira, S; Soriano-De Castro, L B; Sarrias-Giménez, A; Barrera-Pulido, L; Álamo-Martínez, J M; Serrablo-Requejo, A; Blanco-Fernández, G; Nogales-Muñoz, A; Gila-Bohórquez, A; Pacheco, D; Torres-Nieto, M A; Serrano-Díaz-Canedo, J; Suárez-Artacho, G; Bernal-Bellido, C; Marín-Gómez, L M; Barcena, J A; Gómez-Bravo, M A; Padilla, C A; Padillo, F J; Muntané, J

    2015-12-01

    Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10 µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells.

  3. HSP70 mediates survival in apoptotic cells-Boolean network prediction and experimental validation.

    Science.gov (United States)

    Vasaikar, Suhas V; Ghosh, Sourish; Narain, Priyam; Basu, Anirban; Gomes, James

    2015-01-01

    Neuronal stress or injury results in the activation of proteins, which regulate the balance between survival and apoptosis. However, the complex mechanism of cell signaling involving cell death and survival, activated in response to cellular stress is not yet completely understood. To bring more clarity about these mechanisms, a Boolean network was constructed that represented the apoptotic pathway in neuronal cells. FasL and neurotrophic growth factor (NGF) were considered as inputs in the absence and presence of heat shock proteins known to shift the balance toward survival by rescuing pro-apoptotic cells. The probabilities of survival, DNA repair and apoptosis as cellular fates, in the presence of either the growth factor or FasL, revealed a survival bias encoded in the network. Boolean predictions tested by measuring the mRNA level of caspase-3, caspase-8, and BAX in neuronal Neuro2a (N2a) cell line with NGF and FasL as external input, showed positive correlation with the observed experimental results for survival and apoptotic states. It was observed that HSP70 contributed more toward rescuing cells from apoptosis in comparison to HSP27, HSP40, and HSP90. Overexpression of HSP70 in N2a transfected cells showed reversal of cellular fate from FasL-induced apoptosis to survival. Further, the pro-survival role of the proteins BCL2, IAP, cFLIP, and NFκB determined by vertex perturbation analysis was experimentally validated through protein inhibition experiments using EM20-25, Embelin and Wedelolactone, which resulted in 1.27-, 1.26-, and 1.46-fold increase in apoptosis of N2a cells. The existence of a one-to-one correspondence between cellular fates and attractor states shows that Boolean networks may be employed with confidence in qualitative analytical studies of biological networks.

  4. Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells.

    Science.gov (United States)

    Kalaaji, Manar; Mortensen, Elin; Jørgensen, Leif; Olsen, Randi; Rekvig, Ole Petter

    2006-06-01

    Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus.

  5. The pancreatic and duodenal homeobox protein PDX-1 regulates the ductal specific keratin 19 through the degradation of MEIS1 and DNA binding.

    Directory of Open Access Journals (Sweden)

    Johannes von Burstin

    Full Text Available BACKGROUND: Pancreas organogenesis is the result of well-orchestrated and balanced activities of transcription factors. The homeobox transcription factor PDX-1 plays a crucial role in the development and function of the pancreas, both in the maintenance of progenitor cells and in determination and maintenance of differentiated endocrine cells. However, the activity of homeobox transcription factors requires coordination with co-factors, such as PBX and MEIS proteins. PBX and MEIS proteins belong to the family of three amino acid loop extension (TALE homeodomain proteins. In a previous study we found that PDX-1 negatively regulates the transcriptional activity of the ductal specific keratin 19 (Krt19. In this study, we investigate the role of different domains of PDX-1 and elucidate the functional interplay of PDX-1 and MEIS1 necessary for Krt19 regulation. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that PDX-1 exerts a dual manner of regulation of Krt19 transcriptional activity. Deletion studies highlight that the NH(2-terminus of PDX-1 is functionally relevant for the down-regulation of Krt19, as it is required for DNA binding of PDX-1 to the Krt19 promoter. Moreover, this effect occurs independently of PBX. Second, we provide insight on how PDX-1 regulates the Hox co-factor MEIS1 post-transcriptionally. We find specific binding of MEIS1 and MEIS2 to the Krt19 promoter using IP-EMSA, and siRNA mediated silencing of Meis1, but not Meis2, reduces transcriptional activation of Krt19 in primary pancreatic ductal cells. Over-expression of PDX-1 leads to a decreased level of MEIS1 protein, and this decrease is prevented by inhibition of the proteasome. CONCLUSIONS/SIGNIFICANCE: Taken together, our data provide evidence for a dual mechanism of how PDX-1 negatively regulates Krt19 ductal specific gene expression. These findings imply that transcription factors may efficiently regulate target gene expression through diverse, non

  6. Apoptotic phosphorylation of histone H3 on Ser-10 by protein kinase Cδ.

    Directory of Open Access Journals (Sweden)

    Choon-Ho Park

    Full Text Available Phosphorylation of histone H3 on Ser-10 is regarded as an epigenetic mitotic marker and is tightly correlated with chromosome condensation during both mitosis and meiosis. However, it was also reported that histone H3 Ser-10 phosphorylation occurs when cells are exposed to various death stimuli, suggesting a potential role in the regulation of apoptosis. Here we report that histone H3 Ser-10 phosphorylation is mediated by the pro-apoptotic kinase protein kinase C (PKC δ during apoptosis. We observed that PKCδ robustly phosphorylates histone H3 on Ser-10 both in vitro and in vivo. Ectopic expression of catalytically active PKCδ efficiently induces condensed chromatin structure in the nucleus. We also discovered that activation of PKCδ is required for histone H3 Ser-10 phosphorylation after treatment with DNA damaging agents during apoptosis. Collectively, these findings suggest that PKCδ is the kinase responsible for histone H3 Ser-10 phosphoryation during apoptosis and thus contributes to chromatin condensation together with other apoptosis-related histone modifications. As a result, histone H3 Ser-10 phosphorylation can be designated a new 'apoptotic histone code' mediated by PKCδ.

  7. Pro-oxidative action of polyphenols as action mechanism for their pro-apoptotic activity.

    Science.gov (United States)

    Lecci, Raffaella Marina; Logrieco, Antonio; Leone, Antonella

    2014-01-01

    Polyphenols, secondary metabolites widely present in plant kingdom, are known for their positive effects on human health, such as treatments of degenerative disease and cancer. Many dietary polyphenols show anti-inflammatory, immunomodulatory and antioxidant properties and they are proposed as chemopreventive agents for many skin disorders and cancer. Exposure to solar UV radiation is widely considered to cause skin cancer and a consistent carcinogenic dose derived from UVA causes several skin disorders as a consequence of free radicals generation and DNA damages. In this study, verbascoside, isoverbascoside and tyrosol were investigated for their effects on HEKa (Human Epidermal Keratinocytes adult) cell cultures challenged from UVA-rays. Non-toxic doses of each polyphenol were assayed on HEKa before, during and after the exposure to a damaging dose of UVA. Treatment with polyphenols before and after the UVA-irradiation exerted a pro-oxidant effect, while the simultaneous treatment caused a weak decrease of ROS production. The increasing of ROS levels was associated with a proapoptotic effect on HEKa, detected by AnnexinV/Propidiun Iodide, mainly evident in surviving cells treated with the polyphenols after the UVA-irradiation. The pro-apoptotic effect was confirmed by the immunodetection of significant changes in the Bax and Bcl-xL protein levels, leading to apoptotic events. The hypothesis that these polyphenols could trigger the apoptosis pathway mainly in UVA-damaged cells, via ROS increase, is here proposed as action mechanism behind their protective effect.

  8. Assessment of cytotoxic and apoptotic effects of benzaldehyde using different assays.

    Science.gov (United States)

    Ulker, Z; Alpsoy, L; Mihmanli, A

    2013-08-01

    Benzaldehyde (BA) occurs naturally in a number of plants, including cherry, fig and peach fruit and carnation flowers at therapeutic doses. In addition, it is used in cosmetics, personal care products and food as a preservative. In this study, we aimed to determine the cytotoxic and apoptotic effects of different concentrations of BA on cultured human lymphocytes using lactate dehydrogenase assay, cell proliferation (water-soluble tetrazolium salts-1) assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test (apoptotic test) as a group of cytotoxicity tests at 6th and 24th h on human lymphocyte cell culture. The cytotoxicity increased when cells were treated with 10, 25 and 50 μg/mL concentrations of BA (p < 0.05). Moreover, treatment of the cells with the same concentrations significantly decreased the cell number at the 6th and 24th hours (p < 0.05). TUNEL assay results also show that the concentration of BA at 10, 25 and 50 μg/mL caused DNA damage significantly (p < 0.05). According to our results, the toxic and genotoxic effects of BA have to be further evaluated before using in cosmetic and food products.

  9. Bacterial isolates degrading aliphatic polycarbonates.

    Science.gov (United States)

    Suyama, T; Hosoya, H; Tokiwa, Y

    1998-04-15

    Bacteria that degrade an aliphatic polycarbonate, poly(hexamethylene carbonate), were isolated from river water in Ibaraki. Prefecture, Japan, after enrichment in liquid medium containing poly(hexamethylene carbonate) suspensions as carbon source, and dilution to single cells. Four of the strains, 35L, WFF52, 61A and 61B2, degraded poly(hexamethylene carbonate) on agar plate containing suspended poly(hexamethylene carbonate). Degradation of poly(hexamethylene carbonate) was confirmed by gel permeation chromatography. Besides poly(hexamethylene carbonate), the strains were found to degrade poly(tetramethylene carbonate). The strains were characterized morphologically, physiologically, and by 16S rDNA sequence analysis. Strains 35L and WFF52 were tentatively identified as Pseudomonas sp. and Variovorax sp., respectively, while strains 61A and 61B2 constitute an unidentified branch within the beta subclass of the Proteobacteria.

  10. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases

    Directory of Open Access Journals (Sweden)

    Zodwa Dlamini

    2015-11-01

    Full Text Available Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets.

  11. Hydroxytyrosol inhibits hydrogen peroxide-induced apoptotic signaling via labile iron chelation

    Directory of Open Access Journals (Sweden)

    Natalia Kitsati

    2016-12-01

    Full Text Available Although it is known that Mediterranean diet plays an important role in maintaining human health, the underlying molecular mechanisms remain largely unknown. The aim of this investigation was to elucidate the potential role of ortho-dihydroxy group containing natural compounds in H2O2-induced DNA damage and apoptosis. For this purpose, the main phenolic alcohols of olive oil, namely hydroxytyrosol and tyrosol, were examined for their ability to protect cultured cells under conditions of oxidative stress. A strong correlation was observed between the ability of hydroxytyrosol to mitigate intracellular labile iron level and the protection offered against H2O2-induced DNA damage and apoptosis. On the other hand, tyrosol, which lacks the ortho-dihydroxy group, was ineffective. Moreover, hydroxytyrosol (but not tyrosol, was able to diminish the late sustained phase of H2O2-induced JNK and p38 phosphorylation. The derangement of intracellular iron homeostasis, following exposure of cells to H2O2, played pivotal role both in the induction of DNA damage and the initiation of apoptotic signaling. The presented results suggest that the protective effects exerted by ortho-dihydroxy group containing dietary compounds against oxidative stress-induced cell damage are linked to their ability to influence changes in the intracellular labile iron homeostasis.

  12. DNA polymerase-beta is expressed early in neurons of Alzheimer's disease brain and is loaded into DNA replication forks in neurons challenged with beta-amyloid

    NARCIS (Netherlands)

    A. Copani; J.J.M. Hoozemans; F. Caraci; M. Calafiore; E.S. van Haastert; R. Veerhuis; A.J.M. Rozemuller; E. Aronica; M.A. Sortino; F. Nicoletti

    2006-01-01

    Cultured neurons exposed to synthetic beta-amyloid (A beta) fragments reenter the cell cycle and initiate a pathway of DNA replication that involves the repair enzyme DNA polymerase-beta (DNA pol-beta) before undergoing apoptotic death. In this study, by performing coimmunoprecipitation experiments

  13. Chronic hepatitis B serum promotes apoptotic damage in human renal tubular cells

    Institute of Scientific and Technical Information of China (English)

    Cun-Liang Deng; Xin-Wen Song; Hai-Jun Liang; Chen Feng; Yun-Jian Sheng; Ming-Yong Wang

    2006-01-01

    AIM: To investigate the effect of the serum of patients with chronic hepatitis B (CHB) on apoptosis of renal tubular epithelial cells in vitro and to study the role of hepatitis B virus (HBV) and transforming growth factor-β1 (TGF-β1) in the pathogenesis of hepatitis B virus associated glomerulonephritis (HBV-GN).METHODS: The levels of serum TGF-β1 were measured by specific enzyme linked immunosorbent assay (ELISA) and HBV DNA was tested by polymerase chain reaction (PCR) in 44 patients with CHB ,and 20 healthy persons as the control. The normal human kidney proximal tubular cell (HK-2) was cultured together with the sera of healthy persons, CHB patients with HBV-DNA negative(20 cases) and HBV-DNA positive (24 cases) for up to 72 h. Apoptosis and Fas expression of the HK-2 were detected by flow cytometer.RESULTS: The apoptosis rate and Fas expression of HK-2 cells were significantly higher in HBV DNA positive serum group 19.01±5.85% and 17.58±8.35%, HBV DNA negative serum group 8.12±2.80% and 6.96 ± 2.76% than those in control group 4.25±0.65% and 2.33 ± 1.09%, respectively (P < 0.01). The apoptosis rate and Fas expression of HK-2 in HBV DNA positive serum group was significantly higher than those in HBV DNA negative serum (P < 0.01). Apoptosis rate of HK-2 cells in HBV DNA positive serum group was positively correlated with the level of HBV-DNA (r = 0.657). The level of serum TGF-β1 in CHB group was 163.05 ± 91.35 μg/L, significantly higher as compared with 81.40 ± 40.75 μg/L in the control group (P < 0.01).CONCLUSION: The serum of patients with chronic hepatitis B promotes apoptotic damage in human renal tubular cells by triggering a pathway of Fas up-regulation. HBV and TGF-β1 may play important roles in the mechanism of hepatitis B virus associated glomerulonephritis.

  14. Polysaccharide Degradation

    Science.gov (United States)

    Stone, Bruce A.; Svensson, Birte; Collins, Michelle E.; Rastall, Robert A.

    An overview of current and potential enzymes used to degrade polysaccharides is presented. Such depolymerases are comprised of glycoside hydrolases, glycosyl transferases, phosphorylases and lyases, and their classification, active sites and action patterns are discussed. Additionally, the mechanisms that these enzymes use to cleave glycosidic linkages is reviewed as are inhibitors of depolymerase activity; reagents which react with amino acid residues, glycoside derivatives, transition state inhibitors and proteinaceous inhibitors. The characterization of various enzymes of microbial, animal or plant origin has led to their widespread use in the production of important oligosaccharides which can be incorporated into food stuffs. Sources of polysaccharides of particular interest in this chapter are those from plants and include inulin, dextran, xylan and pectin, as their hydrolysis products are purported to be functional foods in the context of gastrointestinal health. An alternative use of degraded polysaccharides is in the treatment of disease. The possibility exists to treat bacterial exopolysaccharide with lyases from bacteriophage to produce oligosaccharides exhibiting bioactive sequences. Although this area is currently in its infancy the knowledge is available to investigate further.

  15. Effect of administration of apoptotic blebs on disease development in lupus mice.

    NARCIS (Netherlands)

    Fransen, J.H.; Berden, J.H.M.; Koeter, C.M.; Adema, G.J.; Vlag, J. van der; Hilbrands, L.B.

    2012-01-01

    INTRODUCTION: Systemic lupus erythematosus (SLE) is an autoimmune disease characterised by the formation of autoantibodies against nuclear components. Disturbed apoptosis and reduced clearance of apoptotic material have been assigned a role in the pathogenesis of SLE. During apoptosis, apoptotic ble

  16. Climacostol reduces tumour progression in a mouse model of melanoma via the p53-dependent intrinsic apoptotic programme

    Science.gov (United States)

    Perrotta, Cristiana; Buonanno, Federico; Zecchini, Silvia; Giavazzi, Alessio; Proietti Serafini, Francesca; Catalani, Elisabetta; Guerra, Laura; Belardinelli, Maria Cristina; Picchietti, Simona; Fausto, Anna Maria; Giorgi, Simone; Marcantoni, Enrico; Clementi, Emilio; Ortenzi, Claudio; Cervia, Davide

    2016-01-01

    Climacostol, a compound produced by the ciliated protozoan Climacostomum virens, displayed cytotoxic properties in vitro. This study demonstrates that it has anti-tumour potential. Climacostol caused a reduction of viability/proliferation of B16-F10 mouse melanoma cells, a rapidly occurring DNA damage, and induced the intrinsic apoptotic pathway characterised by the dissipation of the mitochondrial membrane potential, the translocation of Bax to the mitochondria, the release of Cytochrome c from the mitochondria, and the activation of Caspase 9-dependent cleavage of Caspase 3. The apoptotic mechanism of climacostol was found to rely on the up-regulation of p53 and its targets Noxa and Puma. In vivo analysis of B16-F10 allografts revealed a persistent inhibition of tumour growth rate when melanomas were treated with intra-tumoural injections of climacostol. In addition, it significantly improved the survival of transplanted mice, decreased tumour weight, induced a remarkable reduction of viable cells inside the tumour, activated apoptosis and up-regulated the p53 signalling network. Importantly, climacostol toxicity was more selective against tumour than non-tumour cells. The anti-tumour properties of climacostol and the molecular events associated with its action indicate that it is a powerful agent that may be considered for the design of pro-apoptotic drugs for melanoma therapy. PMID:27271364

  17. Evaluation of anti-apoptotic activity of different dietary antioxidants in renal cell carcinoma against hydrogen peroxide

    Institute of Scientific and Technical Information of China (English)

    Garg; Neeraj; K; Mangal; Sharad; Sahu; Tejram; Mehta; Abhinav; Vyas; Suresh; P; Tyagi; Rajeev; K

    2011-01-01

    Objective:To evaluate the anti-apoptotic and radical scavenging activities of dietary phenolics, namely ascorbic acid,a-tocopherol acetate,citric acid,salicylic acid,and estimate H2O2induced apoptosis in renal cell carcinoma cells.Methods:The intracellular antioxidant potency of antioxidants was investigated.H2O-2-induced apoptosis in RCC-26 was assayed with the following parameters:cell viability(%apoptosis),nucleosomal damage and DNA fragmentation, bcl-2 levels and flow cytometery analysis(ROS production evaluation).Results:Ine anticancer properties of antioxidants such as ascorbic acid,a- tocopherol acetate,citric acid,salicylic acid with perdurable responses were investigated.It was observed that these antioxidants had protective effect(anti-apoptotic activity) against hydrogen peroxide(H2O2) in renal cell carcinoma(RCC-26) cell line.Conclusions:This study reveals and proves the anticancer properties.However,in cancer cell lines anti-apoptotic activity can indirectly reflect the cancer promoter activity through radicals scavenging,and significantly protect nucleus and bcl-2.

  18. Elucidation of the potential disease-promoting influence of IgM apoptotic cell-reactive antibodies in lupus.

    Science.gov (United States)

    Malik, M; Arora, P; Sachdeva, R; Sharma, L; Ramachandran, V G; Pal, R

    2016-06-01

    The undigested remnants of apoptosis are believed to stimulate the generation of autoantibodies in lupus. The biological properties of initiator, disease-specific IgM antibodies that specifically recognize apoptotic cells, readily detected in the sera of lupus patients, remain unclear. Apoptotic cell-reactive IgM monoclonal antibodies (generated from lupus-prone mice), as opposed to control IgM, preferentially stimulated maturation of bone marrow-derived dendritic cells (BMDCs) derived from such mice, relative to BMDCs derived from healthy mice. An influence of both antibody specificity and cell genotype was also apparent in the secretion of signature inflammatory cytokines. Immunization of such antibodies in lupus-prone animals induced increases in total serum IgG levels, with the elicited antibodies also preferentially recognizing moieties on dying cells. An expanded specificity was apparent both upon Western blot on cellular lysate and from the enhanced recognition of dsDNA, Ro60, RNP68 and Sm; the antibody most efficient in mediating autoreactive diversity, while being germline encoded, also induced the highest degree of phenotypic changes on BMDCs. Apoptotic cell-reactive IgM antibodies may therefore be potentially capable of influencing the course of systemic autoimmune disease by affecting both innate and adaptive immunity.

  19. DNA damage and autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely [Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 (United States); Panayiotidis, Mihalis I. [School of Community Health Sciences, University of Nevada, Reno, NV 89557 (United States); Franco, Rodrigo, E-mail: rfrancocruz2@unl.edu [Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 (United States)

    2011-06-03

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  20. Apoptotic cell death and its relationship to gastric carcinogenesis

    Institute of Scientific and Technical Information of China (English)

    Ferda Bir; Nese Calli-Demirkan; A Cevik Tufan; Metin Akbulut; N Lale Satiroglu-Tufan

    2007-01-01

    AIM: To investigate the apoptotic process of cells within the intestinal metaplasia areas co-localizing with chronic gastritis and gastric carcinomas and to analyze the involvement of proteins regulating apoptosis in the process of intestinal metaplasia related gastric carcinogenesis.METHODS: Forty-two gastric carcinoma and seventeen chronic gastritis cases were included in this study. All cases were examined for the existence of intestinal metaplasia. Ten cases randomly selected from each group were processed for TUNEL assay. TUNEL positive cells within the intestinal metaplasia areas, colocalizing either to gastric carcinoma or chronic gastritis,were counted and converted to apoptotic indices.In addition, p53, bcl-2 and bax expression patterns within these tissues were analyzed on the basis of immunohistochemistry.RESULTS: Twenty-eight of the cases were intestinal and 14 of the cases were diffuse type adenocarcinomas.64% (27/42) of the gastric carcinoma cases had intestinal metaplasia. Intestinal metaplasia co-localized more with intestinal type carcinomas compared with diffuse type carcinomas [75% (21/28) vs 42% (6/14),respectively; P≤0.05]. The mean apoptotic index in tumor cells was 0.70±0.08. The mean apoptotic index in intestinal metaplasias co-localizing to tumors was significantly higher than that of intestinal metaplasias co-localizing to chronic gastritis (0.70±0.03 vs 0.09±0.01, respectively; P≤0.05). P53 positivity was not observed in areas of intestinal metaplasia adjacent to tumors or chronic gastritis. Intestinal metaplasia areas adjacent to tumors showed lower cytoplasmic bcl-2 positivity compared to intestinal metaplasia areas adjacent to chronic gastritis [55.5% (15/27) vs 70.5%(12/17), respectively]. On the other hand, intestinal metaplasia areas adjacent to tumors showed significantly higher cytoplasmic bax positivity compared to intestinal metaplasia areas adjacent to chronic gastritis [44.4%(12/27) vs 11.7% (2/17), respectively; P≤0

  1. Novel anti-apoptotic mechanism of A20 through targeting ASK1 to suppress TNF-induced JNK activation.

    Science.gov (United States)

    Won, M; Park, K A; Byun, H S; Sohn, K-C; Kim, Y-R; Jeon, J; Hong, J H; Park, J; Seok, J H; Kim, J M; Yoon, W-H; Jang, I-S; Shen, H M; Liu, Z G; Hur, G M

    2010-12-01

    The zinc-finger protein A20 has crucial physiological functions as a dual inhibitor of nuclear factor-κB (NF-κB) activation and apoptosis in tumor necrosis factor (TNF) receptor 1 signaling pathway. Although the molecular basis for the anti-NF-κB function of A20 has been well elucidated, the anti-apoptotic function of A20 is largely unknown. Here, we report a novel mechanism underlying the anti-apoptotic function of A20: A20 blocks TNF-induced apoptosis through suppression of c-jun N-terminal kinase (JNK) by targeting apoptosis signal-regulating kinase1 (ASK1). First, the ectopic expression of A20 drastically inhibits TNF-induced JNK activation and apoptosis in multiple cell types including those deficient of NF-κB activation. Unexpectedly, the blunting effect of A20 on TNF-induced JNK activation is not mediated by affecting the TNFR1 signaling complex formation. Instead, A20 interacts with ASK1, an important MAPKK kinase in the JNK signaling cascade. More importantly, overexpression of wild-type A20, but not of mutant A20 (ZnF4; C624A, C627A), promotes degradation of the ASK1 through the ubiquitin-proteasome system. Taken together, the results from this study reveal a novel anti-apoptotic mechanism of A20 in TNF signaling pathway: A20 binds to ASK1 and mediates ASK1 degradation, leading to suppression of JNK activation and eventually blockage of apoptosis.

  2. DNA damage in plant herbarium tissue.

    NARCIS (Netherlands)

    Staats, M.; Cuenca, A.; Richardson, J.E.; Ginkel, R.V.; Petersen, G.; Seberg, O.; Bakker, F.T.

    2011-01-01

    Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplif

  3. Apoptotic Cells Are Cleared by Directional Migration and elmo1-Dependent Macrophage Engulfment

    NARCIS (Netherlands)

    van Ham, Tjakko J.; Kokel, David; Peterson, Randall T.

    2012-01-01

    Apoptotic cell death is essential for development and tissue homeostasis [1, 2]. Failure to clear apoptotic cells can ultimately cause inflammation and autoimmunity [3, 4]. Apoptosis has primarily been studied by staining of fixed tissue sections, and a clear understanding of the behavior of apoptot

  4. A comparison of the apoptotic effect of Delta(9)-tetrahydrocannabinol in the neonatal and adult rat cerebral cortex.

    Science.gov (United States)

    Downer, Eric J; Gowran, Aoife; Campbell, Veronica A

    2007-10-17

    The maternal use of cannabis during pregnancy results in a number of cognitive deficits in the offspring that persist into adulthood. The endocannabinoid system has a role to play in neurodevelopmental processes such as neurogenesis, migration and synaptogenesis. However, exposure to phytocannabinoids, such as Delta(9)-tetrahydrocannabinol, during gestation may interfere with these events to cause abnormal patterns of neuronal wiring and subsequent cognitive impairments. Aberrant cell death evoked by Delta(9)-tetrahydrocannabinol may also contribute to cognitive deficits and in cultured neurones Delta(9)-tetrahydrocannabinol induces apoptosis via the CB(1) cannabinoid receptor. In this study we report that Delta(9)-tetrahydrocannabinol (5-50 microM) activates the stress-activated protein kinase, c-jun N-terminal kinase, and the pro-apoptotic protease, caspase-3, in in vitro cerebral cortical slices obtained from the neonatal rat brain. The proclivity of Delta(9)-tetrahydrocannabinol to impact on these pro-apoptotic signalling molecules was not observed in in vitro cortical slices obtained from the adult rat brain. In vivo, subcutaneous administration of Delta(9)-tetrahydrocannabinol (1-30 mg/kg) activated c-jun N-terminal kinase, caspase-3 and cathepsin-D, and induced DNA fragmentation in the cerebral cortex of neonatal rats. In contrast, in vivo administration of Delta(9)-tetrahydrocannabinol to adult rats was not associated with the apoptotic pathway in the cerebral cortex. The data provide evidence which supports the hypothesis that the neonatal rat brain is more vulnerable to the neurotoxic influence of Delta(9)-tetrahydrocannabinol, suggesting that the cognitive deficits that are observed in humans exposed to marijuana during gestation may be due, in part, to abnormal engagement of the apoptotic cascade during brain development.

  5. The inflammatory role of phagocyte apoptotic pathways in rheumatic diseases.

    Science.gov (United States)

    Cuda, Carla M; Pope, Richard M; Perlman, Harris

    2016-08-23

    Rheumatoid arthritis affects nearly 1% of the world's population and is a debilitating autoimmune condition that can result in joint destruction. During the past decade, inflammatory functions have been described for signalling molecules classically involved in apoptotic and non-apoptotic death pathways, including, but not limited to, Toll-like receptor signalling, inflammasome activation, cytokine production, macrophage polarization and antigen citrullination. In light of these remarkable advances in the understanding of inflammatory mechanisms of the death machinery, this Review provides a snapshot of the available evidence implicating death pathways, especially within the phagocyte populations of the innate immune system, in the perpetuation of rheumatoid arthritis and other rheumatic diseases. Elevated levels of signalling mediators of both extrinsic and intrinsic apoptosis, as well as the autophagy, are observed in the joints of patients with rheumatoid arthritis. Furthermore, risk polymorphisms are present in signalling molecules of the extrinsic apoptotic and autophagy death pathways. Although research into the mechanisms underlying these pathways has made considerable progress, this Review highlights areas where further investigation is particularly needed. This exploration is critical, as new discoveries in this field could lead to the development of novel therapies for rheumatoid arthritis and other rheumatic diseases.

  6. Targeting apoptotic pathways in myocardial infarction: attenuated by phytochemicals.

    Science.gov (United States)

    Haidarali, Shaikh; Patil, Chandragouda R; Ojha, Shreesh; Mohanraj, Rajesh; Arya, Dharamvir S; Goyal, Sameer N

    2014-01-01

    Myocardial infarction (MI) is an insidious disease, gently spreading in developed and developing countries. MI is the consequence of hypoxia in myocardial tissue, which may lead to apoptosis, narcosis and followed by cardiac cell death. Activation of apoptotic pathways during MI is frequently reported in clinical, preclinical and post-mortem studies. Several mediators of apoptosis signalling cascades culminate into MI leading to cardiomyocytes death. Such involvements of ischemia-induced apoptosis in MI are widely accepted. Apoptosis is a natural phenomenon for regulating the homeostasis in cellular organelles. Unlike the necrosis, it is a synchronized energy dependent process which is carried out by shrinkage of the cell. This contraction of cells leads to squeezing of nuclei and nuclear chromatin into brusquely demarcated masses. However, such programmed cell death in several tissues, including the myocardium becomes pathogenic under certain conditions. Moreover, reactive oxygen species (ROS) generated oxidative stress also plays a key role in production of apoptosis and several associated signalling alterations which ultimately lead to MI. Recently, certain natural products, especially from the plant kingdom have been evaluated for their anti-apoptotic potential. There is an uprise in the investigations delineating the exact mechanisms through which natural phytochemicals target apoptosis associated MI. This review explores novel signalling pathways and target sites for anti-apoptotic phytochemicals having potential to check the cellular apoptosis consequent to MI. A new vista may explore the prospective treatment of MI by using apoptosis-modulating natural products.

  7. Enhanced apoptotic response to photodynamic therapy after bcl-2 transfection.

    Science.gov (United States)

    Kim, H R; Luo, Y; Li, G; Kessel, D

    1999-07-15

    Apoptosis is a cellular death process involving the sequential activation of a series of caspases, endonucleases, and other enzymes. The initiation of apoptosis can be inhibited by overexpression of bcl-2 and certain other members of a related family of proteins. We examined the effects of bcl-2 overexpression on the apoptotic response to photodynamic therapy (PDT), using aluminum phthalocyanine as the photosensitizing agent. In this study, we compared the immortalized human breast epithelial cell line MCF10A with a subline (MCF10A/bcl-2) transfected with the human bcl-2 gene. The latter was approximately 2-fold more sensitive to the phototoxic effects of PDT. At a 50 mJ/cm2 light dose, photodamage to MCF-10A/bcl-2 resulted in a greater loss of the mitochondrial membrane potential (delta(psi)m), enhanced release of mitochondrial cytochrome c, a more rapid and greater activation of caspase-3, and a greater apoptotic response. Western blot analysis revealed that the transfected cell line showed overexpression of both bcl-2 and bax, and that PDT caused selective destruction of bcl-2, leaving bax unaffected. The greater apoptotic response by the transfected line is, therefore, attributed to the higher bax:bcl-2 ratio after photodamage.

  8. Apoptotic mimicry: an altruistic behavior in host/Leishmania interplay

    Directory of Open Access Journals (Sweden)

    Wanderley J.L.M.

    2005-01-01

    Full Text Available Apoptosis is the most common phenotype observed when cells die through programmed cell death. The morphologic and biochemical changes that characterize apoptotic cells depend on the activation of a diverse set of genes. Apoptosis is essential for multicellular organisms since their development and homeostasis are dependent on extensive cell renewal. In fact, there is strong evidence for the correlation between the emergence of multicellular organisms and apoptosis during evolution. On the other hand, no obvious advantages can be envisaged for unicellular organisms to carry the complex machinery required for programmed cell death. However, accumulating evidence shows that free-living and parasitic protozoa as well as yeasts display apoptotic markers. This phenomenon has been related to altruistic behavior, when a subpopulation of protozoa or yeasts dies by apoptosis, with clear benefits for the entire population. Recently, phosphatidylserine (PS exposure and its recognition by a specific receptor (PSR were implicated in the infectivity of amastigote forms of Leishmania, an obligatory vertebrate intramacrophagic parasite, showing for the first time that unicellular organisms use apoptotic features for the establishment and/or maintenance of infection. Here we focus on PS exposure in the outer leaflet of the plasma membrane - an early hallmark of apoptosis - and how it modulates the inflammatory activity of phagocytic cells. We also discuss the possible mechanisms by which PS exposure can define Leishmania survival inside host cells and the evolutionary implications of apoptosis at the unicellular level.

  9. Cell shape and organelle modification in apoptotic U937 cells

    Directory of Open Access Journals (Sweden)

    MR Montinari

    2009-12-01

    Full Text Available U937 cells induced to apoptosis, progressively and dramatically modified their cell shape by intense blebbing formation, leading to the production of apoptotic bodies. The blebs evolved with time; milder forms of blebbing involving only a region or just the cortical part of the cytoplasm were observed within the first hour of incubation with puromycin; blebbing involving the whole cell body with very deep constrictions is the most frequent event observed during late times of incubation. The ultrastructural analysis of apoptotic cells revealed characteristic features of nuclear fragmentation (budding and cleavage mode and cytoplasmatic modifications. The cytoplasm of blebs does not contain organelles, such as ribosomes or mitochondria. Scarce presence of endoplasmic reticulum can be observed at the site of bleb detachment. However, blebbing is a dispensable event as evaluated by using inhibitor of actin polymerization. In the present study, the progressive modifications of the nucleus, mitochondria, nuclear fragmentation, cytoplasmic blebs formation and production of apoptotic bodies in U937 monocytic cells induced to apoptosis by puromycin (an inhibitor of protein synthesis were simultaneously analyzed.

  10. Apoptotic cell signaling in cancer progression and therapy.

    Science.gov (United States)

    Plati, Jessica; Bucur, Octavian; Khosravi-Far, Roya

    2011-04-01

    Apoptosis is a tightly regulated cell suicide program that plays an essential role in the development and maintenance of tissue homeostasis by eliminating unnecessary or harmful cells. Impairment of this native defense mechanism promotes aberrant cellular proliferation and the accumulation of genetic defects, ultimately resulting in tumorigenesis, and frequently confers drug resistance to cancer cells. The regulation of apoptosis at several levels is essential to maintain the delicate balance between cellular survival and death signaling that is required to prevent disease. Complex networks of signaling pathways act to promote or inhibit apoptosis in response to various cues. Apoptosis can be triggered by signals from within the cell, such as genotoxic stress, or by extrinsic signals, such as the binding of ligands to cell surface death receptors. Various upstream signaling pathways can modulate apoptosis by converging on, and thereby altering the activity of, common central control points within the apoptotic signaling pathways, which involve the BCL-2 family proteins, inhibitor of apoptosis (IAP) proteins, and FLICE-inhibitory protein (c-FLIP). This review highlights the role of these fundamental regulators of apoptosis in the context of both normal apoptotic signaling mechanisms and dysregulated apoptotic pathways that can render cancer cells resistant to cell death. In addition, therapeutic strategies aimed at modulating the activity of BCL-2 family proteins, IAPs, and c-FLIP for the targeted induction of apoptosis are briefly discussed.

  11. Apoptotic cell signaling in cancer progression and therapy†

    Science.gov (United States)

    Plati, Jessica; Bucur, Octavian; Khosravi-Far, Roya

    2011-01-01

    Apoptosis is a tightly regulated cell suicide program that plays an essential role in the development and maintenance of tissue homeostasis by eliminating unnecessary or harmful cells. Impairment of this native defense mechanism promotes aberrant cellular proliferation and the accumulation of genetic defects, ultimately resulting in tumorigenesis, and frequently confers drug resistance to cancer cells. The regulation of apoptosis at several levels is essential to maintain the delicate balance between cellular survival and death signaling that is required to prevent disease. Complex networks of signaling pathways act to promote or inhibit apoptosis in response to various cues. Apoptosis can be triggered by signals from within the cell, such as genotoxic stress, or by extrinsic signals, such as the binding of ligands to cell surface death receptors. Various upstream signaling pathways can modulate apoptosis by converging on, and thereby altering the activity of, common central control points within the apoptotic signaling pathways, which involve the BCL-2 family proteins, inhibitor of apoptosis (IAP) proteins, and FLICE-inhibitory protein (c-FLIP). This review highlights the role of these fundamental regulators of apoptosis in the context of both normal apoptotic signaling mechanisms and dysregulated apoptotic pathways that can render cancer cells resistant to cell death. In addition, therapeutic strategies aimed at modulating the activity of BCL-2 family proteins, IAPs, and c-FLIP for the targeted induction of apoptosis are briefly discussed. PMID:21340093

  12. The RNA-binding protein TIAR is translocated from the nucleus to the cytoplasm during Fas-mediated apoptotic cell death.

    OpenAIRE

    Taupin, J L; Tian, Q.; Kedersha, N; Robertson, M.; Anderson, P

    1995-01-01

    We have determined the structure, intracellular localization, and tissue distribution of TIAR, a TIA-1-related RNA-binding protein. Two related isoforms of TIAR, migrating at 42 and 50 kDa, are expressed in primate cells. Unlike TIA-1, which is found in the granules of cytotoxic lymphocytes, TIAR is concentrated in the nucleus of hematopoietic and nonhematopoietic cells. Because TIAR can trigger DNA fragmentation in permeabilized thymocytes, it is a candidate effector of apoptotic cell death....

  13. Differential nitric oxide synthesis and host apoptotic events correlate with bleaching susceptibility in reef corals

    Science.gov (United States)

    Hawkins, T. D.; Krueger, T.; Becker, S.; Fisher, P. L.; Davy, S. K.

    2014-03-01

    Coral bleaching poses a threat to coral reefs worldwide. As a consequence of the temperature-induced breakdown in coral-dinoflagellate symbiosis, bleaching can have extensive effects on reef communities. However, our understanding of bleaching at a cellular level is limited, and this is particularly true regarding differential susceptibility among coral species. Recent work suggests that bleaching may represent a host innate immune-like response to symbiont dysfunction that involves synthesis of the signalling compound nitric oxide (NO) and the induction of host apoptotic-like cell death. In this study, we examined the activity of apoptosis-regulating enzymes alongside oxidised NO accumulation (a proxy for NO synthesis) in the reef corals Acropora millepora, Montipora digitata, and Pocillopora damicornis during experimental thermal stress. P. damicornis was the most sensitive species, suffering mortality (tissue sloughing) after 5 days at 33 °C but non-lethal bleaching after 9 days at 31.5 °C. A. millepora bleached at 33 °C but remained structurally intact, while M. digitata showed little evidence of bleaching. P. damicornis and A. millepora both exhibited evidence of temperature-induced NO synthesis and, after 5 days of heating, levels of oxidised NO in both species were fivefold higher than in controls maintained at 28.5 °C. These responses preceded bleaching by a number of days and may have occurred before symbiont dysfunction (measured as chlorophyll a degradation and oxidised NO accumulation). In A. millepora, apparent NO synthesis correlated with the induction of host apoptotic-like pathways, while in P. damicornis, the upregulation of apoptotic pathways occurred later. No evidence of elevated NO production or apoptosis was observed in M. digitata at 33 °C and baseline activity of apoptosis-regulating enzymes was negligible in this species. These findings provide important physiological data in the context of the responses of corals to global change and

  14. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  15. Anti-apoptotic signature in thymic squamous cell carcinomas - functional relevance of anti-apoptotic BIRC3 expression in the thymic carcinoma cell line 1889c

    Directory of Open Access Journals (Sweden)

    Bei eHuang

    2013-12-01

    Full Text Available The molecular pathogenesis of thymomas and thymic carcinomas (TCs is poorly understood and results of adjuvant therapy are unsatisfactory in case of metastatic disease and tumor recurrence. For these clinical settings, novel therapeutic strategies are urgently needed. Recently, limited sequencing efforts revealed that a broad spectrum of genes that play key roles in various common cancers are rarely affected in thymomas and thymic carcinomas, suggesting that other oncogenic principles might be important. This made us re-analyze historic expression data obtained in a spectrum of thymomas and thymic squamous cell carcinomas (TSCC with a custom made cDNA microarray. By cluster analysis, different anti-apoptotic signatures were detected in type B3 thymoma and TSCC, including overexpression of BIRC3 in TSCCs. This was confirmed by qRT-PCR in the original and an independent validation set of tumors. In contrast to several other cancer cell lines, the BIRC3-positive TSCC cell line, 1889c showed spontaneous apoptosis after BIRC3 knock-down. Targeting apoptosis genes is worth testing as therapeutic principle in TSCC.

  16. Regulation of apoptotic mediators reveals dynamic responses to thermal stress in the reef building coral Acropora millepora.

    Directory of Open Access Journals (Sweden)

    Mathieu Pernice

    Full Text Available BACKGROUND: Mass coral bleaching is increasing in scale and frequency across the world's coral reefs and is being driven primarily by increased levels of thermal stress arising from global warming. In order to understand the impacts of projected climate change upon corals reefs, it is important to elucidate the underlying cellular mechanisms that operate during coral bleaching and subsequent mortality. In this respect, increased apoptotic cell death activity is an important cellular process that is associated with the breakdown of the mutualistic symbiosis between the cnidarian host and their dinoflagellate symbionts. METHODOLOGY/PRINCIPAL FINDINGS: The PRESENT study reports the impacts of different stressors (colchicine and heat stress on three phases of apoptosis: (i the potential initiation by differential expression of Bcl-2 members, (ii the execution of apoptotic events by activation of caspase 3-like proteases and (iii and finally, the cell disposal indicated by DNA fragmentation in the reef building coral Acropora millepora. In corals incubated with colchicine, an increase in caspase 3-like activity and DNA fragmentation was associated with a relative down-regulation of Bcl-2, suggesting that the initiation of apoptosis may be mediated by the suppression of an anti-apoptotic mechanism. In contrast, in the early steps of heat stress, the induction of caspase-dependent apoptosis was related to a relative up-regulation of Bcl-2 consecutively followed by a delayed decrease in apoptosis activity. CONCLUSIONS/SIGNIFICANCE: In the light of these results, we propose a model of heat stress in coral hosts whereby increasing temperatures engage activation of caspase 3-dependent apoptosis in cells designated for termination, but also the onset of a delayed protective response involving overexpression of Bcl-2 in surviving cells. This mitigating response to thermal stress could conceivably be an important regulatory mechanism for cell survival in

  17. Apoptotic and necrotic brain lesions in a fatal case of carbon monoxide poisoning.

    Science.gov (United States)

    Uemura, K; Harada, K; Sadamitsu, D; Tsuruta, R; Takahashi, M; Aki, T; Yasuhara, M; Maekawa, T; Yoshida, K

    2001-02-15

    A 41-year-old man was accidentally exposed to carbon monoxide (CO) gas and found in a state of cardiopulmonary arrest while he took bath. After admission, he was resuscitated and underwent artificial ventilation in a comatose state and died about 19h later. Computed tomography (CT) examination disclosed bilateral low density area in the basal ganglia and the thalamus, a well-known finding in the CO intoxication. Necropsy, histological examination, DNA ladder assay gave the first line of evidence for the presence of apoptosis as well as necrosis in the human case of CO intoxication. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) positive apoptotic cells were more predominant in the CA2 area than in CA1 area. There is general co-relation between the ratio of TUNEL-positive cells and the DNA laddering on the agarose gel. Basal ganglia and thalamus, which showed bilateral low density area in CT, were revealed to be severe edema. The two types of cell death occurred in the cortex, basal ganglia, hippocampus, thalamus, and cerebellum. Hypoxia caused by CO-hemoglobin formation alone cannot explain the phenomena.

  18. Miltefosine-induced apoptotic cell death on Leishmania major and L. tropica strains.

    Science.gov (United States)

    Khademvatan, Shahram; Gharavi, Mohammad Javad; Rahim, Fakher; Saki, Jasem

    2011-03-01

    The aim of this study was to assess the cytotoxic effects of various concentrations of miltefosine on Leishmania major (MRHO/IR/75/ER) and L. tropica (MHOM/IR/02/Mash10) promastigotes and to observe the programmed cell death features. The colorimetric MTT assay was used to find L. major and L. tropica viability and the obtained results were expressed as 50% inhibitory concentration (IC50). Also, 50% effective doses (ED50) for L. major and L. tropica amastigotes were also determined. Annexin-V FLUOS staining was performed to study the cell death properties of miltefosine using FACS analysis. Qualitative analysis of the total genomic DNA fragmentation was performed by agarose gel electrophoresis. Furthermore, to observe changes in cell morphology, promastigotes were examined using light microscopy. In both strains of L. major and L. tropica, miltefosine induced dose-dependent death with features of apoptosis, including cell shrinkage, DNA laddering, and externalization of phosphatidylserine. The IC50 was achieved at 22 µM and 11 µM for L. major and L. tropica after 48 hr of incubation, respectively. ED50 of L. major and L. tropica amastigotes were 5.7 µM and 4.2 µM, respectively. Our results indicate that miltefosine induces apoptosis of the causative agent of cutaneous leishmaniasis in a dose-dependent manner. Interestingly, L. major did not display any apoptotic changes when it was exposed to miltefosine in concentrations sufficient to kill L. tropica.

  19. The C. elegans apoptotic nuclease NUC-1 is related in sequence and activity to mammalian DNase II.

    Science.gov (United States)

    Lyon, C J; Evans, C J; Bill, B R; Otsuka, A J; Aguilera, R J

    2000-07-11

    The Caenorhabditis elegans nuc-1 gene has previously been implicated in programmed cell death due to the presence of persistent undegraded apoptotic DNA in nuc-1 mutant animals. In this report, we describe the cloning and characterization of nuc-1, which encodes an acidic nuclease with significant sequence similarity to mammalian DNase II. Database searches performed with human DNase II protein sequence revealed a significant similarity with the predicted C. elegans C07B5.5 ORF. Subsequent analysis of crude C. elegans protein extracts revealed that wild-type animals contained a potent endonuclease activity with a cleavage preference similar to DNase II, while nuc-1 mutant worms demonstrated a marked reduction in this nuclease activity. Sequence analysis of C07B5.5 DNA and mRNA also revealed that nuc-1(e1392), but not wild-type animals contained a nonsense mutation within the CO7B5.5 coding region. Furthermore, nuc-1 transgenic lines carrying the wild-type C07B5.5 locus demonstrated a complete complementation of the nuc-1 mutant phenotype. Our results therefore provide compelling evidence that the C07B5.5 gene encodes the NUC-1 apoptotic nuclease and that this nuclease is related in sequence and activity to DNase II.

  20. Cleaved PARP-1, an Apoptotic Marker, can be Detected in Ram Spermatozoa.

    Science.gov (United States)

    Casao, A; Mata-Campuzano, M; Ordás, L; Cebrián-Pérez, J A; Muiño-Blanco, T; Martínez-Pastor, F

    2015-08-01

    The presence of apoptotic features in spermatozoa has been related to lower quality and functional impairment. Members of the poly-ADP-ribose polymerases (PARP) familyare involved in both DNA repair and apoptosis, playing important roles in spermatogenesis. Poly-ADP-ribose polymerase can be cleaved by caspases, and the presence of its cleavage product (cPARP) in spermatozoa has been related to chromatin remodelling during spermatogenesis and to the activation of apoptotic pathways. There are no reports on immunodetection of cPARP in ram spermatozoa; thus, we have tested a commercially available antibody for this purpose. cPARP was microscopically detected in the acrosomal ridge of some spermatozoa (indirect immunofluorescence). A preliminary study was carried out by flow cytometry (direct immunofluorescence, FITC). Ram semen was extended in TALP and incubated for 4 h with apoptosis inducers staurosporine (10 μm) or betulinic acid (200 μm). Both inducers and incubation caused a significant increase in cPARP spermatozoa (0 h, control: 21.4±3.3%, inducers: 44.3±1.4%; 4 h, control: 44.3±2.4%, inducers: 53.3±1.4%). In a second experiment, we compared the sperm fractions after density gradient separation (pellet and interface). The pellet yielded a slightly lower proportion of cPARP spermatozoa (28.5±1.2% vs 36.2±2.0% in the interface; p ram semen, although its presence in untreated samples was weakly related to worse quality (pellet/interface). We suggest to study the relationship of PARP and cPARP levels with between-male differences on sperm fertility.

  1. Apoptosis and DNA damage in human spermatozoa

    Institute of Scientific and Technical Information of China (English)

    R John Aitken; Adam J Koppers

    2011-01-01

    DNA damage is frequently encountered in spermatozoa of subfertile males and is correlated with a range of adverse clinical outcomes including impaired fertilization, disrupted preimplantation embryonic development, increased rates of miscarriage and an enhanced risk of disease in the progeny. The etiology of DNA fragmentation in human spermatozoa is closely correlated with the appearance of oxidative base adducts and evidence of impaired spermiogenesis. We hypothesize that oxidative stress impedes spermiogenesis,resulting in the generation of spermatozoa with poorly remodelled chromatin. These defective cells have a tendency to default to an apoptotic pathway associated with motility loss, caspase activation, phosphatidylserine exteriorization and the activation of free radical generation by the mitochondria. The latter induces lipid peroxidation and oxidative DNA damage, which then leads to DNA fragmentation and cell death. The physical architecture of spermatozoa prevents any nucleases activated as a result of this apoptotic process from gaining access to the nuclear DNA and inducing its fragmentation. It is for this reason that a majority of the DNA damage encountered in human spermatozoa seems to be oxidative. Given the important role that oxidative stress seems to have in the etiology of DNA damage, there should be an important role for antioxidants in the treatment of this condition. If oxidative DNA damage in spermatozoa is providing a sensitive readout of systemic oxidative stress, the implications of these findings could stretch beyond our immediate goal of trying to minimize DNA damage in spermatozoa as a prelude to assisted conception therapy.

  2. Degradation of endocrine disrupting chemicals by genetic transformants with two lignin degrading enzymes in Phlebia tremellosa.

    Science.gov (United States)

    Kum, Hyunwoo; Lee, Sungsuk; Ryu, Sunhwa; Choi, Hyoung T

    2011-10-01

    A white rot fungus Phlebia tremellosa produced lignin degrading enzymes, which showed degrading activity against various recalcitrant compounds. However, manganese peroxidase (MnP) activity, one of lignin degrading enzymes, was very low in this fungus under various culture conditions. An expression vector that carried both the laccase and MnP genes was constructed using laccase genomic DNA of P. tremellosa and MnP cDNA from Polyporus brumalis. P. tremellosa was genetically transformed using the expression vector to obtain fungal transformants showing increased laccase and MnP activity. Many transformants showed highly increased laccase and MnP activity at the same time in liquid medium, and three of them were used to degrade endocrine disrupting chemicals. The transformant not only degraded bisphenol A and nonylphenol more rapidly but also removed the estrogenic activities of the chemicals faster than the wild type strain.

  3. Echinacoside induces apoptotic cancer cell death by inhibiting the nucleotide pool sanitizing enzyme MTH1

    Directory of Open Access Journals (Sweden)

    Dong L

    2015-12-01

    Full Text Available Liwei Dong,1 Hongge Wang,1 Jiajing Niu,1 Mingwei Zou,2 Nuoting Wu,1 Debin Yu,1 Ye Wang,1 Zhihua Zou11Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin Province, People’s Republic of China; 2Department of Psychology, College of Liberal Arts and Social Sciences, University of Houston, Houston, TX, USA Abstract: Inhibition of the nucleotide pool sanitizing enzyme MTH1 causes extensive oxidative DNA damages and apoptosis in cancer cells and hence may be used as an anticancer strategy. As natural products have been a rich source of medicinal chemicals, in the present study, we used the MTH1-catalyzed enzymatic reaction as a high-throughput in vitro screening assay to search for natural compounds capable of inhibiting MTH1. Echinacoside, a compound derived from the medicinal plants Cistanche and Echinacea, effectively inhibited the catalytic activity of MTH1 in an in vitro assay. Treatment of various human cancer cell lines with Echinacoside resulted in a significant increase in the cellular level of oxidized guanine (8-oxoguanine, while cellular reactive oxygen species level remained unchanged, indicating that Echinacoside also inhibited the activity of cellular MTH1. Consequently, Echinacoside treatment induced an immediate and dramatic increase in DNA damage markers and upregulation of the G1/S-CDK inhibitor p21, which were followed by marked apoptotic cell death and cell cycle arrest in cancer but not in noncancer cells. Taken together, these studies identified a natural compound as an MTH1 inhibitor and suggest that natural products can be an important source of anticancer agents. Keywords: Echinacoside, MTH1, 8-oxoG, DNA damage, apoptosis, cell cycle arrest

  4. Development of novel cyclic peptides as pro-apoptotic agents.

    Science.gov (United States)

    Brindisi, Margherita; Maramai, Samuele; Brogi, Simone; Fanigliulo, Emanuela; Butini, Stefania; Guarino, Egeria; Casagni, Alice; Lamponi, Stefania; Bonechi, Claudia; Nathwani, Seema M; Finetti, Federica; Ragonese, Francesco; Arcidiacono, Paola; Campiglia, Pietro; Valenti, Salvatore; Novellino, Ettore; Spaccapelo, Roberta; Morbidelli, Lucia; Zisterer, Daniela M; Williams, Clive D; Donati, Alessandro; Baldari, Cosima; Campiani, Giuseppe; Ulivieri, Cristina; Gemma, Sandra

    2016-07-19

    Our recent finding that paclitaxel behaves as a peptidomimetic of the endogenous protein Nur77 inspired the design of two peptides (PEP1 and PEP2) reproducing the effects of paclitaxel on Bcl-2 and tubulin, proving the peptidomimetic nature of paclitaxel. Starting from these peptide-hits, we herein describe the synthesis and the biological investigation of linear and cyclic peptides structurally related to PEP2. While linear peptides (2a,b, 3a,b, 4, 6a-f) were found inactive in cell-based assays, biological analysis revealed a pro-apoptotic effect for most of the cyclic peptides (5a-g). Cellular permeability of 5a (and also of 2a,b) on HL60 cells was assessed through confocal microscopy analysis. Further cellular studies on a panel of leukemic cell lines (HL60, Jurkat, MEC, EBVB) and solid tumor cell lines (breast cancer MCF-7 cells, human melanoma A375 and 501Mel cells, and murine melanoma B16F1 cells) confirmed the pro-apoptotic effect of the cyclic peptides. Cell cycle analysis revealed that treatment with 5a, 5c, 5d or 5f resulted in an increase in the number of cells in the sub-G0/G1 peak. Direct interaction with tubulin (turbidimetric assay) and with microtubules (immunostaining experiments) was assessed in vitro for the most promising compounds.

  5. Apoptotic and nonapoptotic function of caspase 7 in spermatogenesis.

    Science.gov (United States)

    Lei, Bin; Zhou, Xuming; Lv, Daojun; Wan, Bo; Wu, Huayan; Zhong, Liren; Shu, Fangpeng; Mao, Xiangming

    2017-01-01

    Recent studies have reported that caspase 7 has an apoptotic and nonapoptotic function. However, the relationship between caspase 7 and spermatogenesis remains unknown. This study aimed to investigate the possible function of caspase 7 during normal and abnormal spermatogenesis. The cleaved form of caspase 7 was detected in testis tissues at different postpartum times (5-14 weeks) by qRT-PCR, Western blot and immunohistochemistry (IHC). Then, the mice models of spermatogenic dysfunction were obtained by busulfan (30 mg kg-1 to further evaluate the potential function and mechanism of caspase 7. qRT-PCR and Western blot results showed that caspase 7 expression was gradually elevated from 5 to 14 weeks, which was not connected with apoptosis. IHC results revealed that caspase 7 was mainly located in spermatogenic cells and Leydig cells. In addition, spermatogenic dysfunction induced by busulfan gradually enhanced the apoptosis and elevated the expression of caspase 3, caspase 6, and caspase 9, but decreased the expression of caspase 7 in spermatogenic cells. However, when spermatogenic cells were mostly disappeared at the fourth week after busulfan treatment, caspase 7 expression in Leydig cells was significantly increased and positively correlated with the expression of caspase 3, caspase 6, and caspase 9. Therefore, these results indicate that caspase 7 has a nonapoptic function that participates in normal spermatogenesis, but also displays apoptotic function in spermatogenic dysfunction.

  6. Interaction of late apoptotic and necrotic cells with vitronectin.

    Directory of Open Access Journals (Sweden)

    Ondrej Stepanek

    Full Text Available BACKGROUND: Vitronectin is an abundant plasma glycoprotein identified also as a part of extracellular matrix. Vitronectin is substantially enriched at sites of injured, fibrosing, inflamed, and tumor tissues where it is believed to be involved in wound healing and tissue remodeling. Little is known about the mechanism of vitronectin localization into the damaged tissues. METHODOLOGY/PRINCIPAL FINDINGS: 2E12 antibody has been described to bind a subset of late apoptotic cells. Using immunoisolation followed by mass spectrometry, we identified the antigen recognized by 2E12 antibody as vitronectin. Based on flow cytometry, we described that vitronectin binds to the late apoptotic and necrotic cells in cell cultures in vitro as well as in murine thymus and spleen in vivo. Confocal microscopy revealed that vitronectin binds to an intracellular cytoplasmic structure after the membrane rupture. CONCLUSIONS/SIGNIFICANCE: We propose that vitronectin could serve as a marker of membrane disruption in necrosis and apoptosis for flow cytometry analysis. Moreover, we suggest that vitronectin binding to dead cells may represent one of the mechanisms of vitronectin incorporation into the injured tissues.

  7. PUMA-mediated mitochondrial apoptotic disruption by hypoxic postconditioning.

    Science.gov (United States)

    Li, YuZhen; Guo, Qi; Liu, XiuHua; Wang, Chen; Song, DanDan

    2015-08-01

    Postconditioning can reduce ischemia-reperfusion (I/R)-induced cardiomyocyte apoptosis by targeting mitochondria. p53 upregulated modulator of apoptosis (PUMA) is involved in lethal I/R injury. Here, we hypothesized that postconditioning might inhibit mitochondrial pathway-mediated cardiomyocyte apoptosis by controlling PUMA expression. The cultured neonatal rat cardiomyocytes underwent 3 h of hypoxia and 3 h of reoxygenation. Postconditioning consisted of three cycles of 5 min reoxygenation and 5 min hypoxia after prolonged hypoxia. Hypoxic postconditioning reduced the levels of PUMA mRNA and protein. Concomitantly, the loss of mitochondrial membrane potential, cytochrome c release and caspase-3 activation were decreased significantly by postconditioning. Overexpression of PUMA increased greatly not only the number of apoptotic cardiomyocytes, but also the collapse of mitochondrial membrane potential, cytochrome c release and caspase-3 activation under postconditioning condition. The data suggest that reduction of PUMA expression mediates the endogenous cardioprotective mechanisms of postconditioning by disrupting mitochondrial apoptotic pathway.

  8. Anti-apoptotic action of API2-MALT1 fusion protein involved in t(11;18)(q21;q21) MALT lymphoma.

    Science.gov (United States)

    Hosokawa, Y

    2005-01-01

    At least three distinct chromosomal translocations, t(11;18)(q21;q21), t(1;14)(p22;q32) and t(14;18)(q32;q21) involving the API2 (also known as c-IAP2)-MALT1 fusion protein, BCL10, and MALT1, respectively, have been implicated in the molecular pathogenesis of mucosa associated lymphoid tissue (MALT) lymphoma. Our findings showed that several variants of the API2-MALT1 fusion protein can occur in patients with t(11;18)(q21;q21), and that API2-MALT1 can potently enfance activation of nuclear factor (NF)-kappaB signaling, which may be relevant to the pathogenesis of MALT lymphomas. We also found that MALT1 is rapidly degraded via the ubiquitin-proteasome pathway, as is the case with API2, but upon the synthesis of fusion, API2-MALT1 becomes stable against this pathway. This stability of API2-MALT1 may thus result in inappropriate nuclear factor (NF)-kappaB activation, thereby contributing to the pathogenesis of MALT lymphoma. Recent biochemical and genetic studies have clearly shown that BCL10 and MALT1 form a physical and functional complex and are both required for NF-kappaB activation by antigen receptor stimulation in T and B lymphocytes. It has also been shown that CARMA1, a newly discovered member of the membrane-associated guanylate kinase (MAGUK) families, is critical for antigen receptor-stimulated NF-kappaB activation. It can be assumed that API2-MALT1 can bypass this normal BCL10/MALT1 cellular signaling pathway linked to NF-kappaB activation, thereby inducing antigen receptor-independent proliferation of lymphocytes. Furthermore, BCL10/MALT1- and API2-MALT1-induced NF-kappaB activation may contribute to anti-apoptotic action probably through NF-kappaB-mediated upregulation of apoptotic inhibitor genes. We recently provided direct evidence that API2-MALT1 indeed exerts anti-apoptotic action, in part, through its direct interaction with apoptotic regulators including Smac. Taken together, these findings prompt us to hypothesize that the anti-apoptotic action

  9. Degradation Effect of Gossypol on the Lymphocyte Apoptosis and Nucleic Acid of Duolang Sheep%棉酚对多浪羊淋巴细胞凋亡及其对核酸降解的研究

    Institute of Scientific and Technical Information of China (English)

    刘书东; 左文斌; 贺艳艳; 潘辉; 徐丽君; 顾海洋; 杨威华; 李莲瑞

    2012-01-01

    In this study, the effects of gossypol on Duolang sheep lymphocyte apoptosis and degradation of nucleic acid on were studied by culturing lymphocytes in RPMI 1640 medium, Wright-Giemsa staining, DNA and RNA gel electrophoresis. The results showed that; lymphocyte appeared apoptotic bodies after Wright-Giemsa stained for 4 h; DNA and RNA were detected by Agarose Gel Electrophoresis. The DNA agarose gel electrophoresis showed the typical DNA ladder of apoptosis. The RNA agarose gel electrophoresis appeared 18S rRNA began to degrade at 4th hours. It was concluded that after treated by 1. 35μmol/L of gossypol for 4 h, the lymphocytes could inhibit lymphocyte proliferation of Duolang sheep , and promote apoptosis and degradation of nucleic acid.%主要通过使用RPMI 1640培养基培养淋巴细胞、瑞氏一吉姆萨染色、DNA和RNA电泳等方法来研究棉酚对多浪羊淋巴细胞的凋亡效应和对淋巴细胞核酸影响.研究结果表明,经瑞氏一吉姆萨染色4h淋巴细胞出现凋亡小体;DNA和RNA通过琼脂糖凝胶电泳检测在4h分别出现梯状条带和18S降解.可见,当细胞培养液中棉酚浓度为1.35 umol/L,培养淋巴细胞4h后,可抑制多浪羊淋巴细胞的增殖,并且促进凋亡及其核酸的降解.

  10. Allograft tolerance induced by donor apoptotic lymphocytes requires phagocytosis in the recipient

    Science.gov (United States)

    Sun, E.; Gao, Y.; Chen, J.; Roberts, A. I.; Wang, X.; Chen, Z.; Shi, Y.

    2004-01-01

    Cell death through apoptosis plays a critical role in regulating cellular homeostasis. Whether the disposal of apoptotic cells through phagocytosis can actively induce immune tolerance in vivo, however, remains controversial. Here, we report in a rat model that without using immunosuppressants, transfusion of apoptotic splenocytes from the donor strain prior to transplant dramatically prolonged survival of heart allografts. Histological analysis verified that rejection signs were significantly ameliorated. Splenocytes from rats transfused with donor apoptotic cells showed a dramatically decreased response to donor lymphocyte stimulation. Most importantly, blockade of phagocytosis in vivo, either with gadolinium chloride to disrupt phagocyte function or with annexin V to block binding of exposed phosphotidylserine to its receptor on phagocytes, abolished the beneficial effect of transfused apoptotic cells on heart allograft survival. Our results demonstrate that donor apoptotic cells promote specific allograft acceptance and that phagocytosis of apoptotic cells in vivo plays a crucial role in maintaining immune tolerance.

  11. Quantification of human mitochondrial DNA using synthesized DNA standards.

    Science.gov (United States)

    Kavlick, Mark F; Lawrence, Helen S; Merritt, R Travis; Fisher, Constance; Isenberg, Alice; Robertson, James M; Budowle, Bruce

    2011-11-01

    Successful mitochondrial DNA (mtDNA) forensic analysis depends on sufficient quantity and quality of mtDNA. A real-time quantitative PCR assay was developed to assess such characteristics in a DNA sample, which utilizes a duplex, synthetic DNA to ensure optimal quality assurance and quality control. The assay's 105-base pair target sequence facilitates amplification of degraded DNA and is minimally homologous to nonhuman mtDNA. The primers and probe hybridize to a region that has relatively few sequence polymorphisms. The assay can also identify the presence of PCR inhibitors and thus indicate the need for sample repurification. The results show that the assay provides information down to 10 copies and provides a dynamic range spanning seven orders of magnitude. Additional experiments demonstrated that as few as 300 mtDNA copies resulted in successful hypervariable region amplification, information that permits sample conservation and optimized downstream PCR testing. The assay described is rapid, reliable, and robust.

  12. A chalcone-related small molecule that induces methuosis, a novel form of non-apoptotic cell death, in glioblastoma cells

    Directory of Open Access Journals (Sweden)

    Bhanot Haymanti

    2011-06-01

    Full Text Available Abstract Background Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Results Here we describe a novel chalcone-like molecule, 3-(2-methyl-1H- indol-3-yl-1-(4-pyridinyl-2-propen-1-one (MIPP that induces cell death with the hallmarks of methuosis. MIPP causes rapid accumulation of vacuoles derived from macropinosomes, based on time-lapse microscopy and labeling with extracellular fluid phase tracers. Vacuolization can be blocked by the cholesterol-interacting compound, filipin, consistent with the origin of the vacuoles from non-clathrin endocytic compartments. Although the vacuoles rapidly acquire some characteristics of late endosomes (Rab7, LAMP1, they remain distinct from lysosomal and autophagosomal compartments, suggestive of a block at the late endosome/lysosome boundary. MIPP appears to target steps in the endosomal trafficking pathway involving Rab5 and Rab7, as evidenced by changes in the activation states of these GTPases. These effects are specific, as other GTPases (Rac1, Arf6 are unaffected by the compound. Cells treated with MIPP lose viability within 2-3 days, but their nuclei show no evidence of apoptotic changes. Inhibition of caspase activity does not protect the cells, consistent with a non-apoptotic death mechanism. U251 glioblastoma cells selected for temozolomide resistance showed sensitivity to MIPP-induced methuosis that was comparable to the parental cell line. Conclusions MIPP might serve as a prototype for new drugs that could be used to induce non-apoptotic death in cancers that have become refractory to agents that work through DNA damage and apoptotic mechanisms.

  13. Damage and repair of ancient DNA

    DEFF Research Database (Denmark)

    Mitchell, David; Willerslev, Eske; Hansen, Anders

    2005-01-01

    , and extensive degradation. In the course of this review, we will discuss the current aDNA literature describing the importance of aDNA studies as they relate to important biological questions and the difficulties associated with extracting useful information from highly degraded and damaged substrates derived......Under certain conditions small amounts of DNA can survive for long periods of time and can be used as polymerase chain reaction (PCR) substrates for the study of phylogenetic relationships and population genetics of extinct plants and animals, including hominids. Because of extensive DNA...... degradation, these studies are limited to species that lived within the past 10(4)-10(5) years (Late Pleistocene), although DNA sequences from 10(6) years have been reported. Ancient DNA (aDNA) has been used to study phylogenetic relationships of protists, fungi, algae, plants, and higher eukaryotes...

  14. Phosphorylation of Puma modulates its apoptotic function by regulating protein stability

    OpenAIRE

    M. Fricker; O'Prey, J; Tolkovsky, A. M.; Ryan, K.M.

    2010-01-01

    Puma is a potent BH3-only protein that antagonises anti-apoptotic Bcl-2 proteins, promotes Bax/Bak activation and has an essential role in multiple apoptotic models. Puma expression is normally kept very low, but can be induced by several transcription factors including p53, p73, E2F1 and FOXO3a, whereby it can induce an apoptotic response. As Puma can to bind and inactivate all anti-apoptotic members of the Bcl-2 family, its activity must be tightly controlled. We report here, for the first ...

  15. Regulation of T lymphocyte apoptotic markers is associated to cell activation during the acute phase of dengue.

    Science.gov (United States)

    Torrentes-Carvalho, Amanda; Marinho, Cintia Ferreira; de Oliveira-Pinto, Luzia Maria; de Oliveira, Débora Batista; Damasco, Paulo Vieira; Cunha, Rivaldo Venâncio; de Souza, Luiz José; de Azeredo, Elzinandes Leal; Kubelka, Claire Fernandes

    2014-05-01

    Dengue fever, a public health problem in Brazil, may present severe clinical manifestations as result of an increased vascular permeability and coagulation disorders. T cell activation is a critical event for an effective immune response against infection, including the production of cytokines. We aim to reveal mechanisms that modulate the virus-cell interaction, with an emphasis on cell death. Apoptosis is involved in lymphocyte homeostasis, contributes to the clearance of virus-infected cells but also may play a role in the pathogenesis. Phosphatidylserine exposure on CD8T lymphocytes from dengue patients support early apoptotic processes and loss of genomic integrity, observed by DNA fragmentation in T lymphocytes and indicating late apoptosis. These T cells express activation and cytotoxic phenotypes as revealed by CD29 and CD107a upregulation. Higher frequencies of CD95 were detected in T lymphocytes mainly in those with the cytotoxic profile (CD107a+) and lower levels of anti-apoptotic molecule Bcl-2, suggesting that both CD4+ and CD8+ T cell subsets are more susceptible to apoptosis during acute dengue. The analysis of apoptosis-related protein expression profile showed that not only molecules with pro- but also those with anti-apoptotic functions are overexpressed, indicating that survival mechanisms could be possibly protecting cells against apoptosis caused by viral, immune, oxidative and/or genotoxic stresses. These observations led us to propose that in dengue patients there is an association between T cell susceptibility to apoptosis and the activation state. The mechanisms for understanding the immunopathogenesis during dengue infection are discussed.

  16. The plant defensin NaD1 induces tumor cell death via a non-apoptotic, membranolytic process.

    Science.gov (United States)

    Baxter, Amy A; Poon, Ivan Kh; Hulett, Mark D

    2017-01-01

    Cationic anti-microbial peptides (CAPs) have an important role in host innate defense against pathogens such as bacteria and fungi. Many CAPs including defensins also exhibit selective cytotoxic activity towards mammalian cells via both apoptotic and non-apoptotic processes, and are being investigated as potential anticancer agents. The anti-fungal plant defensin from ornamental tobacco, Nicotiana alata Defensin 1 (NaD1), was recently shown to induce necrotic-like cell death in a number of tumor cell types within 30 min of treatment, at a concentration of 10 μM. NaD1-mediated cell killing within these experimental parameters has been shown to occur via binding to the plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) in target cells to facilitate membrane destabilization and subsequent lysis. Whether NaD1 is also capable of inducing apoptosis in tumor cells has not been reported previously. In this study, treatment of MM170 (melanoma) and Jurkat T (leukemia) cells with subacute (CAPs that have been shown to induce apoptosis through caspase activation, dying cells were not sensitive to a pancaspase inhibitor nor did they display caspase activity or DNA fragmentation over the 24 h treatment time. Furthermore, over the 24 h period, cells exhibited necrotic phenotypes and succumbed to membrane permeabilization. These results indicate that the cytotoxic mechanism of NaD1 at subacute concentrations is membranolytic rather than apoptotic and is also likely to be mediated through a PIP2-targeting cell lytic pathway.

  17. PEGylated apoptotic protein-loaded PLGA microspheres for cancer therapy

    Directory of Open Access Journals (Sweden)

    Byeon HJ

    2015-01-01

    Full Text Available Hyeong Jun Byeon,1 Insoo Kim,1 Ji Su Choi,1 Eun Seong Lee,2 Beom Soo Shin,3 Yu Seok Youn11Department of Pharmaceutical Sciences, School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea; 2Division of Biotechnology, The Catholic University of Korea, Bucheon-si, Republic of Korea; 3Department of Pharmacy, College of Pharmacy, Catholic University of Daegu, Gyeongsan-si, Republic of KoreaAbstract: The aim of the current study was to investigate the antitumor potential of poly(D,L-lactic-co-glycolic acid microspheres (PLGA MSs containing polyethylene glycol (PEG-conjugated (PEGylated tumor necrosis factor–related apoptosis-inducing ligand (PEG-TRAIL. PEG-TRAIL PLGA MSs were prepared by using a water-in-oil-in-water double-emulsion method, and the apoptotic activities of supernatants released from the PLGA MSs at days 1, 3, and 7 were examined. The antitumor effect caused by PEG-TRAIL PLGA MSs was evaluated in pancreatic Mia Paca-2 cell-xenografted mice. PEG-TRAIL PLGA MS was found to be spherical and 14.4±1.06 µm in size, and its encapsulation efficiency was significantly greater than that of TRAIL MS (85.7%±4.1% vs 43.3%±10.9%, respectively. The PLGA MS gradually released PEG-TRAIL for 14 days, and the released PEG-TRAIL was shown to have clear apoptotic activity in Mia Paca-2 cells, whereas TRAIL released after 1 day had a negligible activity. Finally, PEG-TRAIL PLGA MS displayed remarkably greater antitumor efficacy than blank or TRAIL PLGA MS in Mia Paca-2 cell-xenografted mice in terms of tumor volume and weight, apparently due to increased stability and well-retained apoptotic activity of PEG-TRAIL in PLGA MS. We believe that this PLGA MS system, combined with PEG-TRAIL, should be considered a promising candidate for treating pancreatic cancer.Keywords: Poly(D,L-lactic-co-glycolic acid, controlled release, PEGylation, TRAIL, pancreatic cancer

  18. Ca2+, Mg2+-dependent DNase involvement in apoptotic effects in spermatozoa of sea urchin Strongylocentrotus intermedius induced by two-headed sphingolipid rhizochalin.

    Science.gov (United States)

    Sibirtsev, Juriy T; Shastina, Valeria V; Menzorova, Natalia I; Makarieva, Tatyana N; Rasskazov, Valeriy A

    2011-06-01

    Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca(2+), Mg(2+)-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn(2+) blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.

  19. In vivo DNA damaging and apoptotic potential of silver nanoparticles in Swiss albino mice

    OpenAIRE

    Al Gurabi MA; Ali D; Alkahtani S; Alarifi S

    2015-01-01

    Mohammed A Al Gurabi, Daoud Ali, Saad Alkahtani, Saud Alarifi Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia Abstract: Nanoparticles can potentially cause adverse effects on organs, tissue, cell levels, and protein levels because of their physicochemical properties. Silver nanoparticles (AgNPs) are being used on a wide scale in world consumer markets; their potential hazards for humans remain largely unknown. This study aimed to investigate the int...

  20. Repair and degradation systems in irradiated animal cells

    Energy Technology Data Exchange (ETDEWEB)

    Ivannik, B.P.; Proskuryakov, S.Ya.; Ryabchenko, N.I. (Akademiya Meditsinskikh Nauk SSSR, Obninsk. Nauchno-Issledovatel' skij Inst. Meditsinskoj Radiologii)

    It was shown that primary radiosensitivity of DNA depends on the rate of DNA repair. In Zajdela hepatoma cells, cycloheximide administered immediately or 2 h before irradiation of animals does not influence DNA repair. Cycloheximide administered 4 h before irradiation of rats with a dose of 30 Gy arrests DNA repair in thymocytes and Zajdela hepatoma cells. At the same time, in cells of rat lymph nodes and spleen, under similar conditions, cycloheximide does not influence DNA repair and inhibits the secondary DNA degradation.

  1. HDM2 impairs Noxa transcription and affects apoptotic cell death in a p53/p73-dependent manner in neuroblastoma.

    Science.gov (United States)

    Shi, Yun; Takenobu, Hisanori; Kurata, Kenji; Yamaguchi, Yohko; Yanagisawa, Ryu; Ohira, Miki; Koike, Kenichi; Nakagawara, Akira; Jiang, Ling Ling; Kamijo, Takehiko

    2010-08-01

    HDM2, a human homologue of MDM2, is a major negative regulator of p53 function, and increased expression of HDM2 by its promoter polymorphism SNP309 resulted in p53 inactivation and an increased risk of several tumours, including neuroblastoma (NB). Herein, we show that increased expression of HDM2 is related to a worse prognosis in MYCN-amplified NB patients. HDM2 plays an important role in the expression of Noxa, a pro-apoptotic molecule of the Bcl-2 family, which induces NB cell apoptotic death after doxorubcin (Doxo) treatment. Knockdown of HDM2 by siRNA resulted in the upregulation of Noxa at mRNA/protein levels and improved the sensitivity of Doxo-resistant NB cells, although these were not observed in p53-mutant NB cells. Noxa-knockdown abolished the recovered Doxo-induced cell death by HDM2 reduction. Intriguingly, resistance to Doxo was up-regulated by over-expression of HDM2 in Doxo-sensitive NB cells. By HDM2 expression, p53 was inactivated but its degradation was not accelerated, suggesting that p53 was degraded in a proteasome-independent manner in NB cells; downstream effectors of p53, p21(Cip1/Waf1) and Noxa were suppressed by HDM2. Noxa transcription was considerably regulated by both p53 and p73 in NB cells. Furthermore, in vivo binding of p53 and p73 to Noxa promoter was suppressed and Noxa promoter activation was inhibited by HDM2. Taken together, our results may indicate that the HDM2-related resistance to chemotherapeutic drugs of NB is regulated by p53/p73-dependent Noxa expression in NB.

  2. Sequence and expression variations in 23 genes involved in mitochondrial and non-mitochondrial apoptotic pathways and risk of oral leukoplakia and cancer.

    Science.gov (United States)

    Datta, Sayantan; Ray, Anindita; Singh, Richa; Mondal, Pinaki; Basu, Analabha; De Sarkar, Navonil; Majumder, Mousumi; Maiti, Guruparasad; Baral, Aradhita; Jha, Ganga Nath; Mukhopadhyay, Indranil; Panda, Chinmay; Chowdhury, Shantanu; Ghosh, Saurabh; Roychoudhury, Susanta; Roy, Bidyut

    2015-11-01

    Oral cancer is usually preceded by pre-cancerous lesion and related to tobacco abuse. Tobacco carcinogens damage DNA and cells harboring such damaged DNA normally undergo apoptotic death, but cancer cells are exceptionally resistant to apoptosis. Here we studied association between sequence and expression variations in apoptotic pathway genes and risk of oral cancer and precancer. Ninety nine tag SNPs in 23 genes, involved in mitochondrial and non-mitochondrial apoptotic pathways, were genotyped in 525 cancer and 253 leukoplakia patients and 538 healthy controls using Illumina Golden Gate assay. Six SNPs (rs1473418 at BCL2; rs1950252 at BCL2L2; rs8190315 at BID; rs511044 at CASP1; rs2227310 at CASP7 and rs13010627 at CASP10) significantly modified risk of oral cancer but SNPs only at BCL2, CASP1and CASP10 modulated risk of leukoplakia. Combination of SNPs showed a steep increase in risk of cancer with increase in "effective" number of risk alleles. In silico analysis of published data set and our unpublished RNAseq data suggest that change in expression of BID and CASP7 may have affected risk of cancer. In conclusion, three SNPs, rs1473418 in BCL2, rs1950252 in BCL2L2 and rs511044 in CASP1, are being implicated for the first time in oral cancer. Since SNPs at BCL2, CASP1 and CASP10 modulated risk of both leukoplakia and cancer, so, they should be studied in more details for possible biomarkers in transition of leukoplakia to cancer. This study also implies importance of mitochondrial apoptotic pathway gene (such as BCL2) in progression of leukoplakia to oral cancer.

  3. Repeated low-dose 17β-estradiol treatment prevents activation of apoptotic signaling both in the synaptosomal and cellular fraction in rat prefrontal cortex following cerebral ischemia.

    Science.gov (United States)

    Stanojlović, Miloš; Zlatković, Jelena; Guševac, Ivana; Grković, Ivana; Mitrović, Nataša; Zarić, Marina; Horvat, Anica; Drakulić, Dunja

    2015-01-01

    Disturbance in blood circulation is associated with numerous pathological conditions characterized by cognitive decline and neurodegeneration. Activation of pro-apoptotic signaling previously detected in the synaptosomal fraction may underlie neurodegeneration in the prefrontal cortex of rats submitted to permanent bilateral common carotid arteries occlusion (two-vessel occlusion, 2VO). 17β-Estradiol (E) exerts potent neuroprotective effects in the brain affecting, among other, ischemia-induced pathological changes. As most significant changes in rats submitted to 2VO were observed on 7th day following the insult, of interest was to examine whether 7 day treatment with low dose of E (33.3 µg/kg/day) prevents formerly reported neurodegeneration and may represent additional therapy during the early post-ischemic period. Role of E treatment on apoptotic pathway was monitored on Bcl-2 family members, cytochrome c, caspase 3 and PARP protein level in the synaptosomal (P2) fraction of the prefrontal cortex. Furthermore, changes of these proteins were examined in the cytosolic, mitochondrial and nuclear fraction, with the emphasis on potential involvement of extracellular signal-regulated kinases (ERK) and protein kinase B (Akt) activation and their role in nuclear translocation of transcriptional nuclear factor kappa B (NF-kB) associated with alteration of Bax and Bcl-2 gene expression. The extent of cellular damage was determined using DNA fragmentation and Fluoro-Jade B staining. The absence of activation of apoptotic cascade both in the P2 and cell accompanied with decreased DNA fragmentation and number of degenerating neurons clearly indicates that E treatment ensures the efficient protection against ischemic insult. Moreover, E-mediated modulation of pro-apoptotic signaling in the cortical cellular fractions involves cooperative activation of ERK and Akt, which may be implicated in the observed prevention of neurodegenerative changes.

  4. Modulation of mammalian apoptotic pathways by intracellular protozoan parasites.

    Science.gov (United States)

    Rodrigues, V; Cordeiro-da-Silva, A; Laforge, M; Ouaissi, A; Silvestre, R; Estaquier, J

    2012-03-01

    During intracellular parasitic infections, pathogens and host cells take part in a complex web of events that are crucial for the outcome of the infection. Modulation of host cell apoptosis by pathogens attracted the attention of scientists during the last decade. Apoptosis is an efficient mechanism used by the host to control infection and limit pathogen multiplication and dissemination. In order to ensure completion of their complex life cycles and to guarantee transmission between different hosts, intracellular parasites have developed mechanisms to block apoptosis and sustain the viability of their host cells. Here, we review how some of the most prominent intracellular protozoan parasites modulate the main mammalian apoptotic pathways by emphasizing the advances from the last decade, which have begun to dissect this dynamic and complex interaction.

  5. Effects of glucocorticoids on apoptosis and clearance of apoptotic cells.

    Science.gov (United States)

    McColl, Aisleen; Michlewska, Sylwia; Dransfield, Ian; Rossi, Adriano G

    2007-08-17

    The glucocorticoid (GC) drugs are one of the most commonly prescribed and effective anti-inflammatory agents used for the treatment of many inflammatory disorders through their ability to attenuate phlogistic responses. The glucocorticoid receptor (GCR) primarily mediates GC actions via activation or repression of gene expression. GCs directly induce the expression of proteins displaying anti-inflammatory activities. However, the likely predominant effect of GCs is the repression of multiple inflammatory genes that invariably are overexpressed during nonresolving chronic inflammation. Although most GC actions are mediated through regulation of transcription, rapid nongenomic actions have also been reported. In addition, GCs modulate inflammatory cell survival, inducing apoptosis in immature thymocytes and eosinophils, while delaying constitutive neutrophil apoptosis. Importantly, GCs promote noninflammatory phagocytosis of apoptotic cell targets, a process important for the successful resolution of inflammation. Here, the effects and mechanisms of action of GC on inflammatory cell apoptosis and phagocytosis will be discussed.

  6. Effects of Glucocorticoids on Apoptosis and Clearance of Apoptotic Cells

    Directory of Open Access Journals (Sweden)

    Aisleen McColl

    2007-01-01

    Full Text Available The glucocorticoid (GC drugs are one of the most commonly prescribed and effective anti-inflammatory agents used for the treatment of many inflammatory disorders through their ability to attenuate phlogistic responses. The glucocorticoid receptor (GCR primarily mediates GC actions via activation or repression of gene expression. GCs directly induce the expression of proteins displaying anti-inflammatory activities. However, the likely predominant effect of GCs is the repression of multiple inflammatory genes that invariably are overexpressed during nonresolving chronic inflammation. Although most GC actions are mediated through regulation of transcription, rapid nongenomic actions have also been reported. In addition, GCs modulate inflammatory cell survival, inducing apoptosis in immature thymocytes and eosinophils, while delaying constitutive neutrophil apoptosis. Importantly, GCs promote noninflammatory phagocytosis of apoptotic cell targets, a process important for the successful resolution of inflammation. Here, the effects and mechanisms of action of GC on inflammatory cell apoptosis and phagocytosis will be discussed.

  7. Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde.

    Science.gov (United States)

    Ganesan, Murali; Natarajan, Sathish Kumar; Zhang, Jinjin; Mott, Justin L; Poluektova, Larisa I; McVicker, Benita L; Kharbanda, Kusum K; Tuma, Dean J; Osna, Natalia A

    2016-06-01

    Alcohol consumption exacerbates hepatitis C virus (HCV) pathogenesis and promotes disease progression, although the mechanisms are not quite clear. We have previously observed that acetaldehyde (Ach) continuously produced by the acetaldehyde-generating system (AGS), temporarily enhanced HCV RNA levels, followed by a decrease to normal or lower levels, which corresponded to apoptosis induction. Here, we studied whether Ach-induced apoptosis caused depletion of HCV-infected cells and what role apoptotic bodies (AB) play in HCV-alcohol crosstalk. In liver cells exposed to AGS, we observed the induction of miR-122 and miR-34a. As miR-34a has been associated with apoptotic signaling and miR-122 with HCV replication, these findings may suggest that cells with intensive viral replication undergo apoptosis. Furthermore, when AGS-induced apoptosis was blocked by a pan-caspase inhibitor, the expression of HCV RNA was not changed. AB from HCV-infected cells contained HCV core protein and the assembled HCV particle that infect intact hepatocytes, thereby promoting the spread of infection. In addition, AB are captured by macrophages to switch their cytokine profile to the proinflammatory one. Macrophages exposed to HCV(+) AB expressed more IL-1β, IL-18, IL-6, and IL-10 mRNAs compared with those exposed to HCV(-) AB. The generation of AB from AGS-treated HCV-infected cells even enhanced the induction of aforementioned cytokines. We conclude that HCV and alcohol metabolites trigger the formation of AB containing HCV particles. The consequent spread of HCV to neighboring hepatocytes via infected AB, as well as the induction of liver inflammation by AB-mediated macrophage activation potentially exacerbate the HCV infection course by alcohol and worsen disease progression.

  8. Fas transduces dual apoptotic and trophic signals in hematopoietic progenitors.

    Science.gov (United States)

    Pearl-Yafe, Michal; Stein, Jerry; Yolcu, Esma S; Farkas, Daniel L; Shirwan, Haval; Yaniv, Isaac; Askenasy, Nadir

    2007-12-01

    Stem cells and progenitors are often required to realize their differentiation potential in hostile microenvironments. The Fas/Fas ligand (FasL) interaction is a major effector pathway of apoptosis, which negatively regulates the expansion of differentiated hematopoietic cells. The involvement of this molecular interaction in the function of hematopoietic stem and progenitor cells is not well understood. In the murine syngeneic transplant setting, both Fas and FasL are acutely upregulated in bone marrow-homed donor cells; however, the Fas(+) cells are largely insensitive to FasL-induced apoptosis. In heterogeneous populations of lineage-negative (lin(-)) bone marrow cells and progenitors isolated by counterflow centrifugal elutriation, trimerization of the Fas receptor enhanced the clonogenic activity. Inhibition of caspases 3 and 8 did not affect the trophic signals mediated by Fas, yet it efficiently blocked the apoptotic pathways. Fas-mediated tropism appears to be of physiological significance, as pre-exposure of donor cells to FasL improved the radioprotective qualities of hematopoietic progenitors, resulting in superior survival of myeloablated hosts. Under these conditions, the activity of long-term reconstituting cells was not affected, as determined in sequential secondary and tertiary transplants. Dual caspase-independent tropic and caspase-dependent apoptotic signaling place the Fas receptor at an important junction of activation and death. This regulatory mechanism of hematopoietic homeostasis activates progenitors to promote the recovery from aplasia and converts into a negative regulator in distal stages of cell differentiation. Disclosure of potential conflicts of interest is found at the end of this article.

  9. Apoptotic and anti-angiogenic effects of Salvia triloba extract in prostate cancer cell lines.

    Science.gov (United States)

    Atmaca, Harika; Bozkurt, Emir

    2016-03-01

    Plants, due to their remarkable composition, are considered as natural resources of bioactive compounds with specific biological activities. Salvia genus (Lamiaceae) has been used around the world in complementary medicine since ancient times. We investigated the cytotoxic, apoptotic and anti-angiogenic effects of methanolic Salvia triloba extract (STE) in prostate cancer cells. Cell viability was evaluated by XTT; apoptosis was investigated by DNA fragmentation and caspase 3/7 activity assays. Changes in the angiogenic cytokine levels were investigated by human angiogenesis antibody array. Scratch assay was used to determine the cell motility. STE induced cytotoxicity and apoptosis in a concentration-dependent manner in both cancer cells; however, it was not cytotoxic to normal cells. Cell motility was reduced in PC-3, DU-145 and HUVEC cells by STE treatment. ANG, ENA-78, bFGF, EGF, IGF-1 and VEGF-D levels were significantly decreased by -2.9, -3.7, -1.7, -1.7, -2.0 and -1.8 fold in STE-treated DU-145 cells, however, ANG, IL-8, LEP, RANTES, TIMP-1, TIMP-2 and VEGF levels were significantly decreased by -5.1, -2.0, -2.4, -3.1, -1.5, -2.0 and -2.5 fold in PC-3 cells. These data suggest that STE might be a promising candidate for anti-tumor and anti-angiogenic treatment of prostate cancer.

  10. Comparative evaluation of the cytotoxic and apoptotic potential of Poecilocerus pictus and Calotropis gigantea.

    Science.gov (United States)

    Mathen, Caroline; Peter, Shiney Mary; Hardikar, Bhagyashree P

    2011-01-01

    Calotropis gigantea, the giant milkweed, is traditionally used for the treatment of cancer and in Ayurvedic medicine as an anti-helminthic, anti-pyretic, and anti-malarial agent. Poecilocerus pictus, an orthopteran insect, feeds on C. gigantea and both are known to possess cardiac glycosides. The increasing reports on the specific cytotoxicity of cardiac glycosides on human tumor cell lines led us to attempt characterization and comparative evaluation of cardenolides in both the insect and plant extracts for their anti-tumor and apoptotic potential.Chemical characterization using high-performance thin layer chromatography, ultraviolet and infrared spectra analysis confirmed the presence of cardiac glycosides, but differences in the components of the insect extract were indicative of biotransformation. The cytotoxicity studies revealed a more potent trend for the insect extract compared with the plant extract on A549 and COLO205 cells. There was a considerably lesser measure of toxicity on WI38 cells and peripheral blood lymphocytes, whereas B16F1 remained unaffected by both extracts. DNA ploidy analysis on COLO205 indicated that both extracts induced dose-dependent apoptosis. Therefore, both the insect and the plant extracts differentiate between human cancer cells and normal cells and exhibit species specificity. Further investigations are necessary to establish these extracts as promising lead candidates for anti-neoplastic activity.

  11. Tula hantavirus triggers pro-apoptotic signals of ER stress in Vero E6 cells.

    Science.gov (United States)

    Li, Xiao-Dong; Lankinen, Hilkka; Putkuri, Niina; Vapalahti, Olli; Vaheri, Antti

    2005-03-01

    Tula virus is a member of the Hantavirus genus of the family Bunyaviridae. Viruses of this family have an unusual pattern of intracellular maturation at the ER-Golgi compartment. We recently found that Tula virus, similar to several other hantaviruses, is able to induce apoptosis in cultured cells [Li, X.D., Kukkonen, S., Vapalahti, O., Plyusnin, A., Lankinen, H., Vaheri, A., 2004. Tula hantavirus infection of Vero E6 cells induces apoptosis involving caspase 8 activation. J. Gen. Virol. 85, 3261-3268.]. However, the cellular mechanisms remain to be clarified. In this study, we demonstrate that the progressive replication of Tula virus in Vero E6 cells initiates several death programs that are intimately associated with ER stress: (1) early activation of ER-resident caspase-12; (2) phosphorylation of Jun NH2-terminal kinase (JNK) and its downstream target transcriptional factor, c-jun; (3) induction of the pro-apoptotic transcriptional factor, growth arrest- and DNA damage-inducible gene 153, or C/EBP homologous protein (Gadd153/chop); and (4) changes in the ER-membrane protein BAP31 implying cross-talk with the mitochondrial apoptosis pathway. Furthermore, we confirmed that a sustained ER stress was induced marked by an increased expression of an ER chaperone Grp78/BiP. Taken together, we have identified involvement of ER stress-mediated death program in Tula virus-infected Vero E6 cells which provides a new approach to understand the mechanisms in hantavirus-induced apoptosis.

  12. Heat stress induces apoptotic-like cell death in two Pleurotus species.

    Science.gov (United States)

    Song, Chi; Chen, Qiang; Wu, Xiangli; Zhang, Jinxia; Huang, Chenyang

    2014-11-01

    High temperature is an important environmental factor that affects the growth and development of most edible fungi, however, the mechanism(s) for resistance to high temperature remains elusive. Nitric oxide is known to be able to effectively alleviate oxidative damage and plays an important role in the regulation of trehalose accumulation during heat stress in mycelia of Pleurotus eryngii var. tuoliensis. In this paper, we investigated whether heat stress can activate apoptosis-like cell death in mycelia of Pleurotus. Two Pleurotus species were used to detect morphological features characteristic of apoptosis including nuclear condensation, reactive oxygen species accumulation, and DNA fragmentation when exposed to heat stress (42 °C). The results showed that these classical apoptosis markers were apparent in Pleurotus strains after heat treatment. The heat-induced apoptosis-like cell death in Pleurotus was further probed using oligomycin and N-acetylcysteine, both of which were shown to block processes leading to apoptosis. This is the first report that apoptosis-like cell death occurs in Pleurotus species as a result of abiotic stress, and that this process can be inhibited with chemicals that block mitochondrial-induced apoptotic pathways and/or with ROS-scavenging compounds.

  13. Studies on the sonic degradation of deoxyribonucleic acid.

    Science.gov (United States)

    FREIFELDER, D; DAVISON, P F

    1962-05-01

    T7 DNA was partially degraded by x-rays, DNAase, and sonic irradiation. The molecular weight distributions were calculated from sedimentation velocity studies on the resulting preparations. Comparison with the theoretical curve derived by Montroll and Simha showed that the first two degradative methods act grossly at random, whereas sonication is a non-random process resulting in the preferential halving of the DNA molecules in solution.

  14. PKC{eta} confers protection against apoptosis by inhibiting the pro-apoptotic JNK activity in MCF-7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Rotem-Dai, Noa; Oberkovitz, Galia; Abu-Ghanem, Sara [The Schraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences and the Cancer Research Center, Ben - Gurion University, Beer Sheva 84105 (Israel); Livneh, Etta, E-mail: etta@bgumail.bgu.ac.il [The Schraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences and the Cancer Research Center, Ben - Gurion University, Beer Sheva 84105 (Israel)

    2009-09-10

    Apoptosis is frequently regulated by different protein kinases including protein kinase C family enzymes. Both inhibitory and stimulatory effects were demonstrated for several of the different PKC isoforms. Here we show that the novel PKC isoform, PKC{eta}, confers protection against apoptosis induced by the DNA damaging agents, UVC irradiation and the anti-cancer drug - Camptothecin, of the breast epithelial adenocarcinoma MCF-7 cells. The induced expression of PKC{eta} in MCF-7 cells, under the control of the tetracycline-responsive promoter, resulted in increased cell survival and inhibition of cleavage of the apoptotic marker PARP-1. Activation of caspase-7 and 9 and the release of cytochrome c were also inhibited by the inducible expression of PKC{eta}. Furthermore, JNK activity, required for apoptosis in MCF-7, as indicated by the inhibition of both caspase-7 cleavage and cytochrome c release from the mitochondria in the presence of the JNK inhibitor SP600125, was also suppressed by PKC{eta} expression. Hence, in contrast to most PKC isoforms enhancing JNK activation, our studies show that PKC{eta} is an anti-apoptotic protein, acting as a negative regulator of JNK activity. Thus, PKC{eta} could represent a target for intervention aimed to reduce resistance to anti-cancer treatments.

  15. Cytotoxic and apoptotic effects of different extracts of Artemisia biennis Willd. on K562 and HL-60 cell lines

    Directory of Open Access Journals (Sweden)

    Zahra Tayarani-Najaran

    2017-02-01

    Full Text Available Objective(s: Artemisia is a genus of herbs and small shrubs forms an important part of natural vegetation in Iran. It has been reported that several Artemisia species possess anti-proliferative effects. Considering the value of this genus in anti-cancer researches we have chosen Artemisia biennis for cytotoxic and mechanistic studies. Materials and Methods:In this study we have investigated the cytotoxic and apoptotic effects of petroleum ether, dichloromethane, ethyl acetate, ethanol, and ethanol:water (1:1 v/v extracts of A. biennis Willd. on two cancer human cell lines (K562 and HL-60 and J774 as normal cells. Results: CH2Cl2 extract was found to have the highest anti-proliferative effect on cancer cells. IC50 values obtained in AlamarBlue® assay for CH2Cl2 extract were 64.86 and 54.31 µg/ml on K562 and HL-60 cells respectively. In flow cytometry histogram of the cells treated with CH2Cl2 extract, sub-G1 peak was induced. DNA fragmentation, increased in the level of Bax and cleavage of PARP protein all showed the induction of apoptosis with CH2Cl2 extract after 48 hr contact with cells. Conclusion: The results can corroborate the cytotoxic and apoptotic effects of the CH2Cl2 extract of A. biennis on the K562 and HL-60 cancer cell lines.

  16. How does ethanol induce apoptotic cell death of SK-N-SH neuroblastoma cells?*

    Institute of Scientific and Technical Information of China (English)

    Yong Moon; Yongil Kwon; Shun Yu

    2013-01-01

    A body of evidence suggests that ethanol can lead to damage of neuronal cel s. However, the me-chanism underlying the ethanol-induced damage of neuronal cel s remains unclear. The role of mi-togen-activated protein kinases in ethanol-induced damage was investigated in SK-N-SH neurob-lastoma cel s. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide cel viability assay, DNA fragmentation detection, and flow cytometric analysis showed that ethanol induced apoptotic cel death and cel cycle arrest, characterized by increased caspase-3 activity, DNA fragmentation, nuclear disruption, and G 1 arrest of cel cycle of the SK-N-SH neuroblastoma cel s. In addition, western blot analysis indicated that ethanol induced a lasting increase in c-Jun N-terminal protein kinase activity and a transient increase in p38 kinase activity of the neuroblastoma cel s. c-Jun N-terminal protein kinase or p38 kinase inhibitors significantly reduced the ethanol-induced cel death. Ethanol also increased p53 phosphorylation, fol owed by an increase in p21 tumor sup-pressor protein and a decrease in phospho-Rb (retinoblastoma) protein, leading to alterations in the expressions and activity of cyclin dependent protein kinases. Our results suggest that ethanol me-diates apoptosis of SK-N-SH neuroblastoma cel s by activating p53-related cel cycle arrest possibly through activation of the c-Jun N-terminal protein kinase-related cel death pathway.

  17. The forkhead transcription factor FOXO3a controls microglial inflammatory activation and eventual apoptotic injury through caspase 3.

    Science.gov (United States)

    Shang, Yan Chen; Chong, Zhao Zhong; Hou, Jinling; Maiese, Kenneth

    2009-02-01

    Memory loss and cognitive failure are increasingly being identified as potential risks with the recognized increase in life expectancy of the general population. As a result, the development of novel therapeutic strategies for disorders such as Alzheimer's disease have garnered increased attention. The etiologies that can lead to Alzheimer's disease are extremely varied, but a number of therapeutic options are directed against amyloid-beta peptide and inflammatory cell regulation to prevent or halt progressive cognitive loss. In particular, inflammatory microglial cells may have disparate functions that in some scenarios lead to disability through the removal of functional neurovascular cells and in other circumstances foster tissue repair. Given the significance microglial cells hold for neurodegenerative disorders, we therefore examined the function that amyloid (Abeta(1-42)) has upon the microglial cell line EOC 2 and identified a novel role for the forkhead transcription factor FoxO3a and caspase 3. Here we show that Abeta(1-42) leads to progressive injury and apoptotic cell loss in microglial cells that involves both early phosphatidylserine (PS) externalization and late genomic DNA fragmentation over a 24 hour course. Prior to these injury programs, Abeta(1-42) results in the activation and proliferation of microglia as demonstrated by increased proliferating cell nuclear antigen (PCNA) expression and bromodeoxyuridine (BrdU) uptake. Both apoptotic injury as well as the prior activation and proliferation of microglial cells relies upon the presence of FoxO3a, since specific gene silencing of FoxO3a promotes microglial cell protection and prevents the early activation and proliferation of these cells. Furthermore, Abeta(1-42) exposure maintained FoxO3a in an unphosphorylated "active" state and facilitated the cellular trafficking of FoxO3a from the cytoplasm to the cell nucleus to potentially lead to "pro-apoptotic" programs by this transcription factor. One

  18. Evaluation of chemical composition, antioxidant, antibacterial, cytotoxic and apoptotic effects of Aloysia citrodora extract on colon cancer cell line

    Directory of Open Access Journals (Sweden)

    Amir Mirzaie

    2016-06-01

    Full Text Available Background: Aloysia citrodora belongs to the Verbenaceae family of plants, a well-known herbal medicine in Iran. The aim of the present study was to investigate the chemical composition, antioxidant, antibacterial, cytotoxic and apoptotic effect of A. citrodora extract against human colon cancer (HT29 cells by using real-time polymerase chain reaction and flow-cytometry methods. Methods: This experimental study was carried out in Islamic Azad University, East Tehran Branch, from March to September of 2014. At first, the A. citrodora chemical constituents were analyzed by gas chromatography-mass spectrometry (GC-MS technique. In addition, antioxidant assay, antibacterial and anti-cancer effect was performed using 1,1-diphenyl-2-picrylhydrazyl (DPPH, disk diffusion and 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT methods, respectively. The half maximal inhibitory concentration (IC50 value was calculated. We extracted total RNA molecules by using RNX solution, after which cDNA was synthesized. Finally, the pro-apoptotic (Bax and anti-apoptotic (Bcl2 gene expression was performed by real-time polymerase chain reaction and apoptotic effects were analyzed using Flow-cytometry method. Results: GC-MS analysis of Aloysia citrodora extract was shown 37 major components and the most frequent component was belonged to Spathulenol (17.57% and Caryophyllene oxide (15.15% The antioxidant activity of the extract was IC50= 0.6±0.03 mg/ml. The maximum and minimum antibacterial effects of extract were belonged to gram-negative and gram-positive bacteria, respectively. Cytotoxic results revealed that the A.citrodora extract have IC50= 20.1±0.78 mg/ml against colon cancer (HT29 cell line and real-time polymerase chain reaction results showed the expression level of Bax and Bcl2 was increased and decreased respectively in colon cancer cell line (3.470±0.72 (P< 0.05, 0.43±0.35 (P< 0.05. In addition, the flow-cytometry results indicated the 38

  19. Hepatitis E virus ORF2 protein activates the pro-apoptotic gene CHOP and anti-apoptotic heat shock proteins.

    Directory of Open Access Journals (Sweden)

    Lijo John

    Full Text Available BACKGROUND: Hepatitis E virus (HEV is a non-enveloped plus-strand RNA virus that causes acute hepatitis. The capsid protein open reading frame 2 (ORF2 is known to induce endoplasmic reticulum stress in ORF2 expressing cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study we found that HEV ORF2 activates the expression of the pro-apoptotic gene C/EBP homologous protein (CHOP. ORF2 stimulates the CHOP promoter mainly through AARE (amino acid response elements and to a minor extent the ERSE (endoplasmic reticulum stress response elements. Activating transcription factor 4 (ATF4 protein binds and activates the AARE regulatory sites of the CHOP promoter. ORF2 expression also leads to increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α that in turn initiates the translation of ATF4 mRNA. The pro-apoptotic gene CHOP is an important trigger to initiate endoplasmic reticulum stress induced apoptosis. However, the activation of CHOP by ORF2 in this study did not induce apoptosis, nor did BCL2-associated X protein (Bax translocate to mitochondria. Microarray analysis revealed an ORF2 specific increased expression of chaperones Hsp72, Hsp70B', and co-chaperone Hsp40. Co-immunoprecipitation (Co-IP and in silico molecular docking analysis suggests that HEV ORF2 interacts with Hsp72. In addition, Hsp72 shows nuclear accumulation in ORF2 expressing cells. CONCLUSIONS/SIGNIFICANCE: These data provide new insight into simultaneously occurring counter-acting effects of HEV ORF2 that may be part of a strategy to prevent host suicide before completion of the viral replication cycle.

  20. The anti-apoptotic members of the Bcl-2 family are attractive tumor-associated antigens

    DEFF Research Database (Denmark)

    Straten, Per thor; Andersen, Mads Hald

    2010-01-01

    Anti-apoptotic members of the Bcl-2 family (Bcl-2, Bcl-X(L) and Mcl-2) are pivotal regulators of apoptotic cell death. They are all highly overexpressed in cancers of different origin in which they enhance the survival of the cancer cells. Consequently, they represent prime candidates for anti-ca...

  1. Enhanced activation of dendritic cells by autologous apoptotic microvesicles in MRL/lpr mice

    NARCIS (Netherlands)

    Dieker, J.W.; Hilbrands, L.B.; Thielen, A.; Dijkman, H.B.P.M.; Berden, J.H.M.; Vlag, J. van der

    2015-01-01

    INTRODUCTION: Systemic lupus erythematosus is associated with a persistent circulation of modified autoantigen-containing apoptotic debris that might be capable of breaking tolerance. We aimed to evaluate apoptotic microvesicles obtained from lupus or control mice for the presence of apoptosis-assoc

  2. Pro-apoptotic gene regulation in the Caribbean fruit fly, Anastrepha suspensa

    Science.gov (United States)

    Transcriptional activation of pro-apoptotic genes in response to cytotoxic stimuli is a conserved feature of the cell death pathway proposed for metazoans. However, understanding the extent of this conservation in insects, as well as other organisms, has been limited by the lack of known pro-apoptot...

  3. The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal

    Directory of Open Access Journals (Sweden)

    Hafner Martin

    2004-08-01

    Full Text Available Abstract Background Phagocytosis of apoptotic cells is fundamental to animal development, immune function and cellular homeostasis. The phosphatidylserine receptor (Ptdsr on phagocytes has been implicated in the recognition and engulfment of apoptotic cells and in anti-inflammatory signaling. To determine the biological function of the phosphatidylserine receptor in vivo, we inactivated the Ptdsr gene in the mouse. Results Ablation of Ptdsr function in mice causes perinatal lethality, growth retardation and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis. Moreover, eye development can be severely disturbed, ranging from defects in retinal differentiation to complete unilateral or bilateral absence of eyes. Ptdsr -/- mice with anophthalmia develop novel lesions, with induction of ectopic retinal-pigmented epithelium in nasal cavities. A comprehensive investigation of apoptotic cell clearance in vivo and in vitro demonstrated that engulfment of apoptotic cells was normal in Ptdsr knockout mice, but Ptdsr-deficient macrophages were impaired in pro- and anti-inflammatory cytokine signaling after stimulation with apoptotic cells or with lipopolysaccharide. Conclusion Ptdsr is essential for the development and differentiation of multiple organs during embryogenesis but not for apoptotic cell removal. Ptdsr may thus have a novel, unexpected developmental function as an important differentiation-promoting gene. Moreover, Ptdsr is not required for apoptotic cell clearance by macrophages but seems to be necessary for the regulation of macrophage cytokine responses. These results clearly contradict the current view that the phosphatidylserine receptor primarily functions in apoptotic cell clearance.

  4. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Gallati, Sabina, E-mail: sabina.gallati@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Schaller, Andre, E-mail: andre.schaller@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct measurements in

  5. InterProScan Result: CK502663 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available CK502663 CK502663_3_ORF1 86FC71C1D80A728E PFAM PF09230 DFF40 9.5e-28 T IPR015311 Apoptosis, DNA fragmentatio...sm (GO:0005737)|Biological Process: DNA fragmentation involved in apoptotic nuclear change (GO:0006309)|Molecular Function: hydrolase activity (GO:0016787) ...

  6. InterProScan Result: FS908842 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available FS908842 FS908842_1_ORF2 9B061F2AF0FE2CA8 PFAM PF09230 DFF40 5.1e-28 T IPR015311 Apoptosis, DNA fragmentatio...sm (GO:0005737)|Biological Process: DNA fragmentation involved in apoptotic nuclear change (GO:0006309)|Molecular Function: hydrolase activity (GO:0016787) ...

  7. Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo.

    Science.gov (United States)

    Phesse, T J; Myant, K B; Cole, A M; Ridgway, R A; Pearson, H; Muncan, V; van den Brink, G R; Vousden, K H; Sears, R; Vassilev, L T; Clarke, A R; Sansom, O J

    2014-06-01

    Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.

  8. Direct interaction of the molecular scaffolds POSH and JIP is required for apoptotic activation of JNKs.

    Science.gov (United States)

    Kukekov, Nickolay V; Xu, Zhiheng; Greene, Lloyd A

    2006-06-02

    A sequential pathway (the JNK pathway) that includes activation of Rac1/Cdc42, mixed lineage kinases, MAP kinase kinases 4 and 7, and JNKs plays a required role in many paradigms of apoptotic cell death. However, the means by which this pathway is assembled and directed toward apoptotic death has been unclear. Here, we report that propagation of the apoptotic JNK pathway requires the cooperative interaction of two molecular scaffolds, POSH and JIPs. POSH (plenty of SH3s) is a multidomain GTP-Rac1-interacting protein that binds and promotes activation of mixed lineage kinases. JIPs are reported to bind MAP kinase kinases 4/7 and JNKs. We find that POSH and JIPs directly associate with one another to form a multiprotein complex, PJAC (POSH-JIP apoptotic complex), that includes all of the known kinase components of the pathway. Our observations indicate that this complex is required for JNK activation and cell death in response to apoptotic stimuli.

  9. A non-apoptotic role for BAX and BAK in eicosanoid metabolism

    Science.gov (United States)

    Zhang, Tejia; Walensky, Loren D.; Saghatelian, Alan

    2015-01-01

    BCL-2 proteins are key regulators of programmed cell death. The interplay between pro- and anti-apoptotic BCL-2 members has important roles in many cancers. In addition to their apoptotic function, recent evidence supports key non-apoptotic roles for several BCL-2 proteins. We used an unbiased lipidomics strategy to reveal that the pro-apoptotic proteins BAX, and to a lesser extent BAK, regulate the cellular inflammatory response by mediating COX-2 expression and prostaglandin biosynthesis. COX-2 upregulation in response to the bacterial endotoxin lipopolysaccharide is blunted in the absence of BAX, and Bax−/− mouse embryonic fibroblasts display altered kinetics of NFκB and MAPK signaling following endotoxin treatment. Our approach uncovers a novel, non-apoptotic function for BAX in regulation of the cellular inflammatory response and suggests that inflammation and apoptosis are more tightly connected than previously anticipated. PMID:25815636

  10. Pulsed Electromagnetic Field with Temozolomide Can Elicit an Epigenetic Pro-apoptotic Effect on Glioblastoma T98G Cells.

    Science.gov (United States)

    Pasi, Francesca; Fassina, Lorenzo; Mognaschi, Maria Evelina; Lupo, Giuseppe; Corbella, Franco; Nano, Rosanna; Capelli, Enrica

    2016-11-01

    Treatment with pulsed electromagnetic fields (PEMFs) is emerging as an interesting therapeutic option for patients with cancer. The literature has demonstrated that low-frequency/low-energy electromagnetic fields do not cause predictable effects on DNA; however, they can epigenetically act on gene expression. The aim of the present work was to study a possible epigenetic effect of a PEMF, mediated by miRNAs, on a human glioblastoma cell line (T98G). We tested a PEMF (maximum magnetic induction, 2 mT; frequency, 75 Hz) that has been demonstrated to induce autophagy in glioblastoma cells. In particular, we studied the effect of PEMF on the expression of genes involved in cancer progression and a promising synergistic effect with temozolomide, a frequently used drug to treat glioblastoma multiforme. We found that electromagnetic stimulation in combination with temozolomide can elicit an epigenetic pro-apoptotic effect in the chemo- and radioresistant T98G glioblastoma cell line.

  11. DNA from keratinous tissue

    DEFF Research Database (Denmark)

    Bengtsson, Camilla F.; Olsen, Maja E.; Brandt, Luise Ørsted

    2011-01-01

    Keratinous tissues such as nail, hair, horn, scales and feather have been used as a source of DNA for over 20 years. Particular benefits of such tissues include the ease with which they can be sampled, the relative stability of DNA in such tissues once sampled, and, in the context of ancient...... genetic analyses, the fact that sampling generally causes minimal visual damage to valuable specimens. Even when freshly sampled, however, the DNA quantity and quality in the fully keratinized parts of such tissues is extremely poor in comparison to other tissues such as blood and muscle – although little...... systematic research has been undertaken to characterize how such degradation may relate to sample source. In this review paper we present the current understanding of the quality and limitations of DNA in two key keratinous tissues, nail and hair. The findings indicate that although some fragments of nuclear...

  12. DNA from keratinous tissue

    DEFF Research Database (Denmark)

    Bengtsson, Camilla Friis; Olsen, Maia E.; Brandt, Luise Ørsted

    2012-01-01

    Keratinous tissues such as nail, hair, horn, scales and feather have been used as a source of DNA for over 20 years. Particular benefits of such tissues include the ease with which they can be sampled, the relative stability of DNA in such tissues once sampled, and, in the context of ancient...... genetic analyses, the fact that sampling generally causes minimal visual damage to valuable specimens. Even when freshly sampled, however, the DNA quantity and quality in the fully keratinized parts of such tissues is extremely poor in comparison to other tissues such as blood and muscle - although little...... systematic research has been undertaken to characterize how such degradation may relate to sample source. In this review paper we present the current understanding of the quality and limitations of DNA in two key keratinous tissues, nail and hair. The findings indicate that although some fragments of nuclear...

  13. Detection of DNA fragmentation in {gamma}-irradiated rat tongue epithelium

    Energy Technology Data Exchange (ETDEWEB)

    Yasuda, Motoaki; Nakamura, Motoyasu [Hokkaido Univ., Sapporo (Japan). School of Dentistry; Nishioka, Takeshi; Shirato, Hiroki

    1996-03-01

    Apoptosis is the predominant form of cell death and occurs under variety of physiological and pathological conditions. In the present study, we detected the apoptotic cells and S phase cells in rat tongue epithelium in both normal and irradiated conditions. After 24 hours from irradiation, cell cycle arrest was obvious and the number of apoptotic cell reached the maximum level; however there was no detectable DNA fragmentation in basal cells. (author).

  14. PEM fuel cell degradation

    Energy Technology Data Exchange (ETDEWEB)

    Borup, Rodney L [Los Alamos National Laboratory; Mukundan, Rangachary [Los Alamos National Laboratory

    2010-01-01

    The durability of PEM fuel cells is a major barrier to the commercialization of these systems for stationary and transportation power applications. While significant progress has been made in understanding degradation mechanisms and improving materials, further improvements in durability are required to meet commercialization targets. Catalyst and electrode durability remains a primary degradation mode, with much work reported on understanding how the catalyst and electrode structure degrades. Accelerated Stress Tests (ASTs) are used to rapidly evaluate component degradation, however the results are sometimes easy, and other times difficult to correlate. Tests that were developed to accelerate degradation of single components are shown to also affect other component's degradation modes. Non-ideal examples of this include ASTs examining catalyst degradation performances losses due to catalyst degradation do not always well correlate with catalyst surface area and also lead to losses in mass transport.

  15. Mechanisms of humic substances degradation by fungi

    Science.gov (United States)

    Chen, Y.; Hadar, Y.; Grinhut, T.

    2012-04-01

    Humic substances (HS) are formed by secondary synthesis reactions (humification) during the decay process and transformation of biomolecules originating from plants and other dead organisms. In nature, HS are extremely resistant to biological degradation. Thus, these substances are major components in the C cycle and in the biosphere and therefore, the understanding of the process leading to their formation and transformation and degradation is vital. Fungi active in the decomposition process of HS include mainly ascomycetes and basidiomycetes that are common in the upper layer of forest and grassland soils. Many basidiomycetes belong to the white-rot fungi (WRF) and litter-decomposing fungi (LDF). These fungi are considered to be the most efficient lignin degraders due to their nonspecific oxidizing enzymes: manganese peroxidase (MnP), lignin peroxidase (LiP) and laccase. Although bacteria dominate compost and participate in the turnover of HS, their ability to degrade stable macromolecules such as lignin and HS is limited. The overall objectives of this research were to corroborate biodegradation processes of HS by WRF. The specific objectives were: (i) To isolate, identify and characterize HS degrading WRF from biosolids (BS) compost; (ii) To study the biodegradation process of three types of HS, which differ in their structure, by WRF isolated from BS compost; and (iii) To investigate the mechanisms of HA degradation by WRF using two main approaches: (a) Study the physical and chemical analyses of the organic compounds obtained from direct fungal degradation of HA as well as elucidation of the relevant enzymatic reactions; and (b) Study the enzymatic and biochemical mechanisms involved during HA degradation. In order to study the capability of fungi to degrade HS, seventy fungal strains were isolated from biosolids (BS) compost. Two of the most active fungal species were identified based on rDNA sequences and designated Trametes sp. M23 and Phanerochaetesp., Y6

  16. Apoptotic effect of noscapine in breast cancer cell lines.

    Science.gov (United States)

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M

    2016-06-01

    Cancer is a public health problem in the world and breast cancer is the most frequently cancer in women. Approximately 15% of the breast cancers are triple-negative. Apoptosis regulates normal growth, homeostasis, development, embryogenesis and appropriate strategy to treat cancer. Bax is a protein pro-apoptotic enhancer of apoptosis in contrast to Bcl-2 with antiapoptotic properties. Initiator caspase-9 and caspase-8 are features of intrinsic and extrinsic apoptosis pathway, respectively. NF-κB is a transcription factor known to be involved in the initiation and progression of breast cancer. Noscapine, an alkaloid derived from opium is used as antitussive and showed antitumor properties that induced apoptosis in cancer cell lines. The aim of the present study was to determine the apoptotic effect of noscapine in breast cancer cell lines compared to breast normal cell line. Three cell lines were used: i) a control breast cell line MCF-10F; ii) a luminal-like adenocarcinoma triple-positive breast cell line MCF-7; iii) breast cancer triple-negative cell line MDA-MB-231. Our results showed that noscapine had lower toxicity in normal cells and was an effective anticancer agent that induced apoptosis in breast cancer cells because it increases Bax gene and protein expression in three cell lines, while decreases Bcl-xL gene expression, and Bcl-2 protein expression decreased in breast cancer cell lines. Therefore, Bax/Bcl-2 ratio increased in the three cell lines. This drug increased caspase-9 gene expression in breast cancer cell lines and caspase-8 gene expression increased in MCF-10F and MDA-MB-231. Furthermore, it increased cleavage of caspase-8, suggesting that noscapine-induced apoptosis is probably due to the involvement of extrinsic and intrinsic apoptosis pathways. Antiapoptotic gene and protein expression diminished and proapoptotic gene and protein expression increased noscapine-induced expression, probably due to decrease in NF-κB gene and protein expression

  17. Phylogenetic assessment of a new cypermethrin- degrading bacterium Gordonia sp.D2 based on 16S rDNA, gyrB and GyrB sequences%一株氯氰菊酯降解菌16S rDNA,gyrB和GyrB的系统发育分析

    Institute of Scientific and Technical Information of China (English)

    张久刚; 闫艳春

    2008-01-01

    A cypermethrin - degrading bacterium named as Gordonia sp. D2 was isolated from activated sludge of a wastewater treatment biore-actor. At 30 ℃ and pH 7.0, the removal rate of 100 mg cypennethrin L-1 was approximately 52.3% in mineral salts medium within 7.5 days, and extra carbon source addition could enhance the biodegradation. The isolate was identified as Gordonia genus through physio - bio- chemical identification combined with the phylogenetic analyses based on 16S rRNA, gyrB nucleotide and GyrB amino acid sequences. 16S rRNA sequences of strain D2 showed the highest similarity to G. amicalis DSM44461 and G. hydrophobica DSM44015T, with the same value of 98.1% ,meanwhile, D2 shows the highest similarity to G. hydrophobica JCA10086 with the value of 86.8% and 91.1% based on gyrB and GyrB. Three phylogenetic dendrograms were made, and results showed that 16S rRNA gene is suit for identifying the isolate to the genus level, gyrB gene and GyrB amino acid sequences are more adapted for phylogenetic analysis.%从农药厂污水处理池中分离得到一株氯氰菊酯降解菌,在30℃,pH 7.0的条件下,无机盐培养基中100mg/L的氯氰菊酯,经过7.5天,能够降解大约52.3%,外加碳源能够明显提高其降解性能.生理生化实验结合16S rDNA,gyrB和GyrB的系统发育分析,将其归为Gordonia菌属.在16S rDNA水平上,其与G.amicalis DSM44461和G.hydrophobica DSM44015的相似值最高,为98.1%;而在gyrB和GyrB水平上,其与G.hydrophobica JCM10086的相似值最高,分别为86.8%和91.1%.通过对所构建的系统发育树进行评估,表明16S rDNA序列适用于将分离菌株鉴定到属的水平上,而gyrB和GyrB更适用于在属内种的水平上进行系统发育的分析.

  18. Structure of the E6/E6AP/p53 complex required for HPV-mediated degradation of p53

    Science.gov (United States)

    Martinez-Zapien, Denise; Ruiz, Francesc Xavier; Poirson, Juline; Mitschler, André; Ramirez-Ramos, Juan; Forster, Anne; Cousido-Siah, Alexandra; Masson, Murielle; Pol, Scott Vande; Podjarny, Alberto; Travé, Gilles; Zanier, Katia

    2015-01-01

    Summary The p53 pro-apoptotic tumor suppressor is mutated or functionally altered in most cancers. In epithelial tumors induced by “high-risk” mucosal Human Papillomaviruses (hrm-HPVs), including human cervical carcinoma and a growing number of head-and-neck cancers 1, p53 is degraded by the viral oncoprotein E6 2. In this process, E6 binds to a short LxxLL consensus sequence within the cellular ubiquitin ligase E6AP 3. Subsequently, the E6/E6AP heterodimer recruits and degrades p53 4. Neither E6 nor E6AP are separately able to recruit p53 3,5, and the precise mode of assembly of E6, E6AP and p53 is unknown. Here, we solved the crystal structure of a ternary complex comprising full-length HPV16 E6, the LxxLL motif of E6AP and the core domain of p53. The LxxLL motif of E6AP renders the conformation of E6 competent for interaction with p53 by structuring a p53-binding cleft on E6. Mutagenesis of critical positions at the E6-p53 interface disrupts p53 degradation. The E6-binding site of p53 is distal from previously described DNA- and protein-binding surfaces of the core domain. This suggests that, in principle, E6 may avoid competition with cellular factors by targeting both free and bound p53 molecules. The E6/E6AP/p53 complex represents a prototype of viral hijacking of both the ubiquitin-mediated protein degradation pathway and the p53 tumor suppressor pathway. The present structure provides a framework for the design of inhibitory therapeutic strategies against HPV-mediated oncogenesis. PMID:26789255

  19. Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA

    DEFF Research Database (Denmark)

    Boessenkool, Sanne; Epp, Laura S.; Haile, James Seymour

    2012-01-01

    Analyses of degraded DNA are typically hampered by contamination, especially when employing universal primers such as commonly used in environmental DNA studies. In addition to false-positive results, the amplification of contaminant DNA may cause false-negative results because of competition, or...

  20. Microbial Aspects of Anaerobic BTEX Degradation

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Combined with conventional methods, developments in both geochemical (delineation of redox processes) and molecular microbial methods (analysis of 16S rDNA genes and functional genes) have allowed us to study in details microorganisms and genes involved in the anaerobic degradation of benzene, toluene, ethylbenzene and xylene (BTEX) under specific redox conditions. This review summarizes recent research in this field. The potential for anaerobic BTEX degradation is widely spread. Specific groups of microorganisms appear to be involved in degradation under different redox conditions. Members of the Azoarcus/Thauera cluster perform BTEX degradation under denitrifying conditions, Geobacteraceae under Fe (III) reducing conditions and Desulfobacteriaceae under sulfate reducing conditions. The information so far obtained on biochemistry and molecular genetics of BTEX degradation indicates that each BTEX compound is funneled into the central benzyol-CoA pathway by a different peripheral pathway. The peripheral pathways of per BTEX compound show similarities among different physiological groups of microorganisms. We also describe how knowledge obtained on the microbial aspects of BTEX degradation can be used to enhance and monitor anaerobic BTEX degradation.

  1. SIRT1 promotes DNA repair activity in response to radiation

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Jae-Min; Lee, Kee-Ho [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2006-07-01

    Human SIRT1 controls various physiological responses including cell fate, stress, and aging, through deacetylation of its specific substrate protein. In processing DNA damage signaling, SIRT1 attenuates a cellular apoptotic response by deacetylation of p53 tumor suppressor. Ectopically over-expressed SIRT1 resulted in the increase of repair of DNA strand breakages produced by radiation. On the other hand, repression of endogenous SIRT1 expression by SIRT1 siRNA led to the decrease of this repair activity, indicating that SIRT1 can regulate DNA repair capacity of cells with DNA strand breaks.

  2. Enhanced anticancer activity of gemcitabine in combination with noscapine via antiangiogenic and apoptotic pathway against non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Mahavir B Chougule

    Full Text Available BACKGROUND: The aim of this investigation was to evaluate the anticancer activity of Noscapine (Nos and Gemcitabine (Gem combination (NGC against non-small cell lung cancer (NSCLC and to elucidate the underlying mechanism of action. METHODS: Isobolographic method was used to calculate combination index values from cytotoxicity data. In vitro antiangiogenic and apoptotic activity of Nos, Gem and NGC was evaluated. For in vivo studies, female athymic Nu/nu mice were xenografted with H460 tumors and the efficacy of Nos, Gem, or NGC was determined. Protein expressions by immunohistochemical staining were evaluated in harvested tumor tissues. RESULTS: The CI values (<0.59 were suggestive of synergistic behavior between Nos and Gem. NGC treatment showed significantly inhibited tube formation and increased percentage of apoptotic cells. NGC, Gem and Nos treatment reduced tumor volume by 82.9±4.5 percent, 39.4±5.8 percent and 34.2±5.7 percent respectively. Specifically, NGC treatment decreased expression cell survival proteins; VEGF, CD31 staining and microvessel density and enhanced DNA fragmentation and cleaved caspase 3 levels compared to single agent treated and control groups. CONCLUSION: Nos potentiated the anticancer activity of Gem in an additive to synergistic manner against lung cancer via antiangiogenic and apoptotic pathways. These findings suggest potential benefit for use of NGC chemotherapy for treatment of lung cancer.

  3. Norepinephrine-induced apoptotic and hypertrophic responses in H9c2 cardiac myoblasts are characterized by different repertoire of reactive oxygen species generation

    Directory of Open Access Journals (Sweden)

    Anita Thakur

    2015-08-01

    Full Text Available Despite recent advances, the role of ROS in mediating hypertrophic and apoptotic responses in cardiac myocytes elicited by norepinephrine (NE is rather poorly understood. We demonstrate through our experiments that H9c2 cardiac myoblasts treated with 2 µM NE (hypertrophic dose generate DCFH-DA positive ROS only for 2 h; while those treated with 100 µM NE (apoptotic dose sustains generation for 48 h, followed by apoptosis. Though the levels of DCFH fluorescence were comparable at early time points in the two treatment sets, its quenching by DPI, catalase and MnTmPyP suggested the existence of a different repertoire of ROS. Both doses of NE also induced moderate levels of H2O2 but with different kinetics. Sustained but intermittent generation of highly reactive species detectable by HPF was seen in both treatment sets but no peroxynitrite was generated in either conditions. Sustained generation of hydroxyl radicals with no appreciable differences were noticed in both treatment sets. Nevertheless, despite similar profile of ROS generation between the two conditions, extensive DNA damage as evident from the increase in 8-OH-dG content, formation of γ-H2AX and PARP cleavage was seen only in cells treated with the higher dose of NE. We therefore conclude that hypertrophic and apoptotic doses of NE generate distinct but comparable repertoire of ROS/RNS leading to two very distinct downstream responses.

  4. Suppression of interleukin-33 bioactivity through proteolysis by apoptotic caspases.

    Science.gov (United States)

    Lüthi, Alexander U; Cullen, Sean P; McNeela, Edel A; Duriez, Patrick J; Afonina, Inna S; Sheridan, Clare; Brumatti, Gabriela; Taylor, Rebecca C; Kersse, Kristof; Vandenabeele, Peter; Lavelle, Ed C; Martin, Seamus J

    2009-07-17

    Interleukin-33 (IL-33) is a member of the IL-1 family and is involved in polarization of T cells toward a T helper 2 (Th2) cell phenotype. IL-33 is thought to be activated via caspase-1-dependent proteolysis, similar to the proinflammatory cytokines IL-1 beta and IL-18, but this remains unproven. Here we showed that IL-33 was processed by caspases activated during apoptosis (caspase-3 and -7) but was not a physiological substrate for caspases associated with inflammation (caspase-1, -4, and -5). Furthermore, caspase-dependent processing of IL-33 was not required for ST2 receptor binding or ST2-dependent activation of the NF-kappaB transcription factor. Indeed, caspase-dependent proteolysis of IL-33 dramatically attenuated IL-33 bioactivity in vitro and in vivo. These data suggest that IL-33 does not require proteolysis for activation, but rather, that IL-33 bioactivity is diminished through caspase-dependent proteolysis within apoptotic cells. Thus, caspase-mediated proteolysis acts as a switch to dampen the proinflammatory properties of IL-33.

  5. An overview of caspase: Apoptotic protein for silicosis

    Directory of Open Access Journals (Sweden)

    Tumane Rajani

    2010-01-01

    Full Text Available Silicosis is a chronic lung disease characterized by granulomatous and fibrotic lesions, which occurs due to accumulation of respirable silica mineral particles. Apoptosis is an important phenomenon of cell death in silicosis. The relationship between silica dust and its exposure is well established. But, the complex chain of cellular responses, which leads to caspase activation in silicosis, has not been fully discovered. Caspase activation plays a central role in the execution of apoptosis. Silica-induced apoptosis of the alveolar macrophages could potentially favor a proinflammatory state, occurring in the lungs of silicotic patients, resulting in the activation of caspase prior to induction of the intrinsic and extrinsic apoptosis pathways. Recent studies indicated that apoptosis may involve in pulmonary disorders. This review summarizes the current knowledge about the underling mechanism of biochemical pathways in caspase activation that have been ignored so far in silicosis. In addition, caspase could be a key apoptotic protein that can be used as an effective biomarker for the study of occupational diseases. It may provide an important link in understanding the molecular mechanisms of silica-induced lung pathogenesis.

  6. Fragile DNA Repair Mechanism Reduces Ageing in Multicellular Model

    DEFF Research Database (Denmark)

    Bendtsen, Kristian Moss; Juul, Jeppe Søgaard; Trusina, Ala

    2012-01-01

    DNA damages, as well as mutations, increase with age. It is believed that these result from increased genotoxic stress and decreased capacity for DNA repair. The two causes are not independent, DNA damage can, for example, through mutations, compromise the capacity for DNA repair, which in turn...... to DNA damage can undergo full repair, go apoptotic, or accumulate mutations thus reducing DNA repair capacity. Our model predicts that at the tissue level repair rate does not continuously decline with age, but instead has a characteristic extended period of high and non-declining DNA repair capacity...... of compromised cells, thus freeing the space for healthy peers. This finding might be a first step toward understanding why a mutation in single DNA repair protein (e.g. Wrn or Blm) is not buffered by other repair proteins and therefore, leads to severe ageing disorders...

  7. Evidence for involvement of c-Src in the anti-apoptotic action of nitric oxide in serum-deprived RINm5F cells.

    Science.gov (United States)

    Tejedo, J R; Ramírez, R; Cahuana, G M; Rincón, P; Sobrino, F; Bedoya, F J

    2001-11-01

    The mechanism by which nitric oxide (NO) protects from apoptosis is a matter of debate. We have shown previously that phosphorylation of tyrosine residues participates in the protection from apoptosis in insulin-producing RINm5F cells (Inorg. Chem. Commun. 3 (2000) 32). Since NO has been reported to activate the tyrosine kinase c-Src and this kinase is involved in the activation of protein kinase G (PKG) in some cell systems, we aimed at studying the contribution of c-Src and PKG systems in anti-apoptotic actions of NO in serum-deprived RINm5F cells. Here we report that exposure of serum-deprived cells to 10 microM DETA/NO results in protection from degradation of the anti-apoptotic protein Bcl-2, together with a reduction of cytochrome c release from mitochondria and caspase-3 inhibition. Studies with the inhibitors ODQ and KT-5823 revealed that these actions are dependent on both activation of guanylate cyclase and PKG. DETA/NO was also able to induce autophosphorylation and activation c-Src protein both in vivo and in vitro and active c-Src was able to induce tyrosine phosphorylation of Bcl-2 in vitro. The c-Src kinase inhibitor PP1 abrogated the actions of DETA/NO on cGMP formation, PKG activation, caspase activation, cytochrome c release from mitochondria, and Bcl-2 phosphorylation and degradation in serum-deprived cells. We thus propose that activation of c-Src is an early step in the chain of events that signal cGMP-dependent anti-apoptotic actions of NO in mitocohondria.

  8. ARK, the Apaf-1 related killer in Drosophila, requires diverse domains for its apoptotic activity.

    Science.gov (United States)

    Srivastava, M; Scherr, H; Lackey, M; Xu, D; Chen, Z; Lu, J; Bergmann, A

    2007-01-01

    In mammals and Drosophila, apoptotic caspases are under positive control of the CED-4-like proteins Apaf-1 and ARK, respectively. In an EMS-mutagenesis screen, we isolated 33 ark mutants as recessive suppressors of hid-induced apoptosis. The ark mutants are loss-of-function alleles characterized by reduced developmental apoptosis. Using the phenotypic series of these alleles, we identified helical domain I in the nucleotide oligomerization domain as critical for ARK's apoptotic activity. Interestingly, the WD40 region may also have an unanticipated positive requirement for the apoptotic activity of ARK. Considering structural information, we discuss the roles of these domains for assembly and activity of the ARK apoptosome, and propose that the WD40 region is anti-apoptotic in the absence of apoptotic signals, and pro-apoptotic in the presence of such signals. Furthermore, a defined null allele reveals that ark is required for most, but not all apoptosis suggesting the existence of an ARK-independent apoptotic pathway.

  9. Oligomerization and DNA-binding capacity of Pmr, a histone-like protein H1 (H-NS) family protein encoded on IncP-7 carbazole-degradative plasmid pCAR1.

    Science.gov (United States)

    Suzuki, Chiho; Yun, Choong-Soo; Umeda, Takashi; Terabayashi, Tsuguno; Watanabe, Kazuya; Yamane, Hisakazu; Nojiri, Hideaki

    2011-01-01

    Pmr, a histone-like protein H1 (H-NS) family protein encoded on plasmid pCAR1, is a key factor in optimizing gene transcription on both pCAR1 and the host chromosome. To clarify the mode of function of Pmr, we performed gel filtration chromatography analysis and protein-protein cross-linking, and found that Pmr forms homo-oligomers, consisting of its homodimers. We also found, by atomic force microscopy, that Pmr has DNA-bridging capacity. From these results, Pmr was deduced to have features common to H-NS family proteins. Additionally, evaluating protein-DNA affinity is important to clarify the mode of function of Pmr, and hence we performed an electrophoretic mobility shift assay. Though Pmr formed high-order protein-DNA complexes and did not show preference for nucleic acid sequences, the C-terminal region of Pmr did, suggesting that the DNA-binding affinity of Pmr can be evaluated by using its C-terminal region.

  10. Does MW Radiation Affect Gene Expression, Apoptotic Level, and Cell Cycle Progression of Human SH-SY5Y Neuroblastoma Cells?

    Science.gov (United States)

    Kayhan, Handan; Esmekaya, Meric Arda; Saglam, Atiye Seda Yar; Tuysuz, Mehmed Zahid; Canseven, Ayşe Gulnihal; Yagci, Abdullah Munci; Seyhan, Nesrin

    2016-06-01

    Neuroblastoma (NB) is a cancer that occurs in sympathetic nervous system arising from neuroblasts and nerve tissue of the adrenal gland, neck, chest, or spinal cord. It is an embryonal malignancy and affects infants and children. In this study, we investigated the effects of microwave (MW) radiation on apoptotic activity, cell viability, and cell cycle progression in human SH-SY5Y NB cells which can give information about MW radiation effects on neural cells covering the period from the embryonic stages to infants. SH-SY5Y NB cells were exposed to 2.1 GHz W-CDMA modulated MW radiation for 24 h at a specific absorption rate of 0.491 W/kg. Control samples were in the same conditions with MW-exposed samples but they were not exposed to MW radiation. The apoptotic activity of cells was measured by Annexin-V-FITC and propidium iodide staining. Moreover, mRNA levels of proliferative and cell cycle proteins were determined by real-time RT-PCR. The change in cell cycle progression was observed by using CycleTest-Plus DNA reagent. No significant change was observed in apoptotic activity of MW-exposed cells compared to control cells. The mRNA levels of c-myc and cyclin D1 were significantly reduced in MW group (p CDMA modulated MW radiation did not cause apoptotic cell death but changed cell cycle progression.

  11. An NQO1-initiated and p53-independent apoptotic pathway determines the anti-tumor effect of tanshinone IIA against non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Fang Liu

    Full Text Available NQO1 is an emerging and promising therapeutic target in cancer therapy. This study was to determine whether the anti-tumor effect of tanshinone IIA (TSA is NQO1 dependent and to elucidate the underlying apoptotic cell death pathways. NQO1(+ A549 cells and isogenically matched NQO1 transfected and negative H596 cells were used to test the properties and mechanisms of TSA induced cell death. The in vivo anti-tumor efficacy and the tissue distribution properties of TSA were tested in tumor xenografted nude mice. We observed that TSA induced an excessive generation of ROS, DNA damage, and dramatic apoptotic cell death in NQO1(+ A549 cells and H596-NQO1 cells, but not in NQO1(- H596 cells. Inhibition or silence of NQO1 as well as the antioxidant NAC markedly reversed TSA induced apoptotic effects. TSA treatment significantly retarded the tumor growth of A549 tumor xenografts, which was significantly antagonized by dicoumarol co-treatment in spite of the increased and prolonged TSA accumulations in tumor tissues. TSA activated a ROS triggered, p53 independent and caspase dependent mitochondria apoptotic cell death pathway that is characterized with increased ratio of Bax to Bcl-xl, mitochondrial membrane potential disruption, cytochrome c release, and subsequent caspase activation and PARP-1 cleavage. The results of these findings suggest that TSA is a highly specific NQO1 target agent and is promising in developing as an effective drug in the therapy of NQO1 positive NSCLC.

  12. Natural proteasome inhibitor celastrol suppresses androgen-independent prostate cancer progression by modulating apoptotic proteins and NF-kappaB.

    Directory of Open Access Journals (Sweden)

    Yao Dai

    Full Text Available Celastrol is a natural proteasome inhibitor that exhibits promising anti-tumor effects in human malignancies, especially the androgen-independent prostate cancer (AIPC with constitutive NF-κB activation. Celastrol induces apoptosis by means of proteasome inhibition and suppresses prostate tumor growth. However, the detailed mechanism of action remains elusive. In the current study, we aim to test the hypothesis that celastrol suppresses AIPC progression via inhibiting the constitutive NF-κB activity as well as modulating the Bcl-2 family proteins.We examined the efficacy of celastrol both in vitro and in vivo, and evaluated the role of NF-κB in celastrol-mediated AIPC regression. We found that celastrol inhibited cell proliferation in all three AIPC cell lines (PC-3, DU145 and CL1, with IC₅₀ in the range of 1-2 µM. Celastrol also suppressed cell migration and invasion. Celastrol significantly induced apoptosis as evidenced by increased sub-G1 population, caspase activation and PARP cleavage. Moreover, celastrol promoted cleavage of the anti-apoptotic protein Mcl-1 and activated the pro-apoptotic protein Noxa. In addition, celastrol rapidly blocked cytosolic IκBα degradation and nuclear translocation of RelA. Likewise, celastrol inhibited the expression of multiple NF-κB target genes that are involved in proliferation, invasion and anti-apoptosis. Celastrol suppressed AIPC tumor progression by inhibiting proliferation, increasing apoptosis and decreasing angiogenesis, in PC-3 xenograft model in nude mouse. Furthermore, increased cellular IκBα and inhibited expression of various NF-κB target genes were observed in tumor tissues.Our data suggest that, via targeting the proteasome, celastrol suppresses proliferation, invasion and angiogenesis by inducing the apoptotic machinery and attenuating constitutive NF-κB activity in AIPC both in vitro and in vivo. Celastrol as an active ingredient of traditional herbal medicine could thus be

  13. Growth inhibition and pro-apoptotic activity of violacein in Ehrlich ascites tumor.

    Science.gov (United States)

    Bromberg, Natália; Dreyfuss, Juliana L; Regatieri, Caio V; Palladino, Marcelly V; Durán, Nelson; Nader, Helena B; Haun, Marcela; Justo, Giselle Z

    2010-06-07

    The continuing threat to biodiversity lends urgency to the need of identification of sustainable source of natural products. This is not so much trouble if there is a microbial source of the compound. Herein, violacein, a natural indolic pigment extracted from Chromobacterium violaceum, was evaluated for its antitumoral potential against the Ehrlich ascites tumor (EAT) in vivo and in vitro. Evaluation of violacein cytotoxicity using different endpoints indicated that EAT cells were twofold (IC(50)=5.0 microM) more sensitive to the compound than normal human peripheral blood lymphocytes. In vitro studies indicated that violacein cytotoxicity to EAT cells is mediated by a rapid (8-12h) production of reactive oxygen species (ROS) and a decrease in intracellular GSH levels, probably due to oxidative stress. Additionally, apoptosis was primarily induced, as demonstrated by an increase in Annexin-V positive cells, concurrently with increased levels of DNA fragmentation and increased caspase-2, caspase-9 and caspase-3 activities up to 4.5-, 6.0- and 5.5-fold, respectively, after 72 h of treatment. Moreover, doses of 0.1 and 1.0 microg kg(-1) violacein, administered intraperitoneally (i.p.) to EAT-bearing mice throughout the lifespan of the animals significantly inhibited tumor growth and increased survival of mice. In view of these results, a 35-day toxicity study was conducted in vivo. Complete hematology, biochemistry (ALT, AST and creatinine levels) and histopathological analysis of liver and kidney indicated that daily doses of violacein up to 1000 microg kg(-1) for 35 days are well tolerated and did not cause hematotoxicity nor renal or hepatotoxicity when administered i.p. to mice. Altogether, these results indicate that violacein causes oxidative stress and an imbalance in the antioxidant defense machinery of cells culminating in apoptotic cell death. Furthermore, this is the first report of its antitumor activity in vivo, which occurs in the absence of toxicity to

  14. Synthesis of phenstatin/isocombretastatin-chalcone conjugates as potent tubulin polymerization inhibitors and mitochondrial apoptotic inducers.

    Science.gov (United States)

    Kamal, Ahmed; Kumar, G Bharath; Vishnuvardhan, M V P S; Shaik, Anver Basha; Reddy, Vangala Santhosh; Mahesh, Rasala; Sayeeda, Ibrahim Bin; Kapure, Jeevak Sopanrao

    2015-04-07

    A series of phenstatin/isocombretastatin–chalcones were synthesized and screened for their cytotoxic activity against various human cancer cell lines. Some representative compounds exhibited significant antiproliferative activity against a panel of sixty human cancer cell lines of the NCI, with GI50 values in the range of 0.11 to 19.0 μM. Three compounds (3b, 3c and 3e) showed a broad spectrum of antiproliferative efficacy on most of the cell lines in the sub-micromolar range. In addition, all the synthesized compounds (3a–l and 4a–l) displayed moderate to excellent cytotoxicity against breast cancer cells such as MCF-7 and MDA-MB-231 with IC50 values in the range of 0.5 to 19.9 μM. Moreover, the tubulin polymerization assay and immunofluorescence analysis results suggest that some of these compounds like 3c and 3e exhibited significant inhibitory effect on the tubulin assembly with an IC50 value of 0.8 μM and 0.6 μM respectively. A competitive binding assay suggested that these compounds bind at the colchicine-binding site of tubulin. A cell cycle assay revealed that these compounds arrest at the G2/M phase and lead to apoptotic cell death. Furthermore, this was confirmed by Hoechst 33258 staining, activation of caspase 9, DNA fragmentation, Annexin V-FITC and mitochondrial membrane depolarization. Molecular docking studies indicated that compounds like 3e occupy the colchicine binding site of tubulin.

  15. 转基因大豆内外源DNA在少孢根霉发酵中的降解%Degradation of endogenous and exogenous DNA in transgenic roundup ready soybean during fermentation with Rhizopus oligosporus

    Institute of Scientific and Technical Information of China (English)

    杨爱萍; 蒋义武; 祝长青; 何健; 刘欣; 宋尚新

    2012-01-01

    试验旨在研究转基因大豆内、外源基因在少孢根霉发酵过程中的降解变化规律,并探讨其降解规律与相关酶活性的关系.试验采用定性和定量PCR检测各基因的降解变化,同时测定相关酶活.结果表明:内源基因Lectin和外源基因EPSPS在高温蒸煮下降解至800 bp以下,在发酵4d后降解至200 bp以下;发酵过程中外源基因相对含量的变化与核酸酶、脂肪酶、蛋白酶活性的变化相一致.该研究为我国转基因食品加工过程中外源基因的降解及其机理研究提供一定的理论基础.%The purpose of study was to study the degradation of endogenous and exogenous genes and the relationship with associated enzymes activities in the process of fermentation with Rhizopus oligosporus. Qualitative and quantitative PCR were used to analyze the changes of the endogenous and exogenous gene, and the associated enzyme activities were also detected. The results showed that the endogenous gene Lectin and the exogenous gene EPSPS were degraded to less than 800 bp after high-temperature boiling and to less than 200 bp after four days of fermentation. In the process of fermentation, the variation of the relative content of exogenous genes was consistent with the variation of activities of associated nuclease, lipase and protease in soybeans. Therefore, this study provided theoretical foundations for the mechanism of exogenous gene degradation during the transgenic food processing.

  16. Non-apoptotic programmed cell death with paraptotic-like features in bleomycin-treated plant cells is suppressed by inhibition of ATM/ATR pathways or NtE2F overexpression.

    Science.gov (United States)

    Smetana, Ondřej; Široký, Jiří; Houlné, Guy; Opatrný, Zdeněk; Chabouté, Marie-Edith

    2012-04-01

    In plants, different forms of programmed cell death (PCD) have been identified, but they only partially correspond to those described for animals, which is most probably due to structural differences between animal and plant cells. Here, the results show that in tobacco BY-2 cells, bleomycin (BLM), an inducer of double-strand breaks (DSBs), triggers a novel type of non-apoptotic PCD with paraptotic-like features. Analysis of numerous PCD markers revealed an extensive vacuolization, vacuolar rupture, and chromatin condensation, but no apoptotic DNA fragmentation, fragmentation of the nuclei, or sensitivity to caspase inhibitors. BLM-induced PCD was cell cycle regulated, occurring predominantly upon G(2)/M cell cycle checkpoint activation. In addition, this paraptotic-like PCD was at least partially inhibited by caffeine, a known inhibitor of DNA damage sensor kinases ATM and ATR. Interestingly, overexpression of one NtE2F transcriptional factor, whose homologues play a dual role in animal apoptosis and DNA repair, reduced PCD induction and modulated G(2)/M checkpoint activation in BY-2 cells. These observations provide a solid ground for further investigations into the paraptotic-like PCD in plants, which might represent an ancestral non-apoptotic form of PCD conserved among animals, protists, and plants.

  17. DNA-PKcs deficiency sensitizes the human hepatoma HepG2 cells to cisplatin and 5-fluorouracil through suppression of the PI3K/Akt/NF-κB pathway.

    Science.gov (United States)

    Fang, Yuan; Chai, Zongtao; Wang, Dansong; Kuang, Tiantao; Wu, Wenchuan; Lou, Wenhui

    2015-01-01

    The aim of the present study was to investigate the effects of DNA-PKcs deficiency on the chemosensitivity of human hepatoma HepG2 cells to cisplatin (CDDP) and 5-fluorouracil (5-Fu), and to explore the underlying molecular mechanism. After transfection with DNA-PKcs siRNA or control siRNA, HepG2 cells were exposed to combination treatment of CDDP and 5-Fu. The cell viability, DNA damage, cell apoptosis, intracellular reactive oxygen species and glutathione (GSH) level, expression of apoptosis related proteins, activity of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway, and nuclear factor-κB (NF-κB) pathways were assessed. The combination of CDDP and 5-Fu had a synergistic cytotoxic effect in HepG2 cells in terms of the cell viability, DNA damage, apoptosis, and oxidative stress level. DNA-PKcs siRNA could sensitize the HepG2 cells to the combined treatment. DNA-PKcs suppression further reduced the Akt phosphorylation level and Bcl-2 expression in HepG2 cells exposed to CDDP and 5-Fu, but enhanced the expression of pro-apoptotic proteins p53 and caspase-3. Moreover, CDDP could inhibit the transcriptional activity of NF-κB through degradation of IkB-α, while 5-Fu alone seemed in some extent increases the NF-κB activity. The combined treatment with CDDP and 5-Fu resulted in significantly decrease of the transcriptional activity of NF-κB, which was further aggravated by DNA-PKcs siRNA treatment. In conclusion, DNA-PKcs suppression had complementary effects in combination with CDDP and 5-Fu treatment in HepG2 cells, which was associated with suppression of NF-κB signaling pathway cascade, activation of caspase-3 and p53, as well as down-regulation of Bcl-2 and GSH.

  18. Phototherapy-treated apoptotic tumor cells induce pro-inflammatory cytokines production in macrophage

    Science.gov (United States)

    Lu, Cuixia; Wei, Yanchun; Xing, Da

    2014-09-01

    Our previous studies have demonstrated that as a mitochondria-targeting cancer phototherapy, high fluence low-power laser irradiation (HF-LPLI) induces mitochondrial superoxide anion burst, resulting in oxidative damage to tumor cells. In this study, we further explored the immunological effects of HF-LPLI-induced apoptotic tumor cells. When macrophages were co-incubated with apoptotic cells induced by HF-LPLI, we observed the increased levels of TNF-α secretion and NO production in macrophages. Further experiments showed that NF-κB was activated in macrophages after co-incubation with HF-LPLI-induced apoptotic cells, and inhibition of NF-κB activity by pyrrolidinedithiocarbamic acid (PDTC) reduced the elevated levels of TNF-α secretion and NO production. These data indicate that HF-LPLI-induced apoptotic tumor cells induce the secretion of pro-inflammatory cytokines in macrophages, which may be helpful for better understanding the biological effects of cancer phototherapy.

  19. Apoptotic killing of HIV-1-infected macrophages is subverted by the viral envelope glycoprotein.

    Directory of Open Access Journals (Sweden)

    Simon Swingler

    2007-09-01

    Full Text Available Viruses have evolved strategies to protect infected cells from apoptotic clearance. We present evidence that HIV-1 possesses a mechanism to protect infected macrophages from the apoptotic effects of the death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand. In HIV-1-infected macrophages, the viral envelope protein induced macrophage colony-stimulating factor (M-CSF. This pro-survival cytokine downregulated the TRAIL receptor TRAIL-R1/DR4 and upregulated the anti-apoptotic genes Bfl-1 and Mcl-1. Inhibition of M-CSF activity or silencing of Bfl-1 and Mcl-1 rendered infected macrophages highly susceptible to TRAIL. The anti-cancer agent Imatinib inhibited M-CSF receptor activation and restored the apoptotic sensitivity of HIV-1-infected macrophages, suggesting a novel strategy to curtail viral persistence in the macrophage reservoir.

  20. Amelioration of nandrolone decanoate-induced testicular and sperm toxicity in rats by taurine: Effects on steroidogenesis, redox and inflammatory cascades, and intrinsic apoptotic pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, Maha A.E., E-mail: mahapharm@yahoo.com

    2015-02-01

    The wide abuse of the anabolic steroid nandrolone decanoate by athletes and adolescents for enhancement of sporting performance and physical appearance may be associated with testicular toxicity and infertility. On the other hand, taurine; a free β-amino acid with remarkable antioxidant activity, is used in taurine-enriched beverages to boost the muscular power of athletes. Therefore, the purpose of this study was to investigate the mechanisms of the possible protective effects of taurine on nandrolone decanoate-induced testicular and sperm toxicity in rats. To achieve this aim, male Wistar rats were randomly distributed into four groups and administered either vehicle, nandrolone decanoate (10 mg/kg/week, I.M.), taurine (100 mg/kg/day, p.o.) or combination of taurine and nandrolone decanoate, for 8 successive weeks. Results of the present study showed that taurine reversed nandrolone decanoate-induced perturbations in sperm characteristics, normalized serum testosterone level, and restored the activities of the key steroidogenic enzymes; 3β-HSD, and 17β-HSD. Moreover, taurine prevented nandrolone decanoate-induced testicular toxicity and DNA damage by virtue of its antioxidant, anti-inflammatory, and anti-apoptotic effects. This was evidenced by taurine-induced modulation of testicular LDH-x activity, redox markers (MDA, NO, GSH contents, and SOD activity), inflammatory indices (TNF-α, ICAM-1 levels, and MMP-9 gene expression), intrinsic apoptotic pathway (cytochrome c gene expression and caspase-3 content), and oxidative DNA damage markers (8-OHdG level and comet assay). In conclusion, at the biochemical and histological levels, taurine attenuated nandrolone decanoate-induced poor sperm quality and testicular toxicity in rats. - Highlights: • Nandrolone decanoate (ND) disrupts sperm profile and steroidogenesis in rats. • ND upregulates gene expression of inflammatory and apoptotic markers. • Taurine normalizes sperm profile and serum testosterone level

  1. Thraustochytrid protists degrade hydrocarbons

    Digital Repository Service at National Institute of Oceanography (India)

    Raikar, M.T.; Raghukumar, S.; Vani, V.; David, J.J.; Chandramohan, D.

    Rev, 54 (1990) 305-315. 12 Fedorak P M & Westlake D W S, Microbial degradation of aromatics and saturates in Prudhoe Bay crude oil as determined by glass capillary gas chromatography, Can J Microbiol, (1981) 432-443. 13 Atlas R M, Microbial... degradation of petroleum hydrocarbons: An environmental perspective, Microbiol Rev, 45 (1981) 180-209. 14 Atlas R M, Microbial hydrocarbon degradation- Bioremediation of oil spills, J Chem Tech Biotechnol, 52 (1991) 149-156 15 Venkateswaran K, Iwabuchi T...

  2. Investigation of single nucleotide polymorphism loci susceptible to degradation by ultraviolet light.

    Science.gov (United States)

    Machida, Mitsuyo; Taki, Takashi; Shimada, Ryo; Kibayashi, Kazuhiko

    2016-10-01

    DNA in biological fluids is often degraded by environmental factors. Given that single nucleotide polymorphism (SNP) analyses require shorter amplicons than short tandem repeat (STR) analyses do, their use in human identification using degraded samples has recently attracted attention. Although various SNP loci are used to analyze degraded samples, it is unclear which ones are more appropriate. To characterize and identify SNP loci that are susceptible or resistant to degradation, we artificially degraded DNA, obtained from buccal swabs from 11 volunteers, by exposure to ultraviolet (UV) light for different durations (254 nm for 5, 15, 30, 60, or 120 min) and analyzed the resulting SNP loci. DNA degradation was assessed using gel electrophoresis, STR, and SNP profiling. DNA fragmentation occurred within 5 min of UV irradiation, and successful STR and SNP profiling decreased with increasing duration. However, 73% of SNP loci were still detected correctly in DNA samples irradiated for 120 min, a dose that rendered STR loci undetectable. The unsuccessful SNP typing and the base call failure of nucleotides neighboring the SNPs were traced to rs1031825, and we found that this SNP was susceptible to UV light. When comparing the detection efficiencies of STR and SNP loci, SNP typing was more successful than STR typing, making it effective when using degraded DNA. However, it is important to use rs1031825 with caution when interpreting SNP analyses of degraded DNA.

  3. Decreased Apoptotic Rate of Alveolar Macrophages of Patients with Idiopathic Pulmonary Fibrosis

    Directory of Open Access Journals (Sweden)

    Fotios Drakopanagiotakis

    2012-01-01

    and control group. No difference was found between the respiratory function parameters of the two treatment groups after six months. A positive correlation was found between the number of bcl-2 positive stained macrophages and DLCO after treatment. Conclusions. The decreased apoptotic rate of AM of patients with IPF is not associated with decreased expression of apoptosis mediators involved in the external or internal apoptotic pathway.

  4. Evaluating Ethanol-based Sample Preservation to Facilitate Use of DNA Barcoding in Routine Freshwater Biomonitoring Programs Using Benthic Macroinvertebrates

    Science.gov (United States)

    Molecular methods, such as DNA barcoding, have the potential in enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biom...

  5. Phylogenetic and degradation characterization of Burkholderia cepacia WZ1 degrading herbicide quinclorac.

    Science.gov (United States)

    Lü, Zhenmei; Min, Hang; Wu, Shuwen; Ruan, Aidong

    2003-11-01

    Strain WZI capable of degrading quinclorac was isolated from a pesticide manufactory soil and considered to be Burkholderia cepacia, belonged to bacteria, Proteobacteria, beta-Proteobacteria, based on morphology, physio-biochemical properties, whole cell fatty acid analysis and a partial sequencing of 16S rDNA. Strain WZ1 decomposed 90% of quinclorac at original concentration of 1000 mg L(-1) within 11 days. GC/MS analysis showed that the strain degraded quinclorac to 3,7-dichloro-8-quinoline and the cracked residue 2-chloro, 1,4-benzenedicarboxylic acid, indicating that the metabolic pathway was initiated by process of decarboxylation followed by cleavage of the aromatic ring. Stain WZ1 was also able to degrade some other herbicides and aromatic compounds, including 2,4,5-T, phenol, naphthalene and hydrochinone etc. This paper describes for the first time Phylogenetic and degradation characterization of a pure bacterium which, is able to mineralize quinclorac.

  6. p53-independent pRB degradation contributes to a drug-induced apoptosis in AGS cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    The retinoblastoma (RB) tumor suppressor protein, pRB, plays an important role in the regulation of mammalian cell cycle. Furthermore, several lines of evidence suggest that pRB also involves in the regulation of apoptosis. In the present study, the degradation of pRB was observed in apoptotic gastric tumor cells treated with a new potent anti-tumor component, tripchlorolide (TC). The inhibition of pRB degradation by a general cysteine protease inhibitor IDAM resulted in the reduction of the apoptotic cells. Furthermore, the survival of the gastric tumor cells under the TC treatment was enhanced by an over-expression of exogenous pRB. These results suggest that the pRB degradation of the gastric tumor cells under the TC treatment involves in the apoptotic progression. In addition, the same extent of TC-induced pRB-degradation was detected in the gastric tumor cells containing a p53 dominant-negative construct, indicating that this kind of pRB degradation is p53-independent.

  7. Ultrastructural and cytochemical identification of apoptotic cell death accompanying development of the fetal rat olfactory nerve layer.

    Science.gov (United States)

    Pellier, V; Saucier, D; Oestreicher, A B; Astic, L

    1996-07-01

    It has been previously shown that the embryonic olfactory nerve contains, in addition to glial ensheathing cells, a large population of differentiated neurons that migrate from the developing olfactory epithelium, in close association with the olfactory axon fascicles. The purpose of our study was to verify the hypothesis according to which a process of physiological cell death might be involved in the progressive disappearance of these migrating neurons that has been reported during late embryonic stages in several immunocytochemical studies. To do so, we have investigated the development of the olfactory nerve layer in rat embryos by using light and electron microscopy, with special reference to the presence of cell death processes within this structure. We have also applied the histochemical TUNEL method allowing in situ visualization of cells degenerating by apoptosis. In order to determine if neurons were present among dying cells, a procedure of double-labeling was performed by combining the DNA-specific bisbenzimide with two neuronal markers, the protein B-50/GAP-43 and the lectin Ulex europaeus I. Results brought out the precise temporal and spatial patterns of programmed cell death accompanying the morphogenesis of the olfactory nerve layer. A cell death process was observed within the olfactory nerve layer from its onset at embryonic day 13 (E13). While only few pycnotic cells were observed in E13 and E14 embryos, their number increased from E15 to reach a maximum at E16 and then diminished. Few dying cells were also observed along the olfactory axon fascicles when they penetrated the olfactory nerve layer. Degenerating cells appeared strongly TUNEL-labeled and exhibited morphological features of cell death by apoptosis. Double-labeling experiments revealed that some of the apoptotic cells were neurons. These observations indicate that apoptosis may account for the progressive decrease in the number of migrating neurons present within the embryonic

  8. Emodin Induces Apoptotic Death in Murine Myelomonocytic Leukemia WEHI-3 Cells In Vitro and Enhances Phagocytosis in Leukemia Mice In Vivo

    Directory of Open Access Journals (Sweden)

    Yuan-Chang Chang

    2011-01-01

    Full Text Available Emodin is one of major compounds in rhubarb (Rheum palmatum L., a plant used as herbal medicine in Chinese population. Although many reports have shown that emodin exhibits anticancer activity in many tumor cell types, there is no available information addressing emodin-affected apoptotic responses in the murine leukemia cell line (WEHI-3 and modulation of the immune response in leukemia mice. We investigated that emodin induced cytotoxic effects in vitro and affected WEHI-3 cells in vivo. This study showed that emodin decreased viability and induced DNA fragmentation in WEHI-3 cells. Cells after exposure to emodin for 24 h have shown chromatin condensation and DNA damage. Emodin stimulated the productions of ROS and Ca2+ and reduced the level of ΔΨm by flow cytometry. Our results from Western blotting suggest that emodin triggered apoptosis of WEHI-3 cells through the endoplasmic reticulum (ER stress, caspase cascade-dependent and -independent mitochondrial pathways. In in vivo study, emodin enhanced the levels of B cells and monocytes, and it also reduced the weights of liver and spleen compared with leukemia mice. Emodin promoted phagocytic activity by monocytes and macrophages in comparison to the leukemia mice group. In conclusions, emodin induced apoptotic death in murine leukemia WEHI-3 cells and enhanced phagocytosis in the leukemia animal model.

  9. Cytotoxic and pro-apoptotic activities of leaf extract of Croton bonplandianus Baill. against lung cancer cell line A549.

    Science.gov (United States)

    Bhavana, J; Kalaivani, M K; Sumathy, A

    2016-06-01

    The acetone extract (AcE) of the Croton bonplandianus Baill., an exotic weed of the Euphorbiaceae family was studied for cytotoxicity, apoptosis, cell cycle arrest in A549 cell line and antioxidant capacities using MTT assay, acridine orange/ethidium bromide (AO/EB staining), cell cycle analysis and DPPH radical scavenging assay respectively. Based on the cytotoxic activity, the extract was tested for the apoptotic effect using AO/EB and Hoechst 33258 staining. The apoptosis was characterized by chromatin condensation and DNA fragmentation. Further, to determine the stage of cell death, cell cycle analysis was performed by flow cytometry and AcE was found to arrest G2/M phase in a dose dependent manner. The number of cells in G2/M phase increases with concurrent accumulation of cells in sub G₀/G₁phase indicates the induction of apoptosis at G2M phase. The free radical scavenging activity of the AcE against DPPH was considerably significant. The cytotoxic, apoptotic and antioxidant effect of the AcE could be well correlated with the presence of potent free radical scavenging secondary metabolites such as phenols (43 ± 0.05 µg/mL), flavonoids (3.5 ± 0.07 µg/mL) and tannin (0.36 ± 0.1 µg/mL). Our study has shown that A549 cells were more sensitive to AcE with an IC₅₀ of 15.68 ± 0.006 µg/mL compared to the standard drug 2.20 ± 0.008 µg/mL (cisplatin). The results suggest that Croton bonplandianus could serve as a potential source of alternative therapeutic agent for treating cancer. Further research is required to isolate the active principle compound and determination of its anticancer property.

  10. Natural terpenes prevent mitochondrial dysfunction, oxidative stress and release of apoptotic proteins during nimesulide-hepatotoxicity in rats.

    Directory of Open Access Journals (Sweden)

    Brijesh Kumar Singh

    Full Text Available Nimesulide, an anti-inflammatory and analgesic drug, is reported to cause severe hepatotoxicity. In this study, molecular mechanisms involved in deranged oxidant-antioxidant homeostasis and mitochondrial dysfunction during nimesulide-induced hepatotoxicity and its attenuation by plant derived terpenes, camphene and geraniol has been explored in male Sprague-Dawley rats. Hepatotoxicity due to nimesulide (80 mg/kg BW was evident from elevated SGPT, SGOT, bilirubin and histo-pathological changes. Antioxidants and key redox enzymes (iNOS, mtNOS, Cu/Zn-SOD, Mn-SOD, GPx and GR were altered significantly as assessed by their mRNA expression, Immunoblot analysis and enzyme activities. Redox imbalance along with oxidative stress was evident from decreased NAD(PH and GSH (56% and 74% respectively; P<0.001, increased superoxide and secondary ROS/RNS generation along with oxidative damage to cellular macromolecules. Nimesulide reduced mitochondrial activity, depolarized mitochondria and caused membrane permeability transition (MPT followed by release of apoptotic proteins (AIF; apoptosis inducing factor, EndoG; endonuclease G, and Cyto c; cytochrome c. It also significantly activated caspase-9 and caspase-3 and increased oxidative DNA damage (level of 8-Oxoguanine glycosylase; P<0.05. A combination of camphene and geraniol (CG; 1:1, when pre-administered in rats (10 mg/kg BW, accorded protection against nimesulide hepatotoxicity in vivo, as evident from normalized serum biomarkers and histopathology. mRNA expression and activity of key antioxidant and redox enzymes along with oxidative stress were also normalized due to CG pre-treatment. Downstream effects like decreased mitochondrial swelling, inhibition in release of apoptotic proteins, prevention of mitochondrial depolarization along with reduction in oxidized NAD(PH and increased mitochondrial electron flow further supported protective action of selected terpenes against nimesulide toxicity. Therefore

  11. Cytotoxic and apoptotic effects of scorpion Leiurus quinquestriatus venom on 293T and C2C12 eukaryotic cell lines

    Directory of Open Access Journals (Sweden)

    M. A. A. Omran

    2003-01-01

    Full Text Available Scorpion venom toxicity is of major concern due to its influence on human activities and public health. The cytotoxicity and apoptosis induced by scorpion L. quinquestriatus venom on two established eukaryotic cell lines (293T and C2C12 were analyzed. Both cultured cell lines were incubated with varying doses (10, 20, and 50 µg/ml of scorpion venom in serum free medium (SFM for 0.5, 1, 2, 4, and 8 hours at 37°C. The percentage of total lactate dehydrogenase (LDH released in the culture during venom incubation was used as an index of cell damage. Control culture was treated with an equal amount of SFM. Cell injury was recognized morphologically and apoptosis was researched by a Fluorescing Apoptosis Detection System using the principle of TUNEL (TdT-mediated dUTP Nick-End Labelling assay and confirmed by another assay concerning nuclear DNA staining with DAPI stain. Cytotoxicity was remarkable and cell survival highly reduced at the highest tested concentration (50 µg/ml. These effects were rapid and observed within 30 minutes. The apparent initial damage to the nucleus and lysis of the plasmalemma and/or organelle membranes, which was evident by a significant increase in cytosolic LDH release, suggested that this toxin acts at the membrane level. The morphological changes that occurred in apoptotic cells include condensation and compartmentalization of nuclear and cytoplasmic materials into structurally preserved membrane-bound fragments or blebs. The cytotoxic effects are dose and time dependent and cell death by apoptosis was more characteristic of 293T cells than C2C12 cells. The apoptotic effects were more prominent and clear in the early stages of toxicity, while other forms of cell damage such as swelling, rupture, and/or necrosis occurred at later stages.

  12. Caspase-resistant BAP31 inhibits fas-mediated apoptotic membrane fragmentation and release of cytochrome c from mitochondria.

    Science.gov (United States)

    Nguyen, M; Breckenridge, D G; Ducret, A; Shore, G C

    2000-09-01

    BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two identical caspase recognition sites (AAVD.G) that are preferentially cleaved by initiator caspases, including caspase 8. Cleavage of BAP31 during apoptosis generates a p20 fragment that remains integrated in the membrane and, when expressed ectopically, is a potent inducer of cell death. To examine the consequences of maintaining the structural integrity of BAP31 during apoptosis, the caspase recognition aspartate residues were mutated to alanine residues, and Fas-mediated activation of caspase 8 and cell death were examined in human KB epithelial cells stably expressing the caspase-resistant mutant crBAP31. crBAP31 only modestly slowed the time course for activation of caspases, as assayed by the processing of procaspases 8 and 3 and the measurement of total DEVDase activity. As a result, cleavage of the caspase targets poly(ADP-ribosyl) polymerase and endogenous BAP31, as well as the redistribution of phosphatidylserine and fragmentation of DNA, was observed. In contrast, cytoplasmic membrane blebbing and fragmentation and apoptotic redistribution of actin were strongly inhibited, cell morphology was retained near normal, and the irreversible loss of cell growth potential following removal of the Fas stimulus was delayed. Of note, crBAP31-expressing cells also resisted Fas-mediated release of cytochrome c from mitochondria, and the mitochondrial electrochemical potential was only partly reduced. These results argue that BAP31 cleavage is important for manifesting cytoplasmic apoptotic events associated with membrane fragmentation and reveal an unexpected cross talk between mitochondria and the endoplasmic reticulum during Fas-mediated apoptosis in vivo.

  13. Cytotoxic, pro-apoptotic, pro-oxidant, and non-genotoxic activities of a novel copper(II) complex against human cervical cancer.

    Science.gov (United States)

    Frías González, Susana E; Angeles Anguiano, Enrique; Mendoza Herrera, Alberto; Escutia Calzada, Daniel; Ordaz Pichardo, Cynthia

    2013-12-06

    Cisplatin remains one of the most effective current chemotherapeutic agents; however, metal complexes synthesis has increased in order to produce new anti-neoplastic drugs with DNA binding and apoptotic activities in tumor cells and less toxicity for patients. In this study, we evaluated the cytotoxic activity of a novel copper(II) complex (LQM402) against cervical cancer cell lines and found that LQM402 exhibited selective cytotoxicity against HeLa and Ca Ski cells. FITC-annexin assay and DNA fragmentation indicated that apoptosis could be involved in HeLa cell death. Caspase 3/7 and cytochrome c analysis by immunoblotting suggest the intrinsic pathway. LQM402 is a lipid peroxidation inductor according to TBARS production. Additionally, the Ames and micronucleus tests demonstrated non-genotoxic activity for this compound in Salmonella typhimurium and CD1 mice, respectively. Therefore, LQM402 may be a promising and safe anti-cervical cancer compound.

  14. A mitochondrial DNA SNP multiplex assigning Caucasians into 36 haplo- and subhaplogroups

    DEFF Research Database (Denmark)

    Mikkelsen, Martin; Rockenbauer, Eszter; Sørensen, Erik;

    2008-01-01

    Mitochondrial DNA (mtDNA) is maternally inherited without recombination events and has a high copy number, which makes mtDNA analysis feasible even when genomic DNA is sparse or degraded. Here, we present a SNP typing assay with 33 previously described mtDNA coding region SNPs for haplogroup assi...

  15. Chronic NMDA administration to rats increases brain pro-apoptotic factors while decreasing anti-Apoptotic factors and causes cell death

    Directory of Open Access Journals (Sweden)

    Rapoport Stanley I

    2009-09-01

    Full Text Available Abstract Background Chronic N-Methyl-d-aspartate (NMDA administration to rats is reported to increase arachidonic acid signaling and upregulate neuroinflammatory markers in rat brain. These changes may damage brain cells. In this study, we determined if chronic NMDA administration (25 mg/kg i.p., 21 days to rats would alter expression of pro- and anti-apoptotic factors in frontal cortex, compared with vehicle control. Results Using real time RT-PCR and Western blotting, chronic NMDA administration was shown to decrease mRNA and protein levels of anti-apoptotic markers Bcl-2 and BDNF, and of their transcription factor phospho-CREB in the cortex. Expression of pro-apoptotic Bax, Bad, and 14-3-3ζ was increased, as well as Fluoro-Jade B (FJB staining, a marker of neuronal loss. Conclusion This alteration in the balance between pro- and anti-apoptotic factors by chronic NMDA receptor activation in this animal model may contribute to neuronal loss, and further suggests that the model can be used to examine multiple processes involved in excitotoxicity.

  16. Extracellular DNA: the tip of root defenses?

    Science.gov (United States)

    Hawes, Martha C; Curlango-Rivera, Gilberto; Wen, Fushi; White, Gerard J; Vanetten, Hans D; Xiong, Zhongguo

    2011-06-01

    This review discusses how extracellular DNA (exDNA) might function in plant defense, and at what level(s) of innate immunity this process might operate. A new role for extracellular factors in mammalian defense has been described in a series of studies. These studies reveal that cells including neutrophils, eosinophils, and mast cells produce 'extracellular traps' (ETs) consisting of histone-linked exDNA. When pathogens are attracted to such ETs, they are trapped and killed. When the exDNA component of ETs is degraded, trapping is impaired and resistance against invasion is reduced. Conversely, mutation of microbial genes encoding exDNases that degrade exDNA results in loss of virulence. This discovery that exDNases are virulence factors opens new avenues for disease control. In plants, exDNA is required for defense of the root tip. Innate immunity-related proteins are among a group of >100 proteins secreted from the root cap and root border cell populations. Direct tests revealed that exDNA also is rapidly synthesized and exported from the root tip. When this exDNA is degraded by the endonuclease DNase 1, root tip resistance to fungal infection is lost; when the polymeric structure is degraded more slowly, by the exonuclease BAL31, loss of resistance to fungal infection is delayed accordingly. The results suggest that root border cells may function in a manner analogous to that which occurs in mammalian cells.

  17. Syntrophy in Methanogenic Degradation

    NARCIS (Netherlands)

    Worm, P.; Müller, N.; Plugge, C.M.; Stams, A.J.M.; Schink, B.

    2010-01-01

    This chapter deals with microbial communities of bacteria and archaea that closely cooperate in methanogenic degradation and perform metabolic functions in this community that neither one of them could carry out alone. The methanogenic degradation of fatty acids, alcohols, most aromatic compounds, a

  18. Rate of NDF degradation

    DEFF Research Database (Denmark)

    Weisbjerg, Martin Riis; Koukolová, V; Lund, Peter

    2007-01-01

    Degradation profiles for NDF were estimated for 83 samples of grass/grass-clover, 27 samples of cereal whole crop and 14 samples of maize whole crop.......Degradation profiles for NDF were estimated for 83 samples of grass/grass-clover, 27 samples of cereal whole crop and 14 samples of maize whole crop....

  19. Batteries: Imaging degradation

    Science.gov (United States)

    Shearing, Paul R.

    2016-11-01

    The degradation and failure of Li-ion batteries is strongly associated with electrode microstructure change upon (de)lithiation. Now, an operando X-ray tomography approach is shown to correlate changes in the microstructure of electrodes to cell performance, and thereby predict degradation pathways.

  20. Targeting Protective Autophagy Exacerbates UV-Triggered Apoptotic Cell Death

    Directory of Open Access Journals (Sweden)

    Shih-Hwa Chiou

    2012-01-01

    Full Text Available Autophagy is activated by various stresses, including DNA damage, and previous studies of DNA damage-induced autophagy have focused on the response to chemotherapeutic drugs, ionizing radiation, and reactive oxygen species. In this study, we investigated the biological significance of autophagic response to ultraviolet (UV irradiation in A549 and H1299 cells. Our results indicated that UV induces on-rate autophagic flux in these cells. Autophagy inhibition resulting from the knockdown of beclin-1 and Atg5 reduced cell viability and enhanced apoptosis. Moreover, we found that ATR phosphorylation was accompanied by microtubule-associated protein 1 light chain 3B II (LC3B-II expression during the early phases following UV irradiation, which is a well-established inducer of ATR. Knocking down ATR further attenuated the reduction in LC3B-II at early stages in response to UV treatment. Despite the potential role of ATR in autophagic response, reduced ATR expression does not affect autophagy induction during late phases (24 and 48 h after UV treatment. The result is consistent with the reduced ATR phosphorylation at the same time points and suggests that autophagic response at this stage is activated via a distinct pathway. In conclusion, this study demonstrated that autophagy acts as a cytoprotective mechanism against UV-induced apoptosis and that autophagy induction accompanied with apoptosis at late stages is independent of ATR activation.

  1. Autophagy in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Piotr Czarny

    2015-01-01

    Full Text Available DNA damage response (DDR involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1. mTORC1 represses autophagy via phosphorylation of the ULK1/2–Atg13–FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADPribose polymerase 1 (PARP-1, Mre11–Rad50–Nbs1 (MRN complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy.

  2. Androstane derivatives induce apoptotic death in MDA-MB-231 breast cancer cells.

    Science.gov (United States)

    Jakimov, Dimitar S; Kojić, Vesna V; Aleksić, Lidija D; Bogdanović, Gordana M; Ajduković, Jovana J; Djurendić, Evgenija A; Penov Gaši, Katarina M; Sakač, Marija N; Jovanović-Šanta, Suzana S

    2015-11-15

    Biological investigation was conducted to study in vitro antiproliferative and pro-apoptotic potential of selected 17α-picolyl and 17(E)-picolinylidene androstane derivatives. The antiproliferative impact was examined on six human tumor cell lines, including two types of breast (MCF-7 and MDA-MB-231), prostate (PC3), cervical (HeLa), colon (HT 29) and lung cancer (A549), as well as one normal fetal lung fibroblasts cell line (MRC-5). All derivatives selectively decreased proliferation of estrogen receptor negative MDA-MB-231 breast cancer cells after 48 h and 72 h treatment and compounds showed time-dependent activity. We used this cell line to investigate cell cycle modulation and apoptotic cell death induction by flow cytometry, expression of apoptotic proteins by Western blot and apoptotic morphology by visual observation. Tested androstane derivatives affected the cell cycle distribution and induced apoptosis and necrosis. Compounds had different and specific mode of action, depending on derivative type and exposure time. Some compounds induced significant apoptosis measured by Annexin V test compared to reference compound formestane. Higher expression of pro-apoptotic BAX, downregulation of anti-apoptotic Bcl-2 and cleavage of PARP protein were confirmed in almost all treated samples, but the lack of caspase-3 activation suggested the induction of apoptosis in caspase-independent manner. More cells with apoptotic morphology were observed in samples after prolonged treatment. Structure-activity relationship analysis was performed to find correlations between the structure variations of investigated derivatives and observed biological effects. Results of this study showed that some of the investigated androstane derivatives have good biomedical potential and could be candidates for anticancer drug development.

  3. Targeting the Anti-Apoptotic Protein c-FLIP for Cancer Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Safa, Ahmad R., E-mail: asafa@iupui.edu [Department of Pharmacology and Toxicology, Indiana University School of Medicine, 980 W. Walnut Street, R3-C524, Indianapolis, IN 46202 (United States); Indiana University Simon Cancer Center, Indiana University School of Medicine, 980 W. Walnut Street, R3-C524, Indianapolis, IN 46202 (United States); Pollok, Karen E. [Department of Pharmacology and Toxicology, Indiana University School of Medicine, 980 W. Walnut Street, R3-C524, Indianapolis, IN 46202 (United States); Indiana University Simon Cancer Center, Indiana University School of Medicine, 980 W. Walnut Street, R3-C524, Indianapolis, IN 46202 (United States); Herman B. Wells Center for Pediatric Research, 980 W. Walnut Street, R3-C524, Indianapolis, IN 46202 (United States)

    2011-03-29

    Cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) is a major resistance factor and critical anti-apoptotic regulator that inhibits tumor necrosis factor-alpha (TNF-alpha), Fas-L, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis as well as chemotherapy-triggered apoptosis in malignant cells. c-FLIP is expressed as long (c-FLIP{sub L}), short (c-FLIP{sub S}), and c-FLIP{sub R} splice variants in human cells. c-FLIP binds to FADD and/or caspase-8 or -10 in a ligand-dependent and-independent fashion, which in turn prevents death-inducing signaling complex (DISC) formation and subsequent activation of the caspase cascade. Moreover, c-FLIP{sub L} and c-FLIP{sub S} are known to have multifunctional roles in various signaling pathways, as well as activating and/or upregulating several cytoprotective signaling molecules. Upregulation of c-FLIP has been found in various tumor types, and its downregulation has been shown to restore apoptosis triggered by cytokines and various chemotherapeutic agents. Hence, c-FLIP is an important target for cancer therapy. For example, small interfering RNAs (siRNAs) that specifically knockdown the expression of c-FLIP{sub L} in diverse human cancer cell lines augmented TRAIL-induced DISC recruitment and increased the efficacy of chemotherapeutic agents, thereby enhancing effector caspase stimulation and apoptosis. Moreover, small molecules causing degradation of c-FLIP as well as decreasing mRNA and protein levels of c-FLIP{sub L} and c-FLIP{sub S} splice variants have been found, and efforts are underway to develop other c-FLIP-targeted cancer therapies. This review focuses on (1) the functional role of c-FLIP splice variants in preventing apoptosis and inducing cytokine and drug resistance; (2) the molecular mechanisms that regulate c-FLIP expression; and (3) strategies to inhibit c-FLIP expression and function.

  4. DNA Damage in Plant Herbarium Tissue

    Science.gov (United States)

    Staats, Martijn; Cuenca, Argelia; Richardson, James E.; Vrielink-van Ginkel, Ria; Petersen, Gitte; Seberg, Ole; Bakker, Freek T.

    2011-01-01

    Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4–3.8% of fresh control DNA and 1.0–1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens. PMID:22163018

  5. DNA damage in plant herbarium tissue.

    Directory of Open Access Journals (Sweden)

    Martijn Staats

    Full Text Available Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4-3.8% of fresh control DNA and 1.0-1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens.

  6. Microwave-induced inactivation of DNA-based hybrid catalyst in asymmetric catalysis.

    Science.gov (United States)

    Zhao, Hua; Shen, Kai

    2016-03-01

    DNA-based hybrid catalysts have gained strong interests in asymmetric reactions. However, to maintain the high enantioselectivity, these reactions are usually conducted at relatively low temperatures (e.g. DNA-based hybrid catalyst even at low temperatures (such as 5 °C). Circular dichroism (CD) spectra and gel electrophoresis of DNA suggest that microwave exposure degrades DNA molecules and disrupts DNA double-stranded structures, causing changes of DNA-metal ligand binding properties and thus poor DNA catalytic performance.

  7. Silk structure and degradation.

    Science.gov (United States)

    Liu, Bin; Song, Yu-wei; Jin, Li; Wang, Zhi-jian; Pu, De-yong; Lin, Shao-qiang; Zhou, Chan; You, Hua-jian; Ma, Yan; Li, Jin-min; Yang, Li; Sung, K L Paul; Zhang, Yao-guang

    2015-07-01

    To investigate the structure of silk and its degradation properties, we have monitored the structure of silk using scanning electron microscopy and frozen sections. Raw silk and degummed raw silk were immersed in four types of degradation solutions for 156 d to observe their degradation properties. The subcutaneous implants in rats were removed after 7, 14, 56, 84, 129, and 145 d for frozen sectioning and subsequent staining with hematoxylin and eosin (H.E.), DAPI, Beta-actin and Collagen I immunofluorescence staining. The in vitro weight loss ratio of raw silk and degummed raw silk in water, PBS, DMEM and DMEM containing 10% FBS (F-DMEM) were, respectively, 14%/11%, 12.5%/12.9%, 11.1%/14.3%, 8.8%/11.6%. Silk began to degrade after 7 d subcutaneous implantation and after 145 d non-degraded silk was still observed. These findings suggest the immunogenicity of fibroin and sericin had no essential difference. In the process of in vitro degradation of silk, the role of the enzyme is not significant. The in vivo degradation of silk is related to phagocytotic activity and fibroblasts may be involved in this process to secrete collagen. This study also shows the developing process of cocoons and raw silk.

  8. Intermittent degradation and schizotypy

    Directory of Open Access Journals (Sweden)

    Matthew W. Roché

    2015-06-01

    Full Text Available Intermittent degradation refers to transient detrimental disruptions in task performance. This phenomenon has been repeatedly observed in the performance data of patients with schizophrenia. Whether intermittent degradation is a feature of the liability for schizophrenia (i.e., schizotypy is an open question. Further, the specificity of intermittent degradation to schizotypy has yet to be investigated. To address these questions, 92 undergraduate participants completed a battery of self-report questionnaires assessing schizotypy and psychological state variables (e.g., anxiety, depression, and their reaction times were recorded as they did so. Intermittent degradation was defined as the number of times a subject’s reaction time for questionnaire items met or exceeded three standard deviations from his or her mean reaction time after controlling for each item’s information processing load. Intermittent degradation scores were correlated with questionnaire scores. Our results indicate that intermittent degradation is associated with total scores on measures of positive and disorganized schizotypy, but unrelated to total scores on measures of negative schizotypy and psychological state variables. Intermittent degradation is interpreted as potentially derivative of schizotypy and a candidate endophenotypic marker worthy of continued research.

  9. Cytotoxic and apoptotic effects of six herbal plants against the human hepatocarcinoma (HepG2 cell line

    Directory of Open Access Journals (Sweden)

    Nonpunya Apiyada

    2011-10-01

    Full Text Available Abstract Background Six plants from Thailand were evaluated for their cytotoxicity and apoptosis induction in human hepatocarcinoma (HepG2 as compared to normal African green monkey kidney epithelial cell lines. Methods Ethanol-water crude extracts of the six plants were tested with neutral red assay for their cytotoxicity after 24 hours of exposure to the cells. Apoptotic induction was tested in the HepG2 cells with diamidino-2-phenylindole staining. DNA fragmentation, indicative of apoptosis, was analyzed with agarose gel electrophoresis. Alkylation, indicative of DNA damage, was also evaluated in vitro by 4-(4'-nitrobenzyl pyridine assay. Results The extract of Pinus kesiya showed the highest selectivity (selectivity index = 9.6 and potent cytotoxicity in the HepG2 cell line, with an IC50 value of 52.0 ± 5.8 μg/ml (mean ± standard deviation. Extract of Catimbium speciosum exerted cytotoxicity with an IC50 value of 55.7 ± 8.1 μg/ml. Crude extracts from Glochidion daltonii, Cladogynos orientalis, Acorus tatarinowii and Amomum villosum exhibited cytotoxicity with IC50 values ranging 100-500 μg/ml. All crude extracts showed different alkylating abilities in vitro. Extracts of P. kesiya, C. speciosum and C. orientalis caused nuclei morphological changes and DNA laddering. Conclusion The extracts of C. speciosum, C. orientalis and P. kesiya induced apoptosis. Among the three plants, P. kesiya possessed the most robust anticancer activity, with specific selectivity against HepG2 cells.

  10. DNA Nanotechnology

    Science.gov (United States)

    Taniguchi, Masateru; Kawai, Tomoji

    2002-11-01

    DNA is one candidate of promising molecules for molecular electronic devices, since it has the double helix structure with pi-electron bases for electron transport, the address at 0.4 nm intervals, and the self-assembly. Electrical conductivity and nanostructure of DNA and modified DNA molecules are investigated in order to research the application of DNA in nanoelectronic devices. It has been revealed that DNA is a wide-gap semiconductor in the absence of doping. The conductivity of DNA has been controlled by chemical doping, electric field doping, and photo-doping. It has found that Poly(dG)[middle dot]Poly(dC) has the best conductivity and can function as a conducting nanowire. The pattern of DNA network is controlled by changing the concentration of the DNA solution.

  11. SPECIFIC DEGRADATION OF WATERSHEDS

    Institute of Scientific and Technical Information of China (English)

    Boubacar KANE; Pierre Y.JULIEN

    2007-01-01

    An extensive database of reservoir sedimentation surveys throughout continental United States is compiled and analyzed to determine specific degradation SD relationships as function of mean annual rainfall R, drainage area A, and watershed slope S. The database contains 1463 field measurements and specific degradation relationships are defined as function of A, R and S. Weak trends and significant variability in the data are noticeable. Specific degradation measurements are log normally distributed with respect to R, A, and S and 95% confidence intervals are determined accordingly. The accuracy of the predictions does not significantly increase as more independent variables are added to the regression analyses.

  12. DNA Methylation

    OpenAIRE

    Alokail, Majed S.; Alenad, Amal M.

    2015-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication e...

  13. Apoptotic Effects of Hypocrellin A on HeLa Cells

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Hypocrellin A(HA), a photosensitive perylenequinone compound of Hypocrella bambusae , inhibited the proliferation of several tumor cell lines. Human cervical cancer cells, HeLa cells, were used as a model to elucidate the molecular mechanisms of HA-induced tumor cell death. The results show that HA can induce the oligonucleosomal fragmentation of DNA in HeLa cells and also can increase the expression of apoptosis inducer Bax mRNA and that it decreases the expression of apoptosis suppressor, Bcl-2 mRNA, in mitochondria. It can be concluded from the data that HA-induced apoptosis is related to the balance between Bcl-2 and Bax gene expressions.

  14. Cytotoxic and apoptotic activities of extracts of Withania somnifera and Tinospora cordifolia in human breast cancer cells

    Directory of Open Access Journals (Sweden)

    N Maliyakkal

    2013-07-01

    Full Text Available Summary. Withania somnifera (WS and Tinospora cordifolia (TC have been used in the traditional system of medicine in India (Ayurveda for the treatment of cancer. The current study investigated the cytotoxic and apoptotic effects of extracts of WS and TC on human breast cancer cells (MCF7 and MDA MB 231. MTT-based assays revealed dose-dependent cytotoxic effects of the ethanolic extracts of WS and TC in human breast cancer cells, while the aqueous extracts failed to induce significant cytotoxicity. Hoechst 33342 staining and DNA fragmentation assays revealed hallmark properties of apoptosis such as membrane blebbing, nuclear condensation, and DNA fragmentation. The ethanolic extracts of both WS and TC also increased the sub-G0 content, further confirming induction of apoptosis, while WS extracts additionally caused cell cycle arrest in the G2/M phase. Further, the current study also evaluated the cytotoxic effects of WS and TC extracts on human immortalized but, ‘non-cancerous’ cell line (HaCaT. Significantly, the extracts failed to show cytotoxicity or apoptosis in HaCaT cells at the concentration that was cytotoxic to breast cancer cells, indicating less cytotoxic effects of WS and TC against human ‘non-cancerous’ cells. Thus, our study reveals potential anti-cancer activities of the ethanolic and extracts of TC and WS against human breast cancer cells.Industrial relevance. The uses of WS and TC in traditional system of medicine for the management and treatment of cancer have drawn considerable attention. Varieties of pytochemicals and herbal formulations have been developed from plant sources, leading to the scientific interest in the discovery of anticancer agents from crude plant extracts. Medicinal plant extracts have played a significant role in the development of several clinically useful anti-cancer agents. Herbal formulations of the crude extracts could be useful for the treatment of breast cancer with less toxic effects against

  15. The Mannose Receptor Is Involved in the Phagocytosis of Mycobacteria-Induced Apoptotic Cells

    Directory of Open Access Journals (Sweden)

    Teresa Garcia-Aguilar

    2016-01-01

    Full Text Available Upon Mycobacterium tuberculosis infection, macrophages may undergo apoptosis, which has been considered an innate immune response. The pathways underlying the removal of dead cells in homeostatic apoptosis have been extensively studied, but little is known regarding how cells that undergo apoptotic death during mycobacterial infection are removed. This study shows that macrophages induced to undergo apoptosis with mycobacteria cell wall proteins are engulfed by J-774A.1 monocytic cells through the mannose receptor. This demonstration was achieved through assays in which phagocytosis was inhibited with a blocking anti-mannose receptor antibody and with mannose receptor competitor sugars. Moreover, elimination of the mannose receptor by a specific siRNA significantly diminished the expression of the mannose receptor and the phagocytosis of apoptotic cells. As shown by immunofluorescence, engulfed apoptotic bodies are initially located in Rab5-positive phagosomes, which mature to express the phagolysosome marker LAMP1. The phagocytosis of dead cells triggered an anti-inflammatory response with the production of TGF-β and IL-10 but not of the proinflammatory cytokines IL-12 and TNF-α. This study documents the previously unreported participation of the mannose receptor in the removal of apoptotic cells in the setting of tuberculosis (TB infection. The results challenge the idea that apoptotic cell phagocytosis in TB has an immunogenic effect.

  16. Selection of apoptotic cell specific human antibodies from adult bone marrow.

    Directory of Open Access Journals (Sweden)

    Caroline Grönwall

    Full Text Available Autoreactive antibodies that recognize neo-determinants on apoptotic cells in mice have been proposed to have protective, homeostatic and immunoregulatory properties, although our knowledge about the equivalent antibodies in humans has been much more limited. In the current study, human monoclonal antibodies with binding specificity for apoptotic cells were isolated from the bone marrow of healthy adults using phage display technology. These antibodies were shown to recognize phosphorylcholine (PC-associated neo-determinants. Interestingly, three of the four identified apoptotic cell-specific antibody clones were encoded by VH3 region rearrangements with germline or nearly germline configuration without evidence of somatic hypermutation. Importantly, the different identified antibody clones had diverse heavy chain CDR3 and deduced binding surfaces as suggested by structure modeling. This may suggest a potentially great heterogeneity in human antibodies recognizing PC-related epitopes on apoptotic cells. To re-construct the postulated structural format of the parental anti-PC antibody, the dominant clone was also expressed as a recombinant human polymeric IgM, which revealed a substantially increased binding reactivity, with dose-dependent and antigen-inhibitable binding of apoptotic cells. Our findings may have implication for improved prognostic testing and therapeutic interventions in human inflammatory disease.

  17. Homocysteine and its thiolactone may promote apoptotic events in blood platelets in vitro.

    Science.gov (United States)

    Olas, Beata; Malinowska, Joanna; Rywaniak, Joanna

    2010-01-01

    The actions of homocysteine and its major metabolite, cyclic thioester, homocysteine thiolactone on endothelial cells, blood platelets, plasmatic fibrinogen and plasminogen--the important major components of haemostasis, regulating the flowing properties of blood--are complex and sometimes controversial. Homocysteine (Hcys) can promote apoptosis in endothelial cells, but the role of Hcys and its thiolactone in the apoptotic process in blood platelets is unknown. In order to study the appearance of apoptosis in platelets after treatment with the reduced form of Hcys or its thiolactone different markers were chosen: annexin V binding (phosphatidylserine exposure), platelet microparticle formation, mitochondrial membrane depolarization and αIIbβ3 expression in vitro. Apoptotic events and platelet activation were measured by a flow cytometer. In gel-filtered platelets treated with different concentrations of the reduced form of Hcys (25, 50 and 100 µM, 10 min) a significant increase of phosphatidylserine exposure (about 37% at the highest concentration, p < 0.001) and platelet microparticle formation were observed. Homocysteine caused also a dose-dependent depolarization of mitochondrial potential. The same apoptotic markers appeared in HTL-treated platelets (0.2 and 1 µM). Moreover, resveratrol (25 µM), a well known antioxidant, distinctly reduced the level of apoptotic markers. The obtained results indicate that Hcys and its thiolactone may promote in vitro apoptotic events in human gel-filtered platelets.

  18. Apoptotic effects of salinomycin on human ovarian cancer cell line (OVCAR-3).

    Science.gov (United States)

    Kaplan, Fuat; Teksen, Fulya

    2016-03-01

    In this study, we studied the apoptotic and cytotoxic effects of salinomycin on human ovarian cancer cell line (OVCAR-3) as salinomycin is known as a selectively cancer stem cell killer agent. We used immortal human ovarian epithelial cell line (IHOEC) as control group. Ovarian cancer cells and ovarian epithelial cells were treated by different concentrations of salinomycin such as 0.1, 1, and 40 μM and incubated for 24, 48, and 72 h. Dimethylthiazol (MTT) cell viability assay was performed to determine cell viability and toxicity. On the other hand, the expression levels of some of the apoptosis-related genes, namely anti-apoptotic Bcl-2, apoptotic Bax, and Caspase-3 were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, Caspase-3 protein level was also determined. As a result, we concluded that incubation of human OVCAR-3 by 0.1 μM concentration of salinomycin for 24 h killed 40 % of the cancer cells by activating apoptosis but had no effect on normal cells. The apoptotic Bax gene expression was upregulated but anti-apoptotic Bcl-2 gene expression was downregulated. Active Caspase-3 protein level was increased significantly (p < 0.05).

  19. Modafinil abrogates methamphetamine-induced neuroinflammation and apoptotic effects in the mouse striatum.

    Directory of Open Access Journals (Sweden)

    Mariana Raineri

    Full Text Available Methamphetamine is a drug of abuse that can cause neurotoxic damage in humans and animals. Modafinil, a wake-promoting compound approved for the treatment of sleeping disorders, is being prescribed off label for the treatment of methamphetamine dependence. The aim of the present study was to investigate if modafinil could counteract methamphetamine-induced neuroinflammatory processes, which occur in conjunction with degeneration of dopaminergic terminals in the mouse striatum. We evaluated the effect of a toxic methamphetamine binge in female C57BL/6 mice (4 × 5 mg/kg, i.p., 2 h apart and modafinil co-administration (2 × 90 mg/kg, i.p., 1 h before the first and fourth methamphetamine injections on glial cells (microglia and astroglia. We also evaluated the striatal expression of the pro-apoptotic BAX and anti-apoptotic Bcl-2 proteins, which are known to mediate methamphetamine-induced apoptotic effects. Modafinil by itself did not cause reactive gliosis and counteracted methamphetamine-induced microglial and astroglial activation. Modafinil also counteracted the decrease in tyrosine hydroxylase and dopamine transporter levels and prevented methamphetamine-induced increases in the pro-apoptotic BAX and decreases in the anti-apoptotic Bcl-2 protein expression. Our results indicate that modafinil can interfere with methamphetamine actions and provide protection against dopamine toxicity, cell death, and neuroinflammation in the mouse striatum.

  20. Inhibitory effects of apoptotic cell ingestion upon endotoxin-driven myeloid dendritic cell maturation.

    Science.gov (United States)

    Stuart, Lynda M; Lucas, Mark; Simpson, Cathy; Lamb, Jonathan; Savill, John; Lacy-Hulbert, Adam

    2002-02-15

    Dendritic cells (DCs) are the sentinels of the immune system, able to interact with both naive and memory T cells. The recent observation that DCs can ingest cells dying by apoptosis has raised the possibility that DCs may, in fact, present self-derived Ags, initiating both autoimmunity and tumor-specific responses, especially if associated with appropriate danger signals. Although the process of ingestion of apoptotic cells has not been shown to induce DC maturation, the exact fate of these phagocytosing DCs remains unclear. In this paper we demonstrate that DCs that ingest apoptotic cells are able to produce TNF-alpha but have a diminished ability to produce IL-12 in response to external stimuli, a property that corresponds to a failure to up-regulate CD86. By single-cell analysis we demonstrate that these inhibitory effects are restricted to those DCs that have engulfed apoptotic cells, with bystander DCs remaining unaffected. These changes were independent of the production of anti-inflammatory cytokines TGF-beta1 and IL-10 and corresponded with a diminished capacity to stimulate naive T cells. Thus, the ingestion of apoptotic cells is not an immunologically null event but is capable of modulating DC maturation. These results have important implications for our understanding of the role of clearance of dying cells by DCs not only in the normal resolution of inflammation but also in control of subsequent immune responses to apoptotic cell-derived Ags.

  1. Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids.

    Science.gov (United States)

    Lauber, K; Keppeler, H; Munoz, L E; Koppe, U; Schröder, K; Yamaguchi, H; Krönke, G; Uderhardt, S; Wesselborg, S; Belka, C; Nagata, S; Herrmann, M

    2013-09-01

    The phagocytic clearance of apoptotic cells is essential to prevent chronic inflammation and autoimmunity. The phosphatidylserine-binding protein milk fat globule-EGF factor 8 (MFG-E8) is a major opsonin for apoptotic cells, and MFG-E8(-/-) mice spontaneously develop a lupus-like disease. Similar to human systemic lupus erythematosus (SLE), the murine disease is associated with an impaired clearance of apoptotic cells. SLE is routinely treated with glucocorticoids (GCs), whose anti-inflammatory effects are consentaneously attributed to the transrepression of pro-inflammatory cytokines. Here, we show that the GC-mediated transactivation of MFG-E8 expression and the concomitantly enhanced elimination of apoptotic cells constitute a novel aspect in this context. Patients with chronic inflammation receiving high-dose prednisone therapy displayed substantially increased MFG-E8 mRNA levels in circulating monocytes. MFG-E8 induction was dependent on the GC receptor and several GC response elements within the MFG-E8 promoter. Most intriguingly, the inhibition of MFG-E8 induction by RNA interference or genetic knockout strongly reduced or completely abolished the phagocytosis-enhancing effect of GCs in vitro and in vivo. Thus, MFG-E8-dependent promotion of apoptotic cell clearance is a novel anti-inflammatory facet of GC treatment and renders MFG-E8 a prospective target for future therapeutic interventions in SLE.

  2. Apico-basal forces exerted by apoptotic cells drive epithelium folding.

    Science.gov (United States)

    Monier, Bruno; Gettings, Melanie; Gay, Guillaume; Mangeat, Thomas; Schott, Sonia; Guarner, Ana; Suzanne, Magali

    2015-02-12

    Epithelium folding is a basic morphogenetic event that is essential in transforming simple two-dimensional epithelial sheets into three-dimensional structures in both vertebrates and invertebrates. Folding has been shown to rely on apical constriction. The resulting cell-shape changes depend either on adherens junction basal shift or on a redistribution of myosin II, which could be driven by mechanical signals. Yet the initial cellular mechanisms that trigger and coordinate cell remodelling remain largely unknown. Here we unravel the active role of apoptotic cells in initiating morphogenesis, thus revealing a novel mechanism of epithelium folding. We show that, in a live developing tissue, apoptotic cells exert a transient pulling force upon the apical surface of the epithelium through a highly dynamic apico-basal myosin II cable. The apoptotic cells then induce a non-autonomous increase in tissue tension together with cortical myosin II apical stabilization in the surrounding tissue, eventually resulting in epithelium folding. Together our results, supported by a theoretical biophysical three-dimensional model, identify an apoptotic myosin-II-dependent signal as the initial signal leading to cell reorganization and tissue folding. This work further reveals that, far from being passively eliminated as generally assumed (for example, during digit individualization), apoptotic cells actively influence their surroundings and trigger tissue remodelling through regulation of tissue tension.

  3. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double strand break ends

    OpenAIRE

    Zhu, Zhu; Chung, Woo-Hyun; Shim, Eun Yong; Lee, Sang Eun; Ira, Grzegorz

    2008-01-01

    Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remains unknown. We monitored 5’-strand resection at inducible DSB ends and identified proteins required for two stages of resection: initiation and long-range 5’-strand resection. The Mre11-Rad50-Xrs2 complex (MRX) initiates 5’ degradation, whereas Sgs1 and Dna2 degrade 5’-strands exposing long 3’-s...

  4. Degradation of fluorotelomer alcohols

    DEFF Research Database (Denmark)

    Ellis, David A; Martin, Jonathan W; De Silva, Amila O

    2004-01-01

    Human and animal tissues collected in urban and remote global locations contain persistent and bioaccumulative perfluorinated carboxylic acids (PFCAs). The source of PFCAs was previously unknown. Here we present smog chamber studies that indicate fluorotelomer alcohols (FTOHs) can degrade...

  5. Purex diluent degradation

    Energy Technology Data Exchange (ETDEWEB)

    Tallent, O.K.; Mailen, J.C.; Pannell, K.D.

    1984-02-01

    The chemical degradation of normal paraffin hydrocarbon (NPH) diluents both in the pure state and mixed with 30% tributyl phosphate (TBP) was investigated in a series of experiments. The results show that degradation of NPH in the TBP-NPH-HNO/sub 3/ system is consistent with the active chemical agent being a radical-like nitrogen dioxide (NO/sub 2/) molecule, not HNO/sub 3/ as such. Spectrophotometric, gas chromatographic, mass spectrographic, and titrimetric methods were used to identify the degradation products, which included alkane nitro and nitrate compounds, alcohols, unsaturated alcohols, nitro alcohols, nitro alkenes, ketones, and carboxylic acids. The degradation rate was found to increase with increases in the HNO/sub 3/ concentration and the temperature. The rate was decreased by argon sparging to remove NO/sub 2/ and by the addition of butanol, which probably acts as a NO/sub 2/ scavenger. 13 references, 11 figures.

  6. How do polymers degrade?

    Science.gov (United States)

    Lyu, Suping

    2011-03-01

    Materials derived from agricultural products such as cellulose, starch, polylactide, etc. are more sustainable and environmentally benign than those derived from petroleum. However, applications of these polymers are limited by their processing properties, chemical and thermal stabilities. For example, polyethylene terephthalate fabrics last for many years under normal use conditions, but polylactide fabrics cannot due to chemical degradation. There are two primary mechanisms through which these polymers degrade: via hydrolysis and via oxidation. Both of these two mechanisms are related to combined factors such as monomer chemistry, chain configuration, chain mobility, crystallinity, and permeation to water and oxygen, and product geometry. In this talk, we will discuss how these materials degrade and how the degradation depends on these factors under application conditions. Both experimental studies and mathematical modeling will be presented.

  7. Defects in mitochondrial fission protein dynamin-related protein 1 are linked to apoptotic resistance and autophagy in a lung cancer model.

    Directory of Open Access Journals (Sweden)

    Kelly Jean Thomas

    Full Text Available Evasion of apoptosis is implicated in almost all aspects of cancer progression, as well as treatment resistance. In this study, resistance to apoptosis was identified in tumorigenic lung epithelial (A549 cells as a consequence of defects in mitochondrial and autophagic function. Mitochondrial function is determined in part by mitochondrial morphology, a process regulated by mitochondrial dynamics whereby the joining of two mitochondria, fusion, inhibits apoptosis while fission, the division of a mitochondrion, initiates apoptosis. Mitochondrial morphology of A549 cells displayed an elongated phenotype-mimicking cells deficient in mitochondrial fission protein, Dynamin-related protein 1 (Drp1. A549 cells had impaired Drp1 mitochondrial recruitment and decreased Drp1-dependent fission. Cytochrome c release and caspase-3 and PARP cleavage were impaired both basally and with apoptotic stimuli in A549 cells. Increased mitochondrial mass was observed in A549 cells, suggesting defects in mitophagy (mitochondrial selective autophagy. A549 cells had decreased LC3-II lipidation and lysosomal inhibition suggesting defects in autophagy occur upstream of lysosomal degradation. Immunostaining indicated mitochondrial localized LC3 punctae in A549 cells increased after mitochondrial uncoupling or with a combination of mitochondrial depolarization and ectopic Drp1 expression. Increased inhibition of apoptosis in A549 cells is correlated with impeded mitochondrial fission and mitophagy. We suggest mitochondrial fission defects contribute to apoptotic resistance in A549 cells.

  8. Bacteria and lignin degradation

    Institute of Scientific and Technical Information of China (English)

    Jing LI; Hongli YUAN; Jinshui YANG

    2009-01-01

    Lignin is both the most abundant aromatic (phenolic) polymer and the second most abundant raw material.It is degraded and modified by bacteria in the natural world,and bacteria seem to play a leading role in decomposing lignin in aquatic ecosystems.Lignin-degrading bacteria approach the polymer by mechanisms such as tunneling,erosion,and cavitation.With the advantages of immense environmental adaptability and biochemical versatility,bacteria deserve to be studied for their ligninolytic potential.

  9. Dna Sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  10. Polyanhydride degradation and erosion.

    Science.gov (United States)

    Göpferich, A; Tessmar, J

    2002-10-16

    It was the intention of this paper to give a survey on the degradation and erosion of polyanhydrides. Due to the multitude of polymers that have been synthesized in this class of material in recent years, it was not possible to discuss all polyanhydrides that have gained in significance based on their application. It was rather the intention to provide a broad picture on polyanhydride degradation and erosion based on the knowledge that we have from those polymers that have been intensively investigated. To reach this goal this review contains several sections. First, the foundation for an understanding of the nomenclature are laid by defining degradation and erosion which was deemed necessary because many different definitions exist in the current literature. Next, the properties of major classes of anhydrides are reviewed and the impact of geometry on degradation and erosion is discussed. A complicated issue is the control of drug release from degradable polymers. Therefore, the aspect of erosion-controlled release and drug stability inside polyanhydrides are discussed. Towards the end of the paper models are briefly reviewed that describe the erosion of polyanhydrides. Empirical models as well as Monte-Carlo-based approaches are described. Finally it is outlined how theoretical models can help to answer the question why polyanhydrides are surface eroding. A look at the microstructure and the results from these models lead to the conclusion that polyanhydrides are surface eroding due to their fast degradation. However they switch to bulk erosion once the device dimensions drop below a critical limit.

  11. Small DNA pieces in C. elegans are intermediates of DNA fragmentation during apoptosis.

    Directory of Open Access Journals (Sweden)

    P Joseph Aruscavage

    Full Text Available While studying small noncoding RNA in C. elegans, we discovered that protocols used for isolation of RNA are contaminated with small DNA pieces. After electrophoresis on a denaturing gel, the DNA fragments appear as a ladder of bands, approximately 10 nucleotides apart, mimicking the pattern of nuclease digestion of DNA wrapped around a nucleosome. Here we show that the small DNA pieces are products of the DNA fragmentation that occurs during apoptosis, and correspondingly, are absent in mutant strains incapable of apoptosis. In contrast, the small DNA pieces are present in strains defective for the engulfment process of apoptosis, suggesting they are produced in the dying cell prior to engulfment. While the small DNA pieces are also present in a number of strains with mutations in predicted nucleases, they are undetectable in strains containing mutations in nuc-1, which encodes a DNase II endonuclease. We find that the small DNA pieces can be labeled with terminal deoxynucleotidyltransferase only after phosphatase treatment, as expected if they are products of DNase II cleavage, which generates a 3' phosphate. Our studies reveal a previously unknown intermediate in the process of apoptotic DNA fragmentation and thus bring us closer to defining this important pathway.

  12. DDE remediation and degradation.

    Science.gov (United States)

    Thomas, John E; Ou, Li-Tse; All-Agely, Abid

    2008-01-01

    DDT and its metabolites, DDD and DDE, have been shown to be recalcitrant to degradation. The parent compound, DDT, was used extensively worldwide starting in 1939 and was banned in the United States in 1973. The daughter compound, DDE, may result from aerobic degradation, abiotic dehydrochlorination, or photochemical decomposition. DDE has also occurred as a contaminant in commercial-grade DDT. The p,p'-DDE isomer is more biologically active than the o,p-DDE, with a reported half-life of -5.7 years. However, when DDT was repeatedly applied to the soil, the DDE concentration may remain unchanged for more than 20 yr. Remediation of DDE-contaminated soil and water may be done by several techniques. Phytoremediation involves translocating DDT, DDD, and DDE from the soil into the plant, although some aquatic species (duckweed > elodea > parrot feather) can transform DDT into predominantly DDD with some DDE being formed. Of all the plants that can uptake DDE, Cucurbita pepo has been the most extensively studied, with translocation values approaching "hyperaccumulation" levels. Soil moisture, temperature, and plant density have all been documented as important factors in the uptake of DDE by Cucurbita pepo. Uptake may also be influenced positively by amendments such as biosurfactants, mycorrhizal inoculants, and low molecular weight organic acids (e.g., citric and oxalic acids). DDE microbial degradation by dehalogenases, dioxygenases, and hydrolases occurs under the proper conditions. Although several aerobic degradation pathways have been proposed, none has been fully verified. Very few aerobic pure cultures are capable of fully degrading DDE to CO2. Cometabolism of DDE by Pseudomonas sp., Alicaligens sp., and Terrabacter sp. grown on biphenyl has been reported; however, not all bacterial species that produce biphenyl dioxygenase degraded DDE. Arsenic and copper inhibit DDE degradation by aerobic microorganisms. Similarly, metal chelates such as EDTA inhibit the

  13. Apoptotic cell and phagocyte interplay: recognition and consequences in different cell systems

    Directory of Open Access Journals (Sweden)

    Moreira Maria Elisabete C.

    2004-01-01

    Full Text Available Cell death by apoptosis is characterized by specific biochemical changes, including the exposure of multiple ligands, expected to tag the dying cell for prompt recognition by phagocytes. In non-pathological conditions, an efficient clearance is assured by the redundant interaction between apoptotic cell ligands and multiple receptor molecules present on the engulfing cell surface. This review concentrates on the molecular interactions operating in mammalian and non-mammalian systems for apoptotic cell recognition, as well as on the consequences of their signaling. Furthermore, some cellular models where the exposure of the phosphatidylserine (PS phospholipid, a classical hallmark of the apoptotic phenotype, is not followed by cell death will be discussed.

  14. Citohistological Manifestations of the Apoptotic Process in the Prepubescent Mouse Ovary (14-17 days

    Directory of Open Access Journals (Sweden)

    Liliana Petculescu Ciochina

    2015-05-01

    Full Text Available Follicular atresia is a process commonly encountered at ovarian level that limits the number of ovulations and, respectively, the reproductive potential of a female. Ovarian cells death is essential for maintaining homeostasis of this organ and is based on an apoptotic mechanism that ensures selection of the dominant follicle and the disappearance of follicles in excess. The present study evaluates the incidence of morphological changes specific for this process in the prepubescent mouse ovary by applying optical microscopy techniques. Microscopic analysis was performed on sections of ovarian tissue from mice females line NMRI aged 14-17 days. The results show, at this age, significant morphological changes at cellular level, specific for apoptotic process, as condensation and fragmentation of the genetic material, vacuolization of cell cytoplasm, destabilization of cell adhesion junctions and appearance of wide intercellular spaces, invasion of spaces created with leukocytes infiltrated, formation of apoptotic bodies.

  15. Cell-to-Cell stochastic fluctuations in apoptotic signaling can decide between life and death

    CERN Document Server

    Raychaudhuri, S; Nguyen, T; Khan, E M; Goldkorn, T

    2007-01-01

    Apoptosis, or genetically programmed cell death, is a crucial cellular process that maintains the balance between life and death in cells. The precise molecular mechanism of apoptosis signaling and how these two pathways are differentially activated under distinct apoptotic stimuli is poorly understood. We developed a Monte Carlo-based stochastic simulation model that can characterize distinct signaling behaviors in the two major pathways of apoptotic signaling using a novel probability distribution-based approach. Specifically, we show that for a weak death signal, such as low levels of death ligand Fas (CD95) binding or under stress conditions, the type 2 mitochondrial pathway dominates apoptotic signaling. Our results also show signaling in the type 2 pathway is stochastic, where the population average over many cells does not capture the cell-to-cell fluctuations in the time course (~1 - 10 hours) of downstream caspase-3 activation. On the contrary, the probability distribution of caspase-3 activation for...

  16. Hippocampal expression of apoptotic protease activating factor-1 following diffuse axonal injury under mild hypothermia

    Institute of Scientific and Technical Information of China (English)

    Peng Yang; Limin Zhang; Yunhe Zhang; Xifeng Zou; Qunxi Li; Yun Li; Jun Zhu; Jianmin Li; Aijun Fu; Qingjun Liu; Tong Chen; Zelin Sun; Zhiyong Zhang

    2011-01-01

    The influence of mild hypothermia on neural cell apoptosis remains poorly understood. Therefore, the present study established rat models of diffuse axonal injury (DAI) at 33 °C. Morris water maze results demonstrated significantly better learning and memory functions in DAI rats with hypothermia compared with DAI rats with normothermia. Expression of apoptotic protease activating factor-1 in the hippocampal CA1 region was significantly lower in the DAI hypothermia group compared with the DAI normothermia group. Expression of apoptotic protease activating factor-1 positively correlated with latency, but negatively correlated with platform location times and time of swimming in the quadrant area. Results suggested that post-traumatic mild hypothermia in a rat model of DAI could provide cerebral protection by attenuating expression of apoptotic protease activating factor-1.

  17. Apoptosis in Cellular Society: Communication between Apoptotic Cells and Their Neighbors

    Directory of Open Access Journals (Sweden)

    Yuhei Kawamoto

    2016-12-01

    Full Text Available Apoptosis is one of the cell-intrinsic suicide programs and is an essential cellular behavior for animal development and homeostasis. Traditionally, apoptosis has been regarded as a cell-autonomous phenomenon. However, recent in vivo genetic studies have revealed that apoptotic cells actively influence the behaviors of surrounding cells, including engulfment, proliferation, and production of mechanical forces. Such interactions can be bidirectional, and apoptosis is non-autonomously induced in a cellular community. Of note, it is becoming evident that active communication between apoptotic cells and living cells contributes to physiological processes during tissue remodeling, regeneration, and morphogenesis. In this review, we focus on the mutual interactions between apoptotic cells and their neighbors in cellular society and discuss issues relevant to future studies of apoptosis.

  18. Ultrastructural observation of human neutrophils during apoptotic cell death triggered by Entamoeba histolytica.

    Science.gov (United States)

    Sim, Seobo; Kim, Kyeong Ah; Yong, Tai-Soon; Park, Soon-Jung; Im, Kyung-il; Shin, Myeong Heon

    2004-12-01

    Neutrophils are important effector cells against protozoan extracellular parasite Entamoeba histolytica, which causes amoebic colitis and liver abscess in human beings. Apoptotic cell death of neutrophils is an important event in the resolution of inflammation and parasite's survival in vivo. This study was undertaken to investigate the ultrastructural aspects of apoptotic cells during neutrophil death triggered by Entamoeba histolytica. Isolated human neutrophils from the peripheral blood were incubated with or without live trophozoites of E. histolytica and examined by transmission electron microscopy (TEM). Neutrophils incubated with E. histolytica were observed to show apoptotic characteristics, such as compaction of the nuclear chromatin and swelling of the nuclear envelop. In contrast, neutrophils incubated in the absence of the amoeba had many protrusions of irregular cell surfaces and heterogenous nuclear chromatin. Therefore, it is suggested that Entamoeba-induced neutrophil apoptosis contribute to prevent unwanted tissue inflammation and damage in the amoeba-invaded lesions in vivo.

  19. Interaction of apoptotic cells with macrophages upregulates COX-2/PGE2 and HGF expression via a positive feedback loop.

    Science.gov (United States)

    Byun, Ji Yeon; Youn, Young-So; Lee, Ye-Ji; Choi, Youn-Hee; Woo, So-Yeon; Kang, Jihee Lee

    2014-01-01

    Recognition of apoptotic cells by macrophages is crucial for resolution of inflammation, immune tolerance, and tissue repair. Cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) and hepatocyte growth factor (HGF) play important roles in the tissue repair process. We investigated the characteristics of macrophage COX-2 and PGE2 expression mediated by apoptotic cells and then determined how macrophages exposed to apoptotic cells in vitro and in vivo orchestrate the interaction between COX-2/PGE2 and HGF signaling pathways. Exposure of RAW 264.7 cells and primary peritoneal macrophages to apoptotic cells resulted in induction of COX-2 and PGE2. The COX-2 inhibitor NS-398 suppressed apoptotic cell-induced PGE2 production. Both NS-398 and COX-2-siRNA, as well as the PGE2 receptor EP2 antagonist, blocked HGF expression in response to apoptotic cells. In addition, the HGF receptor antagonist suppressed increases in COX-2 and PGE2 induction. The in vivo relevance of the interaction between the COX-2/PGE2 and HGF pathways through a positive feedback loop was shown in cultured alveolar macrophages following in vivo exposure of bleomycin-stimulated lungs to apoptotic cells. Our results demonstrate that upregulation of the COX-2/PGE2 and HGF in macrophages following exposure to apoptotic cells represents a mechanism for mediating the anti-inflammatory and antifibrotic consequences of apoptotic cell recognition.

  20. Interaction of Apoptotic Cells with Macrophages Upregulates COX-2/PGE2 and HGF Expression via a Positive Feedback Loop

    Directory of Open Access Journals (Sweden)

    Ji Yeon Byun

    2014-01-01

    Full Text Available Recognition of apoptotic cells by macrophages is crucial for resolution of inflammation, immune tolerance, and tissue repair. Cyclooxygenase-2 (COX-2/prostaglandin E2 (PGE2 and hepatocyte growth factor (HGF play important roles in the tissue repair process. We investigated the characteristics of macrophage COX-2 and PGE2 expression mediated by apoptotic cells and then determined how macrophages exposed to apoptotic cells in vitro and in vivo orchestrate the interaction between COX-2/PGE2 and HGF signaling pathways. Exposure of RAW 264.7 cells and primary peritoneal macrophages to apoptotic cells resulted in induction of COX-2 and PGE2. The COX-2 inhibitor NS-398 suppressed apoptotic cell-induced PGE2 production. Both NS-398 and COX-2-siRNA, as well as the PGE2 receptor EP2 antagonist, blocked HGF expression in response to apoptotic cells. In addition, the HGF receptor antagonist suppressed increases in COX-2 and PGE2 induction. The in vivo relevance of the interaction between the COX-2/PGE2 and HGF pathways through a positive feedback loop was shown in cultured alveolar macrophages following in vivo exposure of bleomycin-stimulated lungs to apoptotic cells. Our results demonstrate that upregulation of the COX-2/PGE2 and HGF in macrophages following exposure to apoptotic cells represents a mechanism for mediating the anti-inflammatory and antifibrotic consequences of apoptotic cell recognition.

  1. Effect of hrHPV infection on anti-apoptotic gene and pro-apoptotic gene expression in cervical cancer tissue

    Institute of Scientific and Technical Information of China (English)

    Min-Er Tang

    2016-01-01

    Objective:To study the effect of hrHPV infection on anti-apoptotic gene and pro-apoptotic gene expression in cervical cancer tissue.Methods: A total of 56 patients with cervical cancer, 94 cases of patients with cervical intraepithelial neoplasia and 48 cases of patients with chronic cervicitis who were treated in our hospital from May 2013 to December 2015 were selected for study and included in malignant group, precancerous lesion group and benign group respectively. hrHPV infection as well as the expression of anti-apoptotic genes and pro-apoptotic genes in cervical tissue were detected.Results:hrHPV infection rate and viral load in cervical tissue of malignant group were significantly higher than those of precancerous lesion group and benign group; P27 and p16 levels in cervical tissue of malignant group were significantly lower than those of precancerous lesion group and benign group, and K-ras, c-myc, Prdx4 and TNFAIP8 levels were significantly higher than those of precancerous lesion group and benign group; the greater the HPV virus load, the lower the p27 and p16 levels and the higher the K-ras, c-myc, Prdx4 and TNFAIP8 levels in cervical tissue.Conclusions:hrHPV infection can result in tumor suppressor genes p27 and p16 expression deletion and increase the expression of proto-oncogene and apoptosis-inhibiting genes, and it is associated with the occurrence and development of cervical cancer.

  2. ARP2, a novel pro-apoptotic protein expressed in epithelial prostate cancer LNCaP cells and epithelial ovary CHO transformed cells.

    Directory of Open Access Journals (Sweden)

    Jaime Mas-Oliva

    Full Text Available Neoplastic epithelial cells generate the most aggressive types of cancers such as those located in the lung, breast, colon, prostate and ovary. During advanced stages of prostate cancer, epithelial cells are associated to the appearance of androgen-independent tumors, an apoptotic-resistant phenotype that ultimately overgrows and promotes metastatic events. We have previously identified and electrophysiologically characterized a novel Ca(2+-permeable channel activated during apoptosis in the androgen-independent prostate epithelial cancer cell line, LNCaP. In addition, we reported for the first time the cloning and characterization of this channel-like molecule named apoptosis regulated protein 2 (ARP2 associated to a lethal influx of Ca(2+ in Xenopus oocytes. In the present study, LNCaP cells and Chinese hamster ovary cells (CHO cell line transfected with arp2-cDNA are induced to undergo apoptosis showing an important impact on cell viability and activation of caspases 3 and 7 when compared to serum deprived grown cells and ionomycin treated cells. The subcellular localization of ARP2 in CHO cells undergoing apoptosis was studied using confocal microscopy. While apoptosis progresses, ARP2 initially localized in the peri-nuclear region of cells migrates with time towards the plasma membrane region. Based on the present results and those of our previous studies, the fact that ARP2 constitutes a novel cation channel is supported. Therefore, ARP2 becomes a valuable target to modulate the influx and concentration of calcium in the cytoplasm of epithelial cancer cells showing an apoptotic-resistant phenotype during the onset of an apoptotic event.

  3. Role and Association of Inflammatory and Apoptotic Caspases in Renal Tubulointerstitial Fibrosis

    Directory of Open Access Journals (Sweden)

    You Ke

    2016-09-01

    Full Text Available Background/Aims: Caspases, an evolutionary conserved family of aspartate-specific cystein proteases, play pivotal roles in apoptotic and inflammatory signaling. Thus far, 14 mammalian caspases are identified and categorized into 3 distinct sub-types: inflammatory caspases, apoptotic initiator and apoptotic executioner. Caspase-1 is an inflammatory caspase, while caspase-7 belongs to apoptotic executioner. The roles and association of these two distinct types of caspases in renal tubulointerstitial fibrosis (TIF have not been well recognized. Methods: Caspase-1 inhibitor Z-YVAD-FMK and caspase-7 siRNA were used in tubular epithelial cell line NRK-52E (TECs to test their effects on transforming growth factor-beta1 (TGF-β1 stimulation. In vivo, Unilateral ureteral obstruction (UUO animal model was employed in wild-type (WT and caspase-1 knock out (KO (caspase-1-/- mice. Results: In current study, we found that caspase-7 was obviously activated in cultured TECs stimulated by TGF-β1 and in UUO model of WT mice. While in UUO model of caspase-1 KO mice, the increased caspase-7 activation was suppressed significantly along with reduced trans-differentiation and minimized extracellular matrix (ECM accumulation, as demonstrated by western blot, Masson trichrome staining and immunohistochemistry. In addition, pharmacological inhibition of caspase-1 dampened caspase-7 activation and TECs' transdifferentiation induced by TGF-β1 exposure, which was consistent with in vivo study. Notably, caspase-7 gene knock down by specific siRNA abrogated TGF-β1 driven TECs' trans-differentiation and reduced ECM accumulation. Conclusions: Our study associated inflammatory and apoptotic caspases in TIF for the first time and we further confirmed that caspase-1 activation is an upstream event of apoptotic caspase-7 induction in TIF triggered by UUO and in TECs mediated by TGF-β1 induced transdifferentiation.

  4. Yersinia enterocolitica-mediated degradation of neutrophil extracellular traps (NETs).

    Science.gov (United States)

    Möllerherm, Helene; Neumann, Ariane; Schilcher, Katrin; Blodkamp, Stefanie; Zeitouni, Nathalie E; Dersch, Petra; Lüthje, Petra; Naim, Hassan Y; Zinkernagel, Annelies S; von Köckritz-Blickwede, Maren

    2015-12-01

    Neutrophil extracellular trap (NET) formation is described as a tool of the innate host defence to fight against invading pathogens. Fibre-like DNA structures associated with proteins such as histones, cell-specific enzymes and antimicrobial peptides are released, thereby entrapping invading pathogens. It has been reported that several bacteria are able to degrade NETs by nucleases and thus evade the NET-mediated entrapment. Here we studied the ability of three different Yersinia serotypes to induce and degrade NETs. We found that the common Yersinia enterocolitica serotypes O:3, O:8 and O:9 were able to induce NETs in human blood-derived neutrophils during the first hour of co-incubation. At later time points, the NET amount was reduced, suggesting that degradation of NETs has occurred. This was confirmed by NET degradation assays with phorbol-myristate-acetate-pre-stimulated neutrophils. In addition, we found that the Yersinia supernatants were able to degrade purified plasmid DNA. The absence of Ca(2+) and Mg(2+) ions, but not that of a protease inhibitor cocktail, completely abolished NET degradation. We therefore postulate that Y. enterocolitica produces Ca(2+)/Mg(2+)-dependent NET-degrading nucleases as shown for some Gram-positive pathogens.

  5. Anti-apoptotic signaling as a cytoprotective mechanism in mammalian hibernation

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    Andrew N. Rouble

    2013-02-01

    Full Text Available In the context of normal cell turnover, apoptosis is a natural phenomenon involved in making essential life and death decisions. Apoptotic pathways balance signals which promote cell death (pro-apoptotic pathways or counteract these signals (anti-apoptotic pathways. We proposed that changes in anti-apoptotic proteins would occur during mammalian hibernation to aid cell preservation during prolonged torpor under cellular conditions that are highly injurious to most mammals (e.g. low body temperatures, ischemia. Immunoblotting was used to analyze the expression of proteins associated with pro-survival in six tissues of thirteen-lined ground squirrels, Ictidomys tridecemlineatus. The brain showed a concerted response to torpor with significant increases in the levels of all anti-apoptotic targets analyzed (Bcl-2, Bcl-xL, BI-1, Mcl-1, cIAP1/2, xIAP as well as enhanced phosphorylation of Bcl-2 at S70 and T56. Heart responded similarly with most anti-apoptotic proteins elevated significantly during torpor except for Bcl-xL and xIAP that decreased and Mcl-1 that was unaltered. In liver, BI-1 increased whereas cIAP1/2 decreased. In kidney, there was an increase in BI-1, cIAP and xIAP but decreases in Bcl-xL and p-Bcl-2(T56 content. In brown adipose tissue, protein levels of BI-1, cIAP1/2, and xIAP decreased significantly during torpor (compared with euthermia whereas Bcl-2, Bcl-xL, Mcl-1 were unaltered; however, Bcl-2 showed enhanced phosphorylation at Thr56 but not at Ser70. In skeletal muscle, only xIAP levels changed significantly during torpor (an increase. The data show that anti-apoptotic pathways have organ-specific responses in hibernators with a prominent potential role in heart and brain where coordinated enhancement of anti-apoptotic proteins occurred in response to torpor.

  6. Synthesis of apoptotic chalcone analogues in HepG2 human hepatocellular carcinoma cells.

    Science.gov (United States)

    Park, Cheon-Soo; Ahn, Yongchel; Lee, Dahae; Moon, Sung Won; Kim, Ki Hyun; Yamabe, Noriko; Hwang, Gwi Seo; Jang, Hyuk Jai; Lee, Heesu; Kang, Ki Sung; Lee, Jae Wook

    2015-12-15

    Eight chalcone analogues were prepared and evaluated for their cytotoxic effects in human hepatoma HepG2 cells. Compound 5 had a potent cytotoxic effect. The percentage of apoptotic cells was significantly higher in compound 5-treated cells than in control cells. Exposure to compound 5 for 24h induced cleavage of caspase-8 and -3, and poly (ADP-ribose) polymerase (PARP). Our findings suggest that compound 5 is the active chalcone analogue that contributes to cell death in HepG2 cells via the extrinsic apoptotic pathway.

  7. Regulation of apoptotic signal transduction pathways by the heat shock proteins

    Institute of Scientific and Technical Information of China (English)

    LI; Zhengyu; ZHAO; Xia; WEI; Yuquan

    2004-01-01

    The study about apoptotic signal transductions has become a project to reveal the molecular mechanisms of apoptosis. Heat shock proteins (hsps), which play an important role in cell growth and apoptosis, have attracted great attentions. A lot of researches have showed there is a hsps superfamily including hsp90, hsp70, hsp60 and hsp27, etc., which regulates the biological behaviors of cells, particularly apoptotic signal transduction in Fas pathway, JNK/SAPK pathway and caspases pathway at different levels, partly by the function of molecular chaperone.

  8. Drift Degradation Analysis

    Energy Technology Data Exchange (ETDEWEB)

    D. Kicker

    2004-09-16

    Degradation of underground openings as a function of time is a natural and expected occurrence for any subsurface excavation. Over time, changes occur to both the stress condition and the strength of the rock mass due to several interacting factors. Once the factors contributing to degradation are characterized, the effects of drift degradation can typically be mitigated through appropriate design and maintenance of the ground support system. However, for the emplacement drifts of the geologic repository at Yucca Mountain, it is necessary to characterize drift degradation over a 10,000-year period, which is well beyond the functional period of the ground support system. This document provides an analysis of the amount of drift degradation anticipated in repository emplacement drifts for discrete events and time increments extending throughout the 10,000-year regulatory period for postclosure performance. This revision of the drift degradation analysis was developed to support the license application and fulfill specific agreement items between the U.S. Nuclear Regulatory Commission (NRC) and the U.S. Department of Energy (DOE). The earlier versions of ''Drift Degradation Analysis'' (BSC 2001 [DIRS 156304]) relied primarily on the DRKBA numerical code, which provides for a probabilistic key-block assessment based on realistic fracture patterns determined from field mapping in the Exploratory Studies Facility (ESF) at Yucca Mountain. A key block is defined as a critical block in the surrounding rock mass of an excavation, which is removable and oriented in an unsafe manner such that it is likely to move into an opening unless support is provided. However, the use of the DRKBA code to determine potential rockfall data at the repository horizon during the postclosure period has several limitations: (1) The DRKBA code cannot explicitly apply dynamic loads due to seismic ground motion. (2) The DRKBA code cannot explicitly apply loads due to thermal

  9. DNA/chitosan electrostatic complex.

    Science.gov (United States)

    Bravo-Anaya, Lourdes Mónica; Soltero, J F Armando; Rinaudo, Marguerite

    2016-07-01

    Up to now, chitosan and DNA have been investigated for gene delivery due to chitosan advantages. It is recognized that chitosan is a biocompatible and biodegradable non-viral vector that does not produce immunological reactions, contrary to viral vectors. Chitosan has also been used and studied for its ability to protect DNA against nuclease degradation and to transfect DNA into several kinds of cells. In this work, high molecular weight DNA is compacted with chitosan. DNA-chitosan complex stoichiometry, net charge, dimensions, conformation and thermal stability are determined and discussed. The influence of external salt and chitosan molecular weight on the stoichiometry is also discussed. The isoelectric point of the complexes was found to be directly related to the protonation degree of chitosan. It is clearly demonstrated that the net charge of DNA-chitosan complex can be expressed in terms of the ratio [NH3(+)]/[P(-)], showing that the electrostatic interactions between DNA and chitosan are the main phenomena taking place in the solution. Compaction of DNA long chain complexed with low molar mass chitosan gives nanoparticles with an average radius around 150nm. Stable nanoparticles are obtained for a partial neutralization of phosphate ionic sites (i.e.: [NH3(+)]/[P(-)] fraction between 0.35 and 0.80).

  10. Nifedipine treatment reduces resting calcium concentration, oxidative and apoptotic gene expression, and improves muscle function in dystrophic mdx mice.

    Science.gov (United States)

    Altamirano, Francisco; Valladares, Denisse; Henríquez-Olguín, Carlos; Casas, Mariana; López, Jose R; Allen, Paul D; Jaimovich, Enrique

    2013-01-01

    Duchenne Muscular Dystrophy (DMD) is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM) decreased [Ca(2+)]r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the [Ca(2+)]r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB) fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced [Ca(2+)]r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91(phox)/p47(phox) NOX2 subunits) and pro-apoptotic (Bax) genes in mdx diaphragm muscles and lowered serum creatine kinase (CK) levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces [Ca(2+)]r in mdx skeletal muscle cells. The results in this work open new perspectives

  11. Nifedipine treatment reduces resting calcium concentration, oxidative and apoptotic gene expression, and improves muscle function in dystrophic mdx mice.

    Directory of Open Access Journals (Sweden)

    Francisco Altamirano

    Full Text Available Duchenne Muscular Dystrophy (DMD is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM decreased [Ca(2+]r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the [Ca(2+]r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced [Ca(2+]r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91(phox/p47(phox NOX2 subunits and pro-apoptotic (Bax genes in mdx diaphragm muscles and lowered serum creatine kinase (CK levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces [Ca(2+]r in mdx skeletal muscle cells. The results in this work open new

  12. TJ-41 Induces Apoptosis and Potentiates the Apoptotic Effects of 5-FU in Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Suresh Volate

    2009-01-01

    Full Text Available Recent studies suggest that TJ-41, a herbal drug, possesses chemotherapeutic effects. Accordingly, this study was undertaken to investigate the anticarcinogenic effects of TJ-41 on human breast cancer cells lines. TJ-41 inhibited the proliferation of human breast cancer cell lines dose dependently. Flow cytometric analysis showed that this decrease in DNA synthesis is to be associated with induction of apoptosis. In both cell lines, apoptosis was abolished by caspase-9 inhibitor Z-LEHD-fmk but was weakly inhibited by caspase-8 inhibitor Z-IETD-fmk, indicating that caspase-9 activation was involved in TJ-41 induced apoptosis. Additionally, TJ-41 stimulated phosphorylation of c-Jun NH2-terminal kinase (JNK and pretreatment of breast cancer cells with JNK inhibitor SP600125 completely abolished TJ-41 induced apoptosis. Our data also demonstrate that combined treatment of TJ-41 and 5-FU significantly potentiates the apoptotic effects of 5-FU in both breast cancer cell lines. Taken together, these data suggest that TJ-41 might provide a novel chemotherapeutic treatment for breast cancer.

  13. Neuroprotective Effects of Alpha-Mangostin on MPP+-Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells

    Directory of Open Access Journals (Sweden)

    Prachya Janhom

    2015-01-01

    Full Text Available In vitro studies have shown that extracts from mangosteen (Garcinia mangostana Linn. act as antioxidants and cytoprotective agents against oxidative damage. The protective effect of alpha-mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of Parkinson’s disease (PD, has not been investigated. This study aims to investigate whether alpha-mangostin could protect SH-SY5Y neuroblastoma cells from MPP+-induced apoptosis. The effects of alpha-mangostin on MPP+-induced cell death were evaluated with a cell viability assay, staining for nuclear DNA morphology, flow cytometry for apoptotic cells and reactive oxygen species (ROS production, quantitative real-time PCR for the expression of p53, Bax, and Bcl-2, and western blot analysis for cleaved caspase-3. Concomitant treatment with alpha-mangostin attenuated the effect of MPP+ on cell viability and apoptotic cell death. Alpha-mangostin reduced ROS formation induced by MPP+. Bax/Bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha-mangostin and MPP+. The cotreated cells showed a significant decrease in activated caspase-3 compared with MPP+ treatment alone. Our data suggest that cytoprotection of alpha-mangostin against MPP+-induced apoptosis may be associated with the reduction of ROS production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation.

  14. Neuroprotective Effects of Alpha-Mangostin on MPP+-Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells

    Science.gov (United States)

    Janhom, Prachya; Dharmasaroja, Permphan

    2015-01-01

    In vitro studies have shown that extracts from mangosteen (Garcinia mangostana Linn.) act as antioxidants and cytoprotective agents against oxidative damage. The protective effect of alpha-mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of Parkinson's disease (PD), has not been investigated. This study aims to investigate whether alpha-mangostin could protect SH-SY5Y neuroblastoma cells from MPP+-induced apoptosis. The effects of alpha-mangostin on MPP+-induced cell death were evaluated with a cell viability assay, staining for nuclear DNA morphology, flow cytometry for apoptotic cells and reactive oxygen species (ROS) production, quantitative real-time PCR for the expression of p53, Bax, and Bcl-2, and western blot analysis for cleaved caspase-3. Concomitant treatment with alpha-mangostin attenuated the effect of MPP+ on cell viability and apoptotic cell death. Alpha-mangostin reduced ROS formation induced by MPP+. Bax/Bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha-mangostin and MPP+. The cotreated cells showed a significant decrease in activated caspase-3 compared with MPP+ treatment alone. Our data suggest that cytoprotection of alpha-mangostin against MPP+-induced apoptosis may be associated with the reduction of ROS production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation. PMID:26357513

  15. Anti-apoptotic and hepatoprotective effects of gomisin A on fulminant hepatic failure induced by D-galactosamine and lipopolysaccharide in mice.

    Science.gov (United States)

    Kim, Sung-Hwa; Kim, Yeong Shik; Kang, Sam Sik; Bae, Kihwan; Hung, Tran Manh; Lee, Sun-Mee

    2008-02-01

    This study examined the effects of gomisin A, a lignan compound from Schisandra fructus, on D-galactosamine (GalN) and lipopolysaccharide (LPS)-induced hepatic apoptosis and liver failure. Mice were given an intraperitoneal injection of GalN (700 mg/kg) / LPS (10 microg/kg). Gomisin A (25, 50, 100, and 200 mg/kg) was administered intraperitoneally 1 h before the GalN/LPS injection. The liver injury was assessed biochemically and histologically. GalN/LPS increased the serum aminotransferase levels and lipid peroxidation but decreased the reduced glutathione level. The pretreatment with gomisin A attenuated these changes in a dose-dependent manner. The survival rate of the gomisin A group was significantly higher than that of the control. The mitochondria isolated after the mice had been injected with GalN/LPS were swollen, which was attenuated by the gomisin A pretreatment. The elevation of serum tumor necrosis factor-alpha and activation of caspase-3 were observed in the GalN/LPS group, which was attenuated by gomisin A. The gomisin A-pretreated groups showed significantly fewer apoptotic (TUNEL-positive) cells and DNA fragmentation as compared with the GalN/LPS mice. The liver protection afforded by gomisin A is the result of the reduced oxidative stress and its anti-apoptotic activity.

  16. The Effect of Crude Extract of Pandanus conoideus Lamb. var. Yellow Fruit on Apoptotic Expression of the Breast Cancer Cell Line (T47D

    Directory of Open Access Journals (Sweden)

    OKID PARAMA ASTIRIN

    2009-01-01

    Full Text Available A mechanism controlling a growing cancer cells is by a programmed cell death (apoptosis. The wildtype-p53 enable to stop cleaves that follow DNA repair or cell death (apoptosis. The mutation of wt-p53 caused loosing its ability to inhibit cancer cells proliferation. Healing methods like surgery, radiation, immunotherapy and chemotherapy still have some weaknesses, and clinical medicine to cancer is also still has any dissatisfactory. Much of chemotherapy was not given optimal result yet, because no specific action to cancer cells only, but also to the normal cells. These problems encourage important effort to find specific and sensitive anticancer. Empirical evidence indicates that the crude extract of Pandanus conoideus Lamb var. yellow fruit has potential effect as an anticancer. Method of Freshney was used in growing T47D cell line, counting cells was done by direct counting, and apoptotic evaluation was done by TUNEL enzymatic labeling assay. The results of the research demonstrated that the LC50 of yellow fruit extract are 0.25 µL/mL. The percentage of apoptotic of 0.125 µL/mL, 0.0625 µL/mL, and 0.03125 µL/mL are 34.38±2.26, 30.03±3.87 and 21.07±1.14 respectively.

  17. Biliverdin reductase/bilirubin mediates the anti-apoptotic effect of hypoxia in pulmonary arterial smooth muscle cells through ERK1/2 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Song, Shasha [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Wang, Shuang [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Biopharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China); Ma, Jun; Yao, Lan; Xing, Hao; Zhang, Lei; Liao, Lin [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Zhu, Daling, E-mail: dalingz@yahoo.com [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Biopharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China)

    2013-08-01

    Inhibition of pulmonary arterial smooth muscle cell (PASMC) apoptosis induced by hypoxia plays an important role in pulmonary arterial remodeling leading to aggravate hypoxic pulmonary arterial hypertension. However, the mechanisms of hypoxia acting on PASMC apoptosis remain exclusive. Biliverdin reductase (BVR) has many essential biologic roles in physiological and pathological processes. Nevertheless, it is unclear whether the hypoxia-induced inhibition on PASMC apoptosis is mediated by BVR. In the present work, we found BVR majorly localized in PASMCs and was up-regulated in levels of protein and mRNA by hypoxia. Then we studied the contribution of BVR to anti-apoptotic response of hypoxia in PASMCs. Our results showed that siBVR, blocking generation of bilirubin, reversed the effect of hypoxia on enhancing cell survival and apoptotic protein (Bcl-2, procasepase-9, procasepase-3) expression, preventing nuclear shrinkage, DNA fragmentation and mitochondrial depolarization in starved PASMCs, which were recovered by exogenous bilirubin. Moreover, the inhibitory effect of bilirubin on PASMC apoptosis under hypoxic condition was blocked by the inhibitor of ERK1/2 pathway. Taken together, our data indicate that BVR contributes to the inhibitory process of hypoxia on PASMC apoptosis, which is mediated by bilirubin through ERK1/2 pathway. Highlights: • BVR expresses in PASMC and is up-regulated by hypoxia in protein and mRNA levels. • BVR/bilirubin contribute to the inhibitive process of hypoxia on PASMC apoptosis. • Bilirubin protects PASMC from apoptosis under hypoxia via ERK1/2 pathway.

  18. Mitogenomic analyses from ancient DNA

    DEFF Research Database (Denmark)

    Paijmans, Johanna L.A.; Gilbert, M Thomas P; Hofreiter, Michael

    2013-01-01

    analyses (whether using modern or ancient DNA) were largely restricted to the analysis of short fragments of the mitochondrial genome. However, due to many technological advances during the past decade, a growing number of studies have explored the power of complete mitochondrial genome sequences...... (mitogenomes). Such studies were initially limited to analyses of extant organisms, but developments in both DNA sequencing technologies and general methodological aspects related to working with degraded DNA have resulted in complete mitogenomes becoming increasingly popular for ancient DNA studies as well....... To date, at least 124 partially or fully assembled mitogenomes from more than 20 species have been obtained, and, given the rapid progress in sequencing technology, this number is likely to dramatically increase in the future. The increased information content offered by analysing full mitogenomes has...

  19. Motor degradation prediction methods

    Energy Technology Data Exchange (ETDEWEB)

    Arnold, J.R.; Kelly, J.F.; Delzingaro, M.J.

    1996-12-01

    Motor Operated Valve (MOV) squirrel cage AC motor rotors are susceptible to degradation under certain conditions. Premature failure can result due to high humidity/temperature environments, high running load conditions, extended periods at locked rotor conditions (i.e. > 15 seconds) or exceeding the motor`s duty cycle by frequent starts or multiple valve stroking. Exposure to high heat and moisture due to packing leaks, pressure seal ring leakage or other causes can significantly accelerate the degradation. ComEd and Liberty Technologies have worked together to provide and validate a non-intrusive method using motor power diagnostics to evaluate MOV rotor condition and predict failure. These techniques have provided a quick, low radiation dose method to evaluate inaccessible motors, identify degradation and allow scheduled replacement of motors prior to catastrophic failures.

  20. Antifoam degradation testing

    Energy Technology Data Exchange (ETDEWEB)

    Lambert, D. P. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River Ecology Lab. (SREL); Zamecnik, J. R. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River Ecology Lab. (SREL); Newell, D. D. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River Ecology Lab. (SREL); Williams, M. S. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River Ecology Lab. (SREL)

    2015-08-20

    This report describes the results of testing to quantify the degradation products resulting from the dilution and storage of Antifoam 747. Antifoam degradation is of concern to the Defense Waste Processing Facility (DWPF) due to flammable decomposition products in the vapor phase of the Chemical Process Cell vessels, as well as the collection of flammable and organic species in the offgas condensate. The discovery that hexamethyldisiloxane is formed from the antifoam decomposition was the basis for a Potential Inadequacy in the Safety Analysis declaration by the DWPF.

  1. Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins.

    Science.gov (United States)

    Colin, Didier J; Hain, Karolina O; Allan, Lindsey A; Clarke, Paul R

    2015-03-01

    Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

  2. Cytotoxicity and apoptotic effects of nickel oxide nanoparticles in cultured HeLa cells

    Directory of Open Access Journals (Sweden)

    Kezban Ada

    2010-04-01

    Full Text Available The aim of this study was to observe the cytotoxicity and apoptotic effects of nickel oxide nanoparticles on humancervix epithelioid carcinoma cell line (HeLa. Nickel oxide precursors were synthesized by an nickel sulphate-excess ureareaction in boiling aqueous solution. The synthesized NiO nanoparticles (<200 nm were investigated by X-ray diffractionanalysis and transmission electron microscopy techniques. For cytotoxicity experiments, HeLa cells were incubated in50-500 μg/mL NiO for 2, 6, 12 and 16 hours. The viable cells were counted with a haemacytometer using light microscopy.The cytotoxicity was observed low in 50-200 μg/mL concentration for 16 h, but high in 400-500 μg/mL concentration for2-6 h. HeLa cells' cytoplasm membrane was lysed and detached from the well surface in 400 μg/mL concentration NiOnanoparticles. Double staining and M30 immunostaining were performed to quantify the number of apoptotic cells in cultureon the basis of apoptotic cell nuclei scores. The apoptotic effect was observed 20% for 16 h incubation.

  3. The c-Myc Transactivation Domain Is a Direct Modulator of Apoptotic versus Proliferative Signals

    Science.gov (United States)

    Chang, David W.; Claassen, Gisela F.; Hann, Stephen R.; Cole, Michael D.

    2000-01-01

    We have assayed the oncogenic, proliferative, and apoptotic activities of the frequent mutations that occur in the c-myc gene in Burkitt's lymphomas. Some alleles have a modest (50 to 60%) increase in transforming activity; however, the most frequent Burkitt's lymphoma allele (T58I) had an unexpected substantial decrease in transforming activity (85%). All alleles restored the proliferation function of c-Myc in cells that grow slowly due to a c-myc knockout. There was discordance for some alleles between apoptotic and oncogenic activities, but only the T58A allele had elevated transforming activity with a concomitant reduced apoptotic potential. We discovered a novel missense mutation, MycS71F, that had a very low apoptotic activity compared to wild-type Myc, yet this mutation has never been found in lymphomas, suggesting that there is no strong selection for antiapoptotic c-Myc alleles. MycS71F also induced very low levels of cytochrome c release from mitochondria, suggesting a mechanism of action for this mutation. Phosphopeptide mapping provided a biochemical basis for the dramatically different biological activities of the transformation-defective T58I and transformation-enhanced T58A c-Myc alleles. Furthermore, the antiapoptotic survival factor insulin-like growth factor 1 was found to suppress phosphorylation of T58, suggesting that the c-Myc transactivation domain is a direct target of survival signals. PMID:10825194

  4. Anti-apoptotic signaling and failure of apoptosis in the ischemic rat hippocampus

    DEFF Research Database (Denmark)

    Müller, Georg Johannes; Lassmann, Hans; Johansen, Flemming Fryd

    2007-01-01

    Several anti-apoptotic proteins are induced in CA1 neurons after transient forebrain ischemia (TFI), but fail to protect the majority of these cells from demise. Correlating cell death morphologies (apoptosis-like and necrosis-like death) with immunohistochemistry (IHC), we investigated whether a...

  5. Boolean model of Yeast Apoptosis as a tool to study yeast and human apoptotic regulations

    Directory of Open Access Journals (Sweden)

    Laleh eKazemzadeh

    2012-12-01

    Full Text Available Programmed cell death (PCD is an essential cellular mechanism that is evolutionary conserved, mediated through various pathways and acts by integrating different stimuli. Many diseases such as neurodegenerative diseases and cancers are found to be caused by, or associated with, regulations in the cell death pathways. Yeast Saccharomyces cerevisiae, is a unicellular eukaryotic organism that shares with human cells components and pathways of the PCD and is therefore used as a model organism. Boolean modelling is becoming promising approach to capture qualitative behaviour and describe essential properties of such complex networks. Here we present large literature-based and to our knowledge first Boolean model that combines pathways leading to apoptosis (a type of PCD in yeast. Analysis of the yeast model confirmed experimental findings of anti-apoptotic role of Bir1p and pro-apoptotic role of Stm1p and revealed activation of the stress protein kinase Hog proposing the maximal level of activation upon heat stress. In addition we extended the yeast model and created an in silico humanized yeast in which human pro- and anti-apoptotic regulators Bcl-2 family and Valosin-contain protein (VCP are included in the model. We showed that accumulation of Bax in in silico humanized yeast shows apoptotic markers and that VCP is essential target of Akt Signaling. The presented Boolean model provides comprehensive description of yeast apoptosis network behaviour. Extended model of humanized yeast gives new insights of how complex human disease like neurodegenration can initially be tested.

  6. Antiproliferative and pro-apoptotic effects of Uncaria tomentosa in human medullary thyroid carcinoma cells.

    Science.gov (United States)

    Rinner, Beate; Li, Zeng Xia; Haas, Helga; Siegl, Veronika; Sturm, Sonja; Stuppner, Hermann; Pfragner, Roswitha

    2009-11-01

    Medullary thyroid carcinoma (MTC), a rare calcitonin-producing tumor, is derived from parafollicular C-cells of the thyroid and is characterized by constitutive Bcl-2 overexpression. The tumor is relatively insensitive to radiation therapy as well as conventional chemotherapy. To date, the only curative treatment is the early and complete surgical removal of all neoplastic tissue. In this study, the antiproliferative and pro-apoptotic effects of fractions obtained from Uncaria tomentosa (Willd.) DC, commonly known as uña de gato or cat's claw were investigated. Cell growth of MTC cells as well as enzymatic activity of mitochondrial dehydrogenase was markedly inhibited after treatment with different fractions of the plant. Furthermore, there was an increase in the expressions of caspase-3 and -7 and poly(ADP-ribose) polymerase (PARP) fraction, while bcl-2 overexpression remained constant. In particular, the alkaloids isopterpodine and pteropodine of U. tomentosa exhibited a significant pro-apoptotic effect on MTC cells, whereas the alkaloid-poor fraction inhibited cell proliferation but did not show any pro-apoptotic effects. These promising results indicate the growth-restraining and apoptotic potential of plant extracts against neuroendocrine tumors, which may add to existing therapies for cancer.

  7. Different Sensitivities to Apoptotic Induction by Camptothecin between Normal and Senescent Lens Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    Haike Guo; Haiying Jin; Liya Wang; Hongyang Zhang; Xin Yang

    2002-01-01

    Purpose: To investigate whether normal and senescent lens epithelial cells have different defense abilities to apoptotic induction factor in vitro.Methods: Rabbit lens epithelial cells were cultured, passed. When reaching confluence, cells from the first and seventh passage were stained by x-gal staining to detect cell senescence. Cell apoptosis was detected by TUNEL(Roche).10μmol/L camptothecin was used to induce cell apoptosis from the lens epithelial cells of the first and seventh passage to distinguish different sensitivities to apoptotic induction factor between normal and senescent cells.Results: The senescent cells (41.17% ± 5.24% ) were detected in the lens epithelial cell culture of the seventh passage, which are higher than those of the first passage (0.98% ±0. 39% ). There was no apoptotic cell detected in the cell cultures undisturbed. Exposure of the first passage cells to camptothecin resulted in death of approximately 23.87% ± 3.45% of the cells during a 36 hour exposure period. In contrast, significantly more lens epithelial cells died through the apoptosis (38.29% ±4. 01% ) from the seventh passage.Conclusion: Senescent cells increased with cell passage. Senescence lens epithelial cells do not undergo apoptosis if they were not disturbed. But the vulnerabilities to apoptotic induction between health and senescence cells were different.

  8. Modulation of apoptotic pathways of macrophages by surface-functionalized multi-walled carbon nanotubes.

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    Yuanqin Jiang

    Full Text Available Biomedical applications of carbon nanotubes (CNTs often involve improving their hydrophilicity and dispersion in biological media by modifying them through noncovalent or covalent functionalization. However, the potential adverse effects of surface-functionalized CNTs have not been well characterized. In this study, we functionalized multi-walled CNTs (MWCNTs via carboxylation, to produce MWCNTs-COOH, and via poly (ethylene glycol linking, to produce MWCNTs-PEG. We used these functionalized MWCNTs to study the effect of surface functionalization on MWCNTs-induced toxicity to macrophages, and elucidate the underlying mechanisms of action. Our results revealed that MWCNTs-PEG were less cytotoxic and were associated with less apoptotic cell death of macrophages than MWCNTs-COOH. Additionally, MWCNTs-PEG induced less generation of reactive oxygen species (ROS involving less activation of NADPH oxidase compared with MWCNTs-COOH, as evidenced by membrane translocation of p47(phox and p67(phox in macrophages. The less cytotoxic and apoptotic effect of MWCNTs-PEG compared with MWCNTs-COOH resulted from the lower cellular uptake of MWCNTs-PEG, which resulted in less activation of oxidative stress-responsive pathways, such as p38 mitogen-activated protein kinases (MAPK and nuclear factor (NF-κB. These results demonstrate that surface functionalization of CNTs may alter ROS-mediated cytotoxic and apoptotic response by modulating apoptotic signaling pathways. Our study thus provides new insights into the molecular basis for the surface properties affecting CNTs toxicity.

  9. Apoptotic neurons induce proliferative responses of progenitor cells in the postnatal neocortex.

    Science.gov (United States)

    Petrenko, Volodymyr; Mihhailova, Jevgenia; Salmon, Patrick; Kiss, Jozsef Z

    2015-11-01

    Apoptotic cell death is the leading cause of neuronal loss after neonatal brain injury. Little is known about the intrinsic capacity of the immature cerebral cortex for replacing dead cells. Here we test the hypothesis that neuronal apoptosis is able to trigger compensatory proliferation in surrounding cells. In order to establish a "pure" apoptotic cell death model and to avoid the confounding effects of broken blood-brain barrier and inflammatory reactions, we used a diphtheria toxin (DT) and diphtheria toxin receptor (DTR) system to induce ablation of layer IV neurons in the rodent somatosensory cortex during the early postnatal period. We found that DT-triggered apoptosis is a slowly progressing event lasting about for 7 days. While dying cells expressed the morphological features of apoptosis, we could not detect immunoreactivity for activated caspase-3 in these cells. Microglia activation and proliferation represented the earliest cellular responses to apoptotic cell death. In addition, we found that induced apoptosis triggered a massive proliferation of undifferentiated progenitor cell pool including Sox2 as well as NG2 cells. The default differentiation pattern of proliferating progenitors appears to be the glial phenotype; we could not find evidence for newly generated neurons in response to apoptotic neuronal death. These results suggest that mitotically active progenitor populations are intrinsically capable to contribute to the repair process of injured cortical tissue and may represent a potential target for neuronal replacement strategies.

  10. Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dae-Hee, E-mail: leedneo@gmail.com [Departments of Surgery and Pharmacology and Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA (United States); Kim, Dong-Wook [Department of Microbiology, Immunology, and Cancer Biology, University of VA (United States); Jung, Chang-Hwa [Division of Metabolism and Functionality Research, Korea Food Research Institute (Korea, Republic of); Lee, Yong J. [Departments of Surgery and Pharmacology and Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA (United States); Park, Daeho, E-mail: daehopark@gist.ac.kr [School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712 (Korea, Republic of)

    2014-09-15

    Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. - Highlights: • Most GBM cells have been reported to be resistant to TRAIL-induced apoptosis. • Gingerol enhances the expression level of anti-apoptotic proteins by ROS. • Gingerol enhances TRAIL-induced apoptosis through actions on the ROS–Bcl2 pathway.

  11. Regulation of MAP kinase-dependent apoptotic pathway: implication of reactive oxygen and nitrogen species.

    Science.gov (United States)

    Sumbayev, Vadim V; Yasinska, Inna M

    2005-04-15

    Mitogen-activated protein (MAP) kinase signaling cascades are multi-functional signaling networks that influence cell growth, differentiation, apoptosis, and cellular responses to stress. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase that triggers apoptogenic kinase cascade leading to the phosphorylation/activation of c-Jun N-terminal kinases and p38-MAP kinase, which are responsible for inducing apoptotic cell death. This pathway plays a pivotal role in transduction of signals from different apoptotic stimuli. In the present review, we summarized the recent evidence concerning MAP kinase-dependent apoptotic pathway and its regulation in the mammalian cells and organism in vivo. We have shown that the key messengers of regulation of this pathway are the reactive oxygen and nitrogen species. The role of protein oxidation and S-nitrosation in induction of apoptotic cell death via ASK1 is discussed. Also we have outlined other recently discovered signal transduction processes involved in the regulation of ASK1 activity and downstream pathway.

  12. Expression of defender against apoptotic death (DAD-1) in iris and dianthus petals

    NARCIS (Netherlands)

    Kop, van der D.A.M.; Ruys, G.; Dees, D.; Schoot, van der C.; Boer, de A.D.; Doorn, van W.G.

    2003-01-01

    The gene defender against apoptotic death (DAD-1) prevents programmed cell death in animal cells. We investigated the expression pattern of DAD-1 in petals of iris (Iris x hollandica cv. Blue Magic) and carnation (Dianthus caryophyllus cv. Etarro). DAD-1 expression in Iris petals was strongly reduce

  13. Modulation of Apoptotic Signaling by the Hepatitis B Virus X Protein

    Directory of Open Access Journals (Sweden)

    Michael J. Bouchard

    2012-11-01

    Full Text Available Worldwide, an estimated 350 million people are chronically infected with the Hepatitis B Virus (HBV; chronic infection with HBV is associated with the development of severe liver diseases including hepatitis and cirrhosis. Individuals who are chronically infected with HBV also have a significantly higher risk of developing hepatocellular carcinoma (HCC than uninfected individuals. The HBV X protein (HBx is a key regulatory HBV protein that is important for HBV replication, and likely plays a cofactor role in the development of HCC in chronically HBV-infected individuals. Although some of the functions of HBx that may contribute to the development of HCC have been characterized, many HBx activities, and their putative roles during the development of HBV-associated HCC, remain incompletely understood. HBx is a multifunctional protein that localizes to the cytoplasm, nucleus, and mitochondria of HBV‑infected hepatocytes. HBx regulates numerous cellular signal transduction pathways and transcription factors as well as cell cycle progression and apoptosis. In this review, we will summarize reports in which the impact of HBx expression on cellular apoptotic pathways has been analyzed. Although various effects of HBx on apoptotic pathways have been observed in different model systems, studies of HBx activities in biologically relevant hepatocyte systems have begun to clarify apoptotic effects of HBx and suggest mechanisms that could link HBx modulation of apoptotic pathways to the development of HBV-associated HCC.

  14. Inhibition of Citrinin-Induced Apoptotic Biochemical Signaling in Human Hepatoma G2 Cells by Resveratrol

    Directory of Open Access Journals (Sweden)

    Chia-Chi Chen

    2009-07-01

    Full Text Available The mycotoxin citrinin (CTN, a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells. CTN causes cell injury, including apoptosis, but its precise regulatory mechanisms of action are currently unclear. Resveratrol, a member of the phytoalexin family found in grapes and other dietary plants, possesses antioxidant and anti-tumor properties. In the present study, we examined the effects of resveratrol on apoptotic biochemical events in Hep G2 cells induced by CTN. Resveratrol inhibited CTN-induced ROS generation, activation of JNK, loss of mitochondrial membrane potential (MMP, as well as activation of caspase-9, caspase-3 and PAK2. Moreover, resveratrol and the ROS scavengers, NAC and α-tocopherol, abolished CTN-stimulated intracellular oxidative stress and apoptosis. Active JNK was required for CTN-induced mitochondria-dependent apoptotic biochemical changes, including loss of MMP, and activation of caspases and PAK2. Activation of PAK2 was essential for apoptosis triggered by CTN. These results collectively demonstrate that CTN stimulates ROS generation and JNK activation for mitochondria-dependent apoptotic signaling in Hep G2 cells, and these apoptotic biochemical events are blocked by pretreatment with resveratrol, which exerts antioxidant effects.

  15. Boolean Model of Yeast Apoptosis as a Tool to Study Yeast and Human Apoptotic Regulations

    Science.gov (United States)

    Kazemzadeh, Laleh; Cvijovic, Marija; Petranovic, Dina

    2012-01-01

    Programmed cell death (PCD) is an essential cellular mechanism that is evolutionary conserved, mediated through various pathways and acts by integrating different stimuli. Many diseases such as neurodegenerative diseases and cancers are found to be caused by, or associated with, regulations in the cell death pathways. Yeast Saccharomyces cerevisiae, is a unicellular eukaryotic organism that shares with human cells components and pathways of the PCD and is therefore used as a model organism. Boolean modeling is becoming promising approach to capture qualitative behavior and describe essential properties of such complex networks. Here we present large literature-based and to our knowledge first Boolean model that combines pathways leading to apoptosis (a type of PCD) in yeast. Analysis of the yeast model confirmed experimental findings of anti-apoptotic role of Bir1p and pro-apoptotic role of Stm1p and revealed activation of the stress protein kinase Hog proposing the maximal level of activation upon heat stress. In addition we extended the yeast model and created an in silico humanized yeast in which human pro- and anti-apoptotic regulators Bcl-2 family and Valosin-contain protein (VCP) are included in the model. We showed that accumulation of Bax in silico humanized yeast shows apoptotic markers and that VCP is essential target of Akt Signaling. The presented Boolean model provides comprehensive description of yeast apoptosis network behavior. Extended model of humanized yeast gives new insights of how complex human disease like neurodegeneration can initially be tested. PMID:23233838

  16. Role of BK channels in the apoptotic volume decrease in native eel intestinal cells

    DEFF Research Database (Denmark)

    Lionetto, Maria Giulia; Giordano, Maria Elena; Calisi, Antonio

    2010-01-01

    of these channels in the Apoptotic Volume Decrease (AVD) of isolated eel enterocytes, and the possible interaction between BK channels and the progression of apoptosis. The detection of apoptosis was performed by confocal microscopy and annexin V and propidium iodide labelling; cell volume changes were monitored...

  17. The calcimimetic R-568 induces apoptotic cell death in prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Cheng Guangming

    2009-07-01

    Full Text Available Abstract Background Increased serum level of parathyroid hormone (PTH was found in metastatic prostate cancers. Calcimimetic R-568 was reported to reduce PTH expression, to suppress cell proliferation and to induce apoptosis in parathyroid cells. In this study, we investigated the effect of R-568 on cellular survival of prostate cancer cells. Methods Prostate cancer cell lines LNCaP and PC-3 were used in this study. Cellular survival was determined with MTT, trypan blue exclusion and fluorescent Live/Death assays. Western blot assay was utilized to assess apoptotic events induced by R-568 treatment. JC-1 staining was used to evaluate mitochondrial membrane potential. Results In cultured prostate cancer LNCaP and PC-3 cells, R-568 treatment significantly reduced cellular survival in a dose- and time-dependent manner. R-568-induced cell death was an apoptotic event, as evidenced by caspase-3 processing and PARP cleavage, as well as JC-1 color change in mitochondria. Knocking down calcium sensing receptor (CaSR significantly reduced R-568-induced cytotoxicity. Enforced expression of Bcl-xL gene abolished R-568-induced cell death, while loss of Bcl-xL expression led to increased cell death in R-568-treated LNCaP cells,. Conclusion Taken together, our data demonstrated that calcimimetic R-568 triggers an intrinsic mitochondria-related apoptotic pathway, which is dependent on the CaSR and is modulated by Bcl-xL anti-apoptotic pathway.

  18. Studies on the effects of persistent RNA priming on DNA replication and genomic stability

    OpenAIRE

    Stuckey, Ruth

    2014-01-01

    [EN]: DNA replication and transcription take place on the same DNA template, and the correct interplay between these processes ensures faithful genome duplication. DNA replication must be highly coordinated with other cell cycle events, such as segregation of fully replicated DNA in order to maintain genomic integrity. Transcription generates RNA:DNA hybrids, transient intermediate structures that are degraded by the ribonuclease H (RNaseH) class of enzymes. RNA:DNA hybrids can form R-loops, ...

  19. G-CSF protects motoneurons against axotomy-induced apoptotic death in neonatal mice

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    Pitzer Claudia

    2010-02-01

    Full Text Available Abstract Background Granulocyte colony stimulating factor (G-CSF is a growth factor essential for generation of neutrophilic granulocytes. Apart from this hematopoietic function, we have recently uncovered potent neuroprotective and regenerative properties of G-CSF in the central nervous system (CNS. The G-CSF receptor and G-CSF itself are expressed in α motoneurons, G-CSF protects motoneurons, and improves outcome in the SOD1(G93A transgenic mouse model for amyotrophic lateral sclerosis (ALS. In vitro, G-CSF acts anti-apoptotically on motoneuronal cells. Due to the pleiotrophic effects of G-CSF and the complexity of the SOD1 transgenic ALS models it was however not possible to clearly distinguish between directly mediated anti-apoptotic and indirectly protective effects on motoneurons. Here we studied whether G-CSF is able to protect motoneurons from purely apoptotic cell death induced by a monocausal paradigm, neonatal sciatic nerve axotomy. Results We performed sciatic nerve axotomy in neonatal mice overexpressing G-CSF in the CNS and found that G-CSF transgenic mice displayed significantly higher numbers of surviving lumbar motoneurons 4 days following axotomy than their littermate controls. Also, surviving motoneurons in G-CSF overexpressing animals were larger, suggesting additional trophic effects of this growth factor. Conclusions In this model of pure apoptotic cell death the protective effects of G-CSF indicate direct actions of G-CSF on motoneurons in vivo. This shows that G-CSF exerts potent anti-apoptotic activities towards motoneurons in vivo and suggests that the protection offered by G-CSF in ALS mouse models is due to its direct neuroprotective activity.

  20. Pro‑apoptotic effects of pycnogenol on HT1080 human fibrosarcoma cells.

    Science.gov (United States)

    Harati, Kamran; Slodnik, Pawel; Chromik, Ansgar Michael; Behr, Björn; Goertz, Ole; Hirsch, Tobias; Kapalschinski, Nicolai; Klein-Hitpass, Ludger; Kolbenschlag, Jonas; Uhl, Waldemar; Lehnhardt, Marcus; Daigeler, Adrien

    2015-04-01

    Complete surgical resection with clear margins remains the mainstay of therapy for localised fibrosarcomas. Nevertheless, metastatic fibrosarcomas still represent a therapeutic dilemma. Commonly used chemotherapeutic agents like doxorubicin have proven to be effective in pycnogenol and its constituents on human fibrosarcoma cells (HT1080). Ten healthy subjects (six females, four males, mean age 24.8 ± 6 years) received a single dose of 300 mg pycnogenol orally. Blood plasma samples were obtained before and 6 h after intake of pycnogenol. HT1080 cells were treated with these plasma samples. Additionally, HT1080 were incubated separately with catechin, epicatechin and taxifolin that are known as the main constituents of pycnogenol. Vital, apoptotic and necrotic cells were quantified using flow cytometric analysis. Gene expression was analyzed by RNA microarray. The results showed that single application of taxifolin, catechin and epicatechin reduced cell viability of HT1080 cells only moderately. A single dose of 300 mg pycnogenol given to 10 healthy adults produced plasma samples that led to significant apoptotic cell death ex vivo whereas pycnogenol-negative serum displayed no apoptotic activity. Microarray analysis revealed remarkable expression changes induced by pycnogenol in a variety of genes, which are involved in different apoptotic pathways of cancer cells [Janus kinase 1 (JAK1), DUSP1, RHOA, laminin γ1 (LAMC1), fibronectin 1 (FN1), catenin α1 (CTNNA1), ITGB1]. In conclusion, metabolised pycnogenol induces apoptosis in human fibrosarcoma cells. Pycnogenol exhibits its pro-apoptotic activity as a mixture and is more effective than its main constituents catechin, epicatechin and taxifolin indicating that the metabolised components interact synergistically. These results provide experimental support for in vivo trials assessing the effect of the pine bark extract pycnogenol.

  1. New insights into the apoptotic process in mollusks: characterization of caspase genes in Mytilus galloprovincialis.

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    Alejandro Romero

    Full Text Available Apoptosis is an essential biological process in the development and maintenance of immune system homeostasis. Caspase proteins constitute the core of the apoptotic machinery and can be categorized as either initiators or effectors of apoptosis. Although the genes encoding caspase proteins have been described in vertebrates and in almost all invertebrate phyla, there are few reports describing the initiator and executioner caspases or the modulation of their expression by different stimuli in different apoptotic pathways in bivalves. In the present work, we characterized two initiator and four executioner caspases in the mussel Mytilus galloprovincialis. Both initiators and executioners showed structural features that make them different from other caspase proteins already described. Evaluation of the genes' tissue expression patterns revealed extremely high expression levels within the gland and gills, where the apoptotic process is highly active due to the clearance of damaged cells. Hemocytes also showed high expression values, probably due to of the role of apoptosis in the defense against pathogens. To understand the mechanisms of caspase gene regulation, hemocytes were treated with UV-light, environmental pollutants and pathogen-associated molecular patterns (PAMPs and apoptosis was evaluated by microscopy, flow cytometry and qPCR techniques. Our results suggest that the apoptotic process could be tightly regulated in bivalve mollusks by overexpression/suppression of caspase genes; additionally, there is evidence of caspase-specific responses to pathogens and pollutants. The apoptotic process in mollusks has a similar complexity to that of vertebrates, but presents unique features that may be related to recurrent exposure to environmental changes, pollutants and pathogens imposed by their sedentary nature.

  2. SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Herbert, Katharine J.; Cook, Anthony L., E-mail: Anthony.Cook@utas.edu.au; Snow, Elizabeth T., E-mail: elizabeth.snow@utas.edu.au

    2014-06-15

    Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment. - Highlights: • Impaired microRNA biogenesis promotes apoptotic resistance in HaCaT keratinocytes. • TP53 mutations suppress miR-34a-mediated regulation of SIRT1 expression. • SIRT1 inhibition increases p53 acetylation in HaCaTs, restoring apoptosis.

  3. Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis

    Science.gov (United States)

    Millet, Arnaud; Martin, Katherine R.; Bonnefoy, Francis; Saas, Philippe; Mocek, Julie; Alkan, Manal; Terrier, Benjamin; Kerstein, Anja; Tamassia, Nicola; Satyanarayanan, Senthil Kumaran; Ariel, Amiram; Ribeil, Jean-Antoine; Guillevin, Loïc; Cassatella, Marco A.; Mueller, Antje; Thieblemont, Nathalie; Lamprecht, Peter; Mouthon, Luc; Perruche, Sylvain; Witko-Sarsat, Véronique

    2015-01-01

    Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology. PMID:26436651

  4. Abl kinase inhibits the engulfment of apoptotic [corrected] cells in Caenorhabditis elegans.

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    Michael E Hurwitz

    2009-04-01

    Full Text Available The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In Caenorhabditis elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One pathway includes the adaptor protein CED-2 CrkII and the small GTPase CED-10 Rac, and acts to rearrange the cytoskeleton of the engulfing cell. The other pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to extend the surface of the engulfing cell. Although many components required for engulfment have been identified, little is known about inhibition of engulfment. The tyrosine kinase Abl regulates the actin cytoskeleton in mammals and Drosophila in multiple ways. For example, Abl inhibits cell migration via phosphorylation of CrkII. We tested whether ABL-1, the C. elegans ortholog of Abl, inhibits the CED-2 CrkII-dependent engulfment of apoptotic cells. Our genetic studies indicate that ABL-1 inhibits apoptotic cell engulfment, but not through CED-2 CrkII, and instead acts in parallel to the two known engulfment pathways. The CED-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs during the development of the C. elegans gonad. The loss of ABL-1 function partially restores normal DTC migration in the CED-10 Rac pathway mutants. We found that ABI-1 the C. elegans homolog of mammalian Abi (Abl interactor proteins, is required for engulfment of apoptotic cells and proper DTC migration. Like Abl, Abi proteins are cytoskeletal regulators. ABI-1 acts in parallel to the two known engulfment pathways, likely downstream of ABL-1. ABL-1 and ABI-1 interact physically in vitro. We propose that ABL-1 opposes the engulfment of apoptotic cells by inhibiting ABI-1 via a pathway that is distinct from the two known engulfment pathways.

  5. Impact of alginate-producing Pseudomonas aeruginosa on alveolar macrophage apoptotic cell clearance.

    Science.gov (United States)

    McCaslin, Charles A; Petrusca, Daniela N; Poirier, Christophe; Serban, Karina A; Anderson, Gregory G; Petrache, Irina

    2015-01-01

    Pseudomonas aeruginosa infection is a hallmark of lung disease in cystic fibrosis. Acute infection with P. aeruginosa profoundly inhibits alveolar macrophage clearance of apoptotic cells (efferocytosis) via direct effect of virulence factors. During chronic infection, P. aeruginosa evades host defense by decreased virulence, which includes the production or, in the case of mucoidy, overproduction of alginate. The impact of alginate on innate immunity, in particular on macrophage clearance of apoptotic cells is not known. We hypothesized that P. aeruginosa strains that exhibit reduced virulence impair macrophage clearance of apoptotic cells and we investigated if the polysaccharide alginate produced by mucoid P. aeruginosa is sufficient to inhibit alveolar macrophage efferocytosis. Rat alveolar or human peripheral blood monocyte (THP-1)-derived macrophage cell lines were exposed in vitro to exogenous alginate or to wild type or alginate-overproducing mucoid P. aeruginosa prior to challenge with apoptotic human Jurkat T-lymphocytes. The importance of LPS contamination and that of structural integrity of alginate polymers was tested using alginate of different purities and alginate lyase, respectively. Alginate inhibited alveolar macrophage efferocytosis in a dose- and time-dependent manner. This effect was augmented but not exclusively attributed to lipopolysaccharide (LPS) present in alginates. Alginate-producing P. aeruginosa inhibited macrophage efferocytosis by more than 50%. A mannuronic-specific alginate lyase did not restore efferocytosis inhibited by exogenous guluronic-rich marine alginate, but had a marked beneficial effect on efferocytosis of alveolar macrophages exposed to mucoid P. aeruginosa. Despite decreased virulence, mucoid P. aeruginosa may contribute to chronic airway inflammation through significant inhibition of alveolar clearance of apoptotic cells and debris. The mechanism by which mucoid bacteria inhibit efferocytosis may involve alginate

  6. Mycobacterium tuberculosis blocks annexin-1 crosslinking and thus apoptotic envelope completion on infected cells to maintain virulence

    Science.gov (United States)

    Gan, Huixian; Lee, Jinhee; Ren, Fucheng; Chen, Minjian; Kornfeld, Hardy; Remold, Heinz G.

    2017-01-01

    Macrophages infected with attenuated Mycobacterium tuberculosis strain H37Ra become apoptotic, limiting bacterial replication and facilitating antigen presentation. Here, we demonstrate that cells infected with H37Ra became apoptotic after formation of an apoptotic envelope on their surface was complete. This process required exposure of phosphatidylserine on the cell surface followed by deposition of the phospholipid-binding protein annexin-1 and then transglutaminase-mediated crosslinking of annexin-1 via its N-terminal domain. In macrophages infected with virulent strain H37Rv, in contrast, the N-terminal domain of annexin-1 was removed by proteolysis thus preventing completion of the apoptotic envelope, which results in macrophage death by necrosis. Host defense of virulent Mycobacterium tuberculosis thus occurs by failure to form the apoptotic envelope, which leads to macrophage necrosis and dissemination of infection in the lung. PMID:18794848

  7. DNA glue

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V; Astakhova, Irina V.; Malakhov, Andrei D.

    2008-01-01

    Significant alterations in thermal stability of parallel DNA triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in the structure of phenylmethylglycerol inserted as a bulge into DNA (TINA). Insertions of two ortho-TINAs as a pseudo...

  8. Organochlorinated pesticide degrading microorganisms isolated from contaminated soil.

    Science.gov (United States)

    Lovecka, Petra; Pacovska, Iva; Stursa, Petr; Vrchotova, Blanka; Kochankova, Lucie; Demnerova, Katerina

    2015-01-25

    Degradation of selected organochlorinated pesticides (γ-hexachloro